1. No category

2015 Midwestern Section Annual Meeting
March 21-22, 2015
Donald Danforth Plant Science Center
St. Louis, MO
Thanks to our Sponsors!
American Society of Plant Biologists Midwestern Section
Section Officers 2014-2015
Chair
Darron R. Luesse ([email protected])
Southern Illinois University Edwardsville
Vice Chair
Aaron Wyman ([email protected])
Spring Arbor University
Secretary-Treasurer
Gustavo MacIntosh ([email protected])
Iowa State University
Executive Com Rep
Edgar Cahoon ([email protected])
University of Nebraska-Lincoln
Past Chair, ex officio
Edgar Cahoon ([email protected])
University of Nebraska-Lincoln
Local Meeting Organizer
Dmitri A. Nusinow ([email protected])
Donald Danforth Plant Science Center
Short Program
Saturday, March 21
7:00 – 8:00 ...................................................................... Registration/Check-in/Poster Set-up/Breakfast
8:00 – 9:45 ......................................................................................................................... Oral Session I
9:45 – 10:15 .................................................................................................................................... Break
10:15 – 12:00 .................................................................................................................... Oral Session II
12:00 – 1:30 ....................................................................................................................................Lunch
1:30 – 3:30 ...................................................................................................................... Oral Session III
3:30 – 4:00 ...................................................................................................................................... Break
4:00 – 5:00 .................................................................................................................... Keynote Speaker
5:00 – 6:30 ....................................................................................................................... Poster Viewing
6:45 – 8:00 .................................................................................................................................. Banquet
Sunday, March 22
8:00 – 10:00 .................................................................................................................... Oral Session IV
10:00 – 10:30 ............................................................................................................... Business Meeting
10:30 – 12:00 ....................................................................................................................Oral Session V
12:00 – 12:45 .............................................................................................. Announcements and Awards
Meeting Locations ................................................................The Donald Danforth Plant Science Center
http://www.danforthcenter.org/
Registration ................................................................................................................................... Lobby
Keynote, Featured, & Oral Presentations................................................................... AT&T Auditorium
Poster Viewing ................................................................................................................... Lower Atrium
Saturday Lunch .................................................................................................................. Upper Atrium
Saturday Banquet ............................................................................................................... Upper Atrium
Parking .......................................................................................................................................... On site
Note: Vegetarian options will be available as part of all meals provided.
Posters: Posters should be set up before the scheduled poster session at 5 p.m.
Parking: Please park in the lot connected to the Danforth Plant Science Center and BRDG Park
building. Do not park in St Paul's Evangelical Church parking lot just east of the Danforth Campus.
1
Saturday, March 21
Morning
7:00 – 8:00
Registration/Check-in/Poster Set-up/Breakfast ......................... Lobby/ Lower Atrium
8:00
Welcoming Remarks
8:15 – 10:00
Oral Session I .............. Moderator – Edgar Cahoon, University of Nebraska- Lincoln
8:15
Defining the Roles of Serine Palmitoyltransferase-Interacting Proteins in the Regulation
of Sphingolipid Homeostasis
Athen Kimberlin, University of Nebraska-Lincoln – Abstract #T1
8:30
A vesicular trafficking ENTH-domain protein functions in plasma membrane localization
of the receptor kinase FLS2
Carina Collins, University of Missouri-Columbia – Abstract #T2
8:45
Investigation into the CBF Cold Pathway of Soybean (Glycine max) Using
AtRD29a::GFP/GUS Transgenic Soybean
Jennifer Robison, Indiana University-Purdue University Indianapolis – Abstract #T3
9:00
Local versus Systemic Metabolic Changes Induced by the Soybean Aphid and the Effect
on Growth of Corn Earworms on Soybean
Martha Ibore, Iowa State University – Abstract #T4
9:15
Four Hydroxyproline Galactosyltransferases, GALT3, GALT4, GALT5 and GALT6,
Function in Arabinogalactan-Protein Glycosylation, Growth and Development in
Arabidopsis
Debarati Basu, Ohio University – Abstract #T5
9:30
Genetic Characterization and Fine Mapping of the qSD10 Seed Dormancy in rice (Oryza
sativa L.)
Wirat Pipatpongpinyo, South Dakota State University – Abstract #T6
9:45 – 10:15
Break ........................................................................................................... Lower Atrium
(Tour of Donald Danforth Plant Science Center, meet in Lobby)
10:15 – 12:00 Oral Session II ............................ Moderator – Paula McSteen, University of Missouri
10:15
FEATURED SPEAKER: Phosphorylation-dependent regulation of G-protein cycle
during nodule formation in soybean
Sona Pandey, Donald Danforth Plant Science Center, St. Louis, MO
10:45
MSL8 Regulates Opposing Osmotic Challenges in Pollen Hydration and
Germination
Eric S. Hamilton, Washington University in St. Louis – Abstract #T7
11:00
Arabinogalactan-Protein and Cell Wall-Associated Receptor Kinase Interactions: Cellular
Signaling of Root Growth in Arabidopsis
Hayley D. Herock, Ohio University – Abstract #T8
11:15
microRNA160 Regulation of Auxin-Cytokinin Balance during Soybean Root Nodule
Development
Spencer Schreier, South Dakota State University – Abstract #T9
2
11:30
Engineering Resistance to the Bean Pod Mottle Virus Using a Protease Detection System
Matthew D. Helm, Indiana University – Abstract #T10
11:45
Metabolic Characterization of Pennycress (Thlaspi arvense L.) for the Production of Valuable
Fatty Acids
Enkhtuul Tsogtbaatar, The Ohio State University – Abstract #T11
Afternoon
12:00 – 1:30
Lunch, Upper Atrium
1:30 – 3:30
1:30
Oral Session III ..... Moderator – Dmitri A. Nusinow, Donald Danforth Plant Science Center
Genome-Scale Nitrogen Responsive Gene Expression during Maize Development
Jennifer J. Arp, University of Illinois Urbana-Champaign – Abstract #T12
1:45
Maize Nodal Root Growth Response to Water Deficits: Characterization in a Divided-Chamber
Model System
Kara J. Riggs, University of Missouri, Columbia – Abstract #T13
2:00
Gene Expression Microarray of the GPS Treatment Reveals Novel Genes Involved in Early
Gravitropic Signal Transduction
Charles A. Cook, Ohio University – Abstract #T14
2:15
An expansion may be involved in gravitropism in Arabidopsis thaliana
Megan A. Osika, Ohio University – Abstract #T15
2:30
Characterization of vegetative and reproductive defects in the maize tassel-less 4 mutant
Dennis X. Zhu, University of Missouri-Columbia – Abstract #T16
2:45
Formation of Indigo and Indirubin in E. coli expressing a P450 gene from woad
Laxmi Sagwan, Southern Illinois University-Carbondale – Abstract #T17
3:00
Identifying Plant Polyamine Exchangers that Affect Plant Development
Lingxiao Ge, Bowling Green State University – Abstract #T18
3:15
Roles of MAP KINASE17 in Peroxisome Proliferation and NaCl Tolerance in Arabidopsis
thaliana
Elizabeth M. Frick, Washington University in St. Louis – Abstract #T19
3:30 – 4:00
Break ........................................................................................................................ Lower Atrium
4:00
KEYNOTE SPEAKER: Using Proteomics to Study Signaling and Secretion During Responses to
Biotic and Abiotic Stresses
Scott Peck, University of Missouri-Columbia
5:00 – 6:30
5:00 – 5:45
5:45 – 6:30
Poster Session........................................................................................................... Lower Atrium
Presenters of Odd numbered posters will be at their posters
Presenters of Even numbered posters will be at their posters
Evening
6:45 – 8:00
Banquet .................................................................................................................... Upper Atrium
3
Sunday, March 22
Morning
7:00 – 8:00
Breakfast ..................................................................................................... Lower Atrium
8:00 – 10:00
8:00
Oral Session IV ........................ Moderator – Aaron Wyman, Spring Arbor University
Quantitative Trait Loci Mapping For Root Vigor And Days To Flowering In Canola
(Brassica napus L.)
Muhammad Arif Uz Zaman, North Dakota State University – Abstract #T20
8:15
Post-Synthetic Modification of the Plant Cell Wall as a Tool to Study Cell Wall Integrity
Control Involved in Biotic Stress Response
Nathan Reem, Iowa State University – Abstract #T21
8:30
Evolution of inflorescence development in maize and related grasses (Poaceae)
Eden A. Johnson, University of Missouri-Columbia – Abstract #T22
8:45
Loss of Function of RNS2, a Housekeeping RNase T2 enzyme, Causes Alterations in
Cellular Homeostasis and Growth in Arabidopsis
Stephanie Morriss, Iowa State University – Abstract #T23
9:00
Regulation of HD-ZIP III Transcription Factor During Soybean Nodule Development
Suresh Damodaran, South Dakota State University – Abstract #T24
9:15
Preparing for a Proteomics Analysis of Arabidopsis Seedlings Grown in Microgravity
Proma Basu, Ohio University – Abstract #T25
9:30
Putative Histone Readers EML1 and EML3 Differentially Impact Geminivirus Infection
Tami Coursey, Ohio State University – Abstract #T26
9:45
MSH1-Derived Epigenetic Breeding Potential in Tomato
Xiaodong Yang, University of Nebraska-Lincoln – Abstract #T27
10:00 – 10:30 Business Meeting ................................................................................ AT&T Auditorium
10:30 – 12:00 Oral Session V................................... Moderator – Kira Veley, Washington University
10:30
Map-based cloning of Rj4, a gene controlling nodulation specificity in soybean
Fang Tang, University of Kentucky – Abstract #T28
10:45
The Membrane-anchored Ubiquitin-fold (MUB) Protein Family Regulates Ubiquitylation
in Arabidopsis
Xiaolong Lu, Saint Louis University – Abstract #T29
11:00
Next Generation Weed Control System in Cotton
Clayton T. Larue, Monsanto Company – Abstract #T30
11:15
ASPB & The Plant Science Community – A Vision for the Future
Susan Cato, American Society of Plant Biologists
11:30
Development and utilization of high-throughput phenotyping tools to investigate the
genetic basis of drought tolerance in Setaria
Max J. Feldman, Donald Danforth Plant Science Center – Abstract #T31
11:45
Localization of Maize SUCROSE TRANSPORTER1 Reveals Novel Insights into
Phloem Loading and Sucrose Retrieval
Robert F. Baker, University of Missouri-Columbia – Abstract#T32
Afternoon
12:00 – 12:30 Announcements, Awards & Departure ............................................ AT&T Auditorium
4
ORAL ABSTRACTS
ORAL ABSTRACTS (T1 – T32)
T1.
Defining the Roles of Serine Palmitoyltransferase-Interacting Proteins in the Regulation
of Sphingolipid Homeostasis
Kimberlin, Athen1, Yang, Fan1, Han, Gongshe2, Alfano, James1, Dunn, Teresa2, and Cahoon, Edgar1,
University of Nebraska, Lincoln, NE, 2Uniformed Services of the Health Sciences, Bethesda, MD
1
Maintenance of sphingolipid homeostasis is critical for eukaryotic cell growth and regulation of
programmed cell death (PCD). Serine palmitoyltransfersase (SPT) catalyzes the first step in sphingolipid
biosynthesis and is the primary regulatory point for sphingolipid homeostasis. A pair of potential SPT
regulatory proteins, orosomucoid-like proteins (AtORM1 and AtORM2), have been identified in
Arabidopsis. Studies were undertaken with AtORM1 and AtORM2 to determine the role of ORM proteins
in cellular functions and sphingolipid homeostatic regulation, which has yet to be fully defined for plants
and animals. To date, no null T-DNA mutants have been identified for either gene, although viable RNAi
and overexpression lines have been generated for AtORM1 and AtORM2. Based on Q-PCR
measurements, AtORM1 and AtORM2 are expressed throughout Arabidopsis plants, although AtORM2
transcripts are more enriched in pollen, while AtORM1 transcripts are more enriched in vegetative
tissues. AtORM1/2 over-expression lines displayed enhanced resistance to the PCD-inducing mycotoxin
fumonisin B1(FB1) and decreased long chain base (LCB) accumulation. Conversely, AtORM1/2 RNAi
lines showed enhanced FB1 sensitivity and increased LCB accumulation. Interestingly, AtORM1/2 overexpression lines also have altered ceramide synthase activity with Atloh1/Atloh3 activity being higher and
Atloh2 activity being lower, indicating a possible connection between LCB synthesis and downstream
sphingolipid synthesis. AtORM1/2 over-expression lines and AtORM1/2 RNAi lines also display altered
sensitivity to heat stress, altered responses to pathogen interaction, and significant changes in plant size.
This emphasizes the importance of sphingolipid homeostasis and would suggest a complex regulatory
role for AtORM proteins.
T2.
A vesicular trafficking ENTH-domain protein functions in plasma membrane localization
of the receptor kinase FLS2
Collins, Carina, Bond, Lauren, Anderson, Jeffrey, Smith, John, Salamango, Daniel, Peck, Scott, and
Heese, Antje, University of Missouri, Columbia, MO
The plasma membrane (PM) serves as a crucial contact point between a host and potential pathogens.
Plant proteins at this location are required for many aspects of plant defense, from initial microbe
perception to pathogen growth restriction. A complex and dynamic vesicular trafficking network
(including secretion and endocytosis) is essential to ensure the correct localization and level of host
components at the PM necessary for effective immune responses. As such, the receptor kinase Flagellin
Sensing 2 (FLS2) needs to be localized to the PM to perceive its ligand flg22 to initiate a robust immune
response. However, few vesicular trafficking components are known with roles in FLS2 trafficking to and
from the PM. Here, we used a phosphoproteomic screen in Arabidopsis to identify an ENTH-domain
containing protein differentially phosphorylated in response to flg22, thus potentially placing this protein
in the flg22-response pathway. In plants, ENTH-domain proteins are known to function in clathrinmediated vesicle formation at the Trans-Golgi Network (TGN), potentially for delivery of newly
synthesized cargo proteins to the vacuole or PM, or of endocytosed proteins for degradation in the
vacuole or recycling to the PM. Two independent enth mutant alleles showed defects in all investigated
flg22-response and were more susceptible to infection by bacterial Pseudomonas syringae pv. tomato
strains. Total and microsomal FLS2 protein levels were similar between Col-0 and enth mutants.
However, utilizing a simplified PM-enrichment methods, we correlated impaired flg22-signaling to
reduced FLS2 protein levels at the PM. Our data identified this vesicular trafficking ENTH-protein as a
novel positive regulator of innate immunity with roles in regulating correct FLS2 abundance at the PM.
6
T3.
Investigation into the CBF Cold Pathway of Soybean (Glycine max) Using
AtRD29a::GFP/GUS Transgenic Soybean
Robison, Jennifer, Yamasaki, Yuji, and Randall, Stephen, Indiana University-Purdue University
Indianapolis, Indianapolis, IN
As a reported cold intolerant species, the productivity of domestic soybean (Glycine max) is limited by
temperature and other factors. The CBF cold responsive pathway has been well characterized in
Arabidopsis and CBF homologous genes have been discovered in soybean. Under cold stress conditions,
soybean CBF transcript levels increase similarly to cold tolerant species, however the expected CBF
downstream targets appear unresponsive. To investigate the ability of soybean to up-regulate a known
CBF target, plants containing an Arabidopsis RD29a promoter driving GFP/GUS construct were
acquired. Abiotic stress responsive motifs present in the RD29a promoter include CRT/DRE, which
responds to cold; ABRE, which responds to ABA; CGTCA, which responds to wounding; and MBS,
which responds to drought. Two independently transformed soybean homozygote lines expressing
AtRD29aProm::GFP/GUS have been acquired. To evaluate the response of the reporter gene to abiotic
conditions, changes in GUS activity via fluorimetric assay were measured in 12 day old seedling leaves
after 24 hours of either cold or wounding. In both transformed lines, there was a marginal increase in
GUS activity (1.5 - 2 fold) after 24 hours in the cold and a substantial increase of GUS activity (4 - 13
fold) after wounding. These results indicate that the construct is functional in both lines. The limited
increase in GUS activity in response to cold is consistent with soybeans lack of significant acquisition of
cold tolerance.
T4.
Local versus Systemic Metabolic Changes Induced by the Soybean Aphid and the Effect
on Growth of Corn Earworms on Soybean
Ibore, Martha, Kanobe, Charles, Nguyen, Khoi, and MacIntosh, C. Gustavo, Roy J. Carver Department
of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA
Soybean aphids (Aphis glycines) are phloem feeding insect pests of soybean. Aphids diverge assimilates
for their nutrition causing yield losses of up to 40% in susceptible soybean varieties. When attacked by
insects, plants respond by inducing defense mechanisms, mainly through activation of the oxylipin
pathway that produces key phytohormone jasmonic acid (JA), whose precursors are polyunsaturated fatty
acids. JA activates expression of defense genes, including proteinase inhibitors that prevent proteolytic
activity of insects’ digestive proteases. Previous experiments showed that aphids affect fatty acid levels in
soybean plants, suggesting that they can suppress defenses by interfering with the oxylipin pathway and
blocking JA-dependent signaling. Here, we show that levels of palmitic acid, linoleic acid and linolenic
acid in aphid infested soybean leaves significantly increased locally but not systemically. To test whether
aphids are able to block JA signaling, control plants and plants colonized by aphids were wounded and
their transcriptional response evaluated. We found that transcription of Proteinase Inhibitor II (PIN2) and
GH3 genes, both regulated by JA, was significantly repressed in aphid infested and wounded soybean
leaves. These results suggest that by blocking defenses, aphids could help other pests perform better on
soybean. In accordance with this postulate, we found that corn earworms grow better on plants that
previously had aphids. This effect was only observed locally, on aphid-infested leaf, correlating well with
aphid effect on fatty acids. Our results confirm that aphids can block effective anti-herbivore defenses,
and this mechanism of colonization has a positive effect on other pests.
7
T5.
Four Hydroxyproline Galactosyltransferases, GALT3, GALT4, GALT5 and GALT6,
Function in Arabinogalactan-Protein Glycosylation, Growth and Development in
Arabidopsis
Debarati Basu1,2, Lu Tian1,2, Taylor DeBrosse1, Emily Poirier1, Kirk Emch1, Eric Soukup1,3, and Allan M.
Showalter1,2, 1Environmental and Plant Biology, 2Molecular Cellular Biology Program, 3Biological
Sciences, Ohio University, Athens, OH
AGPs are a class of highly glycosylated cell wall glycoproteins, virtually present in all plant cells and in
all plant species, from algae to angiosperms. They have diverse biological roles that span plant
embryogenesis through multiple stages of plant development. Given that the sugar side chains typically
account for more than 90% of the molecular mass of AGPs, they are likely to define the interactive
surface of the molecule and hence its function. Here, we have characterized four galactosyltransferases,
namely AtGALT3 (At3g06440), AtGALT4 (At1g27120), AtGALT5 (At1g74800) and AtGALT6
(At5g62620), all belonging to GT-family-31 that catalyzes addition of galactose (Gal) to hydroxyproline
(Hyp) in AGP protein backbones. Detergent permealised microsomes obtained from Pichia expressing
AtGALT5, and tobacco epidermal cells expressing AtGALT3, AtGALT4 and AtGALT6 specifically
catalyzed incorporation of [14C]Gal from UDP-[14C]Gal to Hyp in two model substrate acceptors,
chemically synthesized [AO]7 and HF-deglycosylated d[AO]51. But unlike AtGALT2, confocal
microscopic analysis of fluorescently tagged AtGALT3-6 in tobacco epidermal cells indicated all four
proteins are only localized in the Golgi vesicles. Additional support that these four GALTs encode an
AGP-Hyp-GALT was provided by two allelic single and double knockout mutants, which demonstrated
significantly lower Hyp-GALT activities and reductions in β-Gal Yariv-precipitated AGPs compared to
wild type plants. Mutant plants showed pleiotropic growth and development phenotypes which were most
severe in the double mutants. In summary, these findings advance our understanding of the biosynthesis
and function of AGPs in plants.
T6.
Genetic Characterization and Fine Mapping of the qSD10 Seed Dormancy in rice (Oryza
sativa L.)
Pipatpongpinyo, Wirat, Feng, Jiuhuan, and Gu, Xing-You, South Dakota State University, Brookings, SD
Seed dormancy, the temporary failure of a viable seed to complete germination under suitable
environmental conditions, provides the resistance of cereal cultivars to the pre-harvest sprouting problem
in crop production. The objectives of this research were to characterize the quantitative trait locus qSD10
on chromosome 10 for seed dormancy and to identify pleiotropic effects of the QTL underlying gene(s) in
an isogenic background. The QTL-containing chromosomal segment was isolated as a single Mendelian
factor from the weedy rice SS18-2 into the background of the cultivated rice line EM93-1 by recurrent
backcrossing with marker-assisted selection. A high-resolution map was developed for the qSD10 region
and recombinants selected for individual marker intervals on the map were evaluated for seed dormancy
by progeny testing. Currently, qSD10 was delimited to a genomic region of <400 Kb flanked by markers
Indel-4 and RM5620. The narrowed qSD10 accounted for ~13% of the phenotypic variance in
germination rate in the progeny lines tested. In addition, the narrowed QTL was also associated with
yield-related traits such as flowering time (r= 0.90), plant height (r= 0.71), panicle length (r= 0.76), and
the number of spikelets per panicle (r=0.67). Research is being conducted to: 1) continue to fine map the
400-kb region to determine if associations between seed dormancy and the other traits arise from the
pleiotropy of a single gene, or the linkage between two or more genes; 2) characterize the gene expression
in different tissues; and 3) confirm selected candidate genes for the function of seed dormancy by an
RNA interference (RNAi) approach.
