1. food and drink
2. food
3. grains and pasta

Plant Biotech Denmark
Annual meeting 2014
January 29 - 30Fa
Faculty of Science
University of Copenhagen
PBD
Plant Biotech Denmark
Cover photo: Distribution of iron in a rice grain analysed by laser-ablation ICP-MS. Colours from blue to red show
increasing iron concentrations. Iron is mainly localized in the aleurone layer as well as in the scutellum of the embryo.
Work by Thomas Hesselhøj Hansen, Daniel Persson, Søren Husted and Jan K. Schjørring, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
2
Programme
WEDNESDAY - January 29, 2014
Page
09.00 - 9.30
Registration, coffee/tea and croissant
09.30 - 9.35
Welcome, by Henrik Brinch-Pedersen, Head of steering
committee, Plant Biotech Denmark
Session 1: Products/Signaling and cellular trafficking
Chair: Anja Thoe Fuglsang
09.35 - 10.20
Keynote talk - Signaling and cellular trafficking:
How LysM effectors contribute to fungal pathogenicity, by
Professor BPHJ (Bart) Thomma, Wageningen University, NL
6
10.20 - 10.40
Enzymatically modified-Gum Arabic using Arabidopsis betaglucuronosyltransferase has altered emulsion property, by
Postdoc Adiphol Dilokpimol, Section for Plant Glycobiology,
Department of Plant and Environmental Sciences, University of
Copenhagen
7
10.40 - 11.00
Super-resolution microscopy for studying vesicular
transport and membrane proteins, by PhD Student Iwona
Ziomkiewicz, Section for Transport Biology, Department of Plant
and Environmental Sciences, University of Copenhagen
8
11.00 - 11.15
Short break, Coffee/tea and fruit
Session 2: Nutrition/Diseases
Chair: Tom Hamborg Nielsen
11.15 - 12.00
Keynote talk - Nutrition:
Root plasticity as a response to the nutritional environment,
by Postdoc Benjamin Gruber, Dept. Physiology and Cell Biology,
IPK Gatersleben, Germany
9
12.00 - 12.20
Transcriptome analysis of the barley seed transfer-cells: a
matter of mineral loading capacity, by Postdoc Behrooz
Darbani, Department of Molecular Biology and Genetics,
Research Centre Flakkebjerg, Aarhus University
10
12.20 - 12.40
Responses to drought and pathogen stress are mediated by
promoter activation of the barley HvNAC6 transcription
factor, by Postdoc Yan-Jun Chen, Section for Plant
Biochemistry, Department of Plant and Environmental Sciences,
University of Copenhagen
11
12.40 - 13.30
Lunch
3
Programme
Session 3: Breeding - quality and productivity/Synthetic and systems biology
Chair: Søren K. Rasmussen
13.30 - 14.15
Keynote talk - Breeding - quality and productivity:
Genome-wide analysis of genetic variation in plants and its
use in plant breeding, by Professor Michele Morgante,
University of Udine, Italy
12
14.15 - 14.35
Hydroxynitrile glucosides in barley, by Postdoc Eva Knoch,
Section for Plant Biochemistry, Department of Plant and
Environmental Sciences, University of Copenhagen
13
14.35 - 14.55
Proteome analysis of the barley aleurone layer for insight
into plant protein secretion and programmed cell death, by
Postdoc Gregorio Barba-Espín, Department of Systems Biology,
Technical University of Denmark
14
14.55 - 15.25
Coffee/tea, cake and fruit
Session 4: Poster session with elevator talks
Chair: Birte Svensson
15.25 - 16.20
3 minute talks based on abstracts (lecture hall)
16.20 - 18.00
Poster session in the Marble Hall – Wine/beer and snacks
18.00 - 22.00
Dinner at Gumle, Thorvaldsensvej 40
4
15
Programme
THURSDAY - January 30, 2014
Session 5: EPSO
Chair: Kåre Lehmann Nielsen
9.00 - 9.30
EPSO – ensuring a future for plant science & scientists in
Europe, by Director Karin Metzlaff, The European Plant Science
Organization (EPSO)
16
Session 6: Plants for healthy food
Chair: Kåre Lehmann Nielsen
9.30 - 10.10
Biofortification to enhance iron, zinc and pro-Vitamin A
concentration in the rice grain, by Senior scientist Inez H
Slamet-Loedin, International Rice Research Institute, The
Philippines
10.10 - 10.40
Coffee/tea, cinnamon roll and fruit
10.40 - 11.15
Technological and physiological functionality of rye and oat
fibre, by Professor Anu Kaukovirta-Norja, VTT Technical
Research Centre of Finland
18
11.15 - 11.50
Heterologous synthesis of omega-3 long chain
polyunsaturated fatty acids in transgenic plants via iterative
metabolic engineering: a terrestrial source of fish oils, by
Professor Johnathan Napier, Rothamsted Research, UK
19
11.50 – 12.25
Celiac Disease and Gluten-Free Foods - Challenges and
Opportunities, by Professor Peter Köhler, The German
Research Centre for Food Chemistry, Leibniz Institut, Germany
20
12.25 - 13.15
Lunch
17
Session 7: Technologies
Chair: Poul Erik Jensen
13.15 - 13.35
Plant miRNAs: Functions and Mechanisms, by PhD student
Laura Arribas Hernandez, Department of Biology, Bioinformatics,
University of Copenhagen
21
13.35 - 13.55
Small RNA regulation & function in nodulation symbiosis, by
PhD student Dennis Berg Holt, Department of Molecular Biology
and Genetics - Plant Molecular Biology, Aarhus University
22
13.55 - 14.35
Recent insights into the enzymatic conversion of
lignocellulosic biomass, by Professor Vincent Eijsink,
Department of Chemistry, Biotechnology and Food Science,
Norwegian University of Life Sciences, Norway
23
14.35 - 15.00
Coffee/tea
15.00 - 17.00
Master class
5
Session 1. Products/Signaling and cellular trafficking
Key Note Talk
How LysM effectors contribute to fungal pathogenicity
Bart P.H.J. Thomma
Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, 6708PB
Wageningen, the Netherlands
[email protected]
Fungi occupy a plethora of niches and play essential roles in diverse environments through
decomposition of organic material as saprophytes or through establishment of symbiotic
relationships with plants and animals that range from mutually beneficial to pathogenic.
During colonization of their niches, fungi secrete effectors that mediate the establishment of
interactions with host organisms. Some of these effectors are widespread among pathogens,
such as LysM effectors that carry no recognizable protein domains other than lysin motifs
(LysMs), well-known carbohydrate-binding protein domains.
Chitin is the major constituent of fungal cell walls and a well-described ‘microbe-associated
molecular pattern’ that is recognized by host cell surface receptors that activate an immune
response. Like fungal LysM effectors, these host chitin receptors contain extracellular LysMs.
We have shown that LysM effectors of various fungal species prevent recognition of chitin by
host immune receptors, although the mechanism to compete for chitin binding with these
host receptors remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal
tomato pathogen Cladosporium fulvum recently revealed intrachain LysM dimerization,
leading to a chitin-binding groove that is deeply buried in the Ecp6 effector protein, mediating
chitin binding with ultra-high affinity.
Likely, in addition to suppression of chitin-triggered immunity, more functions for LysM
effectors exist in fungal pathogens. Recent data from our research on LysM effector
functions will be discussed.
6
Session 1. Products/Signaling and cellular trafficking
Enzymatically modified-Gum Arabic using Arabidopsis beta-glucuronosyltransferase
has altered emulsion property
Adiphol Dilokpimol and Naomi Geshi
Section for Plant Glycobiology, Department of Plant and Environmental Sciences, University
of Copenhagen, Denmark.
Gum Arabic (Acacia gum, E414) is one of the most commonly used food hydrocolloids as an
emulsifier, stabilizer and thickener (e.g., soft drinks, confectionaries, wine). In addition to the
functions described above, Gum Arabic acts as prebiotic and dietary fiber which also helps
reduce caloric intake and certificated as generally recognized as safe (GRAS) by U.S. Food
and Drug Administration (1). Gum Arabic is an exudate plant gum that is obtained from
stems and branches of Acacia senegal and A. seyal trees upon wounding. The structure of
Gum Arabic is complex and heterogeneous comprising of branched polysaccharides (type II
arabinogalactans, type II AGs) and (glyco)proteins. Type II AG is heterogeneous but
commonly composed of beta-1,3-galactan backbone substituted at O6 position with beta-1,6galactan side chains, which can then be further substituted by arabinose and its
oligosaccharides, and less frequently with other sugars, e.g, (4-O-methyl)-glucuronic acid,
rhamnose and fucose (2). At least 10 functionally distinct glycosyltransferases (GTs) are
required for synthesis of type II AG. Among a few characterized AG related GTs, we recently
reported a β-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana (3), which
catalyzes the transfer of glucuronic acid (GlcA) to both β-1,6-galactan side chain and β-1,3galactan main chain in type II AG (3).
Gum Arabic has been modified chemically and/or physically to change the physicochemical
properties and functionality (e.g., SUPERGUMTM, TICAmulsion®, 4). Since increasing
electrolyte groups in the hydrocolloid molecule can advance its solubility and result in
alteration of its physicochemical properties (5), we attempted to improve the physicochemical
properties of Gum Arabic using recombinant AtGlcAT14A. High level of GlcA was
incorporated to Gum Arabic by recombinant AtGlcAT14A. The preliminary results showed
that the particle sizes of oil-emulsion prepared by modified Gum Arabic was smaller than that
of the unmodified gum, which indicates improved emulsifying activity by enzymatically
modified Gum Arabic. Thus, enzymatic treatment is an alternative approach to modify the
structure and properties of Gum Arabic.
References
1 Ellis, M., Egelund, J., Schultz, C. J. et al. (2010). Plant Physiol. 153, 403-419.
2 Tan, L., Varnai, P., Lamport, D. T. A. et al. (2010). J. Biol. Chem. 285, 24575-24583.
3 Knoch, E., Dilokpimol, A., Tryfona, T. et al. (2013). Plant J., doi: 10.1111/tpj.12353.
4 EP1612225-A1, EP1734056-A1, EP1365773-B1.
5 Al-Assaf, S., Phillips, G. O. (2009). Food Sci. Technol. 23, 17-20.
7
Session 1. Products/Signaling and cellular trafficking
Super-resolution microscopy for studying vesicular transport and membrane proteins
Iwona Ziomkiewicz, Johannes Liesche, Thomas Günther Pomorski, Alexander Schulz
Department of Plant and Environmental Sciences, Faculty of Life Sciences, Copenhagen
University
The ability to visualise living cells made fluorescence microscopes one of the basic tools for
cell biologists. With the advent of confocal microscopes, not only the morphology of the cells
and larger organelles could be visualised, but also sub-organelle structures and dynamic
processes in cells. However, resolution of light microscopy principally limits observation to
structures larger than some 200 nm. Smaller structures like vesicles, the cytoskeleton or
macromolecular complexes could only be resolved by electron microscopy which requires a
complicated sample preparation and is not compatible with living cells. Recently, a few
approaches have been developed which circumvent the theoretical resolution limit of the light
microscope and allow for imaging with resolution down to 20 nm (1). We show here how two
different super resolution techniques, 3-dimensional structured illumination microscopy (3DSIM) and direct stochastic optical reconstruction microscopy (dSTORM) help to study
vesicular transport and organization of proteins in plasma membranes of mammalian and
plant cells, respectively.
In collaboration with the group of Jakob B. Sørensen, UCPH, we applied 3D-SIM in other to
understand the role of Vit1a, which is one of the SNARE proteins, in the formation of
exocytotic vesicles in mouse chromaffin cells (1). Vit1a is known to be involved in organelle
fusion, but has also recently been shown to play a role in exocytosis. In order to identify the
vesicle maturation stage in which Vit1a is involved, we performed a series of colocalisation
experiments. Due to their strong secretory activity, chromaffin cells are extremely rich in
secretory vesicles with a size of 50-100 nm. Thus, confocal microscopes cannot discriminate
different vesicle classes. Application of 3D-SIM, which gives two-fold improvement of
resolution in all three dimensions, allowed separation of vesicle classes and localization of
Vit1a to the trans-Golgi network and, partially, to immature vesicles. This technique has also
been shown to be suitable for imaging of plant tissues as it works with moderately thick
specimens (2); therefore 3D-SIM can be a valuable tool for plant biologists studying
membrane trafficking.
The other super-resolution technique, dSTORM gives a resolution down to 20 nm in lateral
direction and vertical of around 100 nm. Here we show one of the first, to our knowledge,
dSTORM images of plant cells. We used this technique and direct immune detection to
visualize organization of ENOD-L9, which is a GPI-anchor protein that exclusively occurs in
the plasma membrane of sieve elements. The extremely high resolution enabled us to observe clustering of the protein into nanodomains and domains and allowed for close approximation of their size to 100-200 nm on average. Moreover dSTORM could be used to resolve
100nm cellulose fibrils in outer epidermis cell walls stained with a cellulose-specific dye.
Super-resolution microscopy is a relatively new technology with only a few reports of its use
in plant science. Our examples make us confident that it will be a useful tool for the study of
membrane proteins, protein-protein interaction and even morphology of macromolecular
complexes.
(1) Weber JP et al. (2010) EMBO J 29: 2477-2490, (2) Schermelleh et al. (2010), J Cell Biol
190: 165-175 (3) Fitzgibbon et al. (2010) Plant Physiol 153: 1453-1463
8
Session 2. Nutrition/Disease
Key Note Talk
Root plasticity as a response to the nutritional environment
Benjamin D. Gruber, Ricardo F.H. Giehl, Nicolaus von Wirén
Molecular Plant Nutrition, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, 06466, Germany. [email protected]
Roots are critical for the adaptation of plants to their environment and undertake roles of
nutrient sensing, foraging and finally uptake. The three dimensional distribution of roots, the
root system architecture (RSA), plays a key role in each of these three processes. Plants
modify the RSA in a nutrient-dependent manner to more efficiently forage for nutrients,
ultimately increasing the roots’ access to sparingly soluble nutrients. In this manner the RSA
is the final trait resulting from a number of particularly plastic processes starting with the
initial sensing of the available nutrient reserves, both internally and externally, working via
signal transduction pathways to finally influence root developmental processes such as root
elongation or lateral root initiation. We are currently working on aspects of nutrient sensing
and the characterisation of the degree of root plasticity exhibited in response to a number of
nutrient deficiencies in Arabidopsis thaliana. Such work is starting to reveal the extent of
plasticity in roots, along with some of the molecular components governing the various steps
leading to modifications of the RSA. However, the difficulty is often in efficiently
characterising root traits in the magnitude required for such studies and in extending such
studies into the field, where roots ultimately remain in the below-ground black box.
9
Session 2. Nutrition/Disease
Transcriptome analysis of the barley seed transfer-cells: a matter of mineral loading
capacity
Shahin Noeparvar1, Behrooz Darbani1, 2, Preben Bach Holm1, Søren Borg1
1
Department of Molecular Biology and Genetics, Research Centre Flakkebjerg, Aarhus
2
University, Forsøgsvej 1, DK-4200 Slagelse, Denmark, Department of Plant and
Environmental Sciences, University of Copenhagen, 1871 Frederiksberg,
Denmark
As the bottleneck of the material loading during seed development, transfer-cell tissue was
microdissected for a transcriptome-wide analysis. The aim was to further improve our
previous proposed roadmap for zinc and iron trafficking and homeostasis in the barley
developing grains (Darbani et al., 2013; Tauris et al., 2009). We analyzed the transfer-cell
region from field-grown plants treated by folia application of either FeSO4 or ZnSO4.
By analyzing 259 million exon-mapped reads, we found ≈17k and ≈20k actively expressed
genes and transcripts, respectively. In response to the iron and zinc treatments, isoform
switching was found as a major mechanism represented by 40% of the differentially
expressed genes and transcripts. Different signaling and metabolic pathways, e.g., flowering
time, hormonal signaling, stress response, intracellular trafficking, DNA damage, apoptosis
and cell cycle, respiration, storage proteins, and retroelements perturbed by the treatments.
Iron showed a wider and immediate effect on the upstream hormonal signaling pathways
compared to the zinc. However, secondary negative feedback loops were the major
response to the both of treatments. Iron treatment also resulted in an earlier response of
ribosomal RNAs. By applying iron and zinc treatments, we also found a wide influence on the
different transporters including auxin efflux transporters, AHA1, arsenite transporter ARSB,
ATM3, different PM, ER, and tonoplast localized Ca transporters, Cu chaperone ATX1,
HMAs, MRPs, PDRs, OPT7, VIT, SWEETs, NRAMPs, ZIPs, YSLs, and different phosphate
and nitrate transporters.
References:
Darbani B, Briat JF, Holm PB, Husted S, Noeparvar S, Borg S. Dissecting plant iron
homeostasis under short and long-term iron fluctuations. Biotechnol Adv. 2013;31(8):1292307.
Tauris B, Borg S, Gregersen PL, Holm PB. A roadmap for zinc trafficking in the developing
barley grain based on laser capture microdissection and gene expression profiling. J Exp
Bot. 2009;60(4):1333-47.
10
Session 2. Nutrition/Disease
Responses to drought and pathogen stress are mediated by promoter activation of the
barley HvNAC6 transcription factor
Yan-Jun (Angie) Chen, David B. Collinge and Michael F. Lyngkjær
Department of Plant and Environmental Sciences, Faculty of Science, University of
Copenhagen, DK-1871 Frederiksberg C, Denmark
HvNAC6, a member of the plant-specific NAC transcription factor family is known as a
positive regulator of plant defence and to affect ABA accumulation in barley. However, little is
known about the spatial and temporal induction of HvNAC6 in response to abiotic and biotic
stresses. Therefore, we identified and characterize the HvNAC6 promoter. Using in silico
analysis, we found abundant putative cis-acting regulatory elements in the HvNAC6 promoter
which are known to respond to abiotic and biotic stresses including ABRE, W-box, GCC box,
etc. Transgenic barley plants carrying a HvNAC6 promoter::GUS reporter construct were
generated to study in vivo activation of HvNAC6 promoter during pathogen infection and
drought stress. The GUS activities were detected by fungal infection with Blumeria graminis
f. sp. hordei (Bgh) in the vascular tissues and the epidermal cells in the leaves. During
drought stress, the GUS activities were detected in the vascular tissues in both root and
leaves and meristem in the lateral roots. Interestingly, the GUS activities had two distinct
peaks during 30 hours after treatments, which predominantly consisted of HvNAC6
expression. The role of HvNAC6 in drought was further studied and we found that HvNAC6
RNAi plants had lower relative water content (RWC) than wild-type during drought stress.
This was associated with a changed expression pattern of an ABA-responsive protein kinase
indicated HvNAC6 might affect ABA accumulation in response to drought. These results
imply that HvNAC6 plays an important role in the crosstalk between abiotic and biotic
stresses.
11
Session 3. Breeding - quality and productivity/
Synthetic - and systems biology
Key Note Talk
Genome-wide analysis of genetic variation in plants and its use in plant breeding.
Michele Morgante
Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine, Via delle Scienze 208,
33100 Udine, Italy
Istituto di Genomica Applicata, Parco Scientifico di Udine, Via J. Linussio 51, 33100 Udine,
Italy
The genomics revolution of the last 15 years has improved our understanding of the genetic
make up of living organisms. The use of genomic tools has allowed us to start to unravel the
genetic make up of traits that are relevant to plant breeding. At the same time a deeper
understanding of what natural variation is at the sequence level has also been achieved,
allowing us to realize that plant genomes are characterised by high levels of structural
variation, consisting of both smaller insertion/deletions, mostly due to recent insertions of
transposable elements, and of larger insertion/deletion similar to those termed in humans
Copy Number Variants (CNVs). These observations indicate that a single genome sequence
might not reflect the entire genomic complement of a species, and prompted us to introduce
the concept of the plant pan-genome, including core genomic features common to all
individuals and a Dispensable Genome (DG) composed of partially shared and/or non
shared DNA sequence elements. Uncovering the intriguing nature of the DG, i.e. its
composition, origin and function, represents a step forward towards an understanding of the
processes generating genetic diversity and phenotypic variation. Additionally, since the DG
clearly appears to be for the most part the youngest and most dynamic component of the pan
genome, it is of great interest to understand whether it is a major contributor to the creation
of new genetic variation in plant evolution as well as in the artificial selection processes of
plant breeding. A new phase in crop evolution of targeted modifications is on the horizon
thanks to the progresses in genomics: we will describe the perspectives in this area using
examples from different crop species.
12
Session 3. Breeding - quality and productivity/
Synthetic - and systems biology
Hydroxynitrile glucosides in barley
Eva Knoch, Pernille S. Roelsgaard, Carl Erik Olsen, Birger L. Møller, Michael F. Lyngkjær
Barley contains five leucine derived hydroxynitrile glucosides (HNGs): epiheterodendrin,
epidermin, sutherlandin, osmaronin and dihydroosmaronin. HNGs are known to be plant
defence compounds that include the cyanogenic glucosides, which can release hydrogen
cyanide (epiheterodendrin is a cyanogenic glucoside). In barley, HNGs constitute more than
90% of the epidermal sugar. The content of HNGs varies between barley cultivars, ranging
from low, over medium to high. It has been suggested that increased HNG content in barley
correlates with increased susceptibility to Blumeria graminis f.sp. hordei, the fungus that
causes powdery mildew (Ibenthal et al., 1993). However, recently it was shown that high
HNG producing barley lines are more resistant to B. graminis infection, possible due to the
toxicity of their breakdown products, and that the compounds are induced in response to B.
graminis infection (Roelsgaard et al., in prep). To learn about the synthesis of HNGs and
their importance in relation to disease susceptibility we have identified candidates for the
HNG biosynthetic enzymes in barley and found that the genes are localized in two gene
clusters. Transient expression of the candidate genes in tobacco results in the production of
HNGs specific to barley. In silico expression analysis indicates that one cluster is
developmentally regulated, while the other is regulated in response to biotic stress. We are
also interested in the regulation of the HNG related genes and will investigate gene
expression during development and biotic stress, especially in response to B. graminis. At
the same time, metabolite levels will be measured and correlated with gene expression to
determine the regulation of HNGs in barley.
References:
Ibenthal W-D, Pourmohseni H, Grosskopf S, Oldenburg H, Shafiei-Azad S (1993) New
approaches towards biochemical mechanisms of resitance/susceptibility of gramineae to
powdery mildew (Erysiphe graminis). 97–106
Roelsgaard P S, et al. (in prep) Hydroxynitrile glucoside levels in barley (Hordeum vulgare)
affect the development and fitness of barley powdery mildew (Blumeria graminis f.sp. hordei)
Abstract: Please use Arial, text size 11, line spacing 1. The abstract, all included, must not
exceed one page. Please do not change margins etc.
13
Session 3. Breeding - quality and productivity/
Synthetic - and systems biology
Proteome analysis of the barley aleurone layer for insight into plant protein secretion
and programmed cell death
Gregorio Barba Espin1, Christina Mark1, Plaipol Dedvisitsakul1, Kinga Zor2, Arto Heiskanen2,
Jenny Emnéus2, Per Hägglund1, Birte Svensson1, Martin Dufva2, Christine Finnie1
1
Department of Systems Biology, Technical University of Denmark.