8
FEATURED SPEAKER
Phosphorylation-dependent regulation of G-protein cycle during nodule formation in soybean
Sona Pandey, Donald Danforth Plant Science Center, St. Louis, MO
Signalling pathways mediated by heterotrimeric G-protein complex comprising Gα, Gβ and Gγ subunits
of the trimer and their regulatory RGS (Regulator of G-protein Signalling) protein are conserved in all
eukaryotes. The role of these proteins in regulation of nodule formation was originally suggested based on
multiple pharmacological experiments. We have confirmed, using molecular genetic analysis, that the
specific Gβ and Gγ proteins of soybean G-protein complex are positive regulators of nodule formation.
We show that during nodulation the G-protein cycle is regulated by the activity of RGS proteins. Lower
or higher expression of RGS proteins due to the RNAi-mediated suppression or overexpression results in
fewer or more nodules, respectively. We also demonstrate the role of nod factor receptor (NFR1)mediated phosphorylation in regulation of heterotrimeric G-protein cycle during nodulation in soybean.
Soybean RGS proteins interact with the nod factor receptor, NFR1, which results in RGS
phosphorylation. These data point to a phosphorylation-based regulation of G-protein signalling during
nodule development in soybean.
T7.
MSL8 Regulates Opposing Osmotic Challenges in Pollen Hydration and Germination
Hamilton, Eric S., Jensen, Greg S., Maksaev, Grigory, Katims, Andrew, Meuhler, Ashley, and Haswell,
Elizabeth S., Washington University in St. Louis, St. Louis, MO
The proper regulation of osmotic forces is essential to pollen’s role in reproduction, the delivery of sperm
cells to the female gametophyte to fertilize the embryo. Most angiosperm species produce pollen that is
desiccated at maturity to survive dispersal. The rehydration of pollen and the germination of a pollen tube
on the stigma present opposing osmotic challenges to the pollen grain. Pollen rehydration creates a
hypoosmotic stress that can compromise pollen viability if it occurs too quickly. Conversely, the
germination and growth of the pollen tube requires the establishment and maintenance of sufficient turgor
pressure to power growth. Although ion channels have been studied extensively for their role in pollen
tube growth, the proteins that assist in regulating these opposing osmotic challenges have not been
uncovered. MscS-Like 8 (MSL8) is a functional mechanosensitive ion channel homologous to the
bacterial mechanosensitive ion channel MscS. MSL8is expressed exclusively in mature pollen of
Arabidopsis, where it is required for full pollen fertility. Knockdown and knockout alleles of MSL8
produce pollen severely compromised by hypoosmotic shock. However, msl8 mutant pollen germinates
better than wild type, although mutant pollen grains and pollen tubes also lyse significantly more than
wild type pollen. Overexpressing MSL8 severely inhibits pollen germination and male fertility. This work
suggests that the proper expression of MSL8 is critical to pollen’s ability to survive rehydration while
being able to produce the osmotic pressure required to germinate and grow a pollen tube, a novel
regulatory function for plant mechanosensitive ion channels.
T8.
Arabinogalactan-Protein and Cell Wall-Associated Receptor Kinase Interactions:
Cellular Signaling of Root Growth in Arabidopsis
Hayley Herock1, Andrew Travers, Lu Tian2,3, Debarati Basu2,3, and Allan Showalter2,3, 1Biological
Sciences, 2Environmental and Plant Biology, 3Molecular Cellular Biology Program, Ohio University,
Athens, OH
Arabinogalactan-proteins (AGPs) are ubiquitous plant cell wall proteins expressed in virtually all plant
cells and reside mainly at the plasma membrane-cell wall interface and in plant exudates. Knock-out
mutants of FLA4 (also known as SOS5), a fasciclinlike arabinogalactan-protein, displayed abnormal
radial swelling of root tips in the presence of elevated salt. AGPs are known to be involved in salt stress,
9
but their precise function and mode of action is unclear. FLA4 is anchored to the plasma membrane by a
glycosylphosphatidylinositol (GPI) anchor, and rest of the protein resides in the cell wall, making it
ideally positioned to transmit information between the cell wall and the cytoplasm. A double mutant
encoding two cell wall associated receptor-like kinases (FEI1 and FEI2) also displayed an identical
phenotype to fla4 (sos5) under identical conditions. In addition, a double mutant encoding two
galactosyltransferases (GALT2 and GALT5) responsible for glycosylation of AGPs displayed an identical
phenotype to fla4 and fei1 fei2 mutants. Since the galt2 galt5 double mutant has a reduced amount of
glycosylated root AGPs, it is hypothesized that glycosylation of FLA4 is required for it to function in the
FLA4/FEI1-FEI2 pathway to signal normal root growth. In order to begin to test this hypothesis, a genetic
cross was performed between fei1 fei2 sos5 triple mutants and galt2 galt5 double mutants to determine
whether all these respective gene products are indeed interacting in a single, linear pathway. We have
obtained quintuple mutant plants and have analyzed their phenotypic characteristics.
T9.
microRNA160 Regulation of Auxin-Cytokinin Balance during Soybean Root NoduleDevelopment
Narasimha Rao Nizampatnam, Spencer Schreier, Suresh Damodaran, and Senthil Subramanian,
Department of Plant Science, South Dakota State University, Brookings, SD
Legume nodules result from coordinated interactions between the plant and nitrogen-fixing rhizobia
bacteria. The phytohormone cytokinin promotes nodule formation. Recent findings suggest that the
phytohormone auxin can inhibit nodule formation. We show that microRNA160 (miR160) is a key
signaling element that regulates auxin-cytokinin balance during nodule development in soybean. miR160
acts by suppressing the levels of ARF10/16/17 family of repressor ARF transcription factors. We
identified through qPCR assays and a fluorescence miRNA sensor that miR160 levels are low in
emerging nodules and high in mature nodules. Over-expression of miR160 (reduced levels of repressor
ARFs) led to enhanced sensitivity to auxin and reduced sensitivity to cytokinin. These roots had
significantly reduced nodule formation suggesting that low miR160 levels favor cytokinin activity to
promote nodule formation. In support of this hypothesis, exogenous cytokinin rescued nodule formation
in moR160 over-expressing roots. Suppression of miR160 levels using a short tandem target mimic
(STTM160) resulted in reduced sensitivity to auxin and enhanced sensitivity to cytokinin. In contrast to
miR160 over-expressing roots, STTM160 roots had increased nodule formation, but nodule maturation
was significantly delayed. This suggested that high miR160 activity favors auxin activity to promote
nodule maturation. In agreement, exogenous auxin restored proper nodule formation and maturation in
STTM160 roots. Therefore, miR160 dictates appropriate sensitivities to auxin and cytokinin to direct
proper nodule formation and maturation.
T10.
Engineering Resistance to the Bean Pod Mottle Virus Using a Protease Detection System
Helm, Matthew D., and Innes, Roger W., Indiana University, Bloomington, IN
Plants must actively defend themselves against diverse pathogens to prevent disease. To do this, plants
utilize a family of disease resistance proteins that contain a nucleotide-binding domain and leucine-rich
repeats (NLR). The NLR protein RPS5 confers disease resistance by detecting the presence of a
pathogen-secreted protease known as AvrPphB. RPS5 detects AvrPphB indirectly by detecting a
conformational change in the AvrPphB substrate PBS1 that is induced by AvrPphB-mediated cleavage.
Recent work has shown that we can change the recognition specificity of RPS5 by replacing the AvrPphB
cleavage site within PBS1 with cleavage sites for other proteases. For example, we have shown that RPS5
can recognize the protease of Turnip Mosaic Virus when the TuMV protease cleavage site is inserted into
PBS1. We are now attempting to use the RPS5-PBS1 system to engineer novel disease resistance traits in
soybean. Specifically, we are engineering resistance to bean pod mottle virus (BPMV). This will be
accomplished by modifying a PBS1 ortholog in soybean to enable recognition of the protease from
10
BPMV. Most soybean cultivars contain an endogenous disease resistance protein that recognizes
AvrPphB, thus we predict that cleavage of the modified soybean PBS1 protein will activate resistance and
limit the spread of the virus. This approach represents a novel NLR-based system for engineering disease
resistance in crop plants and could have far-reaching implications for engineering durable disease
resistance to viral, bacterial, and fungal pathogens.
T11.
Metabolic Characterization of Pennycress (Thlaspi arvense L.) for the Production of
Valuable Fatty Acids
Enkhtuul Tsogtbaatar1, Jean-Christophe Cocuron1,2, Marcos Corchado Sonera3, and Ana P. Alonso1,
1
The Ohio State University, Department of Molecular Genetics, Columbus, OH, 2Center for Applied Plant
Sciences, Columbus, OH, 3University of Puerto Rico, Mechanical Engineering Department, Mayagüez,
Puerto Rico
Pennycress (Thlaspi arvense L.), a plant naturalized to North America, is a promising biodiesel and
industrial crop due to the accumulation of erucic acid in its seeds. Erucic acid is a long-chain fatty acid
and it accounts for maximum of 36% of the total oil. Together these results indicate that total oil and
contents of this industrially relevant fatty acid could be increased to boost the economic competitiveness
of this crop. Understanding the biochemical basis of oil synthesis in pennycress embryos is therefore
timely and relevant to guide future breeding and/or metabolic engineering efforts. For this purpose, a
combination of metabolomics approaches was conducted to assess the active biochemical pathways
during oil synthesis. First, gas chromatography-mass spectrometry profiling of intracellular metabolites
highlighted three main families of compounds: organic acids, amino acids, and sugars/sugar alcohols.
Second, these intermediates were quantified in developing pennycress embryos by liquid chromatography
tandem mass spectrometry in multiple reaction monitoring mode. Finally, partitional clustering analysis
grouped the intracellular metabolites that shared a similar pattern of accumulation over time into eight
clusters. This study underlined that: i) sucrose might be stored rather than cleaved into hexoses; ii)
glucose and glutamine would be the main sources of carbon and nitrogen, respectively; iii) the glycolysis,
the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and the Calvin cycle were active in
developing pennycress embryos.
T12.
Genome-Scale Nitrogen Responsive Gene Expression during Maize Development
Arp, Jennifer, Ibraheem, Farag, and Moose, Stephen, Department of Crop Sciences, University of
Illinois Urbana-Champaign, Urbana, IL
Nitrogen (N) is a key limiting plant nutrient and its availability is expected to have significant impacts on
the expression of genes that function in nitrogen metabolism and growth responses to N. Although a key
target for improving maize yield response to nitrogen, relatively little is known about the gene regulatory
systems that modulate N remobilization. By profiling, N remobilization can be explored as the plant
senesces and moves nutrients from the leaf to the developing ear or grain fill of the seed. Classes of genes
that show coordinated transcriptional responses across different tissues and developmental stages may
indicate key control points in N cycling between source and sink tissues. Of particular interest are the 170
genes which respond to N in opposite directions between the leaf and ear tissue at the same
developmental time point. In total 10,847 of the 29,933 expressed genes were differentially expressed in
response to N in at least one tissue; in any one sample between 3% and 10% of the reads were
differentially expressed. Our analysis shows the N response is context dependent; the developmental
context informs the N response and should be considered in future breeding for improved N
remobilization efficiency. N remobilization genes may need specialized promoters to confer fine-tuned
regulation in the correct developmental context, or we may need to identify the genes which are tipping
points to the balanced nitrogen system as a strategy for improvement.
11
T13.
Maize Nodal Root Growth Response to Water Deficits: Characterization in a DividedChamber Model System
Riggs, Kara J., and Sharp, Robert E., University of Missouri, Columbia, MO
Nodal roots are critical for development of the mature root system in maize and other grasses. Under
drought conditions, nodal roots need to grow through surface soil that may be dry, hard, and hot. These
roots are known to have a superior ability to continue elongation at low water potentials, but the
physiology of this response has been little studied. A divided-chamber experimental system that models
the field situation in which upper soil layers dry was developed to enable studies of nodal root growth
regulation under controlled water deficit conditions. Growth of the primary and seminal root systems
occurs in well-watered conditions while the nodal roots are exposed to precise conditions of low soil
water potential. This system is being used to characterize nodal root growth responses to a range of steady
and reproducible soil water potentials. Two genotypes, selected for differences in response to water stress
in a previous study of the primary root, exhibited different sensitivities of nodal root growth to declining
soil water potential, but similar responses to the water potential of the root growth zone. These results
indicate a difference in root hydraulic properties between the genotypes, leading to a differential ability to
maintain root tip water potential in dry soil. The difference in tissue water potential maintenance was seen
not only between the genotypes but also between the first two developmental nodes of roots. The dividedchamber system provides a powerful approach to investigate the physiological mechanisms regulating
nodal root growth responses to adverse soil conditions.
T14.
Gene Expression Microarray of the GPS Treatment Reveals Novel Genes Involved in
Early Gravitropic Signal Transduction
Cook, C. Adam, and Wyatt, Sarah E., Ohio University, Athens, Ohio
Gravity is a fundamental stimulus that affects plant growth and development. The gravity persistent signal
(GPS) treatment isolates the events of signal transduction in Arabidopsis thaliana. Plants are reoriented
horizontally at 4°C for 1 hour then returned to vertical at room temperature before showing a growth
response. A microarray experiment was designed to identify genes that are regulated during the GPS
treatment. Total RNA was collected from inflorescence stems of 8-10 cm at 2, 4, 10, and 30 min after
reorientation in the cold. The RNA was probed against an Arabidopsis gene expression array with 4
replicates per time point. At 2 min, only 8 genes are differen regulated. At 4 min, 8 transcription factors
are also differential regulated. Quantitative real-time polymerase chain reaction (qPCR) is to be
performed on these genes to independently validate gene expression values obtained from the microarray.
Mutant lines for each gene of interest were obtained and analyzed for phenotype. At 2 to 4 min, genes are
expressed after statolith sedimentation but before the relocalization of auxin in the stem. We identified
genes that have not previously been implied to be gravity-induced. These genes may be involved in a
process or processes of early gravitropic signal transduction.
T15.
An expansion may be involved in gravitropism in Arabidopsis thaliana
Megan Osika, Clare Bruggeman, Marilyn Hayden, and Sarah Wyatt, Ohio University, Athens, OH
The gps6 mutant was identified as a gravitropic mutant of Arabidopsis thaliana through the Gravity
Persistent Signal (GPS) treatment. When subjected to the GPS treatment, the inflorescence stem of gps6
bends out of the plane of gravity. gps6 was isolated from a T-DNA tagged population, and although
regions of the taghave been identified in gps6, the PCR based cloning strategies failed to identify the gene
disrupted, so a deep sequencing strategy was adopted. gDNA was extracted from gps6 and sequenced.
The resulting sequence data was assembled and aligned to the reference genome. Unaligned sequence
reads were assembled and compared against known regions of the T-DNA tag. Four possible candidate
12
genes were identified. PCR primers were designed to amplify these genes ingps6, but only one gene
AT3G03220 (Expansin 13) showed a disruption in sequence. Mutants for EXP-13 were obtained from the
Arabidopsis Biological Resource Center. Phenotypic analysis of these mutants will assist in better
understanding the signal transduction pathway of gravitropism. This work was partially funded by NSF
IOS #1147087 to SEW and an Ohio University Undergraduate Research & Creative Activity Award to
MH.
T16.
Characterization of vegetative and reproductive defects in the maize tassel-less 4 mutant
Zhu, Dennis, and McSteen, Paula, University of Missouri, Columbia, MO
Zea mays (maize) is important both as an agricultural crop and as a genetic model organism. Maize
produces a male reproductive structure called a tassel and female reproductive structure called an ear.
However, tassel-less (tls) mutants are characterized by an absent or reduced tassel. Eight tls loci have
been identified, and two have been cloned. Here, we present the phenotypic characterization and genetic
mapping of the tls4 mutant. tls4 mutants either produce a reduced tassel or no tassel at all. Quantification
of tassel phenotypes shows that tls4 mutants produce shorter branches and fewer spikelets (short branches
that produce florets) than their normal siblings, suggesting that the tls4 gene may be involved in the
initiation of reproductive structures. SEM analysis of immature tassels shows defects in spikelet pair
formation. In addition, tls4 mutants exhibit a number of vegetative phenotypes. Mutant plants are shorter
than normal plants and also produce narrow, rough leaves. Also, the leaves of tls4 mutant plants display
vasculature defects. These pleiotropic phenotypes suggest that tls4 plays a critical role in maize
development. Fine mapping using molecular markers indicates that tls4 maps to a 600kb region of
chromosome 4 containing 29 genes. This region includes a predicted auxin response factor gene. Further
fine mapping and sequencing is ongoing to identify the gene mutated in tls4 plants. As the hormone auxin
is implicated in both leaf patterning and tassel development we propose that the tls4 gene may function in
the auxin signaling pathway.
T17.
Formation of Indigo and Indirubin in E. coli expressing a P450 gene from woad
Sagwan Laxmi, Multani Parminder, and Anterola Aldwin, Southern Illinois University, Carbondale, IL
The plant Isatis tinctoria, commonly known as woad, is a natural source of the blue dye indigo. Indigo
can be produced by oxidation of indole, but the enzyme that catalyzes this reaction in plants is not yet
known. We cloned a P450 gene from I. tinctoria and expressed it in E. coli, which then produced a waterinsoluble blue pigment. Extraction of the pigment with chloroform and subsequent HPLC and UV-Vis
spectrophotometry analysis revealed the presence of a compound with the same retention time and UVVis spectrum as indigo. We also found that another pigment with the same retention time as indirubin is
formed. Indirubin, which is a structural isomer of indigo, is considered to be the active ingredient of a
woad-based herbal remedy for chronic myelogenous leukemia. We now propose that P450 genes in woad
(and perhaps other indigo-producing plants) are involved in the biosynthesis of indigo precursors, as well
as indirubin, and that these genes can be used to increase (or decrease) the production of these
compounds.
T18.
Identifying Plant Polyamine Exchangers that Affect Plant Development
Ge, Lingxiao, Patel, Jigar, Phuntumart, Vipa, and Morris, Paul, Department of Biological Sciences,
Bowling Green State University, Bowling Green, OH
Ubiquitously existent in nature, polyamines play an important role as signaling compounds in plant
responses to both biotic and abiotic stresses. My research has shown that the clade of Arabidopsis and rice
BIDIRECTIONAL AMINO ACID TRANSPORTERS (BATs) function as exchangers of polyamines and
13
amino acids. The method used in this study takes advantage of the genetic resources of the model
organism E. coli. A Double Knockout E. coli strain that is deficient in all polyamine exchangers was
created using standard bacteriophage transduction, and then used to heterologously express candidate
plant transporters AtBAT1 and OsBAT1. Inside-out vesicles of these transgenic E. coli cells were
generated by ultrasound sonication or French press. A radioisotope assay was then performed using these
vesicles to confirm the specificity of the target proteins as polyamine exchangers. To determine the role
of BATs in polyamine homeostasis we tested GFP fusions of BATs by transient expression in Nicotiana
benthamiana. The GFP-tagged AtBAT1 and OsBAT1 all showed plastid localization in tobacco leaves
using confocal microscopy. Furthermore, the overexpression of OsBAT1 in A. thaliana results in delayed
flowering and plants with thicker stems. Thus changes in the expression of a single polyamine transporter
produce changes in development similar to that expected by altering the expression of a transcription
factor.
T19.
Roles of MAP KINASE17 in Peroxisome Proliferation and NaCl Tolerance in Arabidopsis
thaliana
Frick, Elizabeth, and Strader, Lucia, Washington University in St. Louis, St. Louis, MO
Peroxisomes are organelles found in all eukaryotes, from single-celled yeast to plants to humans, and
perform a wide variety of functions. Universally, peroxisomes are the site of fatty acid breakdown
through β-oxidation and hydrogen peroxide detoxification. In plants, peroxisomes are responsible for
many other essential processes, including synthesis of branched chain amino acids, biotin, some vitamins,
auxin and jasmonate, as well as conversion of oils to acetyl-CoA. Although many peroxisome
proliferation factors, such as DRP3A, FIS1A, and PEX proteins, have been identified, how the plant
integrates competing signals from multiple stimuli to control division is not fully understood. We have
identified roles for MAP KINASE17(MPK17) in peroxisome proliferation. The mpk17 mutant displays
numerous peroxisome-related phenotypes, including hypersensitivity to indole-3-butyric acid (IBA) and
altered peroxisome size and number. In the mpk17 mutant, the transcript level of peroxisome division
factor PMD1 is elevated and the pmd1 mutation suppresses mpk17 phenotypes, suggesting that MPK17
effects on peroxisome proliferation are at least partially through regulation of PMD1 levels. In addition to
altered peroxisome number under normal growth conditions, mpk17peroxisomes do not proliferate under
NaCl stress, and mpk17 is partially resistant to NaCl stress. Together, these data suggest a role for
MPK17 in repressing peroxisome division under normal growth conditions, then relieving this repression
to allow peroxisome division under specific stress conditions.