Department of Micro- and Nanotechnology, Technical University of Denmark.
2
Presenting author: [email protected]
The cereal grain aleurone layer is a hormone-reponsive tissue with a specialized role in
protein secretion during germination. In response to the phytohormone gibberellic acid (GA),
the aleurone layer synthesises an array of enzymes that are secreted to the endosperm for
the degradation of storage products. Subsequently, the aleurone cells undergo programmed
cell death (PCD). Another phytohormone, abscisic acid (ABA) can counteract GA. The barley
aleurone layer can be separated from the other grain tissues and retains its specific
responses to GA and ABA in culture, allowing the study of the effect of added signalling
molecules in an isolated system. Using aleurone layers incubated in vitro, application of
hormones, agents disrupting protein secretion, and measurements of redox activity and cell
death are combined with proteome analysis. Numerous proteins with roles in the secretory
pathway and ER stress have been identified. Many proteins passing through the secretory
pathway are glycosylated and N-glycoproteome analysis resulted in identification of over 70
N-glycosylated sites in over 60 barley proteins. We are using the aleurone layer as the basis
for developing a microfluidic system for plant tissue, and can monitor cell death correlated
with redox status in single aleurone layers. Monitoring of redox status in intact aleurone
layers shows an increase in reducing activity induced by GA, which can be dissected into
three components: an intracellular, cell wall- or membrane-associated, and extracellular
secreted component. These treatments and assays are currently being optimised for
incorporation into a microfluidics-based system with optical and electrochemical sensors for
parallel and real-time analysis of PCD in aleurone layers.
14
Session 4: Elevator talks
Biopharming of HBsAg in barley and tomato for development of oral vaccine
against Hepatitis B, by Laeeq Fouzia, Department of Molecular Biology and
Genetics, Research Center Flakkebjerg, Aarhus University
28
Cell wall acetylation and plant fitness: suppressor screening of the REDUCED
WALL ACETYLATION 2 reveal mutants with wild type surface permeability, by
Lorenzo Fimognari, Department of Plant and Environmental Sciences, University of
Copenhagen
33
The plasma membrane-localized Arabidopsis P4-ATPase, ALA10, catalyze
broad-specificity uptake of phospholipids and lysophospholipids, by Rosa
Laura López Marqués, Department of Plant and Environmental Sciences, University
of Copenhagen
37
Novel loci controlling metabolite sensing in Arabidopsis thaliana, by
Frederikke Gro Malinovsky, Department of Plant and Environmental Sciences,
University of Copenhagen
42
Identification of the zinc binding proteins of barley (Hordeum vulgare) grains
grown under three different zinc nutrition regimes: a state of the art label free
differential proteomics, by Giuseppe Dionisio, Department of Molecular Biology
and Genetics, Research Center Flakkebjerg, Aarhus University
47
The transporter HvIRT1 is required for optimal photosystem II activity and
grain manganese loading in barley, by Pai Pedas, Department of Plant and
Environmental Sciences, University of Copenhagen
51
Genetic dissection of vernalization response and winter survival in perennial
ryegrass, by Cristiana Paina, Department of Molecular Biology and Genetics,
Research Center Flakkebjerg, Aarhus University
59
Genome Wide Association Study (GWAS) of manganese use efficiency in
winter barley using chlorophyll a fluorescence, by Florian Leplat, Department of
Plant and Environmental Sciences, University of Copenhagen
62
The potato tuber mitochondrial proteome by Jesper F. Havelund, Department of
Molecular Biology and Genetics, Research Center Flakkebjerg, Aarhus University
70
15
Session 5: EPSO
EPSO – ensuring a future for plant science & scientists in Europe
Director Karin Metzlaff
The European Plant Science Organization (EPSO)
16
Session 6: Plants for healthy food
Biofortification to enhance iron, zinc and pro-Vitamin A concentration in the rice grain
Inez H. Slamet-Loedin and Gerard Barry
International Rice Research Institute (IRRI), Los Baños, Philippines
More than 2 billion people suffer from ‘‘hidden hunger’’ or micronutrient malnutrition. Iron
(Fe), zinc (Zn), and Vit-A deficiencies are among the most prevalent forms of micronutrient
deficiency globally, with women and children most at risk. The most prevalent cause can be
a bland diet overly dependent on starchy staples, such as rice and otherwise low in nutrient
density. A number of studies on Fe biofortification in rice have endeavored by breeding
approaches to increase rice grain iron (and zinc) content to address these problems
(reviewed by Bashir et al., 2013). However, none of these reached the target levels
recommended by nutritionist for these micronutrients: 13-14 µg/g for polished grain Fe, and
24-28 µg/g for Zn, and rice varieties with these improvements are thus not available. We
explored the over-expression and/or ectopic expression of genes for Fe (divalent anion)
chelator, Fe transporter, and Fe storage proteins for roles in enhancing grain Fe in the elite
Asian rice variety IR64; these approaches evaluated both sink and source approaches to
increase the Fe content of the grain. Field trials to evaluate the performance of this GM rice
were conducted in the Philippines and Colombia. A well-defined T-DNA integration
transgenic event showed polished grain Fe of 11 µg/g and Zn of 50 µg/g under these field
conditions. The target for a rice to address vitamin A deficiency should contain around 6 ug/g
beta carotene. Updates on golden rice projects will be presented.
17
Session 6: Plants for healthy food
Technological and physiological functionality of rye and oat fibre
Anu Kaukovirta-Norja, Juhani Sibakov, Nesli Sozer, Emilia Nordlund and Kaisa Poutanen
VTT Technical Research Centre of Finland, [email protected]
Rye and oats are typical Nordic grains, both rich in dietary fibre, the main component of
which in rye is arabinoxylan and in oats beta-glucan. Whole grain rye and oat foods are one
of the cornerstones of the healthy Nordic diet, and dietary fibre is a key component with
many suggested biological functions. Even though in both grains dietary fibre is distributed
throughout the kernel, most of it is located in the bran. This makes rye and oat brans good
sources of dietary fibre for different types of foods. The physiological and technological
properties of rye and oat brans are not solely based on their arabinoxylan and beta-glucan
polymers. Oat and rye bran fractions also contain phytochemicals associated to dietary
fibre, as well as starch and nutritionally interesting proteins. Furthermore, the insoluble
dietary fibre complex which is mainly made of arabinoxylan, cellulose and lignin, can have a
significant influence on the properties of the bran fractions.
The physiological functionality of rye and oat fibres has been extensively studied. So far, the
European Food Safety Authority (EFSA) has permitted health claims concerning normal
bowel function for rye fibre, increased faecal bulk for oat grain fibre, and reduction of blood
glucose values after a meal as well as maintenance of normal cholesterol levels for oat betaglucans. The composition and properties of bran fractions are very much dependent on the
fractionation process and further modification steps. In general, dietary fibre preparations are
challenging food ingredients in terms of formulating foods with appealing sensory quality.
Rye and oat fibres are not an exception. However, in successful cases the bran fractions
can bring technological benefits like structure formation or palatable taste in combination with
beneficial physiological functions. Bioprocessing with enzymes and microbes, in
combination with thermo-mechanical processing offers an efficient tool to tailor
multifunctional, fibre-rich ingredients for different food categories supporting a healthy diet.
18
Session 6: Plants for healthy food
Heterologous synthesis of omega-3 long chain polyunsaturated fatty acids in
transgenic plants via iterative metabolic engineering: a terrestrial source of fish oils
Johnathan A. Napier, Noemi Ruiz-Lopez, Richard P. Haslam, Sarah Usher, Olga Sayanova
Rothamsted Research. Harpenden, UK
We have been evaluating the possibility of producing omega-3 LC-PUFAs in different
transgenic hosts, to provide a sustainable source of these important nutrients. Attempts to
metabolically engineer plants with the primary biosynthetic pathway for LC-PUFAs has been
carried out in both model plants and crop species, allowing insights into factors constraining
the accumulation of these fatty acids. Specifically, a generic bottleneck resides within the
primary LC-PUFA biosynthetic pathway as a result of the “substrate dichotomy” between the
lipid-dependent desaturases and the acyl-CoA-dependent elongases which catalyze the
primary reactions. This bottleneck can be overcome through the use of acyl-CoA dependent
desaturase, though not without impact on phospholipid composition. The use of lipidomic
analyses have allowed us to identify further interventions in this pathway, ultimately leading
to the breakthrough production of a transgenic oilseed crop which contains up to 30%
omega-3 LC-PUFAs in seed oil. The practical challenges associated with implementing
synthetic biology rationales in plant metabolic engineering will also be discussed.
19
Session 6: Plants for healthy food
Celiac Disease and Gluten-Free Foods – Challenges and Opportunities
Peter Koehler
German Research Centre for Food Chemistry, Lise-Meitner-Strasse 34, 85354 Freising,
Germany
Coeliac disease (CD) is a common inflammatory disease of the small intestine that is
triggered by the storage proteins of wheat, rye, barley and possibly oats in genetically
predisposed individuals. This group of proteins (wheat gliadins and glutenins, rye secalins,
barley hordeins, oat avenins) are collectively called gluten in the field of CD. The current
treatment for CD is a lifelong strict gluten-free diet to prevent chronic enteropathy and reduce
the risk of lymphoma and carcinoma. The maximum daily intake of gluten should not exceed
20 mg, corresponding to around one hundredth of a slice of bread. The Codex Alimentarius
as well as European legislation have set the threshold for a “gluten-free” claim to 20 mg
gluten/kg product. For the food industry this situation is a challenge and an opportunity at the
same time. Producing high quality gluten-free foods from coeliac-safe raw materials as well
as from gluten-containing materials rendered gluten-free is an important aim and will lead to
improved choices for CD patients. Enzymes, namely prolyl endopeptidases (PEP), play a
major role either for deglutenising gluten-containing foods or as a possible alternative
therapy of CD (“PEP pill”). Microbial transglutaminase has successfully been used to
produce gluten free beer based on barley malt. Breeding approaches aim at producing socalled synthetic wheat with a low occurrence of CD-active epitopes or at generating ultra-low
gluten (ULG) barley, in which the hordein concentration is below the threshold for gluten-free
foods. For this purpose natural variation among existing cultivars and conventional breeding
can be used. On the other hand genetic engineering approaches such as RNA interference
have also been applied.
20
Session 7: Technologies
Plant miRNAs: Functions and Mechanisms
Laura Arribas Hernandez
Department of Biology, Bioinformatics, University of Copenhagen
microRNAs (miRNAs) are small non-coding RNAs that play a central role in eukaryotic gene
regulation. They form repressive complexes with ARGONAUTE (AGO) proteins, and bring
about sequence specific mRNA repression by recruitment of AGO to complementary mRNA.
In plants, miRNA mediated gene regulation has been shown to play crucial roles in
development and in stress adaptation.
The outcome of miRNA guided repression was long understood to be endonucleolytic
cleavage of target mRNA catalyzed by the AGO protein. A number of recent reports
demonstrate, however, that translational repression without mRNA degradation is also an
important component of miRNA mediated gene regulation. This raises the difficult question of
how the choice between these two radically different modes of regulation is made at the
molecular level.
In animal cells, proteins with glycine-tryptophan (GW) repeats interact directly with AGO, and
direct non-cleavage dependent modes of mRNA regulation. No GW-interactor of AGO has
been identified in the plant miRNA pathway as yet. I will discuss the possible conservation of
this central component of miRNA-mediated gene regulation in plants, and present data on
our progress in the identification of such proteins.
21
Session 7: Technologies
Small RNAs involved in bacterial root symbiosis and its regulation in Lotus japonicus
Dennis Berg Holt, Katharina Markmann, Stig U. Andersen, Vikas Gupta and Jens Stougaard
Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and
Genetics, Aarhus University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark.
Presenting author: [email protected]
Corresponding author: [email protected]
Legume plants are able to form symbiosis with rhizobial bacteria leading to the formation of
nitrogen-fixing nodules. The symbiotic interactions and nodule organogenesis require
complex cellular signaling and reprogramming of root cells, in which miRNAs and other small
RNAs are emerging as regulatory components.
Colleagues and I have identified small RNAs potentially involved in Lotus japonicus root
symbiosis with nitrogen fixing bacteria through analysis of a large-scale ILLUMINA small
RNA sequencing dataset. Candidates are selected based on their expression pattern in the
context of symbiosis, as well as in silico analysis involving prediction of potential mRNA
targets, analysis of target expression data and degradome data from different plant species.
Small RNA expression patterns are confirmed by Stem-Loop qRT-PCR and/or Northern
Blotting. If symbiosis-dependent regulation can be confirmed, small RNA and target
transcript abundances and integrity are manipulated by transgene expression to investigate a
possible role in symbiosis. We are currently performing functional analysis on two symbiosisupregulated miRNA candidates and associated target mRNAs, which we confirmed to be
subject to endonucleolytic cleavage using RACE analysis.
This work is funded by the Danish National Research Foundation and the Oticon Foundation.
22
Session 7: Technologies
Recent insights into the enzymatic conversion of cellulose and other recalcitrant
polysaccharides
Vincent G.H. Eijsink
Norwegian University of Life Sciences (NMBU), Department of Chemistry, Biotechnology and
Food Science, P.O. Box 5003, N-1432 Ås, Norway
The efficiency of the enzymatic conversion of recalcitrant polysaccharides remains an
important limiting factor in biorefinery development. Until recently, degradation of such
polysaccharides was thought to be accomplished by the synergistic action of endo- and exoacting cellulases, i.e. normal glyoside hydrolases. Recently, however, it has been shown that
biomass degrading microbes produce a plethora of enzymes that cleave polysaccharides via
an oxidative mechanism involving molecular oxygen [1-6]. These enzymes were originally
classified as glycoside hydrolase family 61 (GH61) and Carbohydrate-Binding Module family
33 (CBM33). Today they are called Lytic Polysaccharide Monooxygenases (LPMOs) and
they are classified in the CAZy system as Auxiliary Activities family 9 and 10, respectively.
The LPMOs are unique in that they can act on surfaces, such as in crystalline cellulose,
meaning that they do not need to “extract” single polysaccharide chains from their crystalline
contexts, as is the case for glycoside hydrolases. By doing so, LPMOs create better access
to the substrate, which boosts the activity of the hydrolytic enzymes. Indeed, LPMOs
contribute to the efficiency of today’s commercial enzyme cocktails for biomass processing.
1) Vaaje-Kolstad, G. et al., Science, 330, 219-222 (2010)
2) Forsberg, Z. et al., Protein Science, 20, 1479-1483 (2011)
3) Quinlan, R. J. et al., Proc. Natl. Acad. Sci. U. S. A. 108, 15079-15084 (2011)
4) Westereng, B. et al., PLoS One. 6, e27807 (2011)
5) Phillips, C.M. et al., ACS Chem Biol. 6, 1399-1406 (2011)
6) Horn S.J. et al., Biotechnology for Biofuels 5, 45 (2012)
23
Poster abstracts
Products
Dilokpimol et al.
Fouzia et al.
Ruzanski et al.
Zagrobelny et al.
Silvestro et al.
King et al.
Fimognari et al.
Steccanella et al.
Jensen et al.
Enzymatically modified-Gum Arabic using Arabidopsis betaglucuronosyltransferase has altered emulsion property
Biopharming of HBsAg in barley and tomato for development of oral vaccine
against Hepatitis B
Detailed biochemical and structural characterization of plastidic starch
phosphorylase during barley endosperm development
Evolution of the biosynthesis of cyanogenic glucosides in Lepidoptera
Increased brassionosteroid accumulation as a tool for improving biomass
production
Heterologous expression of terpene synthases in the moss, Physcomitrella
patens
Cell wall acetylation and plant fitness: suppressor screening of the REDUCED
WALL ACETYLATION 2 reveal mutants with wild type surface permeability
Linking chlorophyll biosynthesis to photosynthesis
LevB from Bacillus subtilis is a levanase that specifically hydrolyses β2→6
bonds in grass fructan
Signaling and cellular trafficking
Ziomkiewicz et al. Super-resolution microscopy for studying vesicular transport and membrane
proteins
Andersen et al.
Improving analysis of binary protein-protein interactions by chemically induced
dimerization
Poulsen et al.
The plasma membrane-localized Arabidopsis P4-ATPase, ALA10, catalyze
broad-specificity uptake of phospholipids and lysophospholipids
Barba-Espín et al. GA3-induced aleurone layers responding to heat shock or tunicamycin provide
insight into the N-glycoproteome, protein secretion and ER stress
Lopez Marques et ALA2, an Arabidopsis lipid flippase involved in prevacuolar compartment
al.
dynamics
Jørgensen et al.
Regulation of glucosinolate transport
Geshi et al.
Plant O-glycosylation occurs in novel compartments as well as in Golgi
apparatus in the secretory pathway
Malinovsky et al.
Novel loci controlling metabolite sensing in Arabidopsis thaliana
Pireyre et al.
Specific environmental response through protein-protein interactions of MYB
transcription factors
De Porcellinis et
Investigation of the signal transduction cascade that involves the putative
al.
Ser/Thr kinase PmgA in the regulation of photosynthetic apparatus under light
stress in Synechocystis sp. PCC 6803.
Nintemann et al.
Investigating protein dynamics in planta
Larsen et al.
Identifying transporter protein function and elucidating the glucosinolate
transporter complement
Nutrition
Dionisio et al.
Clausen et al.
Baldwin et al.
Long et al.
Pedas et al.
Diseases
Chen et al.
Page
7
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38
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41
42
43
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45
46
Identification of the zinc binding proteins of barley (Hordeum vulgare) grains
grown under three different zinc nutrition regimes: a state of the art label free
differential proteomics.
Manipulation of Phosphate Transporter Gene expression in Brachypodium
distachyon
Cell wall composition in wheat straw: Interactions with nitrogen status
Manganese deficiency severely decreases photosynthesis in maize
The transporter HvIRT1 is required for optimal photosystem II activity and grain
manganese loading in barley
47
Responses to drought and pathogen stress are mediated by promoter
activation of the barley HvNAC6 transcription factor
11
24
48
49
50
51
Poster abstracts
Gjendal et al.
Madsen et al.
Susceptibility and symptom development in different barley genotypes after
infection with Bipolaris sorokiniana
Contribution of glucosinolate transport in establishing a leaf peripheral line of
defense
Breeding – quality and productivity
Knoch et al.
Hydroxynitrile glucosides in barley
Czaban et al.
Comparative genome analysis within the Lolium-Festuca complex
Blennow et al.
The structure of the starch granule affects cereal grain germination
Tanackovic et al.
Comparative analysis of starch biosynthesis in Brachypodium distachyon and
Hordeum vulgare
Byrne et al.
Genome Wide Allele Frequency Fingerprints (GWAFFS) of populations via
genotype by sequencing
Wang et al.
Genotypic differences in heat tolerance mechanisms of wheat (Triticum
aestivum L.)
Paina et al.
Genetic dissection of vernalization response and winter survival in perennial
ryegrass
Skryhan et al.
Redox regulation of starch synthases in Arabidopsis thaliana
Madsen et al.
Non-inhibited barley/wheat-feed enzyme combinations for improved feedstuff
value
Leplat et al.
Genome Wide Association Study (GWAS) of manganese use efficiency in
winter barley using chlorophyll a fluorescence
Linscheid et al.
Transport Engineering to Increase Production of Plant Natural Products
Holme et al.
Use of TALEN technology to induce mutations in barley
Rasmussen et al.
GWAS of seed quality in barley
Nagy et al.
Sequencing and genome comparison of plastid genomes of fertile and malesterile lines in perennial ryegrass (Lolium perenne L.)
Nagy et al.
Sequencing and analysis of mitochondrial genomes of fertile and male-sterile
lines in perennial ryegrass (Lolium perenne L.)
Synthetic- and systems biology
Barba-Espín et al. Proteome analysis of the barley aleurone layer for insight into plant protein
secretion and programmed cell death
Jensen et al.
Elucidating molecular regulatory mechanisms in Arabidopsis
Andersen-Ranberg Expanding the molecular diversity through Synthetic Biology: Using
et al.
combinatorial biochemistry for reconstruction of pathways to high-value and
novel diterpenes
Havelund et al.
The potato tuber mitochondrial proteome
Nielsen et al.
An assembly and expression optimization pipeline for biosynthetic pathway
reconstitution in Escherichia coli
Erthmann et al.
From ecometabolomics to synthetic biology – exploring plasticity of the
triterpenoid biosynthetic pathway
Sønderkær et al.
Development of Methods for Bulk Segregate Analysis in Polyploids to facilitate
Marker Assisted Selection in Tetraploid Potato
Farrell et al.
Expression profile of predicted secreted signal proteins in the perennial
ryegrass transcriptome
Ramos et al.
Inducible microalgae cell wall lysis and characterization for enhance product
recovery
Zygadlo Nielsen et Redirecting photosynthetic reducing power towards bioactive natural product
al.
synthesis
Rydahl et al.
High throughput retrospective antibody screening: a multi-level strategy to
characterise epitope specificity
Lund et al.
Application of a split humanized Renilla luciferase complementation assay to
determine membrane bound protein-protein interactions in the Golgi apparatus
Hansen et al.
Systems biology approach to Lycophytes and its application in evolutionary
transcriptomics
25
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53
13
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55
56
57
58
59
60
61
62
63
64
65
66
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14
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69
70
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75
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77
78
79
80
Poster abstracts
Lauridsen et al.
Crocoll et al.
Petersen et al.
Nissen et al.
Salmean et al.
Development of a protocol for Agrobacterium rhizogenes mediated
transformation of Rhodiola sp. – an approach to enhance the level of bioactive
compounds
Interconnection of Methionine Metabolism and Biosynthesis of Aliphatic
Glucosinolates in Arabidopsis thaliana
Heterologous production of valuable plant natural product in eukaryotic
microalgae
Ostreococcus tauri - a novel expression system for elucidation of the function
of cell wall related genes
Double-blind experiment shed light on protein-glycan interactions
Within each category one abstract has been selected for oral presentation.
26
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82
83
84
85
Poster abstracts
Selection committee for Products:
William Willats, University of Copenhagen
Barbara Halkier, University of Copenhagen
Eva Vincze, Aarhus University
Andreas Blennow, University of Copenhagen
Selection committee for Signaling and cellular trafficking
Jens Stougaard, Aarhus University
Anja Thoe Fuglsang, University of Copenhagen
Hans Thordahl-Christensen, University of Copenhagen
Selection committee for Nutrition
Tom Hamborg Nielsen, University of Copenhagen
Iver Jakobsen, Technical University of Denmark
Henrik Brinch-Pedersen, Aarhus University
Selection committee for Diseases
David Collinge, University of Copenhagen
Michael Lyngkjær, University of Copenhagen
Per Gregersen, Aarhus University
Selection committee for Breeding - quality and productivity
Søren K. Rasmussen, University of Copenhagen
Torben Asp, Aarhus University
Niels sandal, Aarhus University
Selection committee for Synthetic- and systems biology
Kåre Lehmann Jensen, Aalborg University
Poul Erik Jensen, University of Copenhagen
Susanne Jacobsen, Technical University of Denmark
27
Posters: Products
Biopharming of HBsAg in barley and tomato for development of oral vaccine against
Hepatitis B
Fouzia L1,2 , Dionisio G,1 Naz S 2, and Brinch-Pedersen H1*
1-Aarhus University, Research Center Flakkebjerg. Department of Molecular biology and
genetics, Forsøgsvej 1, DK-4200 Slagelse
.