KEYNOTE SPEAKER
Using Proteomics to Study Signaling and Secretion During Responses to Biotic and Abiotic Stresses
Scott Peck, University of Missouri, Columbia, MO
Plants must recognize and respond to a myriad of environmental stresses. Although transcript analyses
(e.g. RNAseq) can provide a tremendous amount of information about molecular responses to these
stresses, there are many cellular changes, including protein modifications or localization, which can only
be determined by directly monitoring the proteome. The Peck lab has developed and employed
quantitative phosphoproteomic and proteomic analytical strategies to address these gaps in our
knowledge.
Plants perceive potential pathogens by recognizing pathogen-associated molecular patterns (PAMPs)
through plasma membrane (PM) receptors. Recognition of flg22, a 22 amino acid PAMP derived from the
bacterial flagellum, by the receptor-like kinase FLS2 induces defense signaling responses and contributes
to innate immunity by restricting bacterial invasion. Quantitative phosphoproteomic analyses of
14
Arabidopsis proteins following PAMP treatments identified numerous candidates for further
investigation. Reverse genetic investigations of these differentially phosphorylated proteins has revealed
functions for a number of these candidates in innate immune responses. Using kinase-substrate pairs we
identified during these studies, we have also demonstrated the efficacy of using bioinformatic predictions
to identify additional components of kinase signalling pathways.
The plasma membrane (PM) is the interface between a cell and its environment. Therefore, PM proteins
play an important role in integrating a cell’s responses to biotic and abiotic stresses. To simplify analyses
of PM proteomes, we have developed a simple and widely applicable strategy for enriching for PM
proteins; and we have used this technique to examine changes in the PM proteome of Arabidopsis during
innate immune responses and in primary maize roots during low water potential (i.e. drought). The
technical strategy as well as some of our recent findings will be discussed.
T20.
Quantitative Trait Loci Mapping For Root Vigor And Days To Flowering In Canola
(Brassica napus L.)
Arif Uz Zaman, Muhammad, Mamidi, Sujan, McClean, Phillip, and Rahman, Mukhlesur, Department of
Plant Sciences, North Dakota State University, Fargo, ND
Winter and spring type canola exhibit completely opposite phenomena in terms of root characteristics and
flowering time. Vernalization requiring late flowering winter types have higher tap root length, diameter
and higher number of primary root branches comparing to early flowering spring type, which leads to a
vigorous root system in winter types. Limited information on genetic control of root system in canola is
available. A F2 population was developed from crossing and reciprocal crosses between winter and spring
type to identify quantitative trait loci (QTL) controlling the root vigor and days to flowering in canola (B.
napus). About 3k SNP markers were derived from the population through genotyping by sequencing. The
final linkage map was constructed with 673 SNPs with a LOD threshold four. One QTL, NRV was
identified on chromosome A01 (24.7 Mbp) for root vigor, and explains 16.3% of the total phenotypic
variation. GBF Interacting Protein 1 (GIP1) and Small Auxin-Up RNAs (SAUR)-like family proteins are
the two candidate genes, related to root growth and development were identified within this QTL region.
Two QTL for days to flowering, DTF1 and DTF2 were identified for days to flowering, accounting for
21.7% and 15% of the total phenotypic variation, respectively. The QTL DTF1was assigned on
chromosome C08 (9.43 Mbp), and DTF2 was assigned on chromosome C04 (14.56 Mbp). Further
research is in progress to understand the complex nature of different root traits in canola and to find out
the genomic region controlling the traits through genome-wide association mapping approach.
T21.
Post-Synthetic Modification of the Plant Cell Wall as a Tool to Study Cell Wall Integrity
Control Involved in Biotic Stress Response
Reem, Nathan1, Pogorelko, Gennady1, Lionetti, Vincenzo2, Bellincampi, Daniela2, and Zabotina, Olga1,
1
Iowa State University, Ames, IA, 2Universita di Roma, Rome, Italy
The plant cell wall is a complex network composed mainly of cellulose and hemicelluloses woven into a
pectin matrix. These polysaccharides, along with lignin and proteins, account for most of the plant’s
biomass and are a critical line of defense against pathogens. It has been shown that oligosaccharides and
other fragmented wall constituents can be sensed by plant cells to contribute to normal growth and
development and to induce defense responses. This sensor mechanism has been dubbed Cell Wall
Integrity (CWI) control, and is a critical new frontier in plant biology and crop improvement. In order to
tease apart the underpinnings of the CWI mechanism, we have pioneered a method called Post-Synthetic
Modification, which involves the expression of cell wall-degrading enzymes targeted to the apoplast in
order to elicit responses. Using this method, fungal-derived feruloyl (FAE) and acetyl esterases (RAE)
were overexpressed in Arabidopsis thaliana and Brachypodium distachyon plants. Arabidopsis and
15
Brachypodium transgenic RAE lines exhibit increased resistance to B. cinerea and B. sorokiniana
infections, respectively, whereas Arabidopsis FAE lines exhibit increased susceptibility to B. cinerea,
indicating a role in CWI. Gene expression analysis shows differential expression of key defense response
genes, and cell wall analysis confirms reduced recalcitrance in FAE plants to enzymatic degradation. Gas
chromatography of wall-bound phenolics indicates an overall reduction in coumarate esters, and a slight
increase of ferulate esters in FAE-expressing plants. These data shed light on the critical importance of
various types of polysaccharide esterification in cell wall integrity.
T22.
Evolution of inflorescence development in maize and related grasses (Poaceae)
Johnson, Eden, Skirpan, Andrea, Matera, Laura, Julius, Ben, and McSteen, Paula, University of
Missouri, Columbia, MO
Meristems control organogenesis in plants through the maintenance of groups of undifferentiated stem
cells. Upon completion of vegetative development, the shoot apical meristem is converted into the
inflorescence (or "flower cluster") meristem. Inflorescence meristems of grass species (Poaceae) will then
give rise to spikelets, which house the male and female floral organs and are the fundamental units of
grass inflorescences. Although solitary spikelets are the ancestral trait shared by several major agroeconomic crops (i.e., wheat and rice), paired spikelets are produced by species in at least three Poaceae
tribes (i.e., Paniceae, Paspaleae, and Andropogoneae), including maize. At least three loci in the
Suppressor of sessile spikelet (Sos) class of mutants have been identified which regulate the production of
paired spikelets in maize. Sos1, Sos2, and Sos3 prevent the formation of the sessile spikelet in the normal
spikelet pair, causing only the ancestral solitary spikelet to form. Each of these mutants also has
characteristic growth defects that indicate additional roles in inflorescence development. Identification of
the Sos genes will further our understanding of the evolution of the derived paired spikelet trait.
Additionally, Mesquite ancestral character state reconstruction is being used to elucidate the number of
times the paired spikelet trait arose in the evolution of the grasses. We plan to use comparative expression
and synteny analyses of Sos and related inflorescence development genes, together with developmental
analyses, to investigate the differences between paired and solitary spikelet development in selected grass
species.
T23.
Loss of Function of RNS2, a Housekeeping RNase T2 enzyme, Causes Alterations in
Cellular Homeostasis and Growth in Arabidopsis
Morriss, Stephanie1, Liu, Xiaoyi2, Floyd, Brice2, Bassham, Diane2, and MacIntosh, Gustavo1, 1Iowa State
University, Department of Biochemistry, Biophysics and Molecular Biology, Ames, IA, 2Iowa State
University, Department of Genetics, Cellular and Developmental Biology, Ames, IA
The T2 family of Ribonucleases is highly conserved across plants, animals and fungi. Members of this
family are characterized by a conserved sequence around the active site. In numerous organisms,
including Arabidopsis, humans, zebrafish and yeast, RNase T2 enzymes are involved in degradation of
ribosomal RNA to maintain cellular homeostasis. The Arabidopsis genome contains five RNase T2 genes
but only one, RNS2, is responsible for degradation of ribosomal RNA. Mutant plants lacking RNS2
activity show disruption of cellular homeostasis evidenced by induction of constitutive autophagy and
accumulation of ribosomal RNA in the vacuole. Here we show that mutations in RNS2 result in disruption
of energy pathways in the cell as evidenced by differential expression of an aldolase, transketolase, and
glyceraldehyde-3-phosphast dehydrogenase. Further study shows that four metabolites of the pentose
phosphate pathway are found at different levels in mutant plants compared to wild type. These
metabolites are ribulose-5-phosphate/xylulose-5-phosphate, fructose-6-phosphate, glucose-6-phosphate
and sedoheptulose-7-phosphate. Lack of RNS2 also results in upregulation of expansins and
xylosyltransferases. RNS2 mutants are larger than wild type plants and have higher water content. We
hypothesize that the induction of constitutive autophagy results in larger vacuoles and in consequence
16
higher water content, which in turn affects rosette size and higher expression of cell wall remodeling
proteins.
T24.
Regulation of HD-ZIP III Transcription Factor During Soybean Nodule Development
Damodaran, Suresh, and Subramanian, Senthil, Department of Plant Science, South Dakota State
University, Brookings, SD
Class III homeodomain leucine zipper (HD-ZIP III) proteins are a group of plant-specific transcriptional
factors (TF) that play key roles in plant development. Spatio temporal activity of HD-ZIP III proteins is
tightly regulated by post-transcriptional and post-translational mechanism to achieve proper plant
development. While these genes are expressed in soybean nodules, their functional roles or how their
activity is regulated are not known. We examined post- transcriptional and post-translational regulation of
two GmHD-ZIP IIIs (GmHD-ZIPIII-1 & GmHD-ZIPIII-2) during soybean nodule development. The
mRNAs of both these HD-ZIP IIIs were found to be regulated by miR166 based on RLM-5’RACE
assays. Analysis of a miR166 sensor using confocal microscopy of mature nodule tissues indicated that
miR166 might limit the expression of HD-ZIP III to the ‘base’ of the nodules in the nodule parenchyma.
However, over- or mis-expression of miR166 using CvMv and ENOD40 promoter showed no visible
changes in nodule development. Experiments are in progress to determine the effect of suppressing
endogenous miR166 using short tandem target mimic. HD-ZIP III protein activity is modulated through
hetero-dimerization with the small lecuine zipper protein, ZPR. GmZPR3d was identified to have highest
interaction levels with GmHD-ZIP III-1 & 2 based on yeast 2 hybrid assays. However, over- or misexpression of GmZPR3d did not show any visible phenotype in nodule development. Experiments are in
progress to determine the effect of suppression of GmZPR3d expression using RNAi. Our results indicate
that HD-ZIP IIIs are actively regulated by post-transcriptional and post-translational mechanisms during
soybean nodule development.
T25.
Preparing for a Proteomics Analysis of Arabidopsis Seedlings Grown in Microgravity
Proma Basu1 [email protected], Sarahann Hutchinson2 [email protected], Darron Luesse2
[email protected], and Sarah E. Wyatt1 [email protected], 1508 Porter Hall, Department of
Environmental and Plant Biology, Ohio University, Athens, OH, 21125 Science Lab Building West,
Department of Biological Sciences, Southern Illinois University Edwardsville, Edwardsville, IL
Proteins are the work horses of the cell. Changes in the genome of any organism manifest themselves
through the differential expression of proteins. Although gene expression studies provide information on
the genes expressed, they do not provide the full picture. After transcription the RNA generated may get
translated to proteins or degraded. Resulting proteins may also be regulated by post-translational
modification. The aim of this study is to identify quantitative and qualitative differences in proteins
expressed in seedlings germinated on Earth or in the spaceflight environment. Arabidopsis Wild Type
Columbia seeds were bulk plated on 0.5X MS plates supplemented with 1% sucrose. The seeds germinate
and grow for three days. Proteins extracted from these seedlings were digested with trypsin overnight, and
labeled with TMT 6-plex reagents. The resulting control and experimental samples were combined and
analyzed by LTQ-Velos Pro Orbitrap LC-MS/MS. The MS/MS spectra was compared to the data in
TAIR10 database to identify the proteins and the modifications on the proteins. Proteins identified with
more than 95% confidence from the peptide sequences are considered valid. The log fold change in
expression of the proteins identified and the differences in post translational modifications between
experimental and control samples may help to further identify components of the signal network involved
in response to gravity.
17
T26.
Putative Histone Readers EML1 and EML3 Differentially Impact Geminivirus Infection
Coursey, Tami, Milutinovic, Milica, Brkljacic, Jelena, and Bisaro, David, Ohio State University,
Columbus, OH
Similar to their host counterparts, plant DNA viruses are subject to chromatin formation. Bound around
histone octamers, DNA accessibility is regulated by post-translational modification (PTM) of exposed
histone tails. While previous work has suggested the deposition of PTMs is important for plant defenses,
we have not explored the role of proteins recognizing/reading these PTMS. Binding specific histone
modifications, histone reader proteins are thought to alter gene expression by recruiting targeted
nucleosome remodeling complexes and transcription factors. Using plant DNA geminiviruses as a model
viral pathogen, we are testing viral interaction with two putative, Agenet domain-containing histone
readers (Emsy-like, EML proteins 1 & 3) sharing homology to Royal Family histone readers (e.g. Tudor
domains). Surprisingly eml plants inoculated with Cabbage leaf curl virus (CaLCuV) exhibited
hypersusceptibility (eml1) or increased tolerance (eml3) not observed in wild-type plants. These
phenotypes were supported by differences in symptomatic plant stunting and viral DNA levels (qPCR).
Quantitative RT-PCR confirmed the presence of EML1 and EML3 expression in wild-type symptomatic
tissue where EMLs are usually lowly expressed. Additionally, transiently expressed EML1 and EML3
bind CaLCuV (chromatin immunoprecipitation, ChIP) and histone H3 (co-immunoprecipitation),
supporting their role as histone readers and their interaction with viral chromatin. We are currently further
testing the impact of EML proteins on CaLCuV infection by measuring changes in the amount of viral
chromatin and transcripts in eml plants. These results suggest Agenet domain-containing EML proteins
are putative histone readers and play specific roles in promoting virus infection or bolstering plant
antiviral defense pathways.
T27.
MSH1-Derived Epigenetic Breeding Potential in Tomato
Xiaodong Yang, Hardik Kundariya, and Sally A. Mackenzie, Department of Agronomy and Horticulture,
University of Nebraska, Lincoln, NE
Evidence is compelling in support of naturally occurring epigenetic influence on phenotype expression in
land plants, although discerning epigenetic contribution is difficult. Agriculturally important attributes
like heterosis, inbreeding depression, phenotypic plasticity and environmental stress response are thought
to have significant epigenetic components, but unequivocal demonstration of this is often infeasible. Here
we investigate gene silencing of a single nuclear gene, MSH1, in the tomato variety ‘Rutgers’ to effect
developmental reprogramming of the plant. The condition is heritable in subsequent generations
independent of the MSH1-RNAi transgene. Crossing these transgene-null, developmentally altered plants
to the isogenic Rutgers wild type results in progeny lines that show enhanced, heritable growth vigor
under both greenhouse and field conditions. This boosted vigor appears to be graft-transmissible and is
partially reversed by treatment with the methylation inhibitor 5-azacytidine, implying influence of mobile,
epigenetic factors and DNA methylation changes. These data provide compelling evidence for the
feasibility of epigenetic breeding in a crop plant.
T28.
Map-based cloning of Rj4, a gene controlling nodulation specificity in soybean
Tang, Fang, Yang, Shengming and Zhu, Hongyan University of Kentucky, Lexington, KY
Leguminous plants have the ability to make their own nitrogen fertilizer by forming a root nodule
symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This biological process plays a
critical role in sustainable agriculture because it reduces the need for external nitrogen input. One
remarkable property of legume–rhizobial symbiosis is its high level of specificity, which occurs at both
inter and intra-species levels and takes place at multiple phases of the interaction, ranging from initial
bacterial infection and nodulation to late nodule development associated with nitrogen fixation.
18
Knowledge of the molecular mechanisms controlling symbiotic specificity will facilitate the development
of new crop varieties with improved agronomic potential for nitrogen-fixing symbiosis. In this study, we
finely mapped the Rj4 locus, a gene controlling nodulation specificity in soybean (Glycine max). The Rj4
allele prevents the host plant from nodulation with many strains of Bradyrhizobium elkanii, which are
frequently present in soils of the southeastern USA. Since B. elkanii strains are poor symbiotic partners of
soybean, cultivars containing an Rj4 allele are considered favorable. We have delimited the Rj4 locus
within a 47-kb genomic region on soybean chromosome 1. The data reported here will facilitate positional
cloning of the Rj4 gene and the development of genetic markers for marker-assisted selection in soybean.
T29.
The Membrane-anchored Ubiquitin-fold (MUB) Protein Family Regulates Ubiquitylation
in Arabidopsis
Lu, Xiaolong1, Korolev, Sergey2, and Downes, Brian1, 1Department of Biology, Saint Louis University ,
St. Louis, MO, 2Department of Biochemistry and Molecular Biology, Saint Louis University, St. Louis,
MO
Ubiquitin is functionally versatile, altering the stability, function, or localization of targeted proteins. To
understand the mechanisms underlying these diverse Ub-roles we have sought to characterize factors that
act before currently known bifurcation into specialized function. Key would be proteins that modulate the
Ub E1 activating and Ub E2 conjugating enzyme activities since these enzymes act together to initiate all
known Ub conjugation events. This work focuses on a regulatory factor called MUB, which interacts
specifically with the Arabidopsis Group VI E2s. MUB was identified as a prenyl-protein that could tether
E2s to the plasma membrane. Here, we report that MUB has a profound biochemical influence on the
activity of Group VI E2s. With the aid of a MUB3/UBC8 co-crystal structure we visualize this interaction
at a resolution of 2.8 angstroms, and have tested structure-based predictions of the biochemical activity.
Specifically, MUB occupies the E2 backside surface known to non-covalently stage activated Ub. What’s
more, MUB inhibits E2~Ub thioester formation. Site-directed mutagenesis has been used to uncouple
MUB/E2 binding from inhibition, leading to our current molecular model that: a MUB “lap bar” domain
is coordinated by E2 binding, which then blocks E1 access to the E2. We propose that inhibition of E1
binding drastically slows the rate of activation, and coupled with inhibition of non-covalent Ub
interaction, should suppress Ub chain formation. These observations are interesting in light of recently
confirmed roles for mono-ubiquitylation in plant cell endocytosis, and the established localization of
MUB E2 complexes at the plasma membrane.
T30.
Next Generation Weed Control System in Cotton
Larue, Clayton, Varagona, Rita, Malven, Marianne, LeClere, Sherry, Brinker, Ron, and Feng, Paul,
Monsanto Company, St. Louis, MO
U.S. farmers have used dicamba on 237 million acres in the past 10 years. Currently, more than 40
products of dicamba or premixes sold by more than 10 companies. It has been used successfully for over
20 years both in professional and homeowner products. Dicamba-tolerant system would address farmer
needs. Next generation weed control system in cotton would help manage difficult broadleaf and
glyphosate-resistant weeds. This presentation will discuss Bollgard II® XtendFlexTM Cotton.
FEATURED SPEAKER
ASPB & The Plant Science Community – A Vision for the Future
Susan Cato, American Society of Plant Biologists, Rockville, MD
19
T31.
Development and utilization of high-throughput phenotyping tools to investigate the
genetic basis of drought tolerance in Setaria
Feldman, Max, Fahlgren, Noah, Gehan, Malia, Shyu, Christine, Wilson, Melinda, and Baxter, Ivan,
Donald Danforth Plant Science Center, St. Louis, MO
Phenotyping has become the rate-limiting step in using large-scale genomic data to understand and
improve agricultural crops. Here, the Bellwether Phenotyping platform for controlled-environment plant
growth and automated, multimodal phenotyping is described. The system has capacity for 1,140 plants,
which pass daily through stations to record fluorescence images, near infrared images, and visible images.
Plant Computer Vision (PlantCV) was developed as an open-source, platform independent quantitative
image analysis community resource. In a four week experiment, wild Setaria viridis, and domesticated
Setaria italica had fundamentally different temporal responses to water deficit. Overall, the Bellwether
Phenotyping platform and PlantCV software detected significant effects of genotype, and environment on
height, biomass, water-use efficiency, color, plant architecture, and near-infrared traits. The genetic
architecture of selected traits will also be discussed.
T32.
Localization of Maize SUCROSE TRANSPORTER1 Reveals Novel Insights into Phloem
Loading and Sucrose Retrieval
Baker, R. Frank1, Leach, Kristen1, Zadrozny, Tara2, Swyers, Michael1, Boyer, Nathanial1, Jackson,
David2, and Braun, David1, 1Division of Biological Sciences, Interdisciplinary Plant Group, Missouri
Maize Center, University of Missouri, Columbia, MO; 2Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY
Plant growth, development, and ultimately crop yield are dependent on the transport of photosynthates
from leaves to non-photosynthetic sink tissues (e.g., tassels). For long-distance transport, sucrose is
loaded into the phloem in leaf minor veins by sucrose transporters (SUTs). In maize, it remains unknown
whether phloem companion cells (CCs) or sieve elements are responsible for sucrose entry into the
phloem. To address this question, we characterized the expression of the maize Sucrose transporter1
(ZmSut1) gene, which was previously proposed to function in sucrose phloem loading based on its
expression and mutant phenotype. For elucidating the ZmSut1expression pattern, we performed RNA in
situ hybridizations on mature leaf tissues. These experiments revealed ZmSut1was expressed in CCs,
supporting a role for this genein phloem loading. Surprisingly, we also detected strong expression in nonconducting cells in leaf veins, suggesting ZmSut1 also functions in sucrose retrieval from the apoplasm in
cells located peripherally to the conducting cells. Analyses of a ZmSut1 promoter:RFP transcriptional
reporter transgene confirmed these findings, and also indicated ZmSut1 expression occurs in both
developing and mature veins throughout the plant. To determine the subcellular localization of the
ZmSUT1 protein, a translational reporter fusion to YFP under the control of the native regulatory
sequences was examined. These studies demonstrated ZmSUT1 localizes to the plasma membrane.
Collectively, these findings indicate ZmSUT1 functions to actively load sucrose into the phloem in the
CCs. Additionally, they provide evidence that ZmSut1 functions to recover sucrose lost to the apoplasm
far from the conducting cells.
20
POSTER ABSTRACTS
POSTER ABSTRACTS (P1 – P65)
P1.
Molecular Basis for Protein Interaction and the Control of Auxin Response Repression
Korasick, David, Srirupa Chatterjee, Gaya Amasinghe, Joseph Jez, and Lucia Strader, Washington
University in St. Louis, St. Louis, MO
Auxin signaling guides plants through nearly every aspect of growth and development. PB1 domain
interactions among AUXIN RESPONSE FACTOR (ARF) and AUXIN/INDOLE 3-ACETIC ACID
(Aux/IAA) proteins regulate auxin-responsive gene transcription. Understanding the driving forces
behind these protein-protein interactions that facilitate auxin signal transduction is vital. The crystal
structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1)
domain that provides both positive and negative electrostatic interfaces for directional protein interaction.
In this work, we investigate the attractive forces that influence ARF-ARF self-interaction. We found
using isothermal titration calorimetry and NMR chemical shift experiments that key residues on both the
basic and acidic faces of the domain contribute to and organize coordinately to stabilize PB1 domain
binding. Our data suggest the positively charged residues on the basic face of the PB1 domain drive the
interaction, whereas the less structured acidic domain face coordinates around these residues to anchor the
binding. These thermodynamic and structural analyses uncover the driving forces and key residues
involved in PB1 domain interactions that universally organize cellular signaling scaffolds. Our data
suggest a revised model of auxin response in which ARF and Aux/IAA multimerization are necessary for
regulation of auxin response.
P2.
Relationship Between Chlorophyll Content and Leaf Optical Properties in Senecing
Tobacco Leaves
Brazil, Elliot, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign,
Urbana, IL
Increasing world demand for food requires an increase in crop yields, especially major sources of
nutrition such as the grain crops maize, rice, and wheat. An avenue for greater yield potential in these
plants is an improvement in leaf photosynthesis. Many of the plants in question have evolved canopies
with a high chlorophyll content at their topmost leaves that does not allow for much penetration of light
past this uppermost leaf level and effectively strangles out any nearby competition for light and other
resources. While this may be useful evolutionarily for the plant, it is not a trait ideal for the cultivation of
row crops. Greater infiltration of light through the canopy so that it can reach more leaves might be
accomplished by altering the way plants transmit and reflect light by modifying chlorophyll levels in their
leaves. Having a better understanding of the tradeoff between chlorophyll, reflectance, and transmittance
will allow for a greater understanding of how a canopy behaves at a given chlorophyll concentration. The
leaf thickness, transmittance, reflectance, and chlorophyll levels of Nicotiana tabacum leaves were
measured and the relationship between these was analyzed. These data and their relationships will
ultimately allow for better modelling of how light interacts with leaf canopies and a greater understanding
of what the ideal chlorophyll content of a leaf would be. This optimal chlorophyll level could serve as a
focus for future breeding and utilizing biotechnology applications to improve canopy photosynthesis.
P3.
Plasma membrane proteomics in the maize primary root elongation zone: novel insights
into root growth adaptation to water stress
Voothuluru, Priyamvada1, Anderson, Jeffrey2, Sharp, Robert1, and Peck, Scott2, 1Division of Plant
Sciences and Interdisciplinary Plant Group, University of Missouri, Columbia, MO, 2Division of
Biochemistry and Interdisciplinary Plant Group, University of Missouri, Columbia, MO
Maintenance of root growth is critical to plant adaptation to drought conditions. Previous studies of the
maize primary root under water stress showed that cell elongation is maintained in the apical region of the
22
growth zone but progressively inhibited further from the apex. These responses involve spatially
differential and coordinated regulation of cellular growth processes, including modifications of both cell
production and cell expansion. As the interface between the cytoplasm and the apoplast (including the cell
wall), the plasma membrane (PM) is likely to play major functions in these growth processes. In addition,
PM proteins may be involved in solute uptake for osmotic adjustment, pH regulation, ion homeostasis and
other critical processes in roots growing under water-stressed conditions. To gain a greater understanding
of the role of PM proteins in root growth adaptation to water stress, a simplified method for enrichment of
PM proteins was used to compare the developmental distribution of PM proteins in the growth zone of
well-watered and water-stressed maize primary roots. The results revealed novel stress-responsive regionspecific changes in protein abundance in water-stressed roots. These changes suggest substantial
modification of ion homeostasis, sugar and nutrient accumulation, and membrane lipid and cell wall
composition in water-stressed roots. The distinct, region-specific abundance patterns have generated
several testable hypotheses and suggest prominent roles for these proteins in root growth regulation under
water-deficit conditions. Integration of these findings with results from previous physiological,
transcriptomic and cell wall proteomics studies reveals novel insights into root growth adaptation to water
stress.
P4.
Plant Defenses and Soybean Aphid Counter-Defenses: an Exploitation of Phytohormone
Signaling
Hohenstein, Jessica, and MacIntosh, Gustavo, Iowa State University, Ames, IA
Soybean aphids (SBA) are specialized phloem-feeding insects that have the potential to cause significant
crop damage and yield reduction. Recent transcriptome analyses of susceptible soybeans identified
differential regulation of phytohormone pathways including jasmonic acid (JA), salicylic acid (SA), and
the abiotic stress hormone abscisic acid (ABA). We are investigating these signals to identify which
pathways confer resistance to SBA and whether aphids induce abiotic stress signals as a decoy response,
thereby antagonizing effective defenses. Knock down of NPR1 expression caused a 37% increase in aphid
populations compared to controls indicating that SA defenses are effective against SBA. We performed
chlorophyll analysis on locally infested tissues to determine if ABA induction is due to general stress
responses and found that aphids produce mild stress. However, exogenous application of ABA resulted in
24% SBA population increase. Furthermore, knock down of SCOF-1, a transcriptional enhancer of
ABRE-dependent gene expression, resulted in significant SBA population decrease. Thus, ABA signaling
positively affects aphid performance. We are also exploring the role phytohormones play in plantmediated interactions between spatially separated pests through gene expression analysis in leaves and
roots of aphid-infested and control plants. In roots, SA, JA, and ET pathways were transiently activated
and differed temporally from those activated in leaves. In conclusion, SA-mediated defenses are effective
against SBA yet aphids are able to induce signals to antagonize phytohormonally-mediated local and
systemic defenses.
P5.
Application of Tobacco Rattle Virus–Based Virus-Induced Gene Silencing (VIGS) to
Characterize Photorespiratory Transporters
Keller, Caroline1, South, Paul1,2, and Ort, Donald1,2, 1University of Illinois, Urbana-Champaign IL,
2
USDA-ARS Photosynthesis Research Unit
Photorespiration is a plant metabolic process that recovers carbon from glycolate produced when
RubisCO fixes oxygen instead of carbon dioxide. This is an energy intensive process that utilizes the
chloroplast, mitochondria, and the peroxisome as well as multiple biochemical transport steps. All the
soluble enzymes involved have been characterized but many of the components facilitating transport
during photorespiration are unknown. Experiments in Arabidopsis thaliana demonstrated the roles of
chloroplastidic DiT1 and DiT2 in nitrogen recycling (Renné et al, 2003 and Schneidereit et al. 2006) and
23
Plgg1 facilitated transport of glycolate and glycerate during photorespiration (Pick et al, 2013). This
project utilizes Virus-induced gene silencing (VIGS), an RNA-mediated system that exploits the plants
anti-viral defense mechanism, to target PLGG1 (a known photorespiratory transporter) and Bass6 (an
unknown and potential photorespiratory transporter). PLGG1 and BASS6 VIGS targets were amplified by
Polymerase Chain Reaction (PCR) and the PCR products were purified and cloned into the pTVR2 VIGS
vector with the BP Clonase Gateway cloning technique. PTRV2 PLGG1 and PTRV2 BASS6 VIGS
vectors were transformed into tobacco plants by Agrobacterium tumefaciens mediated infiltration. Plants
with VIGS encoding PLGG1 and BASS6 targets produced a stunted growth phenotype when compared to
the empty vector control. These results suggest that PLGG1 and BASS6 are important for plant growth
and could contribute to photorespiratory transport. Further characterization of the function of these
transporters will provide insight into key transport steps within this pathway.
P6.
Open-source tools for high-throughput plant phenotyping
Fahlgren, Noah, Gehan, Malia, and Carrington, James, Donald Danforth Plant Science Center, St.
Louis, MO
Demand for food, fuel, and other plant products is expected to increase dramatically over the next
century. At the same time, environmental considerations require that increases in agricultural output must
occur using less water, land, fertilizer, and other inputs per unit of yield. One strategy to sustainably
increase productivity is to develop new crops and cultivars that use resources more efficiently. While
decreasing DNA sequencing costs has enabled rapid genetic screening of crop germplasm, only recently
has the development of robotic imaging platforms and low-cost sensors led to major improvements in
phenotyping throughput. Here we present PlantCV, an open-source framework for analyzing highthroughput plant phenotyping data. We demonstrate the utility of PlantCV and high-throughput
phenotyping by analyzing the phenotypic diversity of a population of Camelina sativa natural accessions
using the Bellwether Phenotyping Platform at the Donald Danforth Plant Science Center. C. sativa is an
oilseed crop from the family Brassicaceae that is an emerging source of oil for fuel and is also being
developed as a production platform for high-value compounds. Analysis of images taken daily for five
weeks was used to measure natural diversity in above ground biomass, growth rates, days to flowering,
and other traits. Additional analysis of seed phenotypes including yield per plant, seed size, and oil
content was used to identify C. sativaaccessions that could enhance breeding efforts. Although PlantCV
was developed for the Bellwether Phenotyping Platform, we demonstrate that PlantCV can be applied to
low-cost phenotyping solutions and encourage community input in future development.
P7.
Adaptation to Permanent Environmental Stress in the Antarctic Alga Chlamydomonas
raudensis: A Study of a Non-model Photosynthetic Organism
Cook, Gregory*, Kalra, Isha*, Stahl, Sarah, Kiss, A.J., and Morgan-Kiss, Rachael, Miami University.
Oxford, OH; *Both authors contributed and will be presenting equally
Most of the earth is composed of permanently cold habitats where a host of photosynthetic psychrophilic
organisms thrive by using unique mechanisms to grow and reproduce under permanent environmental
stresses. Even though there have been extensive studies on model algae and plants, adaptive strategies
employed by non-model organisms are generally poorly understood. Chlamydomonas raudensis
UWO241, isolated from a perennially ice-covered lake in the McMurdo Dry Valley of Antarctica, is a
psychrophilic green alga adapted to a myriad of abiotic stresses. Common environmental pressures
exerted on UWO241 include high salinity, low light, low temperature and nutrient limitations. The
organism utilizes various adaptive strategies to combat these stresses such as enhanced oil/lipid body
production, constitutive up-regulation of key ROS detoxifying enzymes, down regulation of photosystem
I activity and linear electron flow, and efficient energy transfer from light harvesting to photosystem II
reaction center cores. In addition, key enzymes of the water-water cycle appear to be upregulated in the
24
psychrophile and cells maintain low levels of the ROS superoxide even when exposed to high light stress.
An increased understanding of the strategies employed by Chlamydomonas raudensis UWO241 can shed
light on alternative adaptive strategies used by those organisms that inhabit similar niches. Last,
exploitation of novel adaptive mechanisms in extremophilic algae using engineering approaches could
lead to improved stress resistance in less tolerant crop species.
P8.
The Mitochondrial Genome Evolution of the Geranium Family: Elevated Substitution
Rates Decrease Genomic Complexity
Felix Grewe, Field Museum of Natural History, Chicago, IL, and Jeffrey P Mower, University of
Nebraska-Lincoln, Lincoln, NE
The nucleotide substitution rate of plant mitochondrial DNA (mtDNA) is generally very low compared to
the nuclear genome or its plastid counterpart. Mitochondrial sequences from taxa of the geranium family,
however, have an exceptional increase in their substitution rate. To further analyze this unusual finding,
we used next-generation sequencing to obtain complete mtDNAs of seven representative Geraniales taxa.
Our results show that the elevated substitution rates co-occur with an exceptional increase in
mitochondrial genome size. This increase in size is coupled with a radical reduction of complexity
reflected by a massive loss of genes, introns, and RNA editing sites. Of particular note was the loss of
three nad1 introns, including a trans-spliced intron that re-established continuity of nad1 gene
transcription. In parallel, the maturase matR (usually encoded within a nad1 intron) evolved to be a freestanding mitochondrial gene in most Geraniaceae genera. In Pelargonium (which has the fastest evolving
mtDNA), the matR gene is transferred into the nuclear genome where it acquired target signals for protein
re-import into the mitochondria. The mtDNA of Pelargonium has lost its matR gene copy together with
most ribosomal protein and all succinate dehydrogenase genes leading to a highly reduced gene content.
Additionally, the transcripts of the remaining mitochondrial genes lost most RNA editing sites--only a
total of three mitochondrial editing positions remain in Pelargonium. This unique correlation of elevated
substitution rates with increased genome sizes and decreased genomic complexity in the geranium family
represent yet another pathway of mtDNA evolution.
P9.
At14a-Like1 (AFL1) participates in plasma membrane-endomembrane signaling promoting
growth during drought in Arabidopsis thaliana
M. Nagaraj Kumar, Yi-Fang Hsieh, and Paul E. Verslues, Institute of Plant and Microbial Biology,
Academia Sinica, Taipei, Taiwan
Little is known of the upstream sensing and signaling events needed for resistance to drought and other
abiotic stresses. We found that overexpression of At14a-like1 (AFL1), an unknown protein having a small
domain similar to mammalian integrins, greatly enhanced growth and accumulation of the osmoprotective
solute proline without negative effects on unstressed plants. Conversely, inducible RNA-interference
suppression of AFL1 decreased growth and proline accumulation during low water potential while having
no effect on unstressed plants. AFL1 interacted with the endomembrane proteins Protein Disulfide
Isomerase5 (PDI5) and NAI2. These interactions were more prevalent under stress. PDI5 and NAI2
mutants had increased growth and proline accumulation consistent with negative regulatory roles in the
same stress signaling mechanisms as AFL1. AFL1 interaction with the clathrin adaptor protein2-2A
(AP2-2A) as well as structural predictions indicated possible function of AFL1 in cytoskeleton interaction
and endocytosis of clathrin coated vesicles. These functions of AFL1 are currently under investigation.
Interestingly, microarray analysis of plants with increased AFL1 expression found that AFL1 may
promote growth in part by suppression of negative regulatory genes. The effect of AFL1 on expression of
genes involved in cell wall, plasma membrane and endomembrane processes were consistent with the
localization and interactions of AFL1. These data identify new classes of proteins (AFL1, PDI5 and
NAI2) not previously known to be involved in drought signaling. AFL1 structure, protein interactions,
25
trafficking and transcriptome regulation all indicate its involvement in previously uncharacterized
membrane-associated drought signaling.
P10.
Reverse Genetic Analysis of the Rpb4 Gene in Arabidopsis
Williams, Jordyn, Janick-Buckner, Diane, and Buckner, Brent, Truman State University, Kirksville, MO
Rpb4, a subunit of RNA Polymerase II, is a highly conserved protein throughout all eukaryotes. There
have been some studies in yeast that suggest that Rpb4 has a dual function in post-transcriptional
regulation that is evidenced under stress. In a reverse genetic analysis of Rpb4 in T-DNA mutagenized
Arabidopsis thaliana, we identified a mutant exhibiting small, “crinkled” leaves, stunted overall
development, and reproductive sterility. Furthermore, the progeny of self-pollinated heterozygotes do not
segregate in a Mendelian 3:1 ratio suggesting that this mutant exhibits embryo lethality. Interestingly, a
mutation in the maize ortholog of Rpb4 produces a narrow, upward-rolling leaf that is similar to the
crinkled leaf seen in Arabidopsis. These mutant phenotypes could indicate that Rpb4 has a role in plant
development.
P11.
Study of Heat Stress Traits Tolerance in Rapeseed/Canola (Brassica napusL.) based on
Association Mapping
Rahaman, Md. Mizanur, and Rahman, Md. Mukhlesur, North Dakota State University, Fargo, ND
Heat stress causes early abortion of flowers, pollen grain death and pollen sterility of rapeseed/canola.
Seed yield contributing traits like pods per plant, pod length, seeds per pod, seed weight are suffered by
heat. High temperature during flowering time significantly reduces the seed oil and seed protein content.
This study is undertaken to identify heat stress tolerant gene through natural and artificial screening and
molecular study. A total of 160 previously genotyped Brassica napus germplasm of spring type canola
were naturally screened in the field. Screening of the same germplasm under artificial heat stress
simulating condition in a walk-in plant growth chamber is in progress. A controlled experiment has set
with the same germplasm without the heat stress treatment. The phenotypic data of the germplasms will
be aligned with the SNP genotyping data to identify the genomic region controlling the heat tolerant
gene(s) in the wide accessions of B. napus through a genome-wide association mapping. Variable pod
abortion and pollen sterility were identified among the germplasm screened in the field. Under heat stress
simulation condition in the growth chamber, high variation on pollen sterility, flower abortion, yellowing
desiccated pod, pod abortion were observed compared with the control experiment. Several germplasm
were identified tolerant to heat stress in growth chamber heat simulation conditions. The heat stress
tolerant accessions will be used in breeding program to introgress the trait into elite breeding lines.
Genome-wide association mapping will be conducted to find the genomic region controlling the trait.
P12.
Production of Monoraphidium sp. DeK19 in wastewater as a potential biofuel feedstock
Hage, Adam and Holbrook, Gabriel, Northern Illinois University, DeKalb, IL
Microalgae can offset dependency on fossil fuels through the production of biodiesel. Biodiesel can be
produced from these organisms by extracting their lipid contents and subjecting these oils to
transesterification. Monoraphidium are capable of surviving in and treating wastewater, making them a
versatile option for feedstock in alternative fuel production. In order to harness the full potential of a
microalgae-wastewater treatment system, several abiotic parameters must be investigated. The species of
interest in this study, Monoraphidium sp. DeK19, is an alga capable of being used for biodiesel
production in a wastewater treatment system. However, there is a need for improved optimization of
production at a large scale. The current goals are to 1) Identify the type of agitation and level of aeration
needed for optimal Monoraphidium growth in a wastewater environment and compare that to the aeration
situation of a common wastewater treatment facility. 2) Define the dissolved oxygen profiles of algae
26
cultures subjected to varying conditions prior to testing as part of a microalgae-aerobic bacteria symbiotic
system in a wastewater treatment facility. In cultures typical of outdoor wastewater cultivation,
Monoraphidium are capable of surviving at low light (<25 µmol photons m -2s -1) and temperature
(<10ºC) levels. Monoraphidium are also capable maintaining high population densities in a variety of
aeration and agitation conditions. Thus, Monoraphidium sp DeK19 can be grown in wastewater to
produce improved yields with specific growth parameters including; aeration, agitation, and dissolved
oxygen levels.
P13.
Local adaptation of invasive plant to north expansion: analysis from RNA sequencing
Horvath, David, USDA-ARS RRVARC, Sunflower and Plant Biology Research Unit, Fargo ND; Liu,
Dasheng, Shandong Institute of Environmental Science, Jinan, China; Mandadi, Kranthi, Vegetable &
Fruit Improvement Center, Department of Horticultural Sciences, Texas A & M University, College
Station, TX; Li, Peng, Shandong Institute of Environmental Science, Jinan, China; and Liu, Wenming,
College of Life Sciences, Shandong Normal University, Jinan, China
Invasive plant, alligatorweed has recently increased its range northwards in China, and it is unknown if
the range expansion has a genetic or epigenetic basis. It is invasive on at least three continents. Despite its
world-wide invasiveness, less than 100 genes have been sequenced from this species and made publicly
available. We have used a next generation RNA sequencing approach to examine gene expression in
individuals from the northern edge of its range and from the central portion of its range in China to look
for different responses to cold temperatures via a common garden method. Our study presents the first
transcription sequence for alligatorweed. We assembled over 75,000 genes of which over 65,000 had long
open reading frames with similarity to sequences from arabidopsis. Single nucleotide polymorphisms
unique to the northern and southern individuals were identified, and several of these were in genes such as
RESPONSIVE TO DEHYDRATION 21 that could impact the cold response of the plants. We also
identified numerous differences in gene expression associated with the cold response between these two
populations. Gene set and sub-network enrichment analysis indicated differences in the response of
photosynthetic processes and oxidative stress responses were different between the two populations and
we relate these differences to cold adaptation. However, alterations in genes controlling the CREPEAT/DRE BINDING FACTOR (CBF) regulon were not indicated.