2- Lahore College for Women University, Jail Road, Lahore Pakistan
Bio pharming is an emerging trend in the field of genetic engineering. The concept of
traditional vaccine is revolutionized by the introduction of subunit vaccines. In contrast to
traditional vaccine development strategy, a highly immunogenic subunit is exploited for the
development of candidate vaccine.
Hepatitis B is a significant cause of morbidity and mortality in developing countries, where a
major portion of population remain devoid of vaccines against deadly diseases due to lack of
pecuniary funds. Utilization of plants as bioreactors for the development of HBV vaccine can
open a new avenue in this context. Bio pharming for the production of oral vaccine is
desirable from many aspects. It offers a great deal of benefits from immunization stand point,
including easy access, low cost and side effects, enhanced response of immune system at
mucosal site as well as stimulation of humoral immunity.
The plant cell enzymatic machinery can carry out post translational modification quite
effectively as compared to yeast derived vaccine where chemical treatment is carried out for
folding and assembling of immunogen into virus like particles VLPs
The objective of the present study is to investigate the efficacy of tomato and barley as an
expression platform for oral vaccine against HBV. Agroinfiltration with the highly
immunogenic Pres2 and S will be deployed for transient expression assay and as an
alternate to genetic complementation and stable transformation.
28
Posters: Products
Detailed biochemical and structural characterization of the plastidic starch
phosphorylase HvPho1 during barley endosperm development
Christian Ruzanski, Katarzyna Krucewicz, Jose A. Cuesta Seijo, Sebastian Meier,
Monica M Palcic
Carlsberg Laboratory, Gamle Carlsbergvej 10, 1799 København V, Denmark
The production of starch is essential for human nutrition and represents a major metabolic
flux in the biosphere. The Mechanisms underlying the initiation of the biosynthesis of starch
remain unknown. Essentially two main paths exist to produce starch in storage organs like
barley endosperm (Figure 1). Starch synthases use ADP-glucose to produce the two major
components of starch, amylose and amylopectin. Starch phosphorylase (Pho1) uses
glucose-1-phosphate, a precursor for ADP-glucose production, to produce α-1,4-glucans.
The significance of the Pho1 pathway for starch biosynthesis is unclear. To analyze the
importance of barley Pho1 (HvPho1) for starch biosynthesis in barley endosperm we first:
analyzed HvPho1 protein production and enzyme activity level throughout endosperm
development; second: analyzed structural properties of HvPho1. We found that HvPho1 is
present as active protein already at the onset of barley endosperm development. Using
recombinantly purified protein we could show de novo production of α-1,4-glucans using
HvPho1 and glucose-1-phosphate as sole substrate. We speculate that those glucans can
act as initiators for starch granule production in barley endosperm. Structural properties and
oligomeric arrangement of HvPho1 provide clues about the low affinity of HvPho1 to large
polysaccharides like starch or amylopectin. Our results strongly indicate that HvPho1 is a key
factor in starch biosynthesis in barley.
Figure 1 – The pathways of starch synthesis in a barley endosperm cell. The cytosolic and
plastidial compartments are indicated. PGM, phosphoglucomutase; PPiase,
pyrophosphatase; AGPase, ADPglucose pyrophosphorylase; SS, starch synthase; SBE and
DBE, starch-branching enzyme and debranching enzyme; Pho1, starch phosphorylase.
29
Posters: Products
Evolution of the biosynthesis of cyanogenic glucosides in Lepidoptera
Mika Zagrobelny, Nanna Bjarnholt, Søren Bak, Birger Lindberg Møller
Plant Biochemistry Laboratory
Department of Plant and Environmental Sciences (PLEN), University of Copenhagen,
Denmark
An essential component in the co-evolution of plants and insects is the ability to produce and
handle bioactive compounds. To study the molecular mechanism behind the co-adaption in
plant–insect interactions, we have investigated the interactions between Lotus corniculatus
(Fabaceae) and Zygaena filipendulae (Zygaenidae). They both contain the cyanogenic
glucosides (also called α-hydroxynitrile glucosides) linamarin and lotaustralin which liberate
toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only
insects known able to carry out both de novo biosynthesis and sequestration of the same
cyanogenic glucosides from their food plants.
The pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the
same intermediates as the pathway from plants. In both plants and insects, convergent
evolution has led to two P450 enzymes and a glucosyl-transferase acting in sequence to
catalyze cyanogenic glucoside formation. Thus plants and insects have independently found
a way to package a cyanide bomb to fend off herbivores and predators. The first committed
enzyme of the biosynthetic pathway, CYP405, has close homologs in several species of
butterflies and moths, some of which (Heliconiini butterflies) also contain cyanogenic
glucosides. Furthermore the γ-hydroxynitrile glucoside sarmentosin, which is closely related
to cyanogenic glucosides, probably share the first biosynthetic step with the cyanogenic
glucosides. Sarmentosin is found in several species of butterflies and moths, and in a few
species both types of compounds are found. For both types of compounds it has been
hypothesized that some insect species can biosynthesize them. This implies that the ability
to biosynthesize both types of compounds may have evolved in a ditrysian ancestor common
to Papilionidae and Zygaenidae, or even earlier. The ability to produce and handle
hydroxynitrile glucosides would render butterflies and moths capable of colonizing new food
plants containing these same compounds, thus creating competitor-free niches.
Bjarnholt, N., Nakonieczny, M., Kedziorski, A., Debinsky, D.M., Matter, S., Olesen, C.E.,
Zagrobelny, M. 2012: Occurence of sarmentosin and other hydroxynitrile glucosides in
Parnassius (Papilionidae) butterflies and their food plants. J. Chem. Ecol. 38:525-37, DOI:
10.1007/s10886-012-0114-x
Jensen, N.B., Zagrobelny, M., Hjernø, K., Olsen, C.E., Houghton- Larsen, J., Borch, J.,
Møller, B.L., Bak, S. 2011: Convergent evolution in biosynthesis of cyanogenic defence
compounds in plants and insects. Nat. Commun. 2 (273), DOI: 10.1038/ncomms1271.
Zagrobelny, M., Møller, B.L. 2011: Cyanogenic glucosides in the biological warfare between
plants and insects: the Burnet moth-Birdsfoot trefoil model system. Phytochemistry 72,
1585-1592.
30
Posters: Products
Increased brassionosteroid accumulation as a tool for improving biomass production
Daniele Silvestroa, Jozef Mravecb, Poul Erik Jensena, William George Tycho Willatsb
Copenhagen Plant Science Centre, aSection for Molecular Plant Biology, bSection for
Glycobiology,Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
Biomass for the 21st century (B21st) is a unique Danish intersectoral project aiming to
expand our current knowledge and development of biomass resources and technologies
towards future areas of shipping and aviation fuels and chemicals. Within the biological part
of the project we are trying to establish novel biotechnological tools for improving the
production as well as for the specific tailoring the wheat biomass. Brassinosteroids (BR) are
naturally occurring plant hormones involved in regulation of various aspects of plant
development. Based on current knowledge about their function in plants we expect that
elevated BR levels can significantly increase the yield and quality of utilizable biomass. In our
researches we investigate these aspects on the model plant Arabidopsis thaliana and on the
recently introduced model for cereal crops - Brachypodium distachyon. We generated
constructs for overexpression of two BR biosynthetic genes DET2 and BR6OX2 and
transformed these into Arabidopsis and Brachypodium. We also obtained Brachypodium TDNA insertional mutants in DET2 gene to examine the loss of BR synthesis as well. Our
methodology to analyze this plant material involves morphological phenotyping,
determination of BR content by GS/MS, Comprehensive Microarray Polymer Profiling
(CoMPP) and confocal microscopy. Some of the results from our initial investigations will be
presented.
31
Posters: Products
Heterologous expression of terpene synthases in the moss, Physcomitrella patens
Brian King and Henrik Toft Simonsen
Section for Plant Biochemistry, Department of Plant and Environmental Sciences, Faculty of
Science, University of Copenhagen
The moss, Physcomitrella patens, is a promising host for heterologous expression and
characterization of terpenoid biosynthetic pathways as well as industrial production of
valuable terpenoid bioproducts. Moss shares many characteristics with yeast and
filamentous fungi including ease of genetic transformation and targeted gene manipulation
via highly efficient homologous recombination. Unlike yeast and bacteria, moss naturally
produces the biosynthetic precursor isopentenyl pyrophosphate both through the MVA and
MEP pathway. Furthermore, as a true plant, moss possesses the organelles associated with
plant terpenoid metabolism, namely the endoplasmic reticulum and plastids. The genome of
P. patens has been published, and encodes a single functional diterpene synthase – a
bifunctional copalyl and kaurene synthase. Disruption of this gene via homologous
recombination results in a terpenoid-free background, facilitating purification of terpenopids
produced by heterologously expressed terpene synthases from other plants. Kaurene
knockout moss lines also provide a starting point to redirect carbon flux away from native
terpenes and into heterologously expressed pathways. We demonstrate that moss can
express heterologous diterpene and sesquiterpene synthases using the endogenous
kaurene synthase promoter, resulting in production of high levels of terpenoid products.
These examples demonstrate that moss is a useful platform for production of terpenoids.
32
Posters: Products
Cell wall acetylation and plant fitness: suppressor screening of the REDUCED WALL
ACETYLATION 2 reveals mutants with wild type surface permeability
Lorenzo Fimognari1, Majse Nafisi1, Yumiko Sakuragi1
1
Copenhagen University, Department of Plant and Environmental Sciences, Thorvaldsensvej
40, Frederiksberg 1871, Denmark.
The goal of the project is to elucidate the role of plant cell wall acetylation in plant fitness.
Acetylation of plant cell wall polysaccharides attracts increasing attention because it reduces
the production of bioethanol from lignocellulosic materials, and efforts are being made to
reduce the content of cell wall acetylation in plants. Recently the gene REDUCED WALL
ACETYLATION 2 (RWA2) was isolated from the model plant Arabidopsis thaliana and was
shown to be responsible for acetylation of pectins and hemicellulose (Manabe et al. 2011).
The rwa2 mutant (hereafter rwa2) contained a reduced level of acetylation and, in addition,
showed increased resistance to the necrotrophic fungal pathogen Botrytis cinerea (Manabe
et al. 2011) as well as higher surface permeability, which may suggest that the mutants
experience elevated drought stress (Nafisi M., unpublished). These findings suggested that
the RWA2 gene could be a target for future genetic engineering for improved biomass quality
for lignocellulosic biofuel production with added feature of enhanced innate immunity.
However, the possible increased drought stress needed to be overcome for the mutant to
grow robustly in the field where drought resistance is of major importance for plant fitness.
To study this, we randomly mutagenized 30,000 rwa2 seeds and searched for suppressor
mutants that overcome the increased permeability phenotype and successfully isolated 20
suppressor lines. This suggests that the increased surface permeability may be suppressed
by secondary mutations, indicating that may exist a previously unknown genetic interaction
between cell wall acetylation and surface permeability. These mutants hold keys to revealing
genetic markers for better performances with respect to biofuel production without
compromising plant fitness. We are currently carrying out genetic as well as physiological
characterizations of these mutants.
Reference:
Manabe, Y., Nafisi, M., Verhertbruggen, Y., Orfila, C., Gille, S., Rautengarten, C., Cherk, C.,
Marcus, S. E., Somerville, S., Pauly, M., Knox, J. P., Sakuragi, Y., and Scheller, H. V. (2011).
Loss-of-function mutation of reduced wall acetylation2 in Arabidopsis leads to reduced cell
wall acetylation and increased resistance to Botrytis cinerea. PlantPhysiol.155, 1068–1078.
33
Posters: Products
Linking chlorophyll biosynthesis to photosynthesis
Verdiana STECCANELLA1, Mats Hansson2, Poul Erik Jensen1
1
Department of Plant and Environmental Sciences, Molecular Plant Biology Laboratory,
University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
2
Carlsberg Laboratory, Plant Physiology Group, Gamle Carlsberg Vej 10, DK-1799
Copenhagen V, Denmark.
The production of chlorophyll in higher plants is closely regulated in accordance with the
need for the pigment. This ensures both a sufficient supply of the light-absorbing pigments
for a fully functional photosynthetic apparatus but also avoid the accumulation of free
chlorophyll intermediates potentially leading to photo-oxidative damage.
Most steps in the biosynthesis of chlorophyll have been elucidated at the genetic and
biochemical level, but the localization and the subunit composition of some of the
biosynthetic enzymes is yet unclear. In particular the step leading to the formation of the fifth
ring in the chlorophyll molecule is not fully characterized. This step is catalyzed by the so
called aerobic cyclase. We have previously identified one catalytic subunit of this enzyme:
CHL27 (Tottey et al., 2003). CHL27 contains two irons in its active site and in order to
complete its catalytic cycle these irons need to be reduced from Fe3+ to Fe2+. So far no
enzyme involved in this has been identified despite huge efforts from many researchers. This
prompted us to search for alternative ways to reduce the two irons in CHL27. We have used
specific inhibitors of electron transport and mutants affected in regulation of electron flow to
show that this particular step in chlorophyll biosynthesis indeed is directly regulated by the
photosynthetic electron transport chain. This has interesting consequences since there now
is a direct connection between photosynthesis and the biosynthesis of photosynthetic
pigments.
Tottey, Block, Allen, Westergren, Albrieux, Scheller, Merchant & Jensen (2003)
Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the
synthesis of protochlorophyllide. Proc.Natl.Acad.Sci. USA. 100: 16119-16124.
34
Posters: Products
LevB from Bacillus subtilis is a levanase that specifically hydrolyses β2→6 bonds in
grass fructan.
Susanne L. Jensen1, Mikkel B. Diemer1, Maria Lundmark1, Flemming H. Larsen2, Andreas
Blennow1, Helle K Mogensen1, Tom H Nielsen1
1
Dept. of Plant and Environmental Sciences, Faculty of Science, Univ. of Copenhagen
Department of Food Science, University of Copenhagen, Denmark
2
Fructans are non-structural and intracellular polysaccharidesof fructose units which are
linked by either (β2→1) or (β2→6) fructosyl bonds. Fructans from grasses have a complex
structure and contain both types of bonds. To enable degradation of these mixed linkage
fructan we decided to express and characterize a bacterial enzyme, LevB from Bacillus
subtilis, which is supposed to be an endolevanase. LevB was cloned using PCR and
expressed as His6-tagged protein using the Gateway recombinational cloning system. The
expressed protein was purified to near homogeneity. The purified protein was tested using
bacterial fructan and fructans purified from grass (Lolium perenne) shoots as substrates. The
resulting products were analysed by HPAEC and NMR. We showed that the pure LevB
protein degrades specifically the (β2→6) bonds, without hydrolysing the (β2→1) bonds.
When applied together with an exo-inulinase the two enzymes degraded mixed linkage grass
fructans to fructose monomers. Grasses containing fructans may represent a rich source of
carbohydrates for biofuel or other purposes. Our enzyme provides a possibility for mobilizing
this resource. Based on its specificity we suggest that the enzyme can be used to achieve
structural information of the mixed linkage fructans.
35
Posters: Signaling and cellular trafficking
Improving analysis of binary protein-protein interactions by chemically induced
dimerization
Tonni Grube Andersen1, Alexander Schulz1,2, Meike Burow1
1
DNRF Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and
Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871
2
Frederiksberg C, Denmark, Center for Advanced Bioimaging (CAB), Department of Plant
and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871
Frederiksberg C, Denmark
Over the last few decades protein-protein interactions (PPIs) have become evident as an
essential part of many biological processes. This understanding emerged due to the
development of several methods for investigating PPIs, which have greatly increased our
knowledge of biological systems on the molecular level. At present, a combination of two or
more analytical methods with contrasting strengths and weaknesses is the norm for
establishing to what extend PPI occurs in a given system. However, one inherent problem for
virtually all employed approaches aimed at investigating binary PPIs, is the lack of a built-in
control that allows detection of false negatives in the given system. Here we describe an
approach based on integrating chemically induced dimerization (CID) into a binary multimethod PPI protocol to overcome this issue. By implementing the ability to directly induce
PPI between the investigated proteins, we can lower the rate of false negative interactions
and hence increase the sensitivity of the used method. We here show how our approach can
be engineered to be compatible with widely used systems for PPI analysis such as split
ubiquitin-based yeast-two-hybrid systems, fluorescence resonance energy transfer (FRET)
and Bimolecular fluorescence complementation (BiFC) based analytical methods.
36
Posters: Signaling and cellular trafficking
The plasma membrane-localized Arabidopsis P4-ATPase, ALA10, catalyze broadspecificity uptake of phospholipids and lysophospholipids
Lisbeth Rosager Poulsen1, Rosa L. López-Marqués1, Thomas Günther-Pomorski1, Jeffrey F.
Harper2, Reinhard Kunze3, Michael B. Palmgren1
1
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and
2
Environmental Sciences, University of Copenhagen, Denmark Department of Biochemistry
3
and Molecular Biology, University of Nevada, USA, Dahlem Centre of Plant Sciences
(DCPS), Institut für Biologie - Angewandte Genetik, Freie Universität Berlin, Germany
Abstract: Although phospholipids can rapidly diffuse laterally in biological membranes,
movement from one leaflet to the other ("flip-flop" movement) is restricted due to the energy
barrier for the translocation of the polar head group across the hydrophobic lipid bilayer.
Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry,
including membrane budding and vesiculation in the secretory pathway. Generation and
maintenance of the asymmetry requires translocation of specific phospholipids between the
two leaflets of the biological membranes in an energy-dependent manner. The inwardoriented translocation is catalyzed by proteins called flippases, and the primary candidates to
carry out this function are the P4-ATPases.
The P4-ATPase subfamily harbors 12 members in Arabidopsis named ALA1-12 for
Aminophospholipid ATPase [1]. We have previously shown, that two members of the P4
subfamily from Arabidopsis, ALA2 and ALA3, catalyze flipping of phospholipids across
cellular membranes, in this way contributing to vesicle biogenesis in the secretory and
endocytic pathways [2,3]. It has been shown that the P4-ATPases from yeast are involved in
establishing a phospholipid asymmetry across the plasma membrane [4], but so far no
phospholipid transport by a P4-ATPase has been identified in the plant plasma membrane.
Currently we are studying a new and uncharacterized P4-ATPase from Arabidopsis, ALA10.
We have located this pump at the plasma membrane, and shown that it translocates a broad
range of phospholipids. We have heterologously expressed ALA10 in yeast and found out
that the transporter translocates a very broad range of lipids ranging all the way from
sphingomyelin to lysoPC. To prove that this broad specificity is not an artifact of
overexpression in the yeast system, we set up phospholipid translocation assays in wild type
Arabidopsis and knockout plants lacking ALA10 (ala10). We have observed that fluorescent
phospholipid analogues can be taken up by wild-type root cells and incorporated into internal
membranes and that this uptake is delayed in the ala10 knock out lines. By lipid extraction
and TLC (Thin layer chromatography) analysis, we have confirmed that the lipid analogues
are being metabolized in planta after uptake. This is to our knowledge the first evidence that
plants are capable of taking up phospholipids from their surroundings. This uptake could be
physiologically relevant to lipid signaling events, where the modified lipids could be used as
signaling molecules. Alternatively, phospholipids taken up by the root could be used as a
phosphate source during phosphate starvation.
[1] Axelsen and Palmgren (2001), Plant Physiology,126(2):696-706. [2] Poulsen et al. (2008),
The Plant Cell, 20(3):658-76. [3] López-Marqués et al. (2010), Molecular Biology of the Cell,
21(5):791-801. [4] Pomorski et al. (2003), Molecular Biology of the Cell, 14(3):1240-54.
37
Posters: Signaling and cellular trafficking
GA3-induced aleurone layers responding to heat shock or tunicamycin provide insight
into the N-glycoproteome, protein secretion and ER
Gregorio Barba-Espín, Plaipol Dedvisitsakul, Per Hägglund, Birte Svensson, Christine Finnie
The growing relevance of plants for production of recombinant proteins makes understanding
the secretory machinery, including identification of glycosylation sites in secreted proteins, an
important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in
vitro respond to GA3 by secreting an array of proteins and provide a unique system for
analysis of plant protein secretion. Perturbation of protein secretion in GA3-induced aleurone
layers by two independent mechanisms: heat shock and tunicamycin treatment,
demonstrated overlapping effects on both the intracellular and the secreted proteomes.
Proteins in a total of 22 and 178 2D-gel spots changing in intensity in extracellular and
intracellular fractions, respectively, were identified by mass spectrometry. Among these are
proteins with key roles in protein processing and secretion, for example calreticulin, protein
disulphide isomerase, proteasome subunits and isopentenyl diphosphate isomerase. Sixteen
heat shock proteins (HSP) in 29 spots showed diverse responses to the treatments, with only
a minority increasing in response to heat shock. The majority, all of which were small HSPs,
decreased in heat shocked aleurone layers. Additionally, glycopeptide enrichment and Nglycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins; 53 of the
glycoproteins found in extracellular and 36 in intracellular fractions, respectively. This
represents major progress in characterisation of the barley N-glycoproteome since only four
of these sites were previously described. Overall, these findings considerably advance
knowledge of the plant protein secretion system in general and emphasise the versatility of
the aleurone layer as a model system for studying plant protein secretion.
38
Posters: Signaling and cellular trafficking
ALA2, an Arabidopsis lipid flippase involved in prevacuolar compartment dynamics
Rosa Laura López Marqués, Lisa Theorin, Lisbeth Rosager Poulsen, Kristina Faxén, Danny
Møllerup Sørensen, Thomas Günther-Pomorski and Michael Gjedde Palmgren
PUMPKIN center for Membrane Pumps in Cells and Disease, Danish National Research
Foundation, Department of plant and Environmental Sciences, Faculty of Science, University
of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
The secretory pathway is involved in several vital cellular processes, including host-pathogen
interactions, nutrient sensing, and protein sorting. In the past years, a subfamily of P-type
ATPases has been suggested to be involved in vesicle formation. P-type ATPases comprise
a large family of membrane proteins involved in pumping different ionic substrates across
biological membranes. The members of the P4 subfamily (also known as flippases) catalyze
the energy-driven translocation of lipids necessary for establishing transbilayer lipid
asymmetry, a feature necessary for correct functioning of the cells. Deletion of one or more
P4-ATPase genes causes defects in vesicle budding in various organisms and some
members of the yeast family have been shown to interact with the vesiculation machinery.
Unraveling the key features of P4-ATPase functioning is crucial to understand the
mechanisms underlying the whole secretory and endocytic pathways.
In the model plant Arabidopsis, 12 members of the P4-ATPase family have been described
(ALA1-ALA12). A physiological role has, however, only been assigned for two ALA proteins.
ALA1 is located to the plasma membrane and involved in chilling tolerance by a yet unknown
mechanism [1, 2]. ALA3 is located in Golgi membranes and involved in production of vesicles
in root cells specialized in secretion and in the developing pollen tube [3,4]. In order to
expand our knowledge on the physiological relevance of this protein family, we aimed at
characterizing the function of another member, ALA2, localized to the prevacuolar
compartment. For this purpose, we have isolated two Arabidopsis knock out lines on the Col
0 and qrt wild type backgrounds. Under 12-hour light conditions, the mutants develop shorter
stems than the wild type and our preliminary results suggest a defect in flower development
that resembles the phenotype presented by knock-out Auxin Response Factor (ARF) plants.
Biochemical characterization of ALA2 shows that the protein is a very specific
phosphatidylserine transporter and gets activated by the presence of phosphatidyl-inositol-3phosphates, a group of lipids characteristic of prevacuolar compartment membranes.
Overexpression of a GFP:ALA2 fusion in tobacco epidermal cells results in enlarged
prevacuolar compartments, a feature that resembles the overexpression of some hyperactive
ADP-rybosilation factor mutants, such as araQ93L. ARA6 is involved in the first steps of
vesicle formation in the secretory pathway, which suggests that ALA2 might also be involved
in this process. Further characterization of the plant mutant phenotypes is currently in
progress.
[1] López-Marqués RL, Poulsen LR, Palmgren MG. PLoS One. 2012;7(4):e33042. doi:
10.1371/journal.pone.0033042.
[2] Gomès E, Jakobsen MK, Axelsen KB, Geisler M, Palmgren MG. Plant Cell. 2000
Dec;12(12):2441-2454.
[3] Poulsen LR, López-Marqués RL, McDowell SC, Okkeri J, Licht D, Schulz A,
Pomorski T, Harper JF, Palmgren MG. Plant Cell. 2008 Mar;20(3):658-76.
[4] McDowell SC, López-Marqués RL, Poulsen LR, Palmgren MG, Harper JF. PLoS One.
2013 May 7;8(5):e62577. doi: 10.1371/journal.pone.0062577.
39
Posters: Signaling and cellular trafficking
Regulation of glucosinolate transport
Morten Egevang Jørgensen1, Svend Roesen Madsen1, Hussam H. Nour-Eldin1, Dietmar
Geiger2, Rainer Hedrich2, Barbara Ann Halkier1.
1
VKR Research Centre for Pro-Active Plants and DynaMo Center of Excellence, Department
of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
2
Julius-von-Sachs Institute, Molecular Plant Physiology and Biophysics, University of
Würzburg Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
Transport processes are important for the reallocation of defence compounds during
development and predation. A recent identification of two glucosinolate transporters provided
a long-sought molecular tool to study a range of questions regarding the mechanisms
underlying distribution of defence compounds using glucosinolates as a model system1. The
aim of this study was to investigate whether and in what context phosphorylation regulates
the transport activity of AtGTR1 and AtGTR2.
The results indicate a role for phosphorylation in decreasing AtGTR1 and AtGTR2 transport
activity. We used publicly available phosphoproteomics data to search for phosphorylation
sites in AtGTR1 and AtGTR2. Phosphorylation mimics of selected sites showed decreased
transport activity whereas dephosphorylation mimics had wild type transport activity. We
employed co-expression analysis to search for likely kinase candidates. Preliminary oocyte
co-expression experiments indicate that one kinase decreased the transport of both
transporters whereas another kinase decreased the transport of AtGTR2 only.
We hypothesize that AtGTR inactivation by phosphorylation is involved in a defence
response by controlling glucosinolate levels in the apoplastic space. In perspective, the
research could further our understanding of the regulation of transport processes involved in
distribution of defence compounds at the cellular, tissue and organ level.
References
1 Nour-Eldin, H. H. et al. NRT/PTR transporters are essential for translocation of
glucosinolate defence compounds to seeds. Nature 488, 531-534, doi:10.1038/nature11285
(2012).
40
Posters: Signaling and cellular trafficking
Plant O-glycosylation occurs in novel compartments as well as in Golgi apparatus in
the secretory pathway
Naomi Geshi1, Christian Peter Poulsen1, Adiphol Dilokpimol1, Herman Höfte2, Gregory
Mouille2
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
2
INRA, Unite Mixte de Recherche 1318, Institut Jean-Pierre Bourgin, Saclay Plant Sciences,
F-78000, Versailles, France
Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans
specific to plants. AGPs have been implicated in several fundamental developmental
processes such as reproduction, cell proliferation, pattern formation and growth, and in plantmicrobe interaction, however, little is known for the molecular mechanisms. AGPs consist of
a protein backbone O-glycosylated by complex polysaccharides composed mainly of
galactan and arabinose. The main component of AGPs is carbohydrate, which is added to a
protein core catalyzed by large number of glycosyltransferases (GTs) in the secretory
pathway.
We report subcellular localization of AGP GTs from Arabidopsis thaliana, which are not only
found in the Golgi apparatus of ~1 µm diameter, but also found in yet uncharacterized
smaller compartments of ~0.5 µm diameter when expressed as C-terminal fluorescencetagged proteins in Nicotiana benthamiana. This observation conforms with the previous
report that the subcellular localization of GFP-AtGALT31A was found in unidentified
organelles as well as in the Golgi apparatus in T-DNA insertional atgalt31a mutant
complemented with GFP-AtGALT31A (1), thus unlikely to be an artefact from transient
expression. Among three AGP GTs tested, approximately 80% of AtGALT31A was found in
the small compartments, and co-localized with 45 and 40% of AtGALT29A and AtGlcAT14A,
respectively. N-glycosylation enzymes of N-acetylglucosaminyltransferase I (GnTI), αmannosidase II (GMII), and sialyltransferase (ST) were rarely found in the AtGALT31Alocalized small compartments, implying a role of the AtGALT31A-compartments in a part of
AGP (O-glycan) biosynthesis rather than N-glycan processing.
The AtGALT31A-localized small compartments were neither co-localized with SYP61, a
standard marker for trans-Golgi network, nor FM4-64 stained endosome. Further, the
AtGALT31A-localized small compartments were least affected by brefeldin A and
wortmannin. The results indicate a unique feature of the AtGALT31A-localized small
compartments, which is distinct from Golgi apparatus, SYP61 localized subdomain of transGolgi network, FM4-64 stained endosome and wortmannin-vacuolated prevacuolar
compartment. In mammalian cells, both N-and O-glycosyltransferases are co-localized in the
Golgi apparatus, but distinct from each other by forming separate protein complexes. In plant
cells, formation of protein complexes were recently reported for N- and O-glycosylation
enzymes ((2),(3)), but our findings indicate the possibility of an additional level of
organization (localization to spatially distinct compartment) for glycosylation enzymes in the
secretory pathway, which is unique in plant cells.
References
1. Geshi et al. (2013) Plant J. 76, 128–137
2. Schoberer et al. (2013) Plant Physiol. 161, 1737–1754
3. Dilokpimol et al. (2013) BMC Plant Biol., under review
41
Posters: Signaling and cellular trafficking
Novel loci controlling metabolite sensing in Arabidopsis thaliana
Frederikke Gro Malinovsky, Lea Møller Jensen, Meike Burow, Dan Kliebenstein
Plants cannot move away from dangers such as chewing insects, or stressful environments,
so in order to survive the stresses of daily-life they need be able to sense dangers and
respond accordingly. Glucosinolates (GSLs) are secondary metabolites that function as
defence compounds. In our lab we have recently found that Arabidopsis sense and respond
differentially to a group of aliphatic GSLs. Therefore we use them as molecular tools to
determine how sensing and signal tranductions transpires. When applied exogenously one of
these GSLs; 3-hydroxy-propyl (3OHP) causes root meristem growth arrest in all Arabidopsis
accessions tested. But interestingly some of these accessions exhibit a hypersensitivity
towards the hydroxyalkyl GSL manifesting as leaf chlorosis. To identify the molecular
components involved in 3OHP sensing we have screened a population of recombinant
inbred lines segregating for this trait. To date we have identified several QTL across the
genome that are required for the hypersensitivity towards 3OHP. The identification of these
loci will be important for understanding how secondary metabolites are sensed.
42
Posters: Signaling and cellular trafficking
Specific environmental response through protein-protein interactions of MYB
transcription factors
Marie Pireyre, M. E. Møldrup, M. Burow
DNRF Center DynaMo, Department of Plant and Environmental Sciences, University of
Copenhagen.
Glucosinolates are Brassicaceae-specific secondary metabolites involved in plant defense
and immunity. Their synthesis is tightly controlled by a clade of six R2R3 domain MYB
transcription factors regulating the biosynthesis machinery. Mechanical stimuli like wounding
increase expression of these MYBs which in turn leads to distinct changes in glucosinolate
profiles and reduces insect herbivory. The individual MYBs induce production of different
classes of glucosinolates, which is thought to be due to their specific binding to DNA
elements through the R2R3 domain. While the R2R3 domain is highly conserved among the
six MYBs, the C-terminal domain is highly variable. Thus, we propose that MYB specificity
could be modulated by specific protein-protein interactions (PPIs) rather than by DNA binding
specificity.
Recently, all six MYBs have been shown to interact with the MYC2, 3 and 4 transcription
factors which are involved in jasmonate signaling. It remains, however, to be investigated
which properties enable MYBs to engage in protein-protein interactions (PPIs). To learn
more about the influence of the DNA binding and the PPIs on the synthesis of specific
glucosinolates, we have generated stable plants expressing chimeric MYB proteins in myb
knock-out backgrounds and determined the relative abundance of glucosinolate classes for
each chimeric MYB. Under the hypothesis that binding partners provide additional functional
specificity, we have conducted an untargeted Yeast-Two-Hybrid screen to identify putative
specific interactors. This approach has identified membrane-bound partners that might retain
the MYBs in the cytoplasm until rapid activation of the glucosinolate pathway is required.
Testing the mechanisms of MYBs transcription factor specificity could enable us to
understand how the plants modulate their chemotype and thus increase performance
towards pathogens and herbivores.
43
Posters: Signaling and cellular trafficking
Investigation of the signal transduction cascade that involves the putative Ser/Thr
kinase PmgA in the regulation of photosynthetic apparatus under light stress in
Synechocystis sp. PCC 6803
Alice Jara De Porcellinis1, Stephan Klähn2, Lisa Rosgaard1, Wolfgang R. Hess2, and Yumiko
Sakuragi 1
1
Laboratory for Molecular Plant Biology, KU-LIFE, Thorvaldsensvej 40, DK-1871
Frederiksberg, Copenhagen, Denmark.
2
Genetics and Experimental Bioinformatics, University of Freiburg, Faculty of Biology,
Schänzlestr.1, D-79104 Freiburg, Germany.
Cyanobacteria are alternative green platforms for the production of valuable organic
compounds. The elucidation of the transcriptional pathways involved in the carbon fixation
and light harvesting systems represent a crucial step towards the possibility of tweaking the
system in order to enhance the robustness of the production. PmgA is a putative Ser/Thr
kinase that has been correlated with the regulation of both photosystems stoichiometry under
light stress and photomixotrophic growth and sugar accumulation in Synechocystis sp. PCC
6803 [1-4]. The project aims at elucidating the components of the PmgA signal transduction
cascade, particularly focusing on questions as to how PmgA carries its role as a regulator
and which other gene products are involved in the cascade. Two different approaches are
used to address these questions. Global transcriptomic profiling of the pmgA mutant and the
wild type was carried out in order to identify downstream components that are regulated by
PmgA. Results show that photosystem I and phycobilisome related genes are down
regulated in the pmgA mutant as compared to the wild type with concomitant increase of
SyR1 (Synechocystis ncRNA 1), a 130 nt long non-coding transcript. It has been shown that
overexpression of SyR1 causes a severe reduction of pigments [5-6]. Therefore we
speculate the possibility that SyR1 may be involved in the PmgA-mediated regulation of the
photosynthetic apparatus. Moreover another noncoding RNA named ncr0700, is significantly
downregulated in the transcript analysis of pmgA mutant. In order to further study the
interaction occurring between PmgA, Syr1 and ncr0700 and their implications in the
regulation of photosystem stoichiometry and photomixotrophic growth, deletion and
overexpressor lines of the ncRNA have been generated and are currently under study.
[1]Sonoike K, Hihara Y and Ikeuchi M. (2001). Plant Cell Physiol 42(4): 379-384.
[2]Muramatsu M. and Hihara Y. (2003). 216: 446-453.
[3]Muramatsu M., Sonoike K., Hihara Y. (2009). Microbiology 155: 989-996.
[4]Sakuragi Y, Maeda H., DellaPenna D., Bryant D.A. (2006) Plant Physiology 141: 508-521.
[5]Mitschke et al. (2011). PNAS 108: 2124–2129.
[6]Georg J., Voss B., Scholz I., Mitschke J., Wilde A., Hess W.R. (2009) Mol Syst Biol 5:305.
44
Posters: Signaling and cellular trafficking
Investigating protein dynamics in planta
Sebastian J. Nintemann1, Tonni Grube Andersen1, Meike Burow1, Barbara Ann Halkier1
1
DNRF Center DynaMo, Department of Plant and Environmental Sciences, Faculty of
Science, University of Copenhagen, Denmark
Glucosinolates (GLS) are defense compounds found in brassicaceous plants such as the
model plant Arabidopsis thaliana and are known to respond in abundance to environmental
conditions and attack. The complex biosynthetic pathway of these specialized metabolites
serves as a fruitful model system for studying the dynamics of pathway organization. The
glucosinolate pathway involves several subcellular compartments. Moreover, different types
of GLS are synthesized partly by pathway-specific specialized, and partly by more
promiscuous enzymes. Preliminary data suggest that specific interactions between involved
proteins may play a role in GLS biosynthesis.
To study the location of the biosynthetic machinery at the whole plant as well as the cellular
level, we have developed a toolset of fluorophor-tagged biosynthetic enzymes stably
expressed in the native host Arabidopsis under the control of their native promoters. This
toolset will furthermore be used to study the subcellular spatial organization of the enzymes
and to investigate protein-protein interactions by in vivo FRET/FLIM measurements. In
parallel, we utilize a transient expression system in Nicotiana benthamiana to express the
entire GLS biosynthetic machinery, allowing us to further investigate subcellular organization,
protein-protein interactions and to measure the efficiency of GLS biosynthesis. The ability to
include the entire pathway and thus quantify GLS production is a major advantage compared
to in vitro methods or yeast-based analyses.
Together, these tools allow for investigation of protein dynamics in living plant cells. We thus
aim at elucidating the GLS biosynthetic pathway organization and its plasticity in plant
development and stress response by employing these techniques.
45
Posters: Signaling and cellular trafficking
Identifying transporter protein function and elucidating the glucosinolate transporter
complement
Bo Larsen1, Hussam H. Nour-Eldin1, Barbara Ann Halkier 1
1
DynaMo Centre of Excellence, Department of Plant and Environmental Sciences, Faculty of
Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
We use glucosinolates to study intra- and intercellular transport of metabolites to identify the
full glucosinolate transporter complement. By screening a cDNA library with transport genes
containing 10-14 transmembrane domains the first glucosinolate transporters, GRT1 and
GTR2, were discovered1. They are proton-dependent importers, facilitating glucosinolate
translocation via the plant vasculature. To identify glucosinolate exporters and novel
importers, we have built an unprecedented full-length cDNA transporter protein library
including all available Arabidopsis thaliana proteins with 2 or more transmembrane domains.
The library currently contain close to 800 known and putative transporter proteins, i.e. more
than half of all annotated transporters in Arabidopsis. The library is optimized for expression
in Xenopus laevis oocytes and is continuously expanded with protein families of special
interest and transporters identified based on co-expression. To screen for glucosinolate influx
or efflux transporter activity the vitro transcribed transporter are injected as RNA pools into
oocytes to enable high-throughput screening by analyzing the oocytes internal glucosinolate
content by LC-MS. This technology platform is a unique tool for identifying plant metabolite
transporters. Screening the library with plant metabolite extracts and high valued compounds
will lead to discoveries of both academic and commercial interest. The platform will play a
key role in advancing transport engineering and allow us to make super crops with improved
nutritional, commercial and agronomical value.
1. Nour-Eldin HH et.al (2012) NRT/PTR transporters essential for allocation of glucosinolate
defense compounds to seeds Nature. 488, 531-534.
46
Posters: Nutrition
Identification of the zinc binding proteins of barley (Hordeum vulgare) grains grown
under three different zinc nutrition regimes: a state of the art label free differential
proteomics.
Giuseppe Dionisio1, Nasir Mohammed Uddin1, Gary Woffendin2, Jenny Ho2 and Eva Vincze1
1
Aarhus University, Faculty of Science and Technology, Department of Molecular Biology
and Genetics, Forsoegsvej 1, Flakkebjerg, 4200 Slagelse, Denmark
2
Thermo Fisher Scientific Inc. Stafford House, Hemel Hempstead, Hertfordshire, United
Kingdom
Corresponding e-mail: [email protected]
In the search of novel approaches for zinc biofortification of cereal grains, we present a new
pool of zinc binding protein candidates which might act as sink proteins to store zinc in
mature barley grains. Growing barley under three different zinc fertilization regimes resulted
a correlating different Zn levels in the grain. A set of low, medium and high zinc grain has
been selected for a label free differential proteomics approach based on nanoLC-MS/MS
technique. Proteins have been extracted by a modified Osborne fractionation and subjected
to MS/MS identification on a state of the art accurate mass/resolution instrument represented
by the ThermoScientific Q-exactive platform. The MS1 profiles have been differentially
analyzed by TransOmics ver.1.1 (Waters, Mildford, USA) and MS/MS peaks analyzed by
PEAKS ver. 6.0 or the Proteome Discoverer 1.4 Platform. Results indicated that a pool of top
abundant proteins in each Osborne fraction had been differential detected and this fits to an
accumulation profile from low to high zinc containing grains. The log2 abundance parameter
had been created as fold values. Absolute quantification of the amount of the detected
proteins was possible due to a spike protein introduced into each sample for normalization
and quantitative calculations.
In order to validate the involvement of top abundant differential detected proteins as zinc
binding proteins a NTAZn2+-magnetic beads capture technique have been set up and utilized
in order to capture and enrich such proteins. For each Osborne protein fraction of the highest
zinc containing grains a Zn2+-magnetic beads capture step had been performed and bound
proteins processed directly in the beads and hence identified by nanoLC-Q-exactive MS.
Results confirmed previously identified zinc binding proteins by use of low resolution MS
instrument in our group and we identified number of new proteins due to the increased
sensitivity of the Q-exactive MS instrument. We also detected many post translational
modifications and some of them were related to the zinc binding capability of our top
candidates. Furthermore, zinc adducts have been sequenced by MS/MS and possible zinc
binding sites were identified in selected candidates. We propose that the identified major zinc
binding proteins could represent candidates for future zinc biofortification approaches.
47
Posters: Nutrition
Manipulation of Phosphate Transporter Gene expression in Brachypodium distachyon
Signe Sandbech Clausen1, Mette Grønlund1, Ingo Lenk2, Iver Jakobsen1
1
Department of Chemical and Biochemical Engineering, Risø Campus, Technical University
2
of Denmark, P.O. Box 49, DK-4000 Roskilde, Denmark, DLF Trifolium, Højerupvej 31, DK4660 Store Heddinge, Denmark
Phosphorus (P) is an essential nutrient for plant growth. Because P is one of the least plantavailable nutrients in the soil, it is often growth limiting. As a result, most plants engage in a
symbiotic relationship with arbuscular mycorrhizal (AM) fungi. The symbiosis is usually
mutualistic and highly beneficial to P uptake and growth of plants in low P soils. However,
many reports have shown mycorrhiza-induced growth depressions, e.g. in grasses. Such
observations have been explained by the classical carbon drain theory, where the fungus
turns into a functional parasite when it supplies negligible amounts of P to plants. A novel
and alternative hypothesis to the carbon drain theory suggests that growth depressions can
also be caused by P limitation.
In AM plants two phosphate (Pi) uptake routes are present: the mycorrhizal pathway and the
direct pathway. The mycorrhizal pathway can be dominating even when Pi uptake and plant
growth is unaffected and this implies that the direct Pi uptake is reduced. The non-responsive
model grass, Brachypodium distachyon is used to investigate the hypothesis that growthsuppressed mycorrhizal plants will become P limited, when the reduction in direct Pi uptake
is not fully compensated by the ‘hidden’ Pi uptake via the mycorrhizal pathway.
Thirteen phosphate transporter genes have been identified in B. distachyon and their
expression pattern analyzed in non-mycorrhizal and mycorrhizal plants grown at different
phosphate levels. Two putative direct transporter genes, BdPT4 and BdPT8, were downregulated at high phosphate levels in mycorrhizal plants and these have been selected for
further investigation. A transformation approach was used to manipulate the activity of the Pi
transport pathways, by generating over-expression and knockdown (RNAi) lines of the two
transporters. The produced transgenic B. distachyon lines will be used to investigate the
specific roles of the phosphate transporters in Pi uptake. At present initial molecular studies
are being performed to determine the expression profiles of BdPT4 and BdPT8 in the
produced transgenic lines. Based on these profiles, some lines will be selected for additional
molecular studies and elaborate physiological studies. The potential for increasing Pi uptake
efficiency in plants will be evaluated, as Pi uptake efficiency of crops might be improved if a
high activity of the direct uptake pathway in mycorrhizal plants is maintained, making the two
pathways additive instead of complementary. Additionally, two GFP tagged lines
(translational fusion to fluorescent marker) were produced in collaboration with Professor
Maria Harrison at Boyce Thompson Institute for Plant Research, Ithaca, New York, for
subcellular localization studies of the transporter proteins
48
Posters: Nutrition
Cell wall composition in wheat straw: Interactions with nitrogen status
Laëtitia Baldwin, Sylwia Glazowska, Emiko Murozuka, Louise I. Ahl, William G. Willats, Inge
Skrumsager Møller and Jan K. Schjoerring
Department of Plant and Environmental Sciences, Faculty of Science, University of
Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, DENMARK ([email protected])
The supply of nitrogen has a decisive influence on the straw yield of cereal crops. However,
very little information is available on how the composition and structure of the cell walls in the
straw are affected by increasing N status. These aspects are important for the future use of
straw as a lignocellulosic biomass feedstock for production of biomaterials and bioenergy.
The aim of the present work was therefore to study how nitrogen supply affects the cell wall
composition of wheat straw. The final goal is to develop nitrogen strategies to ensure high
straw quality and productivity. The study forms part of a large project entitled: ‘Biomass for
the 21st century (B21st)’, which is a Danish initiative running from 2012 to 2016, focusing on
integrated development of biomass and conversion technologies for biobased fuels and
chemicals.
Winter wheat plants were grown in field experiments with different rates of nitrogen
application (0, 50 and 100 kg N ha-1 applied on top of an early basic dressing of 50 kg N
ha-1). In some treatments a growth regulator with anti-gibberellin action (Moddus) was also
applied. Wheat plants in different developmental stages (from ear emergence to hard dough
growth stages, GS59 and GS87, respectively) were harvested for analysis of the composition
of the cell walls in the stem. Carbon and nitrogen concentrations were measured by IR-MS,
followed by high-throughput screening of plant cell wall structure by use of Comprehensive
Microarray Polymer Profiling (CoMPP) analysis. Finally, specific cell wall components were
quantified. Phenols of lignin component were measured together wih crystalline cellulose
content.