P14.
Effect of Titanium Dioxide Nanoparticles on the Growth, Photosynthetic Efficiency, and
Oxidative Stress in Food Crops
Fleischmann, Paul, Sharma, Nilesh, and Sahi, Shivendra, Western Kentucky University, Bowling Green
KY
In recent times, titanium dioxide nanoparticles (TiO2 NPs) have found a variety of applications including
in medical processes, food production, cosmetics and UV protection. An ever-increasing use in consumer
items paves the way for potential environmental contamination. To ensure environmentally responsible
use and disposal of such NPs, it is necessary to study their fate after introduction into environmental
systems. However, there have been limited studies investigating the transport and toxicity of NPs,
particularly in plants. Some current investigations demonstrate genotoxic and cell damage effects of TiO2
NPs in plant and animal cells. The present study examines the phytotoxic effects of TiO2 NPs in two food
crops: zucchini (Cucurbita pepo) and tomato (Solanum lycopersicum) grown in a controlled hydroponic
system. Insofar, results on photosynthetic parameters suggest that TiO2 NPs do not significantly affect
photosynthetic yield or the total concentration of chlorophyll. Low concentrations of TiO2NPs (500mg/L,
1000mg/L) stimulate growth while higher concentrations (2000mg/L, 4000mg/L) inhibit total biomass.
As a part of this study, expression of cytochrome p450 gene and activities of antioxidative enzyme
(catalase, superoxide dismutase, peroxidase) in the exposed plants are also being analyzed.
27
P15.
High-throughput phenotyping of natural variation in Brachypodium distachyon under
combinations of progressive drought and heat stress
Fahlgren, Noah, Hummel, Tracy, Turnipseed, Stephanie, and Mockler, Todd, Donald Danforth Plant
Science Center, St. Louis, MO
The world population relies on grasses, such as rice, and wheat, as principal sources of calories, but these
crops are not expected to meet global demand by 2050. The geographical distribution of food and
bioenergy crops is limited by several factors including temperature, soil salinity, and water availability.
To tackle the daunting challenge of producing more food and fuel with fewer inputs a variety of strategies
to improve and sustain crop yields will be explored. These strategies may include: mining natural
variation of wild crop relatives to breed crops that require less water; and increasing crop temperature
tolerance to expand the geographical range in which they grow. These research objectives can be
achieved with a variety of methodologies, but they will require both high-throughput DNA sequencing
and phenotyping technologies. A current bottleneck in agricultural science is the ability to efficiently
quantify plant traits (phenotypes) through time. The development of high-throughput phenotyping
technologies and open-source, flexible, and translatable analysis tools that extract agronomically
important traits is the main focus of this research. B. distachyon is a C3 model grass that is closely related
to wheat, and rice, which has similar architectural features. An accession panel of 143 B. distachyon
accessions was screened under, heat, and drought and heat conditions and the resulting imaging data was
examined for traits that approximate water-use-efficiency, biomass accumulation, tiller count, and plant
water content (near-infrared imaging) using new open-source phenomics software tools. Importantly, this
digital physiology data was grounded with manually measured traits.
P16.
Effect of RNAlater® on Proteomic Analysis
Basu, Proma1, Luesse, Darron2, and Wyatt, Sarah1, 1Ohio University, Athens, OH, 2Southern Illinois
University Edwardsville, Edwardsville, IL
Plants manifest adaptation through changes in their proteome. The ideal way to study this response is by
monitoring both transcriptomic and proteomic changes, which is challenging to accomplish from the same
tissue due to the transient nature of transcripts. One way to stabilize the transcripts is using RNAlater®
Solution, but its high salt concentration is predicted to denature the proteins in the sample. The focus of
the current study is to investigate if proteins extracted from RNAlater® treated samples meet the
requirements of a proteomic study. Three day old Arabidopsis Col-0 wild type seedlings were treated with
RNAlater® for 12 hours at RT or flash frozen in liquid nitrogen followed by freezing at -800 C. Proteins
extracted from these samples were reduced, alkylated and digested with trypsin overnight before labelling
with TMT reagents and analyzing by LTQ-Velos Pro Orbitrap LC-MS/MS. An average of 224.03µg
proteins were extracted per replicate from RNAlater® treated samples compared to 236.43µg from liquid
nitrogen samples. 5711 common proteins were identified (with at least 2 unique peptides each and a False
Discovery Rate < 1%) from three replicates of the RNAlater® and liquid nitrogen treated samples. These
metrics show that proteins extracted from RNAlater® treated samples show no significant difference
from those extracted from liquid nitrogen treated samples. Further data analysis to ascertain the effects of
RNAlater® on the seedling proteome is underway. This work was partially funded by NASA grant #
NNX13AM48G to SEW and DRL.
28
P17.
The barren stalk2 Gene Is Required for Axillary Meristem Development in Maize
Yao, Hong1, Skirpan, Andrea2, Wardell, Brian3, Malcomber, Simon3, and McSteen, Paula1, 1University of
Missouri, Columbia, MO, 2Penn State University, University Park, PA, 3California State University, Long
Beach, CA
Axillary meristems (AMs) are groups of stem cells produced in the axils of leaves which generate
vegetative branches (tillers) and inflorescences. Maize has two types of inflorescence, the male tassel
which forms at the tip of the plant and the female ear which is produced from a lateral branch. Previous
studies identified several genes critical for AM development that function in auxin biosynthesis, transport
or signaling. One of these genes is barren stalk1 (ba1), which encodes a basic helix-loop-helix
transcription factor acting downstream of auxin signaling to control AM formation. We have identified a
new mutant, barren stalk2 (ba2), which, due to defects in reproductive AM formation, fails to produce
ears and has fewer branches in the tassel, similar to ba1 mutants. Furthermore, the ba2 mutant has defects
in tiller bud development suggesting that it also plays an essential role in vegetative AMs. The ba2 gene
encodes a protein that co-localizes and heterodimerizes with BA1 in the nucleus. Characterization of the
genetic interaction between ba2 and ba1 demonstrates that ba1 is epistatic to ba2 and shows a dosage
effect in ba2 mutants, providing further evidence that BA1 and BA2 act together in the same pathway.
Characterization of the molecular and genetic interactions between ba2 and other genes required for
regulation of ba1 further supports this hypothesis. We propose that BA2 and BA1 function together
during AM development, and that these mutants provide an essential tool to dissect the gene regulatory
network modulating AM production.
P18.
Investigating Potential Genes Linked to RNA-selective Autophagy in Arabidopsis Thaliana
Ridout, Victoria1, Morriss, Stephanie1, Riaz, Ayesha1, Floyd, Brice2, and MacIntosh, Gustavo1, 1Roy J.
Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames,
IA, 2Department of Genetics, Developmental and Cellular Biology, Iowa State University, Ames, IA
The RNase T2 family of ribonucleases, conserved in all Eukarya, has been connected to an autophagydependent housekeeping turnover of ribosomal RNA, the predominant form of RNA in the cell. While
animals have primarily one RNase T2, plant RNase T2 are diverged into three classes, one of which,
Class II is conserved across plants and retains the housekeeping role. Arabidopsis encodes five RNase T2
(RNS1-5); RNS2 is the only Class II enzyme and is essential for ribosomal turnover. While loss of RNS2
activity causes RNA accumulation in the vacuole and constitutive autophagy, some rRNA degradation
still occurs. RNS4, which most closely resembles RNS2, could have a partially complementary activity
compensating for the loss of RNS2. We also identified two promising candidates for additional steps in
vacuolar rRNA degradation in Arabidopsis: AT1G14250, a nucleoside phosphatase, and AT3G46960, a
DEAD/DEAH box helicase. To examine these mutants, we studied growth under phosphate starvation or
at three different pHs, by measuring root lengths and fresh weights. Phosphate is a nutrient recovered
from RNA by autophagy while pH affects nutrient uptake. The nucleoside phosphatase mutants weighed
more (P<0.05) while root length was not increased. The rns4-3 weighed less (P<0.05). The helicase
mutant was similar to the WT, yet MDC staining revealed the helicase mutant has constitutive autophagy,
suggesting it may be involved in rRNA degradation.
P19.
Adaptive Trait Variation and Genetic Divergence of a Widespread Grass Andropogon
gerardii Across a Great Plains’ Climate Gradient
Galliart, Matthew1, St. Amand, Paul2, Poland, Jesse3, Bello, Nora4, Sabates, Sofia1, Tetreault, Hannah1,
DeLaCruz, Angel1, Bryant, Johnny1, Morgan, Theodore1, Knapp, Mary5, Baer, Sara6, Gibson, David6,
Wilson, Laurel6, Maricle, Brian7, and Johnson, Loretta1, 1Department of Biology, Kansas State
University, Manhattan, KS; 2USDA Hard Winter Wheat Genetics Research Unit, Kansas State University,
29
Manhattan, KS; 3Department of Plant Pathology, Kansas State University, Manhattan, KS; 4Department
of Statistics, Kansas State University, Manhattan, KS; 5Department of Agronomy, Kansas State
University, Manhattan, KS; 6Plant Biology and Center for Ecology, Southern Illinois University,
Carbondale, IL; 7Department of Biological Sciences, Fort Hays State University, Hays, KS
Big bluestem Andropogon gerardii dominates Midwest grasslands, growing from 500 (KS) to 1200 mm
rainfall/yr (IL). Objectives are to 1) investigate ecotype differences in vegetative and reproductive traits,
and 2) characterize genetic divergence among ecotypes. Three ecotypes (central KS [CKS], eastern KS
[EKS], and southern IL [SIL]) were reciprocally planted in Colby, Hays, and Manhattan, KS, and
Carbondale, IL. We evaluated differences in vegetative and reproductive features, predicting ecotypes
would perform best at “home”. Canopy area and height increased west to east, with no evidence for
ecotype differences in Colby and Hays. In Carbondale, SIL increased in height and canopy area relative to
CKS and EKS indicating local adaptation. In Carbondale and Manhattan, CKS flowered earlier than EKS
and SIL. In Colby, CKS was the only ecotype to flower. In Hays, CKS had an estimated 66±9.6%
probability of seed production relative to EKS (15±6.5%) and SIL (15±6.7%). Both SIL and CKS
produced greatest seed at home. Taken together, CKS showed traits consistent with local adaptation to
drought—reduced canopy, short stature, and early flowering. For plants in home sites, PCA shows
variation in vegetative traits sorting with longitude. Stepwise regression of PCA scores on vegetative
traits was associated with seasonal mean precipitation and temperature, elevation, and size of
precipitation events. We characterized genetic divergence using Genotyping-by-Sequencing and
STRUCTURE indicated K=2 clusters. SIL was a distinct group while Kansas ecotypes showed
admixture. Results provide insight into trait variation and genetic divergence and help inform seed
sourcing for restoration in drier climates.
P20.
Putative chloroplast inner membrane protein BASS6 is involved in photorespiratory
metabolism
South, Paul F., and Walker, Berkley J., USDA-ARS Photosynthesis Research Unit, Institute for Genomic
Biology, University of Illinois at Urbana-Champaign, Urbana, IL
In C3 plants roughly 25% of Rubisco reactions is the fixation of oxygen instead of carbon dioxide
resulting in the conversion of RuBP to one molecule of phosphoglycerate and one molecule of glycolate.
C3 plants recover the carbon from oxygenic fixation through the C2 photorespiratory pathway. The C2
pathway is highly compartmentalized involving the chloroplast, peroxisome, and mitochondria. Though
the soluble enzymes involved in photorespiration are well characterized only a few transporters in
photorespiration have been identified such as the glycolate glycerate transporter PLGG1. To identify
additional transporters involved in the C2 photorespiratory pathway we selected known and putative
chloroplast inner membrane proteins and characterized T-DNA insertion lines in Arabidopsis for defects
utilizing a fluorescence based screen. Our results identified the Na / Bile acid symporter BASS6 a
chloroplast inner membrane protein of unknown function. The Arabidopsis bass6-1 T-DNA plants exhibit
a classic photorespiratory phenotype that is rescued at elevated CO2 concentrations, as well as show
reduced photosynthetic rates. Double knockout Arabidopsis line bass6-1plgg1-1 resulted in an additive
growth defect and further reductions in photosynthetic rates compared to either bass6-1 or plgg1-1 single
mutation. In addition, metabolomics analysis and genetic complementation in yeast suggests that BASS6
is involved in the flux of photorespiratory intermediates. We show that BASS6 is involved in
photorespiratory metabolism possibly through redundant or currently unknown transport processes.
Identifying transporters involved in photorespiration can provide insight into how plants maintain
efficient transport of metabolites and co-factors involved in the refixation of carbon, abiotic stress
response and amino acid metabolism.
30
P21.
Analysis of mutations in GERANYLGERANYL DIPHOSPHATE SYNTHASE family
genes
Heintz, Veronica, Luesse, Darron, Hocum, Gabe, and Revelt, Luke, Southern Illinois University
Edwardsville, Edwardsville, IL
In plants, isoprenoids are localized to various organelles in which they undergo reactions resulting in
intermediate compounds used in hormone, chlorophyll, and carotenoid synthesis. The isoprenoid pathway
can follow the mevalonic acid (MVA) pathway in the cytoplasm or the methylerythritol 4-phospate
(MEP) pathway in the plastid which converge resulting in the production of isopentenyl diphosphate
(IPP) and dimethylallyl diphosphate (DMAPP). Geranylgeranyl diphosphate synthase (GGPS) is an
enzyme that produces geranylgeranyl diphosphate (GGPP), the precursor for several isoprenoids.
Examination of the Arabidopsis genome reveals a family of 12 GGPS genes. Previous research has shown
a point mutation in GGPS1 results in a variegated phenotype, while T-DNA insertions in GGPS1 were
lethal to the plant. By studying Arabidopsis lines containing mutations in individual GGPS genes, we
hope to better understand each GGPS gene?s role in the isoprenoid pathway. We have confirmed plants
containing homozygous T-DNA insertions in the GGPS3, GGPS4, GGPS7, GGPS11 and GGPS12 gene.
By using this knockout approach, we looked into the effect of each GGPS-like gene on the overall
phenotype of the plant. We performed a chlorophyll extraction to determine if the T-DNA insertions in
the GGPS family lines affected chlorophyll synthesis. Our preliminary findings show that GGPS4,
GGPS5, and GGPS10 produce lower levels of chlorophyll while GGPS11 produces a normal level of
chlorophyll. We are also interested in over-expressing GGPS3 in a GGPS1 mutant to see if GGPS3,
which is localized to chloroplasts, can rescue the observed variegated phenotype. We have amplified
GGPS3 and are preparing for transformation.
P22.
Role of unsaturated VLCFA in sphingolipids and plants’ response to low temperature stress
Krassovskaya, I, and Markham, JE, University of Nebraska-Lincoln, Lincoln, NE
Temperature is a significant regulator of plant growth and plants have evolved a variety of strategies to
cope with low temperature stress. One such factor appears to be the desaturation of very long-chain fatty
acids (VLCFA) found in sphingolipids. While most plants contain saturated VLCFA in their
sphingolipids, several important cold-tolerant plants are able to synthesize sphingolipids containing
unsaturated VLCFA. The significance of this has only recently been realized through the identification of
acyl lipid desaturase 2 (ADS2) in the model Brassica, Arabidopsis thaliana. ADS2 is an omega-9, acylCoA desaturase that catalyzes the synthesis of 24:1(n-9) and 26:1(n-9) unsaturated VLCFA. Disruption of
ADS2 in Arabidopsis leads to the synthesis of sphingolipids with completely saturated VLCFA and
causes the plant to become highly sensitive to low temperature, triggering spontaneous cell death in
mature leaves. This sensitivity to low temperature can be fully complemented by the synthesis of 24:1 via
elongation from 18:1 precursors, indicating that the biochemical properties of 24:1 are critical for low
temperature resistance in Arabidopsis. The cell death phenotype that results from low temperature
exposure is associated with an increase in ceramide, similar to other spontaneous cell death mutants
(acd5, acd11). Like acd5 and acd11, the cell death phenotype can be completely suppressed by disrupting
the salicylic acid signaling pathway, suggesting that ceramide-induced cell death may have a common
signaling pathway that is sensitive to sphingolipid fluidity in the membrane.
31
P23.
WUSCHEL-RELATED HOMEOBOX Transcription Factors have Conserved Function in
Seed and Seedless Land Plant Development
Youngstrom, Christopher, Irish, Erin, and Cheng, Chi-Lien, University of Iowa, Department of Biology,
Iowa City, IA
Although seedless, ferns belong to the clade that is sister to seed plants and have comparable embryonic
development. In the model fern Ceratopteris richardii,sporophyte development begins with fertilization
of an egg that is hosted in free-living hermaphroditic gametophytes. In both seed and seedless plants
proper axis patterning is critical for subsequent embryonic development. In Arabidopsis, WUSCHELRELATED HOMEOBOX (WOX) transcription factors AtWOX8 and AtWOX9specify apical-basal polarity
in early embryo development, with AtWOX9 serving an additional role in meristem maintenance. Here,
we investigate whether homologs of genes involved in axis patterning retain a conserved function among
embryophytes. We show that the C. richardii homologs of AtWOX8 and AtWOX9,CrWOXA and
CrWOXB, respectively, may play a similar role in sporophyte embryo axis formation and gametophyte
meristem function. In situ hybridization experiments revealed that CrWOXA and CrWOXB transcripts are
expressed in the developing sporophyte, with CrWOXA localizing to apical cells at 24 hours post
fertilization. The functions of CrWOXA and CrWOXB in early embryo and gametophyte meristem in C.
richardii are being studied by overexpression and RNAi knockdown in transgenic C. richardii. Knocking
down levels of CrWOXB transcript altered gametophyte morphology but maintained normal sporophyte
development. We anticipate that overexpression of CrWOXA and CrWOXB will lead to excess sporophyte
development and knocking down of CrWOXA will cause embryo polarity defects which lead to abnormal
or arrested embryos. These results serve as a starting point for molecular dissecting of embryo
development in C. richardii.
P24.
Development of monocot transformation vectors for targeted gene-of-interest expression
during secondary cell wall formation
Cass, Cynthia1,2, Mahoy, Jill2, and Sedbrook, John1,2, 1Illinois State University, Normal, IL; 2Great Lakes
Bioenergy Research Center (GLBRC), Madison, WI
There is a growing effort to use grasses as feedstocks for lignocellulosic derived biofuels. However,
constitutive expression of transgenic modifications to improve digestibility can lead to detrimental effects
on plant health, such as stunting. Therefore, it would be beneficial to have the means to alter gene
expression in a tissue and time specific manner. A good first attempt in this regard would be to isolate and
utilize the promoters of genes which are known to be expressed during secondary cell wall biosynthesis.
Namely, the promoters of enzymes involved in secondary cell wall cellulose and/or lignin monomer
biosynthesis would provide a promising pool of candidates for a secondary cell wall “promoter toolbox”.
Alternatively, enzymes which have been shown to be involved in subsequent lignin polymerization or
side-group modification could also be screened. By using the Brachypodium distachyon derived
promoters for PMT (p-COUMAROYL- CoA MONOLIGNOL TRANSFERASE), CESA7
(CELLULOSE SYNTHASE 7), and CESA8 (CELLULOSE SYNTHASE 8) to drive expression of
reporter genes, we plan to visually characterize their tissue and developmental specific patterns. The
overall goal is to create plant transformation vectors which will express inserted genes of interest in a well
characterized, reproducible manner in the secondary cell wall.
P25.
RNA silencing and epigenetic inheritance
Blevins, Todd, and Pikaard, Craig, Indiana University, Bloomington IN
Multisubunit RNA polymerases IV and V evolved in plants as specialized forms of RNA polymerase II,
synthesizing noncoding RNAs that direct cytosine methylation to corresponding DNA sequences. We’ve
shown that Pol IV recruitment requires chromatin marks that are transgenerationally inherited via the
32
actions of HISTONE DEACETYLASE 6 (HDA6) and DNA METHYLTRANSFERASE 1 (MET1) for
CG maintenance methylation. The resulting chromatin state is not sufficient for silencing, but is required
to maintain silent locus identity, a state which allows recruitment of Pol IV and Pol V, resulting in RNAdirected DNA methylation (RdDM) of cytosines in all sequence contexts, resulting in transcriptional
silencing. Epigenetic memory conferring silent locus identity, and Pol IV/V recruitment, is erased in hda6
or met1 mutants, such that restoration of HDA6 or MET1 activity does not restore siRNA biogenesis or
silencing. In contrast, pol IV or pol V null mutants, or other mutations disrupting the RdDM pathway,
disrupt silencing without erasing silent locus identity because HDA6 and MET1 are unaffected. These
observations show that silent locus specification and silencing are separable steps. An epiallele whose
silent locus identity requires HDA6 and MET1 is a member of a gene pair previously shown to participate
in hybrid incompatibility among geographically isolated Arabidopsis thaliana strains. Our results indicate
that transgenerational epigenetic inheritance of the silent state can be a cause of hybrid incompatibility
and reduced gene flow, thus contributing to speciation in accord with the Bateson-Dobzhansky-Muller
hypothesis.