The main structures probed in the CoMPP analysis were cellulose and different
hemicellulose like xylan and mixed-glucans. There was also binding with pectin and
xyloglucan which are normally less abundant in wheat cell wall. Cell wall structure
glycoproteins were present too. No clear differences between the three nitrogen levels could
be observed. In contrast, there was a strongly increased probing in straw treated with growth
regulator. This was especially noticeable after extraction with NaOH, mainly reflecting noncellulosic polysaccharides
Crystalline cellulose and lignin content were both higher at maturity compared to the other
harvest dates. The concentration of these two components increased with the higher
nitrogen application (150kg.ha-1).
49
Posters: Nutrition
Manganese deficiency severely decreases photosynthesis in maize
Lizhi Long1, Pai Pedas1, Søren Husted1, Lixing Yuan2, Jan Kofod Schjoerring1
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C, DENMARK ([email protected] - [email protected] [email protected] - [email protected])
2
Department of Plant Nutrition, China Agricultural University, Yuanmingyuan West road 2,
Haidian100193 Beijing, CHINA ([email protected])
Manganese (Mn) deficiency is a nutritional disorder, frequently occurring in plants growing on
neutral to alkaline soils. Studies in barley (C-3 plant) have shown that Mn deficiency leads to
a decline in photosynthetic quantum yield efficiency, and reduces the ability of the
photosystem to perform state transitions. As an important C-4 crop species, maize is
presently the second most consumed cereal worldwide and is occupying an increasing
proportion of the global agricultural land. However, very limited information is currently
available on how Mn deficiency affects C-4 photosynthesis. To evaluate the performance and
stability of the photosynthetic apparatus in Mn deficient maize plants, we set up maize plants
in hydroponics for one week at control conditions. Subsequently, four treatments were
induced: two light levels (300 and 600 μE m-2 s-1) in combination with two Mn levels (Mn
sufficient control versus Mn starvation, the latter induced by withholding the external Mn
supply for 10 days).
The experimental treatments resulted in distinct differences in leaf Mn concentration and
photosynthetic performance. At both light intensities, control plants had sufficiently high foliar
Mn concentration to maintain chlorophyll fluorescence values (Fv/Fm), close to the optimal
level around 0.83. Mn-starved plants had about 10 times lower foliar Mn concentration than
the control plants. Despite this very drastic decrease in Mn concentration, neither lower dry
matter weight nor visible symptoms of Mn deficiency were observed in the Mn-starved plants.
Mn-starved plants had significantly lower Fv/Fm values than the control plants, showing
reduced photon use efficiency. State transitions were completely inhibited in Mn-starved
exposed to high light intensity and were more than 50% reduced in Mn-deficient plants
growing at low light intensity. Also the net photosynthetic CO2 assimilation was significantly
reduced in Mn-starved plants. In comparison with control plants, Mn-starved plants were
much less responsive to increasing light intensities and they were light saturated at a much
lower light intensity.
In conclusion, we were able to induce severe Mn deficiency in maize with no visual
symptoms (latent Mn deficiency). Plants with latent Mn deficiency had decreased utilization
of photosynthetically active light (chlorophyll a fluorescence) and were less capable of
responding to increasing light intensities by increasing net photosynthetic CO2 assimilation.
In addition, Mn-starved plants were severely inhibited in their ability to perform
photosynthetic state transitions. Therefore, it is important to prevent Mn latent deficiency in
maize crops in order to avoid yield reductions.
50
Posters: Nutrition
The transporter HvIRT1 is required for optimal photosystem II activity and grain
manganese loading in barley
Pai Pedas1, Lizhi Long1,2, Lixing Yuan2, Søren Husted1, Jan K. Schjoerring1
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C, DENMARK ([email protected] – [email protected] –
[email protected] – [email protected]); 2Department of Plant Nutrition, China Agricultural
University, Beijing 100193, CHINA ([email protected])
Manganese deficiency is a serious plant nutritional disorder in many areas of the world.
There is considerable variability among different barley genotypes with respect to their ability
to grow in soil with low manganese (Mn) avaibility [1]. This is reflected in major differences in
Mn uptake kinetics, where Mn efficient genotypes have up to four times higher uptake
capacity than inefficient genotypes [2]. The variability in Mn uptake among genotypes is
related to the expression level of the Mn transporter HvIRT1 [3]. This plasmamembrane
localized transporter is in agreement with other IRT1 homologs able to transport Mn, iron
(Fe), zinc (Zn) and cadmium (Cd) when expressed in yeast. IRT1 homologs have been
suggested to be the principal transporters for Fe uptake, especially in dicots suffering from
Fe deficeincy [4]. OsIRT1 from rice has also been shown to be important for Fe uptake when
Fe2+ is abundant in waterlogged soils [5]. However, the importance of HvIRT1 for uptake of
Fe and other essential transition elements in monocots, growing in aerated soils is largely
unknown.
The objective of the present study was to examine the in planta role of HvIRT1 by
characterizing barley lines in which the IRT1 gene had been downregulated by RNAi
interference. The level of HvIRT1 transcripts in homozygous barley hvirt1-RNAi lines was
only about 5% of the wild type level, thus showing a very substantial down-regulation.
Marked differences in Mn and Fe uptake were found between wild type, null segregants and
hvirt1-RNAi lines. All plant lines showed a similar straw biomass production and grain yield
when grown in soil without Mn-deficiency. However, the hvirt1-RNAi lines accumulated
approximately 30% less Mn in grain tissue than wild type and corresponding null lines. In
contrast, no differences in grain Fe content was observed. When the plants were grown in an
alkaline, well-aerated soil inducing Mn deficiency, the hvirt1-RNAi lines showed a strong
phenotype with decreased photosynthetic performance and Mn concentration in leaf tissue. It
is suggested that HvIRT1 is controlling the root to shoot Mn translocation and HvIRT1 is
thereby an important target for future plant breeding strategies aiming at developing Mnefficient barley genotypes.
REFERENCES
[1] Hebbern C.A., Pedas P., Schjoerring J.K., Knudsen L., Husted S. 2005. Plant and Soil
272: 233–244
[2] Pedas, P., Hebbern, C.A., Schjoerring, J.K., Holm, P.E., Husted, S. (2005). Plant
Physiology 139: 1411-1420
[3] Pedas, P., Fuglsang, A.T., Jahn, T.P., Schjoerring, J.K., Ytting, C.K., Husted, S. (2008).
Plant Physiology 148: 455-466
[4] Vert G., Grotz N., Dédaldedéchamp F., Gaymard F., Guerinot M.L., Briat J.F., Curie C.
(2002). Plant Cell 14: 1223–1233
[5] Ishimaru Y., Suzuki M., Tsukamoto T., Suzuki K., Nakazono M., Kobayashi T., Wada Y.,
Watanabe S., Matsuhashi S., Takahashi M., Nakanishi H., Mori S., Nishizawa, N.K. (2006).
Plant Journal 45: 335–346
51
Posters: Diseases
Susceptibility and symptom development in different barley genotypes after infection
with Bipolaris sorokiniana
Nele Gjendal and Michael Lyngkjær
Department of Plant and Environmental Sciences, Faculty of Science, University of
Copenhagen, 1871 Frederiksberg C, Denmark
Email: [email protected]
Fungi with a necrotrophic life style are of increasing importance in crop production worldwide. The hemi-biotrophic fungus Bipolaris sorokiniana causes Spot blotch disease in small
gain cereals and disease outbreaks have increased severely in areas that have become
warmer and more humid within the last years. Phytotoxin release by B. sorokiniana is the
causal agent of symptom occurrence in form of necrotic spots, and the number of spots is
seen as an indicator of disease severity. Mlo in barley is a G-protein coupled receptor that
acts as a negative regulator of defense mechanisms. It had been implied that barley mlo
mutants in general are more susceptible to fungal pathogens with a necrotrophic life style,
including B. sorokiniana. However, the role of mlo is not fully understood. We investigated
symptom occurrence, fungal biomass and toxin sensitivity in barley to test the importance of
the mlo mutation on susceptibility to B. sorokiniana in different genetic backgrounds. Our
observations showed that variation in susceptibility was mainly due to the genetic
background and mlo mutations only in some cases contributed to increased susceptibility. By
manipulating light conditions during B. sorokiniana infection we observed that after
incubation in darkness no symptoms occurred on the infected leaves and that these
symptomless plants often had substantial higher amount of fungal biomass, indicating that
symptoms not always correlates with fungal infection. This raises the question when and how
toxins are valuable and needed for fungal infection and colonization, and highlights the need
for a better understanding of the biology of B. sorokiniana.
52
Posters: Diseases
Contribution of glucosinolate transport in establishing a leaf peripheral line of
defense
Svend Roesen Madsen, Hussam Hassan Nour-Eldin and Barbara Ann Halkier
Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and
Environmental Sciences, Faculty of Science, University of Copenhagen, 40 Thorvaldsensvej,
DK-1871 Frederiksberg C, Denmark
Within-leaf distribution of defense compounds is an important part of a plant’s strategy to
fight against insect herbivory and reflects an arms race between these organisms spanning
millions of years. A given distribution pattern reflects a combination of biosynthesis and
transport processes. In Arabidopsis thaliana (Arabidopsis), the strategy has been to
accumulate defense compounds (glucosinolates) along the midvein and at the edge. It is
generally assumed that glucosinolates are synthesized along the vasculature, suggesting
that the edge distribution is established through transport. Recently, two glucosinolatespecific importers, GTR1 and GTR2, were identified in Arabidopsis, and these were shown to
be essential for long distance transport of glucosinolates [1, 2].
Here, we investigated the involvement of GTR-dependent glucosinolate transport processes
in establishing the peripheral line of defense in an Arabidopsis leaf. Our findings show that in
the absence of GTR transport activity, edge distribution patterns are more pronounced and
glucosinolates accumulate more predominantly in leaf tips. Furthermore, we show that
glucosinolates accumulate in the epidermis in a GTR-independent manner. Based on our
results, we propose a model for establishing the glucosinolate distribution within an
Arabidopsis leaf.
1. Nour-Eldin, H.H., et al., NRT/PTR transporters are essential for translocation of
glucosinolate defence compounds to seeds. Nature, 2012.
2. Andersen, T.G., et al., Integration of biosynthesis and long-distance transport establish
organ-specific glucosinolate profiles in vegetative Arabidopsis. Plant Cell, 2013. 25(8): p.
3133-45.
53
Posters: Breeding – quality and productivity
Comparative genome analysis within the Lolium-Festuca complex
Adrian Czaban*, Stephen Byrne, Torben Asp
Department of Molecular Biology and Genetics, Aarhus University, Research Centre
Flakkebjerg, Forsøgsvej 1, 4200 Slagelse, Denmark
*Corresponding author: [email protected]
The Lolium-Festuca complex incorporates species from the Lolium and Festuca genera that
both belong to the Poaceae family. Plants belonging to this complex are very cosmopolitan
and grown in diverse climates. They exhibit wide phenotypic variation in agriculturally
important traits, such as annuality/perenniality, establishment potential, growth speed,
nutritional value, winter hardiness, drought tolerance and resistance to grazing.
The aim of this study was to investigate the genetic background of grasses in the LoliumFestuca complex. To achieve this, we have sequenced both the genomes and
transcriptomes of four representative grass species: Lolium westerwoldicum, Lolium
multiflorum, Festuca pratensis and Lolium temulentum. Libraries of various insert sizes from
each species were sequenced on the Illumina HiSeq platform, with the goal of achieving a
40X genome coverage. We have performed de-novo genome assemblies, de-novo and
genome-guided transcriptome assemblies and utilized the combined data to generate gene
models for each species. This is enabling us to perform a comprehensive comparison of the
gene content in the sequenced genomes and shed a light on the background of their
evolution and phenotypical differences.
54
Posters: Breeding – quality and productivity
The structure of the starch granule affects cereal grain germination
Andreas Blennow1, Shahnoor S. Shaik 1, Massimiliano Carciofi1, Kim H. Hebelstrup2, Helle J.
Martens1
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
2
Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, 4200
Slagelse, Denmark
Cereal grain germination is central for plant early development and efficient germination has
a major role in crop propagation and malting. Endosperm starch is the prime energy reserve
in germination and in this study we hypothesized that optimized starch granule structure, not
only the endosperm starch content per se, is important for germination and seedling
establishment. For that purpose we used barley wild-type (WT) and specifically engineered
degradable hyper-phosphorylated (HP) starch and more resistant amylose-only (AO) starch
barley lines. None of the engineered lines showed severe phenotypic effects on germination
and the WT and HP lines degraded the starch similarly having 30% residual starch after 12
days of germination. However, the AO showed significant degradative resistance having
almost twice as much of residual starch. Interestingly, protein degradation was stimulated for
both HP and AO lines as compared to WT and BG (BG) degradation was moderately
stimulated. AT later germination stages, specific sugars were rapidly consumed in the AO
line due to carbon starvation. α-amylase activity was distinctly suppressed in both the HP
and the AO lines. Pre-germination β-amylase deposition was low in the AO grains and was
generally suppressed in both HP and AO lines over germination. Scanning electron
microscopy of grain sections demonstrated differential patterns of degradation. The data
conclude that correctly structured endosperm starch granules are vital for establishing
correct initiation and maintenance of metabolic dynamics during grain germination and suboptimal granule structure leads to temporal and compensating re-directions of starch, sugar
and protein catabolism.
55
Posters: Breeding – quality and productivity
Comparative analysis of starch biosynthesis in Brachypodium distachyon and
Hordeum vulgare
Vanja Tanackovic1*, Jan T. Svensson1, Alain Buléon2, Mikkel A. Glaring1, Susanne Langgård
Jensen1, Massimiliano Carciofi1, Andreas Blennow1
1
2
Department of Plant and Environmental Sciences, Faculty of Science, KU
INRA, UR1268 Biopolymères Interactions Assemblages, Nantes, France
Brachypodium distachyon is a wild grass that was recently introduced as a model plant for
temperate cereals. In order to explore pre-domesticated grain qualities and possible wild
grass features of cereal grain and starch metabolism, we aimed to establishing
Brachypodium as a model plant for starch metabolism. We identified a comprehensive
number of starch biosynthesis genes including 7 soluble starch synthases (SSs), 2 granule
bound starch synthases (GBSSs), 4 starch branching enzymes (SBEs), 2 glucan- and 1
phosphoglucan- water dikinase (GWD, PWD). All sequences were clustered phylogenetically
in functional groups typical for plants demonstrating very conserved starch biosynthesis.
Plastid targeting sequence motifs and putative carbohydrate-binding modules (CBMs) of the
families CBM20, CBM45, CBM48 and CBM53 were identified. Grain starch micro structure,
granule size, amylopectin chain length distribution, phosphate- and amylose content as well
as grain starch, protein and β-glucan content from Brachypodium were analysed providing
comparative data for Brachypodium with barley (Hordeum vulgare). Starch content was low
and protein and β-glucan content were high as compared to barley grain. Brachypodium
starch granules were relatively small, and wide-angle X-ray scattering (WAXS) and
differential scanning calorimetry (DSC) revealed low crystallinity and high disorder of
Brachypodium starch granules as compared to barley. Amylopectin chain distribution and
amylose were similar in barley and Brachypodium but Brachypodium had less starchphosphate content than barley.
56
Posters: Breeding – quality and productivity
Genome Wide Allele Frequency Fingerprints (GWAFFS) of populations via genotype
by sequencing.
Stephen Byrne1, Czaban A1, Asp T1
1
Department of Molecular Biology and Genetics, Aarhus University, Research Centre
Flakkebjerg, Forsøgsvej 1, 4200 Slagelse, Denmark.
Lolium perenne (perennial ryegrass) breeding programs are primarily based on recurrent
selection to develop improved populations. For accurate phenotyping of many target traits in
these programs, measurements need to be performed on populations planted in swards, as
opposed to single plants. Resolving the allele frequencies within these populations at
multiple SNP locations may allow us to associate the allele SNP frequencies with the
phenotype. In order to test this approach, a strategy is required to accurately determine allele
frequencies in perennial ryegrass populations. We have used a genotyping by sequencing
(GBS) approach (Elshire et al., 2011) that involved genome complexity reduction with
restriction enzymes, barcoding fragments to allow multiplexing, and pooling samples for
sequencing on the Illumina GAII and HiSeq 2000. Different enzymes were tested to generate
a range in the extent of genome complexity reduction. We have applied this approach to
eight perennial ryegrass cultivars to get an insight into expected SNP harvest and the
coverage required to accurately determine allele frequencies. The developed strategies will
ultimately be used to characterise genetic variation in a training population being used to
develop models for genomic selection.
References
ELSHIRE, R. J., GLAUBITZ, J. C., SUN, Q., POLAND, J. A., KAWAMOTO, K., BUCKLER,
E. S. & MITCHELL, S. E. 2011. A Robust, Simple Genotyping-by-Sequencing (GBS)
Approach for High Diversity Species. Plos One, 6.
57
Posters: Breeding – quality and productivity
Genotypic differences in heat tolerance mechanisms of wheat (Triticum aestivum L.)
Xiao Wang 1,2,3, Marija Vignjevic2, , Susanne Jacobsen3 and Bernd Wollenweber2
1
Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of
Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing
Agricultural University, P. R. China
2
Aarhus University, Department of Agroecology, Research Centre Flakkebjerg DK-4200
Slagelse, Denmark
3
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of
Denmark, DK-2800 Kgs. Lyngby, Denmark
Wheat is sensitive to heat stress, especially when occurring at anthesis and during the grain
filling stage. Experiments in order to explore genotypic differences of the impact of heat
stress in ten wheat varieties have been performed. Based on photosynthesis rates and grain
yield, six varieties were selected as tolerant (810, 1110, 579, Terice, Taifun and Vinjet) and
four as heat sensitive (633, 1159, 490 and 1039). The tolerant variety 810 and the sensitive
variety 1039 were selected for further analysis. Higher rates of photosynthetic carbon- and
light-use, were accompanied by lower damage to cell membranes and higher activities of
ascorbate peroxidase and glutathione reductase in variety 810 compared to variety 1039.
Furthermore, proteins related to photosynthesis, glycolysis, stress defence, heat shock
proteins and ATP production were differently expressed in the tolerant and sensitive varieties
under heat stress in relation to the correspondent control, probably indicating the role of
these proteins in heat tolerance mechanisms. Collectively, the results indicate that primarily
the changes in both the amount and activities of enzymes involved in photosynthesis and
antioxidant activity contributed to higher heat tolerance in variety 810 compared to the
sensitive variety 1039.
58
Posters: Breeding – quality and productivity
Genetic dissection of vernalization response and winter survival in perennial ryegrass
Paina C. 1, Byrne S. 1, Asp T. 1
1
Department of Molecular Biology and Genetics, Science and Technology, Aarhus University,
Forsøgsvej 1, 4200 Slagelse, Denmark
Vernalization requirement and winter survival represent high interest agronomical traits in the
breeding programs of temperate grasses. We hereby report the development of a candidate
gene based high throughput Illumina Golden Gate 1,536-plex genotyping assay for perennial
ryegrass, comprising SNP markers developed from transcripts involved in cold response,
cold acclimation, and vernalization response. Based on this assay, a candidate gene based
linkage map was developed and QTLs for winter survival (WS) and vernalization response
measured as growing degree days to heading (GDD) were identified. A positive correlation
was observed between vernalization response and winter survival. Two regions with possible
pleiotropic effects were located on LG4 and LG6. Genes located within the QTL confidence
intervals were used as candidates in candidate gene based association studies.
59
Posters: Breeding – quality and productivity
Redox regulation of starch synthases in Arabidopsis thaliana
Katsiaryna Skryhan1, Mikkel A. Glaring1, Brian B. Nielsen1, Morten M. Nielsen2, Jose A.
Cuesta-Seijo2, Lucia Marri2, Monica M. Palcic2, Andreas Blennow1
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark, 2Carlsberg Laboratory, Gamle
Carlsberg Vej 10, 1799 Copenhagen V, Denmark.
Starch is the major and the most abundant storage carbohydrate in higher plants and it is
also the most important energy source in the human and animal diet. Starch is composed
exclusively of two structurally distinct α-D-glucose polymers, namely amylose and
amylopectin, which are stored inside chloroplasts (transient starch) or amyloplast (storage
starch) in form of insoluble granules. Transient starch is synthesized during the day and is
degraded over the night to meet the constant energy requirements of plant metabolism in the
dark [1]. In the chloroplast, reducing equivalents produced during the day by photosynthesis
are transported from photosystem I via the ferredoxin-thioredoxin system to many target
enzymes. These proteins contain key cysteine residues that make disulfide linkage upon
oxidation [2].
We performed a comprehensive analysis of the redox sensitivity of known starch biosynthetic
enzymes in leaf extracts of Arabidopsis thaliana using native activity gels and enzyme
assays. Our results confirmed that at least two isoforms of starch synthase SS1 and SS3 are
activated by reduction at physiologically relevant potentials [3]. These enzymes were
expressed as 6xHis-tagged proteins in E. coli cells. An analysis of purified recombinant
proteins confirmed their redox sensitivity.
To identify cysteines that are involved in the formation of the regulatory disulfide linkage of
6His-SS1 protein, we generated cys-to-ser site-specific mutants, one for each cysteine
present in the sequence. Enzyme assay data reveal that cys 164, cys 545 and presumably
cys 217 are involved in redox regulation of SS1.
Better understanding of starch synthases activity and their regulation may provide additional
options for starch biosynthesis manipulation.
References:
[1] S. Streb and S. C. Zeeman, “Starch Metabolism in Arabidopsis,” The Arabidopsis book: 9,
2012. Published by the American Society of Plant Biologist.
[2] P. Schürmann and B. B. Buchanan, “The ferredoxin/thioredoxin system of oxygenic
photosynthesis,” Antioxidants & Redox Signaling: 10/7, 1235–74, 2008.
[3] M. A. Glaring, K. Skryhan, O. Kötting, S. C. Zeeman, and A. Blennow, “Comprehensive
survey of redox sensitive starch metabolising enzymes in Arabidopsis thaliana,” Plant
Physiology and Biochemistry: 58, 89–97, 2012.
60
Posters: Breeding – quality and productivity
Non-inhibited barley/wheat-feed enzyme combinations for improved feedstuff value
Claus Krogh Madsen, Giuseppe Dionisio, Henrik Brinch-Pedersen
Department of Molecular Biology and Genetics, Aarhus University, Research Centre
Flakkebjerg.
The utilization of feed by animals is never fully efficient and nutrients must be supplied in
some level of excess in order to meet the physiological requirements of the animals. The
level of excess must be as small as possible in order to minimize resource use and the
leaching of nutrients from agricultural systems. Feed enzymes including microbial phytases,
xylanases and proteases are deployed to promote feed utilization thus reducing the need to
overcompensate. Unfortunately the action of feed enzymes is countered by inhibitors which
have evolved in plants as part of their protection against microbes. This four year GUDP
funded project aims to integrate plant breeding, genetics and protein chemistry in order to
develop perfect matches of feed crop genotype and feed enzymes. Preliminary experiments
have demonstrated that the degree of enzyme inhibition is variable between seed batches of
different cultivars. To which extend this is influenced by cultivar/genotype or environmental
factors is examined in field experiments. In order to advance plant breeding and enzyme
development it is important to identify the genes responsible for the genetic component of the
variation. While some inhibitor genes are known the full complement may be larger and
undiscovered genes may underlie some the variation between cultivars. A functional
screening will therefore be performed in order to unravel the specific inhibitors of applied
feed enzymes. Wheat and barley grain expression libraries will be created in P. pastoris for
this purpose and queried with selected feed enzymes in combination with chromogenic
substrates. Differential proteomics and qRT-PCR will be used to connect the observed
differences in inhibition to the candidate genes.The project consortium consists of Aarhus
University, Sejet Plant Breeding, DLG and Novozymes.
61
Posters: Breeding – quality and productivity
Genome Wide Association Study (GWAS) of manganese use efficiency in winter
barley using chlorophyll a fluorescence
Florian Leplat1, Pai Pedas1, Søren Husted1, Søren K. Rasmussen1
1
Department of Plant and Environmental Sciences, Faculty of Sciences, University of
Copenhagen
Nowadays, breeders extend the range of traits to be studied. Besides yield, disease
resistances, and the main economical traits, nutrient use efficiency has become of major
interest. On top of the macronutrient use efficiency studies, micronutrient utilization has to be
further studied and implemented to crop breeding program. In our study we focused on
manganese (Mn) which represents a major plant nutritional disorder in winter cereals in
Denmark [1]. Manganese deficiency causes significant yield reductions and is difficult to
diagnose, typically due to the lack of visual symptoms. Studies have shown differences
between genotypes in terms of tolerance to growth at low Mn availability in soil [2,3],
indicating a strong genetic component for this trait.
The objective of this work is to investigate the genetic background underlying Mn use
efficiency in winter barley. We aim to combine phenotyping tools and quantitative genetic
methods to reveal the quantitative loci involved in the use of Mn. A complete phenotypic
scoring of the trait for subsequent genetic analysis was a major prerequisite for Genome
Wide Analysis Study (GWAS).
Recently, chlorophyll a fluorescence measurements have been used to diagnose Mn use
efficiency [4]. We extended this technic to phenotype a wide range of cultivars. We used a
collection of 248 winter barley varieties originating from the whole of Europe and various
breeder origins. The collection was genotyped with 9k SNP iSelect array. During two
consecutive years, we used two different fields in Denmark and one in Sweden indicating low
Mn availability. Soil pot experiments in greenhouse have also been carried out to induce Mn
deficiency in winter barley plants.
As a result, GWAS was performed using a mixed linear model statistic approach, taking into
account the population structure and the kinship matrix. Significant SNPs were detected
significantly associated to Mn use efficiency trait along the 7 chromosomes of winter barley
genome. The experiment has produced promising data and shows the potential of combining
plant nutrition tools with SNP genome wide approach for plant breeding purposes.
[1] Husted S., Laursen K.H., Hebbern C.A., Schmidt S.B., Pedas P., Haldrup A., Jensen
P.E., (2009). Manganese deficiency leads to genotype-specific changes in fluorescence
induction kinetics and state transitions. Plant Physiology, 150: 825-833.
[2] Hebbern C.A., Pedas P., Schjoerring J.K., Knudsen L., Husted S., (2005). Genotypic
differences in manganese efficiency: field experiments with winter barley (Hordeum vulgare
L.). Plant and Soil, 272: 233–244.
[3] Pedas P., Hebbern C.A., Schjoerring J.K., Holm P.E., Husted S., (2005). Differential
capacity for high-affinity manganese uptake contributes to differences between barley
genotypes in tolerance to low manganese availability. Plant Physiology, 139: 1411-1420.
[4] Schmidt S.B., Pedas P., Laursen K.H., Schjoerring J.K., Husted S., (2013). Latent
manganese deficiency in barley can be diagnosed and remediated on the basis of
chlorophyll a fluorescence measurements. Plant Soil, 372: 417-429.
62
Posters: Breeding – quality and productivity
Transport Engineering to Increase Production of Plant Natural Products
Nora Linscheid, Hussam Nour-Eldin
DynaMo Center of Excellence, Department of Plant and Environmental Sciences, Faculty of
Science, University of Copenhagen, Thorvaldsensvej 40, 1871Frederiksberg C, Denmark
Plants produce a vast array of natural compounds that are beneficial to humans as
pharmaceuticals, flavouring and colouring agents, and in many other applications. Increasing
the accumulation of such biological compounds in plants is of high interest for industry and
agriculture. However, genetic engineering approaches that target biosynthetic pathways
often suffer from a limitation on prospected yield due to inherent feedback inhibition
mechanisms or toxic effects exerted by over-accumulation of final products.
Targeting plant membrane transporters potentially represents an important strategy to
remove a given end-product from its place of synthesis and thereby boost biosynthesis by
alleviating feedback inhibition and toxic effects. Development of transport engineering
strategies to increase production of valuable plant natural products has been hampered by
lack of molecular knowledge about transporters involved in transport pathways of plant
specialized metabolites. The recent identification of essential transporters in the
glucosinolate transport pathway can be employed as a model system to develop and
evaluate novel transport engineering approaches.
In my Master project I will develop two transport engineering approaches and evaluate their
ability to boost biosynthesis of glucosinolates in planta. The overall goal is to increase either
or both source and sink strengths by i.) replacing native glucosinolate transporters with
hyperactive mutant versions, and/or ii.) source/sink-specifically over-expressing of native and
hyperactive glucosinolate transporters. Analysis of the outcomes of these two approaches
will constitute an important step towards establishing generic transport engineering strategies
for the over-accumulation of plant natural compounds in target tissues.
63
Posters: Breeding – quality and productivity
Use of TALEN technology to induce mutations in barley
Inger B Holme1, Toni Wendt1,2, Colby G Starker3, Michelle Christian3, Daniel F. Voytas3,
Henrik Brinch-Pedersen1
1
Aarhus University, Department of Molecular Biology and Genetics, Research Centre
Flakkebjerg, Slagelse, Denmark, 2Present address: Carlsberg Laboratory, Valby,
Copenhagen, 3Department of Genetics, Cell Biology and Development and Center for
Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA
Until recently the technologies available for plant transformation did not allow for the
generation of specific alterations in existing genes. Various tools for altering existing genes
have, however, recently successfully been developed in model plants. Common to these
tools is that they can be designed to create a double strand DNA break at specific sites in the
genome. These double strand breaks will subsequently be repaired by the plant cell's own
DNA repair mechanisms and depending on the transformation conditions this repair can lead
to mutations or homologous recombination at the specific site. One of these tools is named
Transcription Activator-like Effector nucleases (TALENs). In the last two years we have
developed and implemented the TALEN tool in barley. We have designed TALEN constructs,
which can induce double strand breaks at a specific site in the barley genome and
transformed barley with these constructs. Analysis of the resulting primary transformants
show that the TALENs are active and induce DNA breaks that lead to mutations at the
intended locations in the barley genome (Wendt et al., 2013). Currently, we are optimizing
the system using various vector constructs to enhance the TALEN expression in the cells.
Wendt T., Holm PB., Starker CG., Christian M., Voytas DF., Brinch-Pedersen H, Holme IB.
(2013). TAL effector nucleases induce mutations at a pre-selected location in the genome of
primary barley transformants. Plant Mol Biol. 83, 279-285.
64
Posters: Breeding – quality and productivity
GWAS OF SEED QUALITY IN BARLEY
Søren K. Rasmussen1 – Xiaoli Shu1,2 – Anna Maria Torp1
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark, e-mail: [email protected]
2
Key Lab of the Ministry of Agriculture for Nuclear Agricultural Sciences, Institute of Nuclear
Agricultural Sciences, Zhejiang University, Hangzhou 310029, P.R. China
Keywords: amylose, amylopectin, beta-glucan, genome-wide association scan, phytic acid,
resistant starch, waxy, high-amylose
Taking advantage of the recent development in SNP genotyping of cereal crops and
establishment of germplasm like the European Barley Core collection [1] we want to carry out
gene-discovery by genome-wide association scan in order to identify novel genes and QTL
controlling the concentration of important compounds in the cereal grain. In a collection of
205 European barley varieties we found a large variation in the resistant starch content but
no statistically significant correlation between RS content and β-glucan content was found in
a subset of 61 varieties. Ten SNP markers out of 40 associated with RS were located in
genes with a known role in starch biosynthesis [2]. Analysis of 254 barley varieties showed
negative correlation between amylose and beta-glucan and between amylopectin and betaglucan. Many SNP were identified and some of these corroborated earlier mapped QTL for
these compounds and were located in loci and chromosomal regions not previously identified
to control the content of these three compounds in the grain [3]. These examples shows the
potential of obtaining SNP markers for dissecting know QTL as well for identification of new
genes directly involved in the biosynthesis or accumulation of these compound in the starchy
endosperm. The study also revealed the interplay between the different pathways.
References
1. A. Tondelli, X. Xu, M. Moragues, F. Schnaithmann, R. Sharma, C. Ingvardsen, J.
Comadran, B. Thomas, J. Russell, R. Waugh, A. Schulman, K. Pillen, S. K. Rasmussen, B.
Kilian, L. Cattivelli, A. J. Flavell: Structural and temporal variation in the genetic diversity of a
European collection of spring 2-row barley cultivars and utility for association mapping of
quantitative traits. Plant Genome 6: doi: 10.3835/plantgenome2013.03.0007, 2013
2. Shu, X., Backes, G., Rasmussen, S. K.: Genome-wide association study of resistant
starch (RS) phenotypes in a barley variety collection. J. Agric. Food Chem. 60,
10302−10311, 2012
3. Shu, X., Rasmussen, S. K.: Quantification of amylose, amylopectin and β-glucan in search
for genes controlling the three major quality traits in barley by genome-wide association
studies (submitted)
65
Posters: Breeding – quality and productivity
Sequencing and genome comparison of plastid genomes of fertile and male-sterile
lines in perennial ryegrass (Lolium perenne L.)
Istvan Nagy, Md. Shofiqul Islam, Ian Max Møller, Stephen Byrne, Torben Asp
Department of Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, DK-4200
Slagelse
Chloroplast genomes in higher plants are typically represented by monomeric circular
molecules of an average size of 150 kilobases. Most of the known angiosperm choloroplast
genomes share a common macro-structure. This is comprised of a large single-copy region
followed by a cruciform structure with two 20 to 25 kb long highly similar inverted repeats that
are separated by a 10 to 18 kb long unique region. Chloroplast genomes contain around 110
to 120 unique genes. Gene content, gene structure as well as the linear order of coding
sequences shows a relatively high degree of conservation between higher plants. We
established full-length chloroplast sequences for two fertile and two cytoplasmic male sterile
(CMS) lines of perennial ryegrass. In addition, pyrosequencing reads of an average length of
about 400 nucleotides were mapped to a common reference backbone provided by the
published chloroplast sequence of the ryegrass variety Cashel. In all of the four sequenced
lines, average short read coverages of 10 to 15 were obtained along the whole chloroplast
genome that facilitated a reliable variant detection. Detailed analysis of Single Nucleotide
Variants suggests that in all investigated genotypes two slightly different chloroplast
genomes might exist in parallel. Furthermore, in comparison to the fertile genotypes, the
number of single nucleotide substitutions detected was around 5 times higher in male-sterile
lines. Sequence comparisons suggest that faster accumulation of mutations is probably not
the main factor of the relatively high sequence-level diversity detected in the chloroplast
genomes of the CMS lines. A more likely hypothesis is that due to the transgressive
maternal inheritance, the organellar genomes that have been fixed in the cytoplasm of the
investigated CMS lines originate from distantly related taxa.
66
Posters: Breeding – quality and productivity
Sequencing and analysis of mitochondrial genomes of fertile and male-sterile lines in
perennial ryegrass (Lolium perenne L.)
Istvan Nagy, Md. Shofiqul Islam, Ian Max Møller, Stephen Byrne, Torben Asp
Department of Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, DK-4200
Slagelse
The mitochondrial genomes of higher plants represent several characteristics that
discriminate them from other eukaryotic mitochondrial genomes. Plant mitochondrial
genomes are large and variable in size with substantial amounts of non-coding regions. A
couple of sequence parts of plant mitochondrial genomes represent “promiscuous” DNA of
nuclear and plastid origin, as well as sequence regions that are likely be obtained through
horizontal transfer from mitochondria of other species. While angiosperm mitochondrial
genomes exhibit extremely slow rates of nucleotide substitutions, they are subjected to a
rapid structural evolution due to frequent intra- and intermolecular recombinations among
large repeated sequences. Such structural rearrangements within and between circular
master molecules usually generate heterogeneous pools of mitochondrial sub-genomes of
different sizes and configurations. The complete mitochondrial sequence and a circular
molecular map for the fertile ryegrass line F1-30 has been recently published by our group.
Here we report on completed mitochondrial genome sequencing projects in further three
ryegrass genotypes. Complete genome sequences were generated in two cytoplasmic malesterile (CMS) lines and in a fertile maintainer line by de novo hybrid Next Generation
Sequencing approaches using Roche 454 Pyrosequencing and Pacific Biosciences Single
Molecule Reads technologies. Genome sequencing reveals the co-existence of
mitochondrial sub-genomes with extensive structural rearrangements in all investigated
ryegrass genotypes. A proposed general mechanism for building subgenomic molecules as
well as the significance of structural changes in the manifestation of male sterility will be
discussed in detail.
67
Posters: Synthetic- and systems biology
Elucidating molecular regulatory mechanisms in Arabidopsis
Lea Møller JENSENa, Barbara Ann HALKIERa, Daniel KLIEBENSTEINb, and Meike BUROWa
a
Department of Plant Biology and Biotechnology, Faculty of Science, University of
Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark
b
Department of Plant Science, University of California, Davis, One Shields Ave, CA 95616,
US
Various regulatory mechanisms mediated by RNA, proteins, and metabolites are known to
take part in regulatory networks. Research on regulation is important for our understanding of
how organisms control their metabolism. In this study, we investigate regulatory genes and
mechanisms in the model plant Arabidopsis thaliana by utilizing our knowledge on the
production of a group of secondary metabolites, namely aliphatic glucosinolates.
Most of the biosynthetic genes of the aliphatic glucosinolate pathway have been identified
(1), however, the knowledge concerning their regulation is more limited. AOPs, biosynthetic
enzymes functioning in the last part of the pathway have been suggested to be involved in
regulation of glucosinolate production (2, 3). To shed light on the regulatory roles of AOPs,
we investigate the regulatory network surrounding them by generating different transgenic
lines.
Furthermore, we utilize different A. thaliana accessions having different glucosinolate levels
and thereby different regulatory networks. The regulatory capacity of the AOPs varies among
accessions depending on the allelic status of another biosynthetic locus (GS-ELONG) and
other yet unknown regulators controlling aliphatic glucosinolate biosynthesis. This shows that
the regulatory function of a gene is dependent on the regulatory network it is a part of, which
makes it possible to find new regulatory players and novel regulatory mechanisms.
1. I. E. Sonderby, F. Geu-Flores, B. A. Halkier, Biosynthesis of glucosinolates - gene
discovery and beyond. Trends Plant Sci. 15, 283 (May, 2010).
2. A. M. Wentzell et al., Linking metabolic QTLs with network and cis-eQTLs controlling
biosynthetic pathways. Plos Genetics 3, 1687 (Sep, 2007).
3. D. J. Kliebenstein, J. Gershenzon, T. Mitchell-Olds, Comparative quantitative trait loci
mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana
leaves and seeds. Genetics 159, 359 (Sep, 2001).
68
Posters: Synthetic- and systems biology
Expanding the molecular diversity through Synthetic Biology: Using combinatorial
biochemistry for reconstruction of pathways to high-value and novel diterpenes.
Johan Andersen-Ranberg1,2, Britta Hamberger1,2, , Birger Lindberg Møller1,2 and Björn
Hamberger1,2*
1
Department of Plant and Environmental Sciences, Copenhagen University, Denmark
UNIK Center for Synthetic Biology, Copenhagen University, Denmark
* [email protected]
2
Diterpenes (C-20) are a subgroup of terpenoids with more than 14000 different compounds
known. The main source of the structurally complex diterpene molecules is from plants,
where they act as e.g. hormones or defence compounds. In plants, diterpenes are formed by
cyclization governed by diterpene synthases (diTPS) which can typically be divided into two
classes (type II and type I). These work in tandem by catalysing the reaction leading from the
universal precursor geranylgeranyl-pyrophosphate (GGPP) into multicyclic diterpenes.
In this project we studied the plant species Tripterygium wilfordii, Coleus forskohlii and
Euphobia peplus, accumulating the pharmacologically active diterpene compounds triptolide,
forskolin and ingenol-3-angelate, respectively. From deep transcriptome sequencing data 30
candidate diTPS genes were identified and isolated. A commonly used method for functional
characterization of diTPS uses coupled in vitro assay with purified diTPS enzymes and
GGPP as substrate. However, for this study, heterologous expression of diTPS genes in
Nicotiana benthamiana was established. This method enabled rapid functional testing of
combinations of diTPS from all three plant species and correlation with already characterized
diTPS. With this system it was possible to reconstruct native biosynthetic routes to high value
diterpene compounds and to build novel biosynthetic routes introducing product specificity
and new-to-nature diterpene compounds including stereochemical control.
Structural elucidation of novel, potentially new-to-nature diterpenoids requires purification of
substantial amounts of the target molecules. We achieved medium scale production of the
metabolites in the N. benthamiana system, by upregulating the DXS (DOXP synthase) and
GGPPS (geranylgeranylpyrophosphate synthase) enzymes in the MEP (non-mevalonate)
pathway. Amounts and purity of purified diterpenes using preparative gas chromatography
was sufficient for structural elucidation by NMR. This streamlined procedure allowed us to
explore the biosynthetic capacities of diTPS and to isolate diterpenes with very similar
properties but different structure. Identification of diTPS involved in the biosynthesis of high
value diterpenes paves the way for biotechnological production and increases our
understanding of the evolution of diTPS and the molecular structure of their chemically
diverse products.
69
Posters: Synthetic- and systems biology
The potato tuber mitochondrial proteome
Jesper F. Havelund1,2, Fernanda Salvato3, Mingjiea Chen3, R.S.P. Rao3, Adelina RogowskaWrzesinska2, Ole N. Jensen2, David R. Gang4, Jay J. Thelen3, Ian M. Møller1
1
Department of Molecular Biology and Genetics, Science and Technology, Aarhus University,
Forsøgsvej 1, DK-4200 Slagelse, Denmark
2
Department of Biochemistry and Molecular Biology, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
3
Department of Biochemistry, University of Missouri-Columbia, 109 Bond Life Sciences
Center, Columbia, MO 65211, USA
4
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164
USA.
Mitochondria are called the powerhouses of the cell. To better understand the role of
mitochondria in maintaining and regulating metabolism in storage tissues, highly purified
mitochondria were isolated from dormant potato tubers (Solanum tuberosum L. cv. Folva)
and their proteome investigated. Proteins were resolved by one-dimensional gel
electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid
chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different
search programs, a total of 1060 non-redundant proteins were identified in a quantitative
manner using normalized spectral counts including as many as five-fold more “extreme”
proteins (low mass, high pI, hydrophobic) than previous mitochondrial proteome studies. We
estimate that this compendium of proteins represents a high coverage of the potato tuber
mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression
spanned 1800-fold and included nearly all components of the electron transport chain,
tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71
pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new
proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate
dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the
dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of
prominent post-translational modifications (PTM) revealed that >50% of the identified
proteins harbor at least one modification. The most prominently observed class of PTMs was
oxidative modifications. This study reveals approximately 500 new or previously unconfirmed
plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive
identification of mitochondrial proteins and their modified forms.
References:
Salvato, F., Havelund, J.F., Chen, M., Rao, R.S.P., Rogowska-Wrzesinska, A., Jensen, O.N.,
Gang, D.R., Thelen, J.J. and Møller, I.M (2014). The potato tuber mitochondrial proteome,
Plant Physiology, Accepted.
70
Posters: Synthetic- and systems biology
An assembly and expression optimization pipeline for biosynthetic pathway
reconstitution in Escherichia coli
Morten Thrane Nielsen, Johan Andersen-Ranberg, Susanna Seppälä, Karina Marie Madsen,
Ulla Christensen, Björn Hamberger, Birger Lindberg-Møller and Morten Nørholm
Microbial expression systems have proven an invaluable tool for isolation and
characterization of enzymes involved in secondary metabolism. More recently, microbial cell
factories have emerged as a solution for sustainable production of natural product based fine
chemicals. However, reconstitution of biosynthetic pathways leading to natural products in
microbial cell factories remains a formidable challenge. Here, we describe a gene assembly
and expression optimization pipeline that addresses some aspects of this challenge. The
pipeline is build upon the simple and versatile uracil-excision cloning technology and a new
Bio-Brick standard, DNA-Bricks. The DNA-Brick pipeline facilitates single-step, standardized,
sequence and ligase-independent, assembly of multiple DNA fragments. Particularly suitable
applications include 1) gene discovery, 2) pathway optimization by comparison of
orthologous enzymes, 3) balancing of enzyme levels using i.e. ribosomal binding site
variation. As proof of concept, we reconstituted the biosynthetic route for a novel plant
derived diterpene precursor in Escherichia coli and utilized the DNA-Brick pipeline to
optimize supply of the universal diterpene precursor, geranyl-geranyl-pyrophosphate,
resulting in dramatically increased diterpene production. We envision, that the DNA-Brick
pipeline will accelerate biosynthetic pathway reconstitution in microbial cell factories and
thereby contribute to determine the scope of natural products that can be efficiently produced
by cell factories.
71
Posters: Synthetic- and systems biology
From ecometabolomics to synthetic biology – exploring plasticity of the triterpenoid
biosynthetic pathway
Pernille Ø. Erthmanna, Vera Kuzinaa, Bekzod Khakimovb, Jörg M. Augustina, Søren Baka
a
Department of Plant and Environmental Sciences, University of Copenhagen
Department of Food Science, University of Copenhagen
b
Triterpenoid saponins are natural plant defense compounds rather widespread in plants.
They are amphipathic molecules that may interact with sterols in membranes, and induce
pore formation and cell death. The wild crucifer Barbarea vulgaris shows resistance towards
flea beetles (Phyllotreta nemorum)1, a severe pest in crucifer crops like oil seed rape
(Brassica napus). An ecometabolomic approach identified saponins to correlate with flea
beetle resistance in B. vulgaris2. In addition a transcriptomic 454-dataset was used to create
a quantitative trait loci (QTL) map which identified specific saponins to lie in the QTL for flea
beetle resistance3. The proposed saponin pathway branch-off from the sterol biosynthesis
with 2,3-oxidosqualene as the shared precursor. 2,3-oxidosqualene is cyclized by
oxidosqualene cyclases (OSC) to a number of backbone structures, oxidized by cytochromes
P450 (CYP), and glycosylated by glycosyltransferases (UGT) to create the vast structural
diversity.