P26.
Characterization of PALs Transcriptional Regulation in Maize
Alers-Velazquez, Roberto1, Li, Wei1, Valentin Morales, Jasmin1, Prada Salcedo, Luis1, Gray, John2,
Grotewold, Erich1, and Doseff, Andrea1, 1The Ohio State University, Columbus, OH; 2University of
Toledo, Toledo, OH
Phenylalanine ammonia-lyase (PAL) is a key player in the phenylpropanoid pathway regulating stress
defense mechanisms. Maize PALs are encoded by 9 genes, located in 3 different chromosomes. Cluster
analysis based on amino acid composition, defined 3 clusters: Cluster 1: PAL2, PAL1, PAL9; Cluster 2:
PAL3, PAL5, PAL6; and Cluster 3: PAL4, PAL7, PAL8. Yet, the underlying mechanisms regulating PALs
transcription remain unrecognized. Thus, the goal of this study was to investigate the transcriptional
regulation of PALs. To identify transcriptional factors (TFs) that bind to PAL promoters, we used yeast
one hybrid assays (Y1H). The promoters corresponding to 9 PALs were cloned into the pDONR-P4P1R
entry vector and subcloned into the pMW#2 and pMW#3 yeast vectors, containing HIS3 or LacZ reporter
genes. These promoters were then used as baits in Y1H to screen a full-length cDNA library recently
generated by our group, containing 1901 TF and co-regulators from maize. A total of 158 novel PALinteracting TFs were identified. Of these TFs, 77 were previously linked to stress responses or lignin
biosynthesis. Yeast strains containing each of the 9 PAL promoters were re-transformed individually with
each of the 77 TFs to detect shared and specific TF-promoter interactions. These studies revealed 5 TFs
that bind to all 9 PAL promoters. In addition, 12 TFs were shown to bind to all three PALs in cluster 1; 6
TFs bind to all PALs in cluster 2 and 8 TFs bind all PALs cluster 3 promoters. The specificity of these cisinteractions were also investigated using beta-galactosidase assays. Further studies will investigate the
nature of the PALs cis-regulatory elements and the combination of TFs required for altered PAL
expression. These findings provide new insights into the transcriptional regulatory network responsible of
PALs expression.
P27.
Do Plants Use Mechanical Signals To Sense Pathogens?
Veley, Kira, and Haswell, Elizabeth, Washington University in St. Louis, St. Louis, MO
A great deal is known about the signaling pathways that plants employ to sense and respond to molecular
patterns associated with pathogenic infection. For example, plant cells specifically recognize molecules
that result from cell wall damage (triggering DAMP-triggered immunity), pathogen-associated molecules
(triggering PAMP-triggered immunity), and pathogen-derived effector proteins (triggering effectortriggered immunity). We are interested in a related but less well-studied question: Do mechanical signals
also contribute to plant-pathogen interactions? In addition to the established signals listed above,
mechanical signals such as increased membrane tension (e. g. caused by the formation of a fungal
33
appressorium) could also be used as a signal by the plant. Increased membrane tension can be detected by
mechanosensitive ion channels. Here we present several lines of evidence supporting the hypothesis that
the Arabidopsis mechanosensitive ion channel MscS-Like (MSL)10 is involved in immune response
signaling: 1) Overexpression of MSL10 is associated with reduced cell expansion, H2O2 accumulation,
and cell death; 2) these phenotypes are relieved by growth at high temperatures; and 3) these phenotypes
are associated with immune response-related gene expression patterns. We believe this work is a first step
toward understanding how mechanical signaling may be used by plants to sense pathogen infection.
P28.
Mesocosm growth of Monoraphidium dek19 for production of biodiesels
Kephart, Anthony, and Holbrook, Gabriel, Northern Illinois University, DeKalb, IL
Algae are a promising source of triacylglycerol biomass for use in generating biodiesels. Many species
are capable of using municipal wastewater as a nutrient source, lowering production costs and reducing
eutrophication effects on the watershed by sequestering nitrates and phosphates. If wastewater supports
vigorous algal growth, the number of suitable sites is then dependent on microalga species performance in
a variety of physical, chemical, biological and climatic environments at scale. There is evidence that
Monoraphidium is a chlorophyte capable of tolerating the annual cold temperature and lower light
conditions of the Midwest in comparison to more typical Southern locations favored for mass algal
growth projects. In order to verify its capacity to perform as feedstock for biodiesel, it must be shown to
function at industrial scale. Most contemporary work has shown their ability to grow to high cell density
and the effect of various growth conditions in 1 or 2L laboratory cultures. Here we show the effects of
scaling-up on productivity of the species Monoraphidium sp. dek19 at low temperatures. M. sp. dek19
was grown in 100 gallon cultures in triplicate at 19.51±1.47°C, 42.7μE, and 10:14 D/N cycle. We detail
some chemical changes of the medium due to algal growth, and quantify triglyceride metabolite yield.
P29.
Linking the Magic 8 to Gravitropism
Held, Jeremy, Cook, Adam, Pugh, Josiah, Benson, Chris, and Wyatt, Sarah E., Ohio University, Athens,
OH
Gravitropism describes a plant’s growth response to a change in the gravity vector. Gravity is perceived
in a plant by special amyloplasts in the root cap columella and the starch sheath of the stems. Asymmetric
distribution of auxin then leads to the growth response. Less is known about the transduction stage
between gravity perception and the growth response. The Gravity Persistent Signal (GPS) treatment
involves reorienting Arabidopsis thaliana plants 90o with respect to gravity at a temperature of 4°C for 60
minutes. During this time, inflorescence stems will not bend; the transduction phase is effectively
inhibited until the plants are returned to room temperature. Total RNA was isolated from inflorescence
stems at 2min, 4min, 10min, and 30min while oriented horizontally at 4°C. An Agilent gene expression
microarray was used to select for genes with a log fold change (LFC) greater than 1 or less than -1 and
with p-values less than 0.05. Eight genes, with six being over-expressed and two being under-expressed,
were found to be significant at the 2 minute time point. These genes, termed the “Magic 8,” have become
the focus of a large-scale mutant analysis. Seeds of Arabidopsis containing T-DNA insertions in the genes
have been obtained and bred to homozygosity. RT-PCR will be used to confirm the gene deletion in the
mutants. qRT-PCR will confirm expression levels for each gene in inflorescence tissue. Phenotype
analyses will reveal any gravitropic effect of these genes. Partially supported by NSF IOS #1147087.
34
P30.
Membrane-Anchored Ubiquitin-Fold Protein (MUB) Interaction Candidates (MUBi)
Examined for Co -Expression, -Localization, and Direct Interaction In Vitro
Lu. Xiaolong, and Downes, Brian, Saint Louis University, St. Louis, MO
Monoubiquitylation is the addition of one ubiquitin to a target protein and is believed to impart regulatory
functions. A combination of in vitro and in vivo experiments indicate that a group of ubiquitin-like
proteins called MUBs localize to the plasma membrane and prevent the recharging of E2s, which should
promote monoubiquitylation. The proteins targeted for, and the cues triggering monoubiquitylation are
unknown. In this study candidate MUB-interacting (MUBi) proteins are discussed and prioritized
according to co-expression and subcellular localization. N-terminal GST tagged MUBi protein candidates
lacking membrane association domains are tested for in vitro interaction with an N-terminally tagged HisMUB3, in reciprocal pull downs. The results of these pull-downs will be discussed in light of the
physiological pathways they implicate. In vitro yeast-two-hybrid using a split ubiquitin reporter and in
planta fluorescent assays will be used to validate the interaction between MUBi and MUB pairs. In
eukaryotes MUBs are conserved protein families. MUBs could be key proteins regulating the interaction,
processing, or endocytosis of plasma membrane proteins representing a conserved signaling pathway for
eukaryotic cells. If it is found that MUBs coordinate the ubiquitylation of specific MUBi substrates,
MUBs may be reclassified as E3 ligases.
P31.
Specific Mutations in the SERK1 LRR-RLK Gene Activate Floral Abscission in
Arabidopsis by a HAE/HSL2 Independent Process
Taylor, Isaiah, Baer, John and Walker, John C., Division of Biological Sciences and Interdisciplinary
Plant Group, University of Missouri, Columbia, MO
Arabidopsis floral organ shedding, or abscission, occurs rapidly following pollination. Plants containing
mutations in the paralogous leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes HAESA
and HAESA-like 2 (HAE/HSL2) are completely defective in abscission and retain sepals, petals, and
stamen throughout their lives. Through multiple suppressor screens of a hae hsl2 mutant, we isolated 4
lines with semi-dominant point mutations in the SERK1 gene exhibiting restored abscission. SERK1
encodes a well-studied member of the SERK family of co-receptor protein kinases implicated as general
LRR-RLK transactivators. The mutant alleles of serk1 identified in our screen suppress the abscission
defect of multiple hae hsl2 mutants, but a T-DNA mutant allele of serk1 encoding a truncated kinase
domain does not rescue hae hsl2. Three of the serk1 mutations map to a short loop on the N-lobe of the
SERK1 protein kinase domain. These mutant kinases exhibit wildtype autophosphorylation levels in
vitro. Transgenic expression of one of these mutant alleles, serk1-7, suppresses the hae hsl2 abscission
defect. A fourth mutant, serk1-10, contains a mutation that maps to the extracellular LRR domain.
Analysis of the published crystal structure of the SERK1-BL-BRI1 extracellular domain complex, along
with location of a known hyperactive mutant of the related protein BAK1/SERK3, suggests serk1-10
mutant may exhibit gain of function. Taken together, these results suggest there is a HAE/HSL2
independent signaling pathway activated in these mutants, possibly through hyperactivation of SERK1.
These mutants will inform our understanding of abscission, as well as SERK1 activity, an important
general signaling regulator.
P32.
Toward Improving Photosynthetic Efficiency in Sugarcane
Grennan, Aleel1, Kannan, Baskaran2, Zhang, Xiaoguo2, Kim, Jae Yoon2, Karan, Ratna2, Altpeter, Fredy2,
Long, Stephen1, Ort, Donald1, 1University of Illinois, Urbana, IL; 2University of Florida, Gainesville, FL
Sugarcane (Saccharum sp. Hybrids) and sorghum (Sorghum bicolor) are among the most productive
plants in production making them ideal candidates for biofuel feedstocks. Despite their high
photosynthetic rates it still falls short of the modeled theoretical C4 NADP-ME maximum. Modeling has
35
shown that increases in potential yield can only be achieved by increasing the photosynthetic efficiency of
conversion of intercepted solar energy. One approach we are taking to reach this goal of photosynthetic
improvement is to alter the light environment within the leaf by changing chloroplast size. Using RNAi
sugarcane lines with increased chloroplast size were developed and planted in a replicated field trial at our
field site in Citra, FL in 2014. Under field conditions no significant difference in photosynthetic
performance was observed, however there was a significant increase in biomass on both a fresh and dry
weight basis. Lightsheet microscopy demonstrated a change in light penetration within the leaf, but not
enough to account for the increase in biomass. Preliminary data using second harmonic generation
microscopy suggests changes in transitory starch, specifically amylopectin, in lines with increased
chloroplast size, which could be a possible explanation for the biomass increase. Other plastid types are
also being examined. Sorghum lines with increased chloroplast size are being analyzed for similar
biomass increase.
P33.
The Maize TFome - development of a transcription factor open reading frame collection for
functional genomics
Gray, John1, Burdo, Brett2, BIOL 3020 Students1, Li, Tai1, Goetting-Minesky, Mary P.1, Velliquette,
David1, Thomas, Julie1, Wittler, Bettina2, Hunt, Matthew2, Gentzel, Irene2, dos Santos Brito, Michael2,
Mejía-Guerra, Maria Katherine2, Connolly, Layne N.2, Qaisi, Dalya2, Casas, Maria I.2, Doseff, Andrea
I.2, and Grotewold, Erich2, 1University of Toledo, Toledo, OH; 2The Ohio State University, Columbus,
OH
Gene regulatory networks are central to all cellular processes. In plants they help link molecular targets
with agronomic traits of functional value. Transcription Factors (TF) and co-regulators (CoReg) represent
~7% of the maize genome, and are key regulators of plant metabolism. To define the gene regulatory
networks (GRNs) that regulate metabolism of maize phenolic compounds, we initiated The Grass
Transcription Factor ORFeome Project (TFome). We report the development and release of a publicly
available maize TF ORF collection (TFome) of 2,017 unique gene clones in recombination-ready vectors
that facilitate mobilization of the TF sequences into different expression vectors. The collection includes
several hundred co-regulators (CoREG), which we classified into well defined families, and for which
propose here a standard nomenclature, as we have previously done for TFs. Strategies were developed to
overcome the limitations associated with cloning ORFs from a genome that remains incompletely
annotated. This required, in many instances, combining genome-wide expression data with gene synthesis
approaches. The strategies developed will aid the development of similar resources in other plant species.
Information on all the clones generated is available through the GRASSIUS knowledgebase.
P34.
Progress toward the identification of the gene responsible for the raggedseedling378
phenotype
Fabbri, Marissa1, Henn, Rosalyn1, Yeh, Cheng-ting2, Hu, Alvis2, Timmermans, Marja3, Schnable,
Patrick2, Buckner, Brent1, and Janick-Buckner, Diane1, 1Truman State University, Kirksville, MO; 2Iowa
State University, Ames, IA; 3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
The EMS-induced maize mutant raggedseedling378 (rgd378) exhibits a variable and quite severe
phenotype. Mutant plants have small filamentous leaves and a split coleoptile, or produce no shoot at all.
The root system of rgd378 plants is also altered, developing fewer seminal roots and a shorter primary
root compared to wild-type siblings. Histological analysis identified alterations to mesophyll cells and
vascular bundle spacing in leaves and metaxylem abnormalities in primary roots. Bulked segregant RNAseq (BSR-seq) analysis was used to map the gene responsible for the rgd378 phenotype to a large interval
on chromosome 3. SnpEff analysis was used to locate genetic variants in the approximately 1000 genes
within this interval. Genes with genetic variants associated with the rgd378plants but not the wild-type
36
allele and that are likely to cause high or moderate structural alterations on their protein product are
currently being evaluated as possible candidate genes for rgd378.
P35.
The identification of biosynthetic genes in orphan species
Kilgore, Matthew B., Donald Danforth Plant Science Center, St. Louis, MO
Secondary metabolites are often restricted in their distribution to different groups of organisms. For this
reason, attempts to study these often useful and interesting forms of metabolism require an ability to work
in a diversity of orphan species. Methods for gene discovery with low investment and high efficiency are
needed to effectively identify the biosynthetic genes in these diverse pathways. During this work, a
workflow for efficiently identifying biosynthetic genes is developed and applied to Amaryllidaceae
alkaloid biosynthesis. Genes discovered during this work include a cytochrome p450 capable of phenolphenol coupling 4’-O-methylnorbelladine to noroxomaritidine, a norbelladine 4’-O-methyltransferase
(NpN4OMT) and a short chain dehydrogenase/reductase capable of forming norbelladine from tyramine
and 3,4-dihydroxybenzaldehyde. These enzymatic discoveries support the future application of this
workflow to other biosynthetic pathways and organisms.
P36.
Production and Characterization of a Population of Epigenetic NILs
Heller, Nicholas, Lucas, Christine, Barber, Wesley, and Moose, Stephen, University of Illinois, Urbana,
IL
The heritability of many phenotypes is not fully explained by genomic DNA sequence despite the
creation of high-density markers. Epigenetic variation might be a source of the “missing heritability” and
contribute to the inheritance of complex traits. In maize (Zea mays) 85% of the genome is transposable
elements which contribute to epigenetic variation by chromatin remodeling, often via small RNAs
(sRNAs) and DNA methylation. To understand the epigenetic regulation of phenotypic inheritance, an
inbred population carrying mediator of paramutation1 (mop1) was used. This recessive mutation results
in a dysfunctional RNA-dependent RNA polymerase2 (RDR2) and therefore a global decrease in 24-nt
sRNA molecules, which are usually associated with the maintenance of transposon silencing. Exposing
the genome to this condition may generate epigenetic variants in a nearly identical genetic population. In
addition, the red fluorescent protein (RFP) was introduced as a reporter gene fused to the tissue-specific
promoter for FLOURY2 (FL2) which encodes the abundant alpha-zein seed storage proteins. The
population was grown in a nitrogen-deficient nursery to expose active nitrogen-utilization genes to altered
regulation via epigenetic variation. A screening in 2014 revealed a high frequency of variants in
developmental pathways as well as a wide range of RFP expression after the genome had been exposed to
mop1. Future work will expand this system to utilize the Illinois Long Term Selection Lines. The results
to date indicate that these populations will reveal insight into the epigenetic regulation of gene expression
and the inheritance of this regulation.
P37.
Identifying the Key Genes that Play a Role in Starch Degradation in Alfalfa Taproots
Gipson, Monique, and Gana, Ache Joyce, Chicago State University, Chicago, IL
Medicago sativa L. commonly known as alfalfa is an important forage legume. Information validating the
pathway of non-photosynthetic starch degradation pathway in alfalfa taproots triggered by defoliation is
scarce. Isoamylase, β-Amylase, Phosphoglucan WaterDikinase, Glucan Water Dikinase, Maltose
Transporter, Disproportionating Enzyme 1, Like Starch Excess 4 and Starch Excess 4 are proteins that
play a role in starch degradation in photosynthetic tissues of plants. The role of these proteins in alfalfa
taproots is unknown. The aim of this research entails: identifying the genes that facilitates starch
degradation in alfalfa taproots. To investigate this objective NCBI BLAST and PLAZA databases were
37
used to identify the legume versions of the Arabidopsis starch degradation genes. Degenerate primers
were designed from the conserved regions of these genes from multiple sequence alignment. PCR was
performed with alfalfa gDNA and the primers synthesized for the starch degradation genes ISA, AMY,
PWD, GWD, MEX1, DPE1, and SEX4. PCR revealed product size of 790bp for PWD, 1370bp for DPE1,
and 380bp for MEX1, whereas PCR findings for the remaining genes were inconclusive. Gel purified
bands were sequenced. Sequence match was obtained for MEX1 PCR band verifying its authenticity to be
the maltose transporter. Expression of these genes in alfalfa taproots following defoliation would
demonstrate if they play roles in starch degradation. The information gained from this study should bridge
the gap in our knowledge between the classical pathways of starch degradation in photosynthetic tissues
when compared to degradation of stored starch in the non-photosynthetic tissues such as in alfalfa
taproots.
P38.
Analysis of scp1 in Gravity Persistent Signal 5 (gps5) of Arabidopsis thaliana
Jared Ross, Elisa Morales, and Darron Luesse, Southern Illinois University Edwardsville, Edwardsville,
IL
The direction of gravity plays an important role in plant development. It impacts nutrient absorption,
water absorption, and light capture by informing growth direction of branches and roots. Under normal
conditions, plants reorient their inflorescence stems when challenged with a 90 degree reorientation. In
the cold, this response does not occur. However, after subsequent return to a vertical orientation at room
temperature, the stem manifests the cold gravistimulation by transiently curving to the side. To leverage
this response for information about the signaling pathway responsible for gravitropism, a mutant screen
was performed to isolate lines that showed aberrant responses. The gravity persistent signal 5 (gps5)
mutant was identified due to its hypergravitropic phenotype under these conditions. Deep sequencing of
this mutant revealed a T-DNA insertion in the gene SCP1. The goal of this work is to determine if the
insertion in SCP1 is the cause of the hypergravitropic phenotype. We have followed two-pronged
approach to answer this question. First, a homozygous GABI-Kat line insertion in SCP1 was isolated
from a pool of available seed. Second, we are preparing constructs for transformation rescue of the
phenotype with genomic SCP1. Identification of the cause of the gps5 phenotype will provide information
about how plants initiate and maintain signaling during gravitropism. Analysis second generation
homozygous SCP1 is being performed to determine the cold gravity response of scp1 mutant
inflorescence stems.
P39.
Investigate the Regulation of Mitogen-activated Protein Kinase Phosphatase 1 (MKP1) in
PAMP Signaling and Resistance to Bacteria
Jiang, Lingyan1, Wan, Ying1, Anderson, Jeffrey1, Ulm, Roman2, and Peck, Scott1, 1University of Missouri,
Columbia, MO; 2University of Geneva, Geneva, Switzerland
The first layer of plant immunity against microbial pathogens depends on recognition of conversed
pathogen-associated molecular patterns (PAMPs) mediated by pattern recognition receptors (PRRs),
initiating many intracellular responses. One important PAMP response is protein phosphorylation
including mitogen-activated protein kinases (MAPKs). MAP kinase phosphatases (MKPs) are important
negative regulators of MAPKs. Work with non-plant organisms has shown that MAPKs control their own
negative regulators, establishing negative feedback loops to attenuate the response. However, in plants,
the regulation of MKPs is less understood. Arabidopsis MKP1 was shown to be a negative regulator of
innate immune signaling. MKP1 was also shown to be phosphorylated and activated by MPK6 in vitro,
suggesting that phosphorylation may be an important mechanism for regulating MKP1. To test this
hypothesis, phosphorylation site mutants were generated. Phosphorylation site mutants were not able to
complement the mkp1 phenotype, indicating that phosphorylation of those sites is required for the
function of MKP1. One hypothesis is that PAMP treatment induces the phosphorylation of MKP1 and
38
that phosphorylation stabilizes the protein, and this supposition is supported by the result that both PAMP
and MG132 treatment increased MKP1 protein level. In addition, to investigate the possible roles of
distinct subdomains in the function of MKP1, domain truncation mutants were generated. Preliminary
results indicate that the gelsolin but not the camodulin binding domains are required for the function of
MKP1. Because the gelsolin domain binds to actin that mediates the protein localization, suggesting that
different protein localizations may also contribute to the regulation of MKP1.
P40.
Isotopically Nonstationary 13C Flux Analysis of Changes in Arabidopsis thaliana Leaf
Metabolism due to High Light Acclimation
Ma, Fangfang1, Jazmin, Lara2, Young, Jamey2, and Allen, Doug1,3, 1Donald Danforth Plant Science
Center, St. Louis, MO; 2Department of Chemical & Biomolecular Engineering, Vanderbilt University,
Nashville, TN; 3USDA-ARS, St. Louis, MO
Improving plant productivity is an important aim for metabolic engineering. Efforts to improve
photosynthetic efficiency would be aided by more quantitative descriptions of primary metabolism in
leaves. Metabolic flux analysis (MFA) can quantify plant central carbon metabolism based upon a
combination of extracellular flux measurements and intracellular isotope labeling measurements taken
temporally. We performed in vivo isotopic labeling of Arabidopsis thaliana rosettes with 13CO2 and
estimated fluxes throughout leaf photosynthetic metabolism by Isotopically nonstationary metabolic flux
analysis (INST-MFA). Leaves were acclimated to either 200 or 500 µmol m-2s-1 light. The results
provide a comprehensive description of changes in carbon partitioning and overall photosynthetic flux in
response to high-light acclimation of leaves.
P41.
Genomic Analysis of Polymorphisms Conferring Storage Protein Deficiency in Common
Bean
Pandurangan, Sudhakar1, Crosby, William2, Pauls, K. Peter3, and Marsolais, Frederic1,4, 1Department
of Biology, Western University, London, Canada; 2Department of Biological Sciences, University of
Windsor, Windsor, Canada; 3Department of Plant Agriculture, University of Guelph, Guelph, Canada;
4
Genomics and Biotechnology, Southern Crop Protection and Food Research Centre, Agriculture and
Agri-Food Canada, London, Canada
A series of genetically related lines of common bean (Phaseolus vulgaris) integrate a progressive
deficiency in major storage proteins, the 7S globulin phaseolin and lectins. To understand the genomic
basis for variations in protein profiles previously determined by proteomics, the four genotypes were
submitted to short-fragment genome sequencing using an Illumina HiSeq 2000 platform. Reads were
aligned to reference sequences from G19833, BAT-93 and OAC-Rex, and a genomic clone of the lectin
locus from an arcelin-5 genotype. The results of the analyses identified polymorphisms responsible for
the lack of specific storage proteins, as well as those associated with large differences in storage protein
expression.
39
P42.
Golden Gate Cloning in Building Multigene Constructs for Photorespiration Bypass in C3
Plants
W. Liu, Helen1, Keller, Caroline1, South, Paul2,3, and Ort, Don2,3, 1University of Illinois at UrbanaChampaign, Department of Crop Sciences, Urbana, IL; 2USDA-ARS Photosynthesis Research Unit,
Urbana, IL; 3Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign,
Champaign, IL
Plant synthetic biology is an important and fast growing area of research. Plant synthetic biology provides
the opportunity to improve current agricultural production despite limited land resources and water
availability for an increasing human population. Golden Gate cloning system provides a more efficient
way to assemble and transform multigene constructs into desired hosts. We used a modular cloning
system to assemble multiple genes into one construct targeting a bypass in the photorespiratory pathway
of C3 plants. Golden Gate cloning allowed us to use cloning vectors as well as parts to assemble
multigene constructs that can be transformed into C3 dicot plants. We first generated constructs with
individual transcription units and then into more than 20 multigene constructs. We transformed our
multigene constructs into Arabidopsis by floral dips and into Nicotiana tabacum. Genotyping by
polymerase chain reaction of extracted genomic DNA was performed to confirm that our large multigene
constructs have been successfully transformed into C3 dicot plants. The generating of multigene
constructs through Golden Gate cloning systems and verifying the successful transformation into C3
plants is the first step into the increase of crop productivity and yield through the development of
improved synthetic biology strategies.
P43.
Genotypic Variation in Lateral Root Growth between Maize Lines Experiencing Mild
Water Deficits
Tyler Dowd1,3, David Braun2,3, and Robert Sharp1,3, 1Division of Plant Sciences, 2Division of Biological
Sciences, 3Interdisciplinary Plant Group, University of Missouri, Columbia, MO
Water deficit is the most limiting constraint to crop production worldwide. In sub-optimal conditions the
capability of roots to mine the soil for available water is essential for survival and productivity. The
formation and elongation of lateral roots is an important process determining the architecture of the root
system, and is highly plastic in response to soil drying. To gain a better understanding of lateral root
growth responses to mild water deficits, two maize lines (Zea mays L. cv B73 and cv FR697) were
studied under a range of steady-state water deficits. Plants were grown in an enclosed tube system with
media pre-calibrated to precise water potentials (Ψw) in a controlled environment chamber at 90% relative
humidity (RH). The RH of the chamber in combination with the enclosed system created microclimates
for shoot growth at near saturation humidity, thereby reducing evapotranspiration and allowing
photosynthetically active growth in a stable Ψwenvironment. Under mild water deficit the genotypes
responded differently, with an increase in primary axis lateral length in FR697 and a decrease in primary
axis lateral length in B73. In addition, the mechanisms each genotype employed to modify their root
system also varied. FR697 exhibited increased elongation of existing lateral axes, resulting in an
enhanced root system, while B73 reduced the number of lateral axes formed, leading to a decrease in total
lateral root length. The differential responses exhibited indicate potential for further exploration of lateral
root phenotypes for developing varieties more suitable for growth in water-limited environments.
40
P44.
Genes with Lipidation Signals Modulated by Splicing (GLiSMS): A Broadly Conserved, but
Uncharacterized Mechanism Determining the Conditional Membrane Localization of
Functionally Diverse Proteins
Rajapaksha, Nadeesha, King, Jeremy, and Downes, Brian, Department of Biology, Saint Louis
University, St. Louis, MO
Over 60% of intron-containing plant genes undergo alternative splicing (AS), producing a vast repertoire
of mRNA isoforms raising questions are the functional significance of AS, and how it is regulated. For
example; How AS regulates protein sorting, when exposed to different conditions, is not well understood,
and is a focus of this study. Sorting is determined by physical protein characteristics including signal
sequences and posttranslational modifications, thus prone to AS regulation. Examination of plant
prenylated proteins reveals an AS mechanism that switches between membrane and cytosolic sorting,
which we now refer to as GLiSMS (for Genes with Lipidation Signals Modulated by Splicing). In
prenylation, a lipid anchor is covalently added to the C-terminal “CaaX” motif of protein products.
GLiSMS use splicing to regulate the availability of prenylation signals in affected genes. Here, using
plant MUB (Membrane-anchored Ubiquitin-fold) mRNAs as an example, in 43 plant species, we
determined that the mechanism is indeed broadly conserved. We assessed MUB GLiSMS conservation in
terms of species distribution, splice-type, and flanking sequences. Based on these preliminary results, we
are currently scripting PERL algorithms to analyse the full transcriptomes of available plants species for
all prenylated proteins. The execution of these scripts should massively increase our data set, further
define GLiSMS, and give us the opportunity to further understand the regulatory sequence contexts.
P45.
Transformation Rescue of gravity persistent signal 5 with an Arabidopsis Aldose-1Epimerase to Determine its Role in the Signal Transduction of Gravitropism
Vierling, Michael, Morales, Elisa, and Luesse, Darron, Southern Illinois University Edwardsville,
Edwardsville, IL
Gravitropism is the capability of a plant to perceive the gravitational vector and to coordinate its growth
accordingly. The inflorescence stem exhibits negative gravitropism in that it grows in the opposite
direction of the gravitational vector, while the roots exhibit positive gravitropism growing in the same
direction as the gravitational vector. This response to gravity corresponds to a series of physiological and
molecular responses. To better understand the signal transduction steps of this process, a mutant screen
was performed to isolate lines that showed an aberrant response after a cold gravity stimulation. The
gravity persistentsignal5 (gps5) mutant was identified due to its hypergravitropic response. Deepsequencing of this mutant revealed it has four T-DNA insertions in addition to one deletion that was
previously identified. One of these insertions is in a gene predicted to encode a protein with the conserved
domains of an aldose-1-epimerase.To determine if this insertion is responsible for the gps5 phenotype we
have taken two approaches. The first involves isolation of a homozygous T-DNA line from the SALK
collection. Preliminary results indicate wild-type gravitropic response in this mutant. The second
approach is transformation rescue of gps5 with a wild-type copy of the epimerase. The gene is currently
being transferred into the pEARLEYGATE 100 plasmid for overexpression in gps5.
P46.
Beyond the wall: characterizing the role of boron in the meristem
Durbak, Amanda1, Phillips, Kim1, O'Neill, Malcolm2, Pike, Sharon1, Gassmann, Walter1, and McSteen,
Paula1, 1University of Missouri, Columbia, MO; 2Complex Carbohydrate Research Center, University of
Georgia, Athens, GA
Boron (B) is an essential plant micronutrient, and deficiency results in significant defects in plant
development. The maize tassl-less1 (tls1) gene encodes a B transporter in the aquaporin family, and tls1
mutants have altered reproductive development, as well as defects in vegetative growth. tls1 mutants were
41
found to have an overall reduction in B content, with the greatest reduction observed in young
inflorescences (flowering branches), indicating that meristematic tissue is particularly sensitive to B
deficiency. Microscopy on tls1 vegetative and reproductive apices revealed that tls1 mutants have smaller
meristems, suggesting that B plays a role in meristem maintenance. B is best known for its role in crosslinking the cell wall polysaccharide rhamnogalacturon-II (RG-II). The level of dimerized RG-II in tls1
mutants was significantly reduced in young tls1 tassels (male inflorescence), indicating that meristem
defects may result from altered cell wall properties. However, there was no difference in RG-II crosslinking in tls1 leaves or roots, suggesting that B may have other functions. Historically, there have been
reports of B interacting with hormones, namely auxin and cytokinin. tls1 mutants have root defects
similar to those seen in cytokinin signaling mutants, and our analysis of double mutants between tls1 and
cytokinin or auxin mutants supports an interaction between B and hormones. We propose that cross-talk
between auxin, cytokinin, and boron plays a critical role in plant development, and current work is
focused on using molecular markers and genetics to dissect the relationship between these hormones and
boron in the meristem.
P47.
Determining Optimal Conditions for Growth and Lipid Production by the Green Microalga
Monoraphidium sp. Dek19 in Wastewater
Kirchner, Nicholas, Grayburn, W. Scott, and Holbrook, Gabriel, Northern Illinois University, DeKalb, IL
Microalgae can potentially serve a dual purpose as both a renewable fuel source and an environmental
cleanser. Monoraphidium sp. Dek19 is a locally isolated alga that can produce extractable lipids. The
current goals are to determine 1) how to separate an alga out of a mixed culture, 2) the optimal abiotic
conditions for growth of Monoraphidium sp. Dek19 in both splitter and final wastewater effluent, 3) the
rate of nutrient depletion to better estimate time of harvest, 4) an effective method of imaging and
quantifying lipids using Nile red fluorescence. Sucrose gradients can separate Monoraphidium sp. Dek19
out of a mixed culture when centrifuged at 1,845xg for 35 minutes, all the while remaining viable. This
could provide an effective method to purify and isolate the desired species. Monoraphidium sp. Dek19
still grows effectively at colder temperatures (≤10ºC) and low light (≤10 µmol photons m -2s -1), but at a
slower rate. This could indicate that year-round growth is a possibility in wastewater treatment facilities
of the Midwestern USA as effluent temperatures fluctuate between 10-15ºC. However, monocultures of
Monoraphidium sp. Dek19 exhibit acceptable growth rates at 22 ºC with a light intensity of 45-55 µmol
photons m -2s -1 . Future research will investigate whether it is possible to compare neutral lipid production
between algal cultures grown at different temperatures using splitter and final effluent via Nile red
fluorescence.
P48.
Impact of Genetic Perturbations to Photorespiration on Rubisco Activation State
Barber, Beau1, Ort, Donald2, and Walker, Berkley2, 1Carl R. Woese Institute for Genomic Biology,
University of Illinois at Urbana-Champaign, Urbana, IL; 2USDA-ARS Photosynthesis Research Unit,
Urbana, IL
Plant growth depends on photosynthesis for biomass production. The first step of photosynthesis is
carbon dioxide fixation through Ribulose-1,5-bisphosphate (RuBP) carboxylation. RuBP carboxylation is
catalyzed by the enzyme, Ribulose bisphosphate carboxylase-oxygenase (Rubisco). Rubisco is vital to
photosynthesis, but also catalyzes RuBP oxygenation, producing toxic phosphoglycolate and causing
photorespiration as a result. Photorespiration converts phosphoglycolate into 3-phosphoglycerate at the
expense of energy and fixed carbon. Photorespiration also deactivates Rubisco, reducing the
photosynthetic assimilation rate. Although there is evidence indicating photorespiration as a cause of
Rubisco deactivation, little knowledge exists concerning the mechanism of Rubisco deactivation by
photorespiration. Previous research indicates that the Rubisco activation state is potentially affected by
certain photorespiratory metabolites. Since different metabolites are metabolized by different enzymes in
42
photorespiration, specific metabolites will accumulate in the absence of photorespiratory enzymes and
affect Rubisco activity. We investigated the impact of genetic disruptions to photorespiration on Rubisco
activity by performing CO2 gas exchange measurements on knockout lines of Arabidopsis thaliana. The
results of the gas exchange measurements indicated that the in vivo Rubisco activity decreased in A.
thaliana plants lacking key photorespiratory enzymes. These results support our hypothesis that enzyme
deficiencies impact the Rubisco deactivation potentially due to certain metabolite accumulations. To
determine if certain metabolite accumulations cause Rubisco deactivation, we will next analyze the
photorespiratory metabolite profiles and in vitro Rubisco activation state.
P49.
The raggedseedling378 leaf developmental mutant exhibits enrichment for genes involved in
stress responses
Henn, Rosalyn1, Fabbri, Marissa1, Yeh, Cheng-ting2, Hu, Alvis2, Timmermans, Marja3, Schnable,
Patrick2, Buckner, Brent1, and Janick-Buckner, Diane1, 1Truman State University, Kirksville, MO; 2Iowa
State University, Ames, IA; 3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
The maize raggedseedling378 (rgd378) mutant exhibits very small filamentous leaves and is highly
susceptible to fungal infection. We performed bulked segregant RNA-seq analysis on shoot tissue of 7
day old rgd378 and wild-type sibling plants to locate the chromosomal interval in which the rgd378 gene
resides, as well as to perform differential gene expression analysis. We identified over 5,400 differentially
regulated genes in mutant compared to wild-type shoot tissue. Gene set enrichment analysis was carried
out utilizing BiNGO and PlantGSEA to identify over-and under-represented GO categories of genes.
Genes that function in glycolysis, fatty acid catabolism and responses to both abiotic and biotic stresses
were over-represented in rgd378 RNA-seq samples, whereas the wild-type samples were enriched in
genes that function in photosynthesis, chloroplast biogenesis, DNA replication, cell division and leaf
development. These studies are being used to inform and narrow our candidate gene search.
P50.
Preparation for Proteomic Analysis of Gravitropic Signal Transduction in Arabidopsis
thaliana Seedlings under Microgravity Conditions
Hutchinson, Sarah, and Luesse, Darron, Southern Illinois University Edwardsville, Edwarsville, IL
A notable characteristic of plant physiology is the ability of plants to sense the direction of the gravity
vector and organize their body plan along it. This ability, known as gravitropism, is the sum result of
three distinct processes: Stimulus Perception, Signal Transduction, and Differential Growth. Of the three,
little is actually known about the mechanics of signal transduction. The ability to compare gene and
protein expression in plants grown both with and without gravity represents a critical step in
understanding how the process works. Previously we reported on the optimization of an experimental
procedure for analyzing the proteomes of Arabidopsis thaliana seedlings grown under Earth-bound and
microgravity conditions. Here we present an update on the extended stasis time of planted seeds and the
results of a trial run using the improved procedure under exact Earth-bound conditions. Furthermore, we
report on our initial examination of microgravity’s potential effect on root physiology through the
exposure of 12-day old gravity mutants and their reference ecotypes to clinostat conditions. Preliminary
data indicates prolonged exposure (~8hr.) of seedlings to clinorated, altered gravity produces a negative
gravitropic response of varying strength in primary auxiliary roots of Columbia WT and WS WT.
P51.
Plant Cell Fate: A Conversation Between Nuclei and Organelles
Wilson, Margaret E., and Haswell, Elizabeth S., Washington University in Saint Louis, St. Louis, MO
Plastid biogenesis and differentiation are linked to cellular and organismal development and the
consequences of abnormal plastid function are often manifested at a whole-plant level. The profound
effect that impaired plastid homeostasis can have on plant development is seen in an Arabidopsis mutant
43
lacking functional versions of two plastid-localized mechanosensitive ion channels, Mechanosensitive
Channel of Small Conductance-Like2 (MSL2) and MSL3. MSL2 and MSL3 serve as osmotic safety
valves, releasing osmolytes when plastids swell under hypoosmotic stress and in their absence (msl2 msl3
mutants) plastids experience constitutive hypoosmotic stress resulting in numerous developmental defects
including abnormal leaf morphology and meristematic callus formation. Recently, we have shown that a
previously unidentified plastid-based osmoregulatory pathway is activated in msl2 msl3 mutants, resulting
in an abscisic acid (ABA)-dependent accumulation of the amino acid proline (Pro). Consequently, we are
beginning to identify the underlying causes and potential signaling pathways contributing to the wholeplant phenotypes of msl2 msl3 mutants. We have found that the meristematic callus formation phenotype
observed in msl2 msl3 mutant plants requires both ABA biosynthesis and the accumulation of high levels
of reactive oxygen species (ROS). Additionally, both ROS accumulation and callus formation are
enhanced in the absence of Pro biosynthesis, suggesting a role for Pro biosynthesis in the modulation of
ROS levels in msl2 msl3 mutants. These recent findings establish the foundation for an exploration into
the relationship between plastid homeostasis and plant development.
P52.
Identifying the gene responsible for the carbohydrate partitioning defective7 mutation of
Zea mays
Braun, David, Brush, Parker, Barron, Brady, Hibbard, Jaime, Baker, R. Frank, and Leach, Kristen,
University of Missouri, Columbia, MO
Carbohydrate partitioning is the biological process in which carbohydrates (e.g., sucrose) are transported
from photosynthetic source tissues (e.g., mature leaves) to non-photosynthetic sink tissues (e.g.,
developing leaves, ears, and roots). Although this process is essential for plant growth and development,
the regulation of carbohydrate partitioning and the genes involved are not well understood. The
carbohydrate partitioning defective7 (cpd7) mutant of maize was identified by pale-green coloration of
the mature leaves and progressive anthocyanin accumulation within these pale-green regions. Staining
these regions for starch revealed hyper-accumulation of starch in cpd7-1 mutant leaves as compared to
leaves from wild-type siblings, indicating the Cpd7 gene plays a role in carbohydrate partitioning. Two
additional mutants with similar phenotypes, cpd7-2 and cpd7-3, were verified to be allelic to cpd7-1
through complementation testing and mapping. To identify the causative mutation, a positional cloning
strategy was undertaken, delimiting a 60,000 bp region on chromosome 9, containing two candidate
genes. A complementation test between cpd7 and a Mutator transposable element insertion in one of the
two genes has led to the identification of the putative causative gene, which is currently being sequenced
in cpd7-1, cpd7-2, and cpd7-3. This information will aid in the identification of genes involved in
controlling whole-plant carbohydrate partitioning.
P53.
Microarray Reveals Novel Role in Gravity Signaling for ATAIB and WRKY18
Cook, C. Adam, Benson, Chris, and Wyatt, Sarah E., Ohio University, Athens, OH
Gravity is a fundamental stimulus that affects plant growth and development. The gravity persistent signal
(GPS) treatment isolates the events of signal transduction in Arabidopsis thaliana. Plants are reoriented
horizontally at 4°C for 1 hour, then returned to vertical at room temperature before showing a growth
response. A microarray experiment was designed to identify genes that are regulated during the GPS
treatment. Total RNA was collected from inflorescence stems of 8-10 cm at 2, 4, 10, and 30 min after
reorientation in the cold. The RNA was probed against an Arabidopsis gene expression array with 4
replicates per time point. At 4 min, 8 transcription factors are also differentially regulated. Quantitative
real-time polymerase chain reaction (qPCR) was performed on these genes to independently validate gene
expression values obtained from the microarray. Mutant lines for each gene of interest were obtained,
bred to homozygosity and analyzed for phenotype. These genes have not been previously implicated to be
44
gravity-induced. These genes may be involved in a process or processes of early gravitropic signal
transduction. Partially supported by NSF IOS #1147087.