A 454 transcriptomic and an Illumina genomic dataset were mined for OSC, CYPs and
UGTs. The catalytic activities of five UGTs were characterized by in vitro assays in E. coli,
and found to glycosylate at the C3 or/and the C28 of selected triterpene backbones4. An
analysis of dN/dS site and branch models revealed that the UGT catalyzing the 3-Oglucosylation is under positive selection, suggesting that this UGT has evolved to become
specific to the saponins. The activities of four CYPs were studied by in vitro assays in yeast,
and shown to oxidize at the C28 position of the triterpene backbone. To reveal the function of
the OSCs the Cowpea Mosaic Virus-Hyper Translatable System (CPMV-HT) was used. This
enable production of the triterpenoid backbone structures α-amyrin, β-amyrin and lupeol in
various amounts from the common precursor 2,3-oxidosqualene. In addition, the OSCs were
found to lie within the QTL for resistance. By combinatorial expression in N. benthamiana of
OSCs, CYPs and UGTs using the CPMV-HT system, a number of known and new-to-nature
saponin structures were generated.
A combined metabolomics, genomics, transcriptomics, and genetic approach identified
saponins as determinants for flea beetle resistance in B. vulgaris, and provided the
necessary molecular tools for in planta production of known and new-to-nature structures by
synthetic biology. This ability to combine OSCs, CYPs and UGTs in planta by combinatorial
biochemistry paves the way for a rational design triterpenes for structure-activity
relationships for development of bioactive triterpenes with specificity to different herbivores
and diseases, and demonstrates the plasticity of the pathway.
References:
Agerbirk, N., C. E. Olsen, B. M. Bibby, H. O. Frandsen, L. D. Brown, J. K. Nielsen & J.
a. A. Renwick. (2003): A saponin correlated with variable resistance of Barbarea vulgaris to
the diamondback moth Plutella xylostella. Journal of chemical ecology. Vol. 29, pp. 14171433.
2
Kuzina, V., C. T. Ekstrom, S. B. Andersen, J. K. Nielsen, C. E. Olsen & S. Bak (2009):
Identification of Defense Compounds in Barbarea vulgaris against the Herbivore Phyllotreta
nemorum by an Ecometabolomic Approach. Plant physiology. Vol. 151, pp. 1977-1990.
3
Kuzina, V., J. K. Nielsen, J. M. Augustin, A. M. Torp, S. Bak & S. B. Andersen (2011):
Barbarea vulgaris linkage map and quantitative trait loci for saponins, glucosinolates,
1
72
Posters: Synthetic- and systems biology
hairiness and resistance to the herbivore Phyllotreta nemorum. Phytochemistry. Vol. 72, pp.
188-198.
4
Augustin, J. M., S. Drock, T. Shinoda, K. Sanmiya, J. K. Nielsen, B. Khakimov, C. E.
Olsen, E. H. Hansen, V. Kuzina, C. T. Ekstrøm, T. Hauser & S. Bak. (2012): UDPGlycosyltransferase from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3O-Glycosylation in Saponin-Mediated Insect Resistance. Plant physiology, Vol. 160, pp.
1881-1895.
73
Posters: Synthetic- and systems biology
Development of Methods for Bulk Segregate Analysis in Polyploids to facilitate Marker
Assisted Selection in Tetraploid Potato
Mads Sønderkær1, Kacper Kaminski1,2, Mette S. Andersen1 Hanne Grethe Kirk3, & Kåre L.
Nielsen1
1
Aalborg University, Department of Biotechnology, Chemistry and Environmental
Engineering, Aalborg, Dk-9000, Denmark
2
Department of Agroecology, Faculty of Science and Technology, Aarhus University, Blichers
Allé 20, 8830 Tjele, Denmark
3
Danish Potato Breeding Foundation, Grindstedvej 55, 7184 Vandel, Denmark
E-mail: [email protected], [email protected]
Breeding for increased space and resource efficient crops is important to feed the world's
increasing population and support a societal shift from a fossil fuel-based to a bio-based
society. Crops with storage organs in the soil, like potato (S. tuberosum), produce twice the
amount of calories per hectare with the same or less input compared to cereals, but breeding
tools have been limited and thus a high unexploited potential is likely to present in the elite
germplasm. The completion of the genome sequence of potato has enabled applications
such as bulk segregate analysis (BSA) to rapidly identify genomic loci of agronomical interest
and associated molecular markers to use in e.g. marker assisted selection (MAS) and
Genomics Selection (GS). We have developed a next generation sequencing-assisted ultrarapid BSA method which we apply on a population of a ~ 5,000 individuals derived from a
poly parental cross consisting of 19 tetraploid breeding clones. The population is derived
from elite germplasm and is being phenotyped for six major traits in potato.
Here we present the BSA method developed, initial results from different data sets, and the
development of genetic markers based on re-sequencing data of the 19 breeding clones.
These markers will be used to perform BSAs on the off spring enabling the development of a
MAS and GS platform for potato.
74
Posters: Synthetic- and systems biology
Expression profile of predicted secreted signal proteins in the perennial ryegrass
transcriptome
Jacqueline D. Farrell1, Stephen Byrne,1 Torben Asp1
1
Department of Molecular Biology and Genetics, Aarhus University, Research Centre
Flakkebjerg, Forsøgsvej 1, 4200 Slagelse, Denmark
Perennial ryegrass (Lolium perenne L.) is an important grass species for both recreation and
agriculture purposes in temperate regions worldwide. Denmark is the world’s largest
exporter of grass seed and therefore it plays an important role in the country’s economy. The
goals of this study were to perform RNA-sequencing on multiple tissues from a highly inbred
genotype to develop a reference transcriptome. The de novo transcriptome assembly of the
inbred genotype created 186,141 transcripts with an average length of 830 bp. Within the
inbred reference transcriptome 78,627 predicted open reading frames were found of which
24,434 were predicted as complete. We also conducted a de novo transcriptome assembly
with a heterozygous genotype to enable SNP discovery. We were able to identify 2,357
putative secreted proteins in our transcriptome database. As we had sequenced RNA from
multiple tissues we were able to get an insight into the expression profiles of these putative
secreted signal proteins.
75
Posters: Synthetic- and systems biology
Inducible microalgae cell wall lysis and characterization for enhance product recovery
Erick Ramos1, Yumiko Sakuragi1
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C
Photosynthetic microorganisms such as microalgae are sunlight-driven cell factories that can
sustainably produce a wide range of molecules from sun light, CO2 and water. Many algae
present a cell wall that provides a formidable barrier necessary for survival in aquatic
environments. Unfortunately, this barrier affects the recovery of products of interest in algal
biotechnology. Downstream process contributes to the 60% of the total production cost since
extraction and separation is an energy-intense process associated with the cell wall making
production not economically feasible. The main limitation is breaking the cell wall to recover
the products inside the cell; therefore strategies to weaken the cell wall are needed. Fragile
cell walls could reduce solvent and energy inputs needed for high value products thus reduce
the energy and cost of microalgae downstream processing. In this project two approaches
are taken to address this problem, first by engineering microalgae with an inducible lysis
device for enhance product recovery and second by studying the patterns of occurrence of
cell wall components during cell growth and under stress which could potentially provide
useful tools in downstream algal biomass processing.
The first approach is to use synthetic biology tools to engineer Chlamydomonas reinhardtii
with an inducible lysis system to facilitate product release by nuclear transforming the cells
with inducible promoters fused with a cell wall degrading gene and a reporter gene driven by
a constitutive promoter. Engineered microalgae will response to a chemical stress by
expression a cell wall degrading enzyme that will form pores in the cell wall and released the
accumulated recombinant protein that can be measure from the media. The second
approach addresses the fact that microalgal cell walls are complex and poorly understood, it
has been observed variations in cell walls in some microalgae species grown under different
conditions can be dramatic. Cell wall characterization will be carried out for microalgae
species that are commercially important such as Dunaliella salina, Haematococcus pluvialis
and Nannochloropsis. Biochemical and CoMMP analysis will be done at different growth
stages and under different stress conditions to revel changes in the composition of the
carbohydrates in the cell wall.
The microalgae programmed lysis system has many uses to release products; moreover it
could potentially be applied in robust industrial microalgae. Along with the inducible lysis
system, the knowledge collected from the characterization of the cell wall changes of
different microalgae species will be useful tools to reduce cost and energy in downstream
process.
76
Posters: Synthetic- and systems biology
Redirecting photosynthetic reducing power towards bioactive natural product
synthesis
Agnieszka Zygadlo Nielsena, Lærke Münter Lassena, Artur Wlodarczyka, Silas Busck Mellora,
Maria Henriques de Jesusa, Thiyagarajan Gnanasekarana, Birger Lindberg Møllerb and Poul
Erik Jensena
Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Interdisciplinary
Research Center “bioSYNergy”, VILLUM Research Center “Plant Plasticity”, aSection for
Molecular Plant Biology, bPlant Biochemistry Laboratory, Department of Plant and
Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871
Frederiksberg C, Copenhagen, Denmark
Photosynthesis provides the energy and carbon building blocks required for synthesis of a
wealth of bioactive natural products of which many have potential uses as pharmaceuticals.
Photosynthesis in plants or cyanobacteria provides ATP and NADPH as well as carbon
sources for primary metabolism. Cytochrome P450 monooxygenases (P450s) in the plant
endoplasmic reticulum (ER) are essential in the synthesis of many bioactive natural products,
powered by single electron transfers from NADPH. P450s are present in low amounts and
the reactions proceed relatively slowly due to limiting concentrations of NADPH. We have
recently demonstrated that it is possible to break the evolutionary compartmentalization of
energy generation and P450-catalysed biosynthesis, by relocating an entire P450 dependent
pathway to the chloroplast and driving the pathway by direct use of the reducing power
generated by photosystem I in a light-dependent manner. This demonstrates the potential of
transferring pathways for structurally complex high-value natural products and directly
tapping into the reducing power generated by photosynthesis to drive the P450s using water
as the primary electron donor.
Current work is directed towards establishing stable transgenic lines of both plants and
cyanobacteria with multi-enzyme pathways expressed in the thylakoids and produce highvalue bioactive compounds directly driven by light. We explore different strategies to optimize
product formation. One approach is scaffolding which aim at more efficient channelling of the
substrate and intermediates. For this we utilize the protein binding properties of PDZ
domains for building a modular synthetic scaffold that spatially recruits the necessary
enzymes in a desirable manner In another approach, we investigate the interaction of P450
enzymes with different ferredoxin proteins in order to optimise the electron transfer between
photosystem I and the P450s. We have found differential interactions between chloroplast
ferredoxins and ER-derived P450s, and these may be harnessed to improve partitioning of
electrons from photosynthesis towards bioactive compound biosynthesis.
77
Posters: Synthetic- and systems biology
High throughput retrospective antibody screening: a multi-level strategy to
characterise epitope specificity
Maja G. Rydahl*1, Olivier Tranquet2, Marie-Christine Ralet2, Valérie Echasserieau2, Jonatan
Fangel1, Henriette Lodberg Petersen1, David Domozych3, William G. T. Willats1
1
Copenhagen University, Faculty of Life Sciences, Department of Plant Biology and
Biotechnology, Section for Plant Glycobiology, Thorvaldsensvej 40, 1870 Frederiksberg C,
Copenhagen, Denmark. 2 INRA, UR1268 Biopolymers, Interactions, Assemblies, rue de la
Géraudière, Nantes cedex 03, France. 3 Department of Biology and Skidmore Microscopy
Imaging Center, Skidmore College, Saratoga Springs, New York, 12866, USA.
Monoclonal antibodies (mAbs) are powerful tools for the in situ detection of cell wall
components. However, the overwhelming majority of mAbs are raised against epitopes
occurring in the walls of angiosperms, creating a bias towards cell wall components of land
plants. There is increasing interest in the study of the cell walls of early divergent plant
groups including the charophytic and chlorophytic algae. This interest is driven largely by the
pivotal phylogenetic position of these algae in relation to plant, and plant cell wall evolution.
Given the paucity of relevant mAbs we have untaken to produce new sets of antibodies
directed against algal cell walls – and to do this we are using a novel biology-driven
approach.
The starting point for most mAb production is usually the selection of a target molecule
(antigen). Although this strategy has certainly been successful in many cases, it ideally
requires an extensive prior knowledge of the cell walls concerned. This risks that unknown or
quantitatively minor but important components may be overlooked as potential targets. A
biology-driven approach is based on immunisation with crude cell wall preparations, which as
far as possible represent whole cell wall glycomes. Using three main approaches, mAbs is
then retrospectively screened for biologically interesting binding profiles, by: 1) carbohydrate
microarrays populated with cell wall extracts from phylogenetically diverse species, 2)
immunocytochemical binding profile and 3) epitope determination using defined carbohydrate
microarrays. By combining these already well described and recognised methods, the novel
specificity of the epitopes is thereby determined in a high throughput manner.
An initial study using this biology driven approach, indicates that we have produced and
identified mAbs that recognise biologically significant structures at the cellular and/or
evolutionary level. Among others, mAbs directed at starch and ulvan structures has been
identified and are today studied further. This biology driven strategy could potentially create a
new paradigm for the production of plant cell wall directed antibodies.
78
Posters: Synthetic- and systems biology
Application of a split humanized Renilla luciferase complementation assay to
determine membrane bound protein-protein interactions in the Golgi apparatus
Christian Have Lund1, Jennifer R. Bromley1,2,3, Anne Stenbæk1, Casper Søgaard1, Henrik
Scheller2,3,4 & Yumiko Sakuragi1.
1. University of Copenhagen, Department of Plant and Environmental Sciences,
Frederiksberg, DK-1871, Denmark. 2. Joint BioEnergy Institute, Feedstocks Division,
Emeryville, CA 94608, USA. 3. Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, CA 94720, USA. 4. Department of Plant and Microbial Biology,
University of California.
Increasing evidence suggests that enzymes and proteins involved in cell wall biosynthesis
form protein complexes [1-3]. Even though conventional biochemical protein-protein
interactions approaches have proven to be successful in identifying protein-protein
interaction (PPI) between Golgi localized proteins they have their limitations. Membrane
proteins are in general very difficult to extract from their membranes without interrupting
interactions. Therefore, in situ PPI methods have been applied to test interaction between
proteins inside the Golgi lumen in plants. Bimolecular fluorescence complementation (BiFC)
has been used to detect PPI among proteins involved in pectin and xyloglucan biosynthesis
[4-6], but BiFC has one major limitation that the split YFP parts irreversible assembles
leading to high number of false positives. Förster resonance energy transfer (FRET)
successfully detected PPI within pectin biosynthesis [5], but FRET can be quite labor
intensive and limits its use.
Due to the limitations in the before mentioned methods we have adapted a split humanized
Renilla luciferase complementation assay (SLCA) in Nicotiana benthamiana, to perform
binary interaction screening in a mid- to high-throughput manner. We have demonstrated
that SLCA successfully shows in situ interaction between the positive controls
(homodimerization of ARAD1) with limited background signal. In addition, we used SLCA to
screen for PPI among xyloglucan biosynthetic proteins where we confirmed already
published interaction and identified novel interactions. This suggests that SLCA may be used
to analyze in situ protein-protein interactions between Golgi resident proteins in an easy and
mid- to high-throughput fashion in planta.
1. Oikawa, A., et al., Golgi-localized enzyme complexes for plant cell wall biosynthesis.
Trends in Plant Science, 2013. 18(1): p. 49-58. 2. Scheller, H.V., et al., Biosynthesis of
pectin. Physiologia Plantarum, 2007. 129(2): p. 283-295. 3. Mohnen, D., Pectin structure and
biosynthesis. Current Opinion in Plant Biology, 2008. 11(3): p. 266-277. 4. Atmodjo, M.A., et
al., Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin
biosynthetic homogalacturonan:galacturonosyltransferase complex. Proceedings of the
National Academy of Sciences, 2011. 108(50): p. 20225-20230. 5. Harholt, J., et al., ARAD
proteins associated with pectic Arabinan biosynthesis form complexes when transiently
overexpressed in planta. Planta, 2012: p. 1-14. 6. Chou, Y.-H., G. Pogorelko, and O.A.
Zabotina, Xyloglucan Xylosyltransferases XXT1, XXT2, and XXT5 and the Glucan Synthase
CSLC4 Form Golgi-Localized Multiprotein Complexes. Plant Physiology, 2012. 159(4): p.
1355-1366.
79
Posters: Synthetic- and systems biology
Systems biology approach to Lycophytes and its application in evolutionary
transcriptomics
Bjoern Oest Hansen1,Marek Mutwil1
1
Max-Planck-Institute for Molecular Plant Physiology, 14476 Potsdam, Germany
Expression atlas efforts, such as AtGenExpress for Arabidopsis, provide invaluable resource
for a studied organism [1]. High-throughput expression data, usually in form of microarrays,
can be used to study gene expression, composition of biological pathways and gene function
prediction. However, designing a microarray platform is non-trivial and prohibitively
expensive. Recent advances in RNA sequencing technology and algorithmic breakthroughs
have made it possible to generate affordable and biologically sound expression data.
We are currently generating expression atlas for Selaginella moellendorfii, a model species
for Lycophytes, and important ingredient of Chinese medicine. The tissue atlas will include all
major tissue types and environmental perturbations, such as cold, heat, high light stress and
others. Each sample will be subjected to three analyses: RNA sequencing, secondary
metabolite composition and cell wall composition.
The data will be analyzed using state of art bioinformatical approaches and will generate coexpression network that can group functionally related genes together [2]. In addition, by
comparing gene expression with metabolome analysis and cell wall composition, we will be
able to generate hypotheses linking transcripts with metabolites and polysaccharides. The
pipeline we are establishing can be applied to any organism.
Combined with publicly available RNA-seq data from other species we can identify
duplicated modules of genes within and across species, which seem to be involved in a
diverse range of cellular functions, such as protein synthesis, biotic stress, and most
prominently, cell wall biosynthesis.
Genome-wide phylogenetic analyses can be used to determine phylogenetic events and
evolutionary periods that have created gene families. Our method of analysing RNA-Seq
data can detect duplication of whole biological pathways. Combining this with phylogenetic
analysis can reveal evolutionary periods that created the duplicated pathways. This, in future,
will be used to link morphological and molecular traits that made plants as they are today.
[1] Schmid, M. et al (2005). Nat Genet. 37(5):501-6. [2] Persson, S. et al. (2005). PNAS USA,
102(24), 8633-8638;
80
Posters: Synthetic- and systems biology
Development of a protocol for Agrobacterium rhizogenes mediated transformation of
Rhodiola sp. – an approach to enhance the level of bioactive compounds.
Uffe Bjerre Lauridsen1, Martin Himmelboe2, Josefine Nymark Hegelund3, Renate Müller4,
Henrik Lütken5
Crop Sciences Section, Department of Plant and Environmental Sciences, Faculty of
Science, University of Copenhagen, Højbakkegård Allé 9-13, DK-2630 Taastrup, Denmark
[email protected], [email protected], [email protected], [email protected],
[email protected]
1
5
Rhodiola sp., commonly known as roseroot, has for centuries been utilised in biomedicine
against depression and for improving mental abilities. Specifically the root of R. rosea,
containing the two bioactive compounds salidroside and rosavin, has been used. Due to
excessive collecting, the natural populations have been declining. Natural transformation with
root-loci (rol)-genes from a wildtype Agrobacterium rhizogenes causes hairy roots (HRs) to
develop from the site of infection. Many HRs exhibit a higher content of secondary
metabolites compared to wildtype roots. The purpose of this study is to obtain HR cultures
containing rol-genes from Rhodiola sp. for future sustainable production of bioactive
compounds. Stems and leaves of R. rosea and two accessions of R. pachyclados were
sterilized and subsequently inoculated with A. rhizogenes. The inoculated plants were cocultivated with the bacteria for 3 days in darkness, washed and moved to antibioticcontaining media to remove residual bacteria. The development of HRs was monitored
concurrently. For inoculated R. pachyclados the percentages of stems explants developing
HRs were approx. 43% and 36% of accession 1 and 2, respectively, with no HRs developing
on the leaves. On the control explants HR development occurred on approx. 53% and 39%
of the stem segments and on approx. 0% and 1% of the leaves from accession 1 and 2,
respectively. The control roots were indistinguishable from the roots of the inoculated
explants, making it difficult to select transformants solely on the HR phenotype. The HR
development on inoculated R. rosea explants were approx. 20% and 17% of stem segments
and leaves, respectively. In R. rosea no control explants developed roots. The large
background of root growth in R. pachyclados indicates that there may only be few successful
events of natural transformation among the A. rhizogenes inoculated explants. The results
are more promising for R. rosea where HR development was found exclusively on inoculated
explants. The next step is to verify the transformation using PCR. The transformation method
resulted in development of HRs from R. rosea¸ solely for the inoculated explants. It is,
however, at this point uncertain if the obtained roots from R. pachyclados indeed are
transformed HRs.
81
Posters: Synthetic- and systems biology
Interconnection of Methionine Metabolism and Biosynthesis of Aliphatic
Glucosinolates in Arabidopsis thaliana
Christoph Crocoll1, Barbara Ann Halkier1
1
DynaMo Center of Excellence, Section of Molecular Plant Biology, Department of Plant and
Environmental Sciences, Faculty of Science, University of Copenhagen, Denmark
Methionine occupies a central position in the plant’s cellular metabolism. Methionine is not
only a simple building block for protein biosynthesis but serves also as precursor or
intermediate in several other pathways. In fact, 80 % of the free methionine is found as its
metabolite S-adenosyl-methionine (SAM) which is a major methyl-group donor in transmethylation reactions[1]. The central role of methionine and its metabolites is reflected in a
tight regulation and very limited availability of free methionine in plants (~5-15 nmol/g fresh
weight).
Plant species of the Brassicaceae family, including the model plant Arabidopsis, have
succeeded in adding another level of complexity by producing methionine-derived
specialized bioactive compounds known as aliphatic glucosinolates (GLs). GLs can also be
derived from other amino acids and are in general important key players in the plant’s natural
defense system against herbivores and microorganisms.
A major player in the tight regulation of methionine metabolism has been identified as the
first committed step in the de novo biosynthesis of methionine catalyzed by cystathionine
gamma-synthase (CGS). Two regulatory motifs have been identified in the N-terminal part of
CGS which allow for regulation on both the transcriptional and post-transcriptional level.
Mutations in one of the regulatory motifs result in over-accumulation of methionine to up to
60-fold higher levels than found in wild-type Arabidopsis plants[2]. Analysis of the levels of
aliphatic glucosinolates in these mutants showed an increase of up to 4-fold compared to
wild-type plants while GLs derived from tryptophan showed no such over-accumulation.
Expectantly, other methionine metabolites such as SAM were also found to over-accumulate
in these mutants.
Using molecular and bioimaging tools we try to identify key players that are responsible for
the re-allocation of methionine from the tightly regulated methionine metabolism into the
biosynthesis pathway of aliphatic glucosinolates. In addition, the acquired knowledge might
also help to eliminate bottlenecks in engineering of aliphatic glucosinolates into heterologous
host systems.
1. Ravanel, S., et al., Methionine metabolism in plants: chloroplasts are autonomous for de
novo methionine synthesis and can import S-adenosylmethionine from the cytosol. Journal of
Biological Chemistry, 2004. 279(21): p. 22548-57.
2. Inaba, K., et al., Isolation of an Arabidopsis thaliana Mutant, mto1, That Overaccumulates
Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation).
Plant Physiology, 1994. 104(3): p. 881-887.