P54.
Emerging Properties of Gene Regulatory Networks and Grids
Ouma, Wilberforce Zachary1,2, Yousefi, Mohammadmahdi R3, Lopez, Edgar2, Doseff, Andrea, I.4,5, and
Grotewold, Erich2,4, 1Molecular, Cellular, and Developmental Biology (MCDB) Graduate Program, The
Ohio State University, Columbus, OH; 2Center for Applied Sciences (CAPS), The Ohio State University,
Columbus, OH; 3Department of Electrical and Computer Engineering, The Ohio State University,
Columbus, OH; 4Department of Molecular Genetics, The Ohio State University, Columbus, OH;
5
Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care, and Sleep, Heart, and
Lung Research Institute, The Ohio State University, Columbus, OH
Gene regulatory networks provide a holistic view of the transcription factor (TF) - target gene interactions
and regulation. This system-wide approach offers an unparalleled understanding of how an organism
functions by not only revealing the direct effects a TF exerts on its target genes, but also how it indirectly
affects the entire system through a cascading effect. Maize (Zea mays) lacks a well-characterized
genome-wide Protein-DNA Interactions (PDI) profile (interactome). However, the interactomes of model
organisms such as Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, and Mus
musculus, have been largely established, offering excellent opportunities to construct and analyze these
regulatory grids to discover common properties. Using these properties as a basis for comparison, our aim
is to ascertain how much more information is needed to construct a basic gene regulatory grid for maize,
which is the first step to creating a maize gene regulatory network. Regulatory grids are static
representations of the network and encompass all possible interactions between transcription factors and
genes, but do not specify their relationship. We mined the literature data of binding sites of transcription
factors (TFs) in each of the aforementioned genomes. This data could be chromatin immunoprecipitationsequencing (ChIP-seq), chromatin immunoprecipitation microarray (ChIP-ChIP), or yeast one-hybrid
experiments from online sources such as modEncode and Flymine. With this information, we
reconstructed gene regulatory grids for the model organisms. Statistical and topological network
properties inherent in each of the grids (such as presence of hubs, network motifs, node degree
distribution and connected components) were determined using Matlab and R statistical environment.
These properties will be compared to the maize and Arabidopsis thaliana network properties.
P55.
Identification of Mutations in the gravity persistent signal 5 Mutant of Arabidopsis
Morales, Elisa, Ross, Jared, Vierling, Michael, and Luesse, Darron, Southern Illinois University
Edwardsville, Edwardsville, IL
Gravity dictates critical spatial information about a plant’s surroundings and provides important cues
about growth orientation. It provides the correct orientation in which the primary shoots and roots should
grow for efficient nourishment acquisition. During gravitropism the roots respond positively to gravity by
growing downward, into the soil to obtain water and nutrients. The stems respond negatively to gravity
growing upward, towards the sunlight to maximize its absorption (Morita, 2010). When an Arabidopsis
thalianaplant is exposed to a change in gravity in a cold temperature environment (4°C), no response is
seen. However, when returned to room temperature in an upright position, it will curve in response to the
change of gravity that occurred at the colder temperature (Muday, et. Al.2002). This is known as the
gravity persistent signal (GPS) response. The gps5 mutant was identified due to an enhanced GPS
response. Deep genomic sequencing and microarray analysis have indicated five candidate mutations in
the gps5 genome that may be the cause of the hypergravitropic phenotype. The goal of this work is to
determine which gene is responsible. For each candidate, SALK lines were obtained and homozygous
mutants were confirmed. Influorescence stem, hypocotyl, and root gravitropism were measured in an
attempt to re-create the gps5 phenotype. Preliminary results suggest that none of the insertion lines show
45
a hyper gravitropic responseTransformation rescue experiments have also been initiated for each gene.
Identification of the gps5 mutant will provide important information for understanding the signaling
events that regulate gravitropism.
P56.
The TOC159 mutant of Arabidopsis thaliana Accumulates Reduced Levels of
Polyunsaturated Fatty Acids
Afitlhile, Meshack, Western Illinois University, Macomb, IL
We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2)
accumulates normal levels of fatty acids, and transcripts of galactolipid synthesis enzymes. The MGD1
gene was downregulated and the ppi2-2 mutant accumulates decreased levels of
monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was
impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was
upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant
accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. A gene that
encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which
suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to
bilayer forming plastid lipids was decreased in the cold-acclimated wild type but not in the ppi2-2 mutant.
This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of
18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective
Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty
acids.
P57.
Phylogenomic and Structural Analysis of the TRAF family of Coregulators in Maize
Scott, Michael Walter1, McFarland, Frank1, Grotewold, Erich2, Doseff, Andrea I.2,and Gray, John1,
1
University of Toledo, Toledo, OH; 2The Ohio State University, Columbus, OH
The TNF receptor (TNFR) associated factor (TRAF) protein family is characterized by the presence of a
conserved coiled-coil domain TRAF/MATH domain (for meprin and TRAF-C homology). This domain is
required for homo- or heterodimerization and interaction with receptor or cytoplasmic signaling proteins
including transcription factors (TFs). This family of proteins is present in plants but the biological role of
only a few plant TRAF proteins have been characterized. In Arabidopsis the SEVEN IN ABSENTIA
(SINA) clade of TRAF-like proteins has been implicated in proteasome-mediated regulation of
transcription factors such as NAC1, CUC2, and AP2. In rice, OsDIS1 has been identified as a negative
regulator in the drought tolerance response. We performed a survey of the TRAF family of proteins in
maize and have identified at least 46 members. The maize TRAF proteins could be divided into two major
clades and various subclades based on the presence or absence of MATH, and BTB (for BR-C, ttk and
bab) or POZ (for Pox virus and Zinc finger) domains. The gene structure of 30 members of the TRAF
family could be confirmed from full length cDNA clones present in the maize TFome collection. The
expression profile of maize TRAF genes was summarized from RNA-seq data. To investigate the
structure of TRAF proteins in maize, the XTALPRED algorithm was employed to select members with a
high crystalizability index. Progress on the overexpression and purification of selected maize TRAF
proteins in their native conformation will be presented. This project was supported by NSF grant IOS1125620.
46
P58.
Zea mays Sucrose Transporter2 Contributes to Plant Growth, Development, and
Agronomic Yield
Leach, Kristen, and Braun, David, Division of Biological Sciences, Interdisciplinary Plant Group,
Missouri Maize Center, University Of Missouri, Columbia, MO
During daylight, plants often have excess photosynthetic productivity, resulting in the accumulation of
photosynthates, including sucrose that is transiently stored in the vacuole. At night, or as photosynthesis
becomes limiting because of environmental conditions, plants can remobilize sucrose from the vacuole
into the cytoplasm to sustain the plant’s metabolism and growth. Based on homology to other SUT2
transporter proteins, Sucrose transporter2 (ZmSut2) is hypothesized to function as a sucrose/H+
symporter located on the tonoplast membrane and to export sucrose temporarily stored in the vacuole. To
understand the biological importance ofZmSut2, we identified several Mutator transposon insertions into
the gene and characterized the resulting mutants using molecular and phenotypic analyses. From
preliminary studies in the greenhouse in which wild-type and mutant plants were grown in the same pot,
we observed that the mutants had a slower growth rate and were generally of a smaller stature. Based on
these growth differences, wild-type and mutant plants were grown under increasing population densities
in the field to investigate whether increased competition (i.e., reduced light interception) would
exacerbate phenotypic differences. The plant growth rate tended to be slower in the mutants compared to
their wild-type siblings throughout plant development, and this difference became more exaggerated at
higher densities. Ear and kernel size were also significantly smaller in the mutants at higher plant
densities. These findings suggest that ZmSut2 plays an important role by remobilizing sucrose out of the
vacuole for subsequent use in growing tissues, and that ZmSut2 function makes an important contribution
to maize development and agronomic yield.
P59.
Metabolic Engineering of Camelina for Improved Biofuel Functionality
Erdozáin Salón, Sandra, Nazarenus, Tara, Kumssa, Tadele, and Cahoon, Edgar, University of
Nebraska-Lincoln, Lincoln, NE
Camelina sativa (false flax) is an emerging Brassicaceae oilseed crop in the Great Plains and Pacific
Northwest of the United States. Growing interest in camelina is largely due to its potential for biodiesel
production in geographic areas that are not well-suited for soybean cultivation. Because it is not grown
for food use in the US, camelina can serve as a production platform for biofuels. Genetic transformation
of camelina can be achieved by simple floral vacuum infiltration of Agrobacterium. Consequently,
metabolic engineering of camelina can be conducted in a rapid and non-labor intensive manner. A major
limitation of camelina oil for biofuels is its low oxidative stability arising from a high linoleic acid (18:2)
and linolenic acid (18:3) content. To improve the oxidative stability of camelina oil, we have devised
metabolic engineering strategies to generate oils enriched in oleic acid (18:1) and low in the
polyunsaturated fatty acids (PUFAs) linoleic and linolenic acids. Metabolic engineering strategies and
results from field evaluation studies will be presented.
P60.
Proteomics & Mass Spectrometry: Essential Tools for Systems Biology
Sophie Alvarez, Michael J. Naldrett, and Bradley S. Evans, Donald Danforth Plant Science Center,
Proteomics & Mass Spectrometry Facility, St. Louis, MO
Nowadays mass spectrometry (MS) is well established as an integrated tool for studying biological
systems, in particular in the identification and quantification of perturbations from the environment, such
as host-microbe interaction and the adaptative and/or defensive mechanisms induced. Protein and
metabolite contents which are the final products of genome expression correlate intrinsically with how
different species and strain genotypes interact with the environment. Qualitative MS-based proteomics
tools are routinely used in defining protein-protein interaction networks and protein signaling pathways,
47
whilst quantitative methods can reveal the dynamics of cellular networks in response to modifications
with the environment. Though proteomic approaches are more advanced, approaches are rapidly
developing for the study of metabolites and metabolic networks with the emergence of higher resolution
mass spectrometers. The Proteomics & Mass Spectrometry Facility (PMSF) has been involved in
facilitating the progress of various research projects for profiling proteins and targeted small molecules
from different species and strains under various conditions. The main tools used for protein and small
molecule analyses and the state of the art equipment at the PMSF are presented here.
P61.
Chloroplast to Nucleus Signalling: Role in Insect Resistance
Guthrie, Katherine1, Bhattacharya, Oindrila2, Ortiz, Irma2, and Walling, Linda2, 1Northwest Missouri
State University, Maryville, MO; 2University of California Riverside, Riverside, CA
After wounding of tomato leaves, Leucine aminopeptidase A (Lap-A) accumulates in the chloroplast
stroma. It creates a signal that moves to the nucleus to up-regulate the production of proteinase inhibitors
and polyphenol oxidases and down-regulate another set of genes. This mode of regulation (chloroplast to
nucleus) is called retrograde signaling. Recently, transcription factors were found to move from the
chloroplast to the nucleus to control defense genes. These proteins have nuclear localization signals
(NLS) and chloroplast transit peptides (cTP) suggesting dual localization. The purpose of this project was
to identify transcription factors that may be involved in LAP-A’s retrograde signal. In tomato, WRKY
transcription factor 5 (SlWRKY5) has a NLS and cTP and its residence in the plastid stroma was
confirmed by proteomics. Given the role of the Arabidopsis homolog of WRKY5 in defense signaling,
WRKY5 is likely a mobile signal. The goal was to monitor the movement of both LAP-A and WRKY5
after wounding. To this end, the SlWRKY5 and Lap A cDNAs were amplified using gene specific primers
and PCR and inserted into Gateway vectors, pEarleyGate 101 and 102 to make yellow and cyan
fluorescent fusion proteins, respectively. These vectors will be delivered into leaves using gold particle
bombardment. The location of the fusion proteins after wounding will be determined using confocal
microscopy. If the protein moves, its role in retrograde signaling of these proteins will be further
investigated. This knowledge will be important in the creation of insect-resistant tomato crops and
prevention of crop loss.
P62.
Three Arabinogalactan Protein Specific Hyp-O-Galactosyltransferase in Arabidopsis
Regulates Tip Growth and Abiotic Stress Tolerance
Lu Tian and Debarati Basu, Ohio University, Athens, OH
Hydroxyproline-O-galactosyltransferase (Hyp-O-GALT) is a prominent post translational modification
displayed by extracellular glycoproteins in plants. The hydroxyproline residues in arabinogalactan
proteins (AGPs), a plant cell wall glycoprotein, are glycosylated with O-linked galactose by Hyp-OGALTs. Numerous evidences demonstrated that AGPs are critical for plant growth and developmental
processes but the exact molecular role of AGP glycans in plant growth and development is yet to be
elucidated. Here we have identified three members of GT31 family, (GALT3-At3g06440, GALT4At1g27120 and GALT6-At5g62620) which encodes AGP specific Hyp-O-GALT by heterologous
expression of these three GALTs in tobacco leaf epidermal cells. Transcript profiling by real-time PCR
revealed an overlapping but distinct expression patterns suggesting their specificities. Transiently
expressed GALT3, GALT4 and GALT6 fluorescent protein fusions were exclusively localized within the
Golgi vesicles. Biochemically, the disruption of any of the three GALT genes led to significant reduction
in both AGP specific GALT activity as well as b-Yariv precipitatable AGPs. Both primary root and
pollen tube growth of the knock-out mutants were less sensitive to the inhibitory effect of b-Yariv
reagent. Additionally, loss-of-function mutants of these three GALTs caused defects in root hair growth
and seed coat mucilage under optimal conditions and as well as displays conditional phenotypes of
impaired root growth and defective anisotropic growth at root tip under salt stress.
48
P63.
Arabidopsis MAPK Phosphatase 1 (MKP1) regulates the abundance of plant-derived
chemical signals that induce virulence gene expression in Pseudomonas syringae
Anderson, Jeffrey1, Wan, Ying1, Kim, Young-Mo2, Pasa-Tolic, Ljiljana2, Metz, Thomas2, and Peck, Scott1,
1
University of Missouri, Columbia, MO; 2Pacific Northwest National Laboratory, Richland, WA
A primary layer of plant immunity involves plasma membrane-localized receptors that perceive
conserved molecular features of microbes termed pathogen-associated molecular patterns (PAMPs). To
counteract PAMP-induced defenses, the bacterial pathogen Pseudomonas syringae uses a type III
secretion system (T3SS) to deliver effector proteins into plant cells. These effectors suppress host
defenses, thereby allowing the bacteria to escape detection and proliferate. Although the T3SS is critical
for virulence, it is not constitutively present in bacteria and must be produced during infection. Although
putative plant-derived signals that induce T3SS-encoding genes in P. syringae have been proposed based
largely on in vitro experiments, whether these signals actually affect outcomes of P. syringae-plant
interactions has been unclear due to the lack of mutants altering their abundance in the host. We recently
found that an Arabidopsis mutant mkp1 (mapk phosphatase 1) is more resistant to P. syringae infection,
and that bacteria are unable to efficiently deliver T3SS effectors into mkp1 cells [1]. To investigate the
mechanism(s) responsible for these phenotypes, we performed a metabolomic comparison of exudates
from wild type and mkp1 plants and discovered that several T3SS-inducing metabolites were decreased in
mkp1. Exogenously adding the T3SS-inducing metabolites during infection suppressed both the decreased
effector delivery and enhanced resistance phenotypes of mkp1. These results indicate that mkp1 is more
resistant due to a lack of chemical cues that initiate bacterial virulence.
P64.
Danforth Plant Science Center’s Integrated Microscopy Facility
R. Howard Berg, Donald Danforth Plant Science Center, Integrated Microscopy Facility, St. Louis, MO
The Integrated Microscopy Facility offers training and full service in both light and electron microscopy.
Our dedicated staff has extensive experience in preparing and imaging plant cells, and our services are
offered to the plant science community generally. The facility's Leica SP8-X confocal microscope is
optimized for high resolution live cell imaging and includes a white light laser (delivering 200 laser lines,
470-670 nm), a high speed resonance scanner (8,000 lines scanned per second), five detectors (including
three HyD detectors with doubled sensitivity), and custom-adjusted detection windows for emission
bandpasses. For electron microscopy samples are prepared by ultra-rapid freezing using a Bal-Tec high
pressure freezer. After freeze substitution they are embedded in resin for thin section transmission
electron microscopy using a Leo 912 AB energy filtered TEM. This instrument is equipped for elemental
analysis by electron energy loss spectroscopy (EELS) of thin sections. We consider the facility to be a
national resource for plant scientists and welcome your inquiries.
P65.
MMF1 regulates the photoperiodic control of hypocotyl elongation in Arabidopsis thaliana
He Huang1, Rebecca Nolan1, Jessica Goldsworthy2, Tom Liu3, Sophie Alvarez1,and Dmitri A. Nusinow1;
Donald Danforth Plant Science Center, St. Louis, MO; 2Michigan State University, East Lansing, MI;
3
Ladue Horton Watkins High School, St. Louis, MO
1
Plants alter growth and development to respond to changing environmental conditions, such as day
length. This plasticity is the result of the integration of the circadian clock with specific signaling
pathways to regulate physiology. However, the components that mediate the photoperiodic control of
growth are poorly understood. Using affinity purification and mass spectrometry (AP-MS), we discovered
a new protein that directly binds to both clock and light signaling factors, named MASS SPEC
IDENTIFIED MODULATING GROWTH FACTOR 1 (MMF1). MMF1 is a conserved, plant-specific,
nuclear-localized factor that cycles with a peak at dusk. mmf1 mutant seedlings have elongated
49
hypocotyls in a day-length specific manner, compared to wild type. Conversely, constitutive
overexpression of MMF1 shortens hypocotyls, regardless of day length. Affinity purification of MMF1
co-precipitates the circadian-clock associated Evening Complex, red light photoreceptors, and the COP1SPA complex. Biochemical and molecular assays demonstrate that MMF1 binds directly to EARLY
FLOWERING 4 (ELF4) and PHYTOCHROME B. Mutational analysis combined with AP-MS show that
phyB is necessary for MMF1 association with clock and light signaling components. Growth assays
performed under specific wavelengths suggests that MMF1 regulates red light signaling pathways.
Consistently, modulating MMF1 levels alter the expression of genes known to be downstream of the
phytochrome signaling pathways, including LONG HYPOCOTYL IN FAR RED 1 (HFR1) and the
homeobox transcription factor, ATHB-2 under short day conditions. These results define MMF1 as a new
clock and phytochrome-binding factor that participates in growth regulation under photoperiodic
conditions. Targeting MMF1 for manipulation could lead to improved growth responses to changing
environmental conditions.
50
INDEX OF REGISTRANTS
[Registered by March 10, 2015]
Last_Name
Afitlhile
Ahmed
Alers-Velazquez
Allen
Alsdurf
Alvarez
Anderson
Anterola
Arif Uz Zaman
Arp
Ayers
Baker
Barber
Bartlem
Basu
Basu
Benson
Berg
Braun
Brazil
Brenner
Buckner
Cahoon
Collins
Cook
Cook
Correia
Coursey
Damodaran
Deng
Dowd
Downes
Durbak
Erdozain Salon
Fabbri
Fahlgren
Fleishmann
First_Name
Meshack
Sheaza
Roberto
Doug
Jake
Sophie
Jeff
Aldwin
Muhammad
Jennifer
Jessica
Robert
Beau David
Derek
debarati
Proma
Chris
Howard
David
Elliot
Caitlin
Brent
Edgar
Carina
Gregory
Adam
Bianca
Tami
Suresh
Molian
Tyler
Brian
Amanda
Sandra
Marissa
Noah
Paul
Organization
Western Illinois University
Bowling Green State University
The Ohio State University
USDA-ARS
Kansas State University
Donald Danforth Plant Science Center
University Of Missouri
Southern Illinois University Carbondale
North Dakota State University
University Of Illinois at Urbana-Champaign
University Of Illinois at Urbana-Champaign
University Of Missouri-Columbia
University of Illinois at Urbana-Champaign
KWS Gateway Research Center
Ohio University
Ohio University
Ohio University
Danforth Plant Science Center
University Of Missouri
University of Illinois
Saint Louis University Department of Biology
Truman State University
University Of Nebraska
University of Missouri
Miami University
Ohio University
Ohio University
Ohio State University
South Dakota State University
Monsanto Company
University of Missouri
Saint Louis University
University Of Missouri
University Of Nebraska-Lincoln
Truman State University
Donald Danforth Plant Science Center
Western Kentucky University
51
Frick
Galliart
Gana
Ge
Gehan
Gilbert
Gipson
Gray
Green
Grennan
Guthrie
Hage
Hamilton
Hartmann
Haswell
Heese
Heintz
Held
Heller
Helm
Henn
Herock
Hohenstein