82
Posters: Synthetic- and systems biology
Heterologous production of valuable plant natural product in eukaryotic microalgae
Annette Petersen og Hussam Hassan Nour-Eldin
Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and
Environmental Sciences, Faculty of Science, University of Copenhagen, 40 Thorvaldsensvej,
DK-1871 Frederiksberg C, Denmark
From a commercial perspective microalgae are highly attractive hosts for microbial
production of valuable plant natural products. So far heterologous expression in eukaryotic
microalgae has been characterized by single gene expression of e.g. valuable proteins. In
comparison the capacity of these host organisms for expressing multigene plant biosynthetic
pathways remains non-explored. This can in part be attributed to a limited molecular tool-set
for nuclear expression of multiple genes of interest. In this study we have developed a set of
powerful pathway expression vectors optimized for nuclear expression in Chlamydomonas
reinhardtii. As proof of concept we use them to establish the biosynthetic pathway of
glucosinolates in Chlamydomonas reinhardtii. The chosen pathway comprises 6 genes which
need to be expressed in the same strain. The two expression vectors allow easy screening
for complete pathway insertion and ensure high expression levels of the inserted pathway.
We present the strategy and the initial results. The advantages of the established expression
system are discussed.
83
Posters: Synthetic- and systems biology
Ostreococcus tauri – a novel expression system for elucidation of the function of cell
wall related genes
Karen S. Nissen1, Bodil Jørgensen1, Elisabeth Johansen1, Maria D. Mikkelsen1, Jonatan U.
Fangel1, Peter Ulvskov1, William G. T. Willats1
1
Copenhagen University, Faculty of Life Sciences, Department of Plant Biology and
Biotechnology, Section for Plant Glycobiology, Thorvaldsensvej 40, 1870 Frederiksberg C,
Copenhagen, Denmark.
Plant cell walls are diverse and highly complex structures that provide support, protection
and define shape and size of plant cells. Identification of gene function in a system with such
complex background is challenging. In addition, the multicellularity and genetic redundancy
of multicellular plants, pose a challenge for the elucidation of the genetic basis of cell wall
structures.
Unicellular plants as algae have great potential as model organisms for studying the biology
of photosynthetic organisms. The unicellular green algae Ostreococcus tauri, belonging to
the class Prasinophyceae, has been described as the smallest eukaryote having a size of
~1µm [1]. The cellular simplicity, low genetic redundancy and small genome of Ostreococcus
tauri make it a suitable system for analyzing basic cellular processes.
Recently, Ostreococcus tauri has been applied as an expression system for studies of the
plant circadian clocks, where Luciferase reporter lines have helped to characterize clock
gene function and revealed evolutionary conserved features of circadian regulatory networks
[2]. The identification of a simple cell wall in Ostreococcus tauri [3] has further made this
organism an eligible system for studying cell wall related genes. We aim to implement
Ostreococcus tauri as a new expression system for cell wall related genes, to gain insight
into the genetic mechanisms underlying plant cell wall diversity and evolution.
1. Chretiennotdinet, M.J., et al., A New Marine Picoeucaryote - Ostreococcus tauri gen et spnov (Chlorophyta, Prasinophyceae). Phycologia, 1995. 34(4): p. 285-292.
2. Corellou, F., et al., Clocks in the Green Lineage: Comparative Functional Analysis of the
Circadian Architecture of the Picoeukaryote Ostreococcus. Plant Cell, 2009. 21(11): p. 34363449.
3. Personal communication, Peter Ulvskov, unpublished data.
84
Posters: Synthetic- and systems biology
Double-blind experiment shed light on protein-glycan interactions
Armando Salmeán*1 Alexia Guillouzo2, Robert Larocque2, Henriette L. Pedersen1 Murielle
Jam2, Mirjam Czjzek2, Gurvan Michel2, Cécile Hervé*2 and William G.T. Willats1
* Authors contributed equally [email protected], [email protected]
1
Plant Cell wall Evolution and Diversity, section for Plant Glycobiology, Department of Plant
and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871
Frederiksberg C (Denmark)
2
Marine Glycobiology, Station Biologique de Roscoff, UPMC - Centre National de la
Recherche Scientifique, Place Georges Teissier, 29680 Roscoff (France)
CNRS/UPMC, UMR 7139 Marine Plants and Biomolecules, Station Biologique de Roscoff, F29682 Roscoff, France
Algal cell walls are highly diverse, complex and heterogeneous. Algae are phylogenetically
distant from land plants and have evolved independently from each other, thus they contain
unique and original polysaccharides in their cell wall including compounds with industrial
relevance such as agar, agarose, alginates and carrageenans. Comprehensive Microarray
Polymer Profiling (CoMPP) has proofed a powerful tool to analyse the glycome of plant cell
walls, notably through the production and use of particular cell-wall probes. However due to a
lack of specific algal probes we can only obtain a partial view of the cell-wall architecture in
algae with this technique.
On the other hand polysaccharides contained in the algal cell walls are efficiently utilized as
carbon and energy sources by marine bacteria. Therefore this biochemical carbohydrate
diversity from marine macroalgae is mirrored by a variety of marine bacterial enzymes that
specifically recognize and degrade these substrates, a high throughput method for
heterologous expression of bacterial CAZymes has been developed in the Station Biologique
de Roscoff (France) however most of them stay uncharacterized. These proteins can
potentially be used as probes for CoMMP analysis.
For that reason we combined both techniques collating this double blind to identify and
characterize new marine probes. The results of this double blind experiment rendered with
the identification of novel putative probes which contribute resolving the fine structure of algal
cell walls.
85
About Plant Biotech Denmark
Plant Biotech Denmark is a non-profit network of Danish public plant biotechnology research
groups. The objective is to strengthen Danish plant biotechnology research.
Steering Committee
Henrik Brinch-Pedersen (chairman)
Senior Scientist
Department of Molecular Biology and Genetics
Aarhus University
Email: [email protected]
Svend Christensen
Professor, Head of Department of Plant and Environmental Sciences
University of Copenhagen
Email: [email protected]
Søren K. Rasmussen
Professor
Department of Plant and Environmental Sciences
University of Copenhagen
Email: [email protected]
Birte Svensson
Professor
Systems Biology, Enzyme and Protein Chemistry
Technical University of Denmark
Email: [email protected]
Kåre Lehmann Nielsen
Associate Professor
Department of Biotechnology, Chemistry and Environmental Engineering
Aalborg University
Email: [email protected]
Secretariat
Solveig Krogh Christiansen
Senior Advisor
Plant Biotech Denmark
c/o University of Copenhagen
Thorvaldsensvej 40
1871 Frederiksberg C
Email: [email protected]
Inga Christensen Bach
Communications Officer
Plant Biotech Denmark
c/o University of Copenhagen
Thorvaldsensvej 40
1871 Frederiksberg C
Email: [email protected]
86
87
88
Laura
Henrik
Christian
Meike
Yan-Jun (Angie)
Svend
Solveig Krogh
Mette
Signe Sandbech
David
Maria
Christoph
Adrian
Behrooz
Louise
Thomas
Alice
Adiphol
Malene
Giuseppe
Christoph
Tonni Grube
Johan
Torben
Raquel
Marielle
Inga
Søren
Laetitia
Gregorio
Christian
Mickael
Andreas
Søren
henrik
Gianluca
PhD student
Senior scientist
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Communications Officer
associate professor
post doc
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chef
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Akademisk medarbejder
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First_name
GEZIEL
ALI ABDUREHIM
Mette Sondrup
Position
PhD Student
PhD student
Post doc
Department
Plant and Environmental Sciences
Plant and Environmental Sciences
Biotechnology, Chemistry and
Environmental Engineering
Andersen
Plant and Environmental Sciences
Andersen-Ranberg
Plant and Environmental Sciences
Asp
Molecular Biology and Genetics
Azevedo
Department of biology
Babineau
Agroecology
Bach
Plant and Environmental Sciences
Bak
Plant and Environmental Sciences
Baldwin
Plant and Environmental Sciences
Barba-Espin
Dept Systems Biology
Berg Oehlenschlæger Plant and Environmental Sciences
Blaise
Molecular Biology and genetics
Blennow
Plant and Environmental Sciences
Borg
Molecular Biology and Genetics
Boserup
Bretani
plant, food and envirorment
biotechnology (Milano, Italy)
Brey
Plant and Environmental Sciences
Brinch-Pedersen
Molecular Biology and Genetics
Bukh
Plant and Environmental Sciences
Burow
Plant and Environmental Sciences
Chen
Agroecology
Christensen
Plant and Environmental Sciences
Christiansen
Clausen
Plant and Environmental Sciences
Clausen
Kemiteknik
Collinge
Plant and Environmental Sciences
Constantin
Plant and Environmental Sciences
Crocoll
Plant and Environmental Sciences
Czaban
Molecular Biology and Genetics
Darbani
Molecular Biology and Genetics
de Bang
Plant and Environmental Sciences
de Bang
Plant and Environmental Sciences
De Porcellinis
Plant and Environmental Sciences
Dilokpimol
Plant and Environmental Sciences
Dinesen
Plant and Environmental Sciences
Dionisio
Molecular Biology and genetics
Dockter
Last_name
AGUILAR
AHMED
Andersen
University of Copenhagen
Aarhus University
University of Copenhagen
University of Copenhagen
Aarhus University
University of Copenhagen
Plant Biotech Denmark
University of Copenhagen
Technical University of Denmark
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aarhus University
Aarhus University
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aarhus University
Carlsberg Laboratory
University of Copenhagen
University of Copenhagen
Aarhus University
University of Copenhagen
Aarhus University
University of Copenhagen
University of Copenhagen
University of Copenhagen
Technical University of Denmark
University of Copenhagen
Aarhus University
University of Copenhagen
Aarhus University
mad.dk
Copenhagen, Frederikensberg
University_Company
University of Copenhagen
University of Copenhagen
Aalborg University
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89
Vincent G.H.
Mette
Thomas
Barbara Ann
Bjoern Oest
Nikolaj
Jesper
Jesper F.
Laura Arribas
Kurt
Jesper
Preben Bach
Inger
Dennis Berg
Irina Alexandra
Simon
Susanne
Ahmed
Iver
senior scientist
Associate Professor
Prof
PhD student
PhD student
Associate Professor
PhD Student
PhD student
Senior Advisor
Postdoc
Adjunct professor
Senior scientist
PhD student
PhD student
Postdoc
Associate professor
Breeding manager
Forskningsspecialist
Postdoctoral researcher
Dennis
PhD student
Pernille Østerbye
Post Doc
Jonatan
PhD student
Jacqueline
Center Manager
Bente
PhD
Lorenzo
Associate Professor
Christine
Sr Staff Sci, Head of IB R&DJens
Ph.D. student
Jens
Associate professor
Anja Thoe
PhD student
Joel
PhD fellow
Nethaji
Associate professor
Naomi
postdoc
Bhim
Ph.D.- student
Nele
PhD fellow
Sylwia
seniorforsker
Per
Postdoc
Benjamin D.
Professor
Hjortsholm
Holck
Holm
Holme
Holt
Ionescu
Ip Cho
Jacobsen
Jahoor
Jakobsen
Hansen
Harholt
Havelund
Hernandez
Günther-Pomorski
Halkier
Hansen
Grønlund
Eriksson
Erthmann
Fangel
Farrell
Faurby
Fimognari
Finnie
Frisbæk Sørensen
Frydenvang
Fuglsang
Fürstenberg-Hägg
Gallage
Geshi
Ghaley
Gjendal
Głazowska
Gregersen
Gruber
Eijsink
Technical University of Denmark
Norwegian University of Life
Sciences (NMBU)
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aarhus University
University of Copenhagen
University of Copenhagen
Technical University of Denmark
DuPont IB
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aarhus University
Chem. & Biochem. Engineering
Plant and Environmental Sciences
Molecular Biology and Genetics
Molecular Biology and Genetics
Molecular Biology and Genetics
Plant and Environmental Sciences
Plant and Environmental Sciences
Department of Systems Biology
DAPB
University of Copenhagen
Aarhus University
Aarhus University
Aarhus University
University of Copenhagen
University of Copenhagen
Technical University of Denmark
Nordic Seed
Technical University of Denmark
University of Copenhagen
University of Copenhagen
Max Planck Institute for Molecular
Plant Physiology
Plant and Environmental Sciences
University of Copenhagen
Plant and Environmental Sciences
University of Copenhagen
Molecular Biology and Genetics
Aarhus University
Department of Biology, Bioinformatics University of Copenhagen
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Molecular Biology and Genetics
Molecular Plant Nutrition, Leibniz
Institute of Plant Genetics and Crop
Plant Research (IPK), Gatersleben
Dept of chemical and biochemical
engineering
Plant and Environmental Sciences
Plant and Environmental Sciences
Department of Chemistry,
Biotechnology and Food Science
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Molecular Biology and Genetics
Plant and Environmental Sciences
Plant and Environmental Sciences
Systems Biology
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
90
Natascha
Astrid
Peter Skov
Katarzyna
Rasmus
Fouzia
Daniela
Marlen
Bo
Lærke Münter
Uffe
Tomas
Florian
Nora
Lizhi
Rosa Laura
Christian Have
Michael
Henrik
Claus Krogh
Research assistant
Plant Breeder
phd scholor
Postdoc
PhD student
PhD Student
post doc
Research assistant
PhD Student
PhD
Student
PhD student
Associate Professor
PhD student
Lektor
Associate Professor
Post. Doc.
Brian
Hanne Grethe
Eva
Peter
Postdoc
Breeder
Postdoc
Professor
PhD Student
PhD student
Master student
Kaukovirta-Norja
Anu
Professor
Krucewicz
L. Hjortshøj
Laeeq
Lai
Landschreiber
Larsen
Lassen
Lauridsen
Laursen
Leplat
Linscheid
Long
Lopez Marques
Lund
Lyngkjær
Lütken
Madsen
Krahl Hansen
Kristensen
Kristensen
King
Kirk
Knoch
Koehler
Jakubauskas
Jensen
Jensen
Jensen
Jensen
Jensen
Jensen
Johansen
Justesen
Jørgensen
Jørgensen
Jørgensen
Kaminski
MSc student of BiochemistryDainius
Senior Scientist
Christian Sig
Head of Department
Erik
Lea
PhD Student
Maria Stumph
Professor
Poul Erik
Susanne Langgård
staff scientist
Elisabeth
Post. Doc.
Bo
Associate professor
Bodil
Associate Professor
Kirsten
Morten Egevang
PhD Candidate
Kacper Piotr
Molecular Biology and Genetics
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Molecular Biology and Genetics
Enzymology
Plant and Environmental Sciences
German Research Centre for Food
Chemistry
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Molecular Biology and Genetics
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Biotechnology, Chemistry and
Environmental Engineering
VTT Technical Research Centre of
Finland
Plant and Environmental Sciences
Plant and Environmental Sciences
University of Copenhagen
University of Copenhagen
University of
Copenhagen/Carlsberg Laboratory
The Carlsberg Laboratory
Sejet Plantbreeding I/S
Aarhus University
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aarhus University
University of Copenhagen
LKF Vandel
University of Copenhagen
Freising, Germany
University of Copenhagen
DLF-TRIFOLIUM A/S
Aarhus University
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aalborg University
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
91
Mohammed Saddik
Jozef
Renate
Birger Lindberg
Ian Max
Svenning
Merethe
Istvan
Johnathan A.
Mari-Anne
Catherine S.
Ea Høegh Riis
Kåre Lehmann
Associate Professor
postdoc
Professor
Professor
Professor
phd
PhD student
Postdoc
Professor
Associate Professor
Lab technician
Student
Lektor
Post Doctorial Fellow
Morten Thrane
PhD student
Sebastian
guest researcher
Mamoru
PhD student
Karen Stoltenberg
Department Manager
Finn
Direktør
Peter
PhD student
Lene Irene
Associate Professor
Stefan
Head of Molecular Breeding Jihad
PhD student
Cristiana
post doc
harriet
postdoc
Eirini
Associate Professor
Pai
Associate Professor
Carsten
Mikkelsen
Mileck
Mirza
Morgante
Maria Dalgaard
Maya
Nadia
Michele
Post-Doc
Master's Student
PhD
Professor
Nielsen
Nintemann
Nishimoto
Nissen
Okkels
Olesen
Olsen
Olsson
Orabi
Paina
parsons
Pateraki
Pedas
Pedersen
Nielsen
Motawia
Mravec
Müller
Møller
Møller
Möller
Mørch Frøsig
Nagy
Napier
Newman
Nielsen
Nielsen
Madsen
Malik
malinovsky
Manstretta
Mark
Mellor
Metzlaff
PhD student
Svend Roesen
Seed Enhancement ScientisAli Hafeez
postdoc
frederikke gro
MsC student
Raffaele
PhD Student
Christina
PhD student
Silas
Director
Karin
Molecular Biology and Genetics
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
New Technology
Plant and Environmental Sciences
Production and Process Technology
Plant and Environmental Sciences
Plant and Environmental Sciences
DTU Systems Biology
Plant and Environmental Sciences
The European Plant Science
Organization (EPSO)
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Dipartimento di Scienze Agrarie ed
Ambientali, Università di Udine
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Molecular Biology and Genetics
Plant and Environmental Sciences
Plant and Environmental Sciences
Molecular Biology and Genetics
Rothamsted Research
Plant and Environmental Sciences
Plant and Environmental Sciences
Biotechnology, Chemistry and
Environmental Engineering
Biotechnology, Chemistry and
Environmental Engineering
Plant and Environmental Sciences
Plant and Environmental Sciences
University of Copenhagen
University of Copenhagen
Carlsberg Laboratory
University of Copenhagen
Chr Hansen A/S
ActiFoods ApS
University of Copenhagen
University of Copenhagen
Nordic Seed A/S
Aarhus University
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aalborg University
[email protected]
[email protected]
[email protected]
[email protected]
University of Copenhagen
University of Copenhagen
University of Copenhagen
Istituto di Genomica Applicata,
Italy
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aarhus University
University of Copenhagen
University of Copenhagen
Aarhus University
Harpenden, UK
University of Copenhagen
University of Copenhagen
Aalborg University
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
University of Copenhagen
Syngenta Seeds AB
University of Copenhagen
University of Copenhagen
Technical University of Denmark
University of Copenhagen
92
Birte
Elsa
Pernille
Mads
Claus
Academic employee
Associate Professor
PhD student
Postdoc
seniorforsker
Professor
PhD exchange student
PhD
ceo
Professor
PhD Researcher
Post Doc
PhD student
PhD student
Senior scientist
PhD Student
Professor
PhD
Professor
PhD
Ph.D.
Post doc.
Senior advisor
PhD student
Scientist
Associate professor
Owner
PhD student
Professor
Research assistent
Associate Professor
postdoc
PostDoc
Stefan
Maria
Anne-Mette
Annette
Marie
Gert
Lisbeth Rosager
Morten
Erick
Søren K
Aleksander
Fred
Milena
Christian
Maja
Christina
Yumiko
Armando
Raquel
Niels
Jan K.
Daiana
Lizette
Bjarne
Alexander
Pratik
Daniele
Agnieszka
Katsiaryna
Inez H
Verdiana
Jens
Abida
Ph.D. student
PhD student
Lab technician
Pentzold
Plant and Environmental Sciences
Perestrello Ramos HenPlant and Environmental Sciences
Petersen
Plant and Environmental Sciences
Petersen
Plant and Environmental Sciences
Pireyre
Plant and Environmental Sciences
Poulsen
Poulsen
Plant and Environmental Sciences
Poulsen
Ramos
Plant and Environmental Sciences
Rasmussen
Plant and Environmental Sciences
Riise Hansen
Plant Biology and biotechnology
Rook
Plant and Environmental Sciences
Roux
Plant Molecular Biology
Ruzanski
Enzymology, Carlsberg Laboratory
Rydahl
Plant and Environmental Sciences
Rønn Ingvardsen
Molecular Biology and Genetics
Sakuragi
Plant and Environmental Sciences
Salmeán
Plant and Environmental Sciences
Sánchez Pérez
Plant and Environmental Sciences
Sandal
Molecular Biology and Genetics
Schjoerring
Plant and Environmental Sciences
Schmidt
Molecular Biology and genetics
Schneider
Schou
Schulz
Plant and Environmental Sciences
Shah
Plant and Environmental Sciences
Silvestro
Plant and Environmental Sciences
Siwoszek
Plant and Environmental Sciences
Skryhan
Plant and Environmental Sciences
Slamet-Loedin
International Rice Research Institute
Steccanella
Plant and Environmental Sciences
Stougaard
Molecular Biology and Genetics
Sultan
Department of Systems Biology,
Enzyme and Protein Chemistry
Svensson
BioSys
Sverrisdóttir
Biotechnology, Chemistry and
Environmental Engineering
Sølvhøj Roelsgaard
Plant and Environmental Sciences
Sønderkær
Biotechnology, Chemistry and
Environmental Engineering
Saabye
Research and technology politics
Danish Agriculture and Food
Council
University of Copenhagen
Aalborg University
Technical University of Denmark
Aalborg University
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
GPO
University of Copenhagen
Breedex
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
Carlsberg Laboratory
University of Copenhagen
Aarhus University
University of Copenhagen
University of Copenhagen
University of Copenhagen
Aarhus University
University of Copenhagen
Aarhus University
Carlsberg Laboratory
CEBIO
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
The Philippines
University of Copenhagen
Aarhus University
Technical University of Denmark
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
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[email protected]
93
Postdoc
PhD student
Associate Professor
post doc
postdoc
postdoc
Scientist
PhD student
PhD student
postdoc
PhD student
Master student
Professor
Professor
Associate Professor
PhD senior scientist
PhD student
PhDstudent
MSc. Stud.
Associate Professor
Vanja
Sebastian
Bart P.H.J.
Hans
Anna Maria
Stine
Mohammad Nasir
Silvia
Daniel
Eva
Xiao
Toni
Artur
Bernd
deyang
Fen
Mika
Shakhira
Xin
Iwona
Agnieszka
Jeppe Thulin
Tanackovic
Theobald
Thomma
Thordal-Christensen
Torp
Tuvesson
Uddin
Vidal-Melgosa
Vik
Vincze
Wang
Wendt
Wlodarczyk
Wollenweber
xu
Yang
Zagrobelny
Zakhrabekova
Zhan
Ziomkiewicz
Zygadlo Nielsen
Østerberg
Plant and Environmental Sciences
Agroecology
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant Physiology
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Molecular Biology and Genetics
Plant and Environmental Sciences
Plant and Environmental Sciences
Plant and Environmental Sciences
Laboratory of Phytopathology
Plant and Environmental Sciences
Plant and Environmental Sciences
Development, Plant breeding
Molecular Biology and Genetics
Plant and Environmental Sciences
University of Copenhagen
University of Copenhagen
Wageningen University
University of Copenhagen
University of Copenhagen
Lantmännen Lantbruk
Aarhus University
University of Copenhagen
University of Copenhagen
Aarhus University
University of Copenhagen
Carlsberg Laboratory
University of Copenhagen
Aarhus University
University of Copenhagen
University of Copenhagen
University of Copenhagen
Carlsberg Research Center
University of Copenhagen
University of Copenhagen
University of Copenhagen
University of Copenhagen
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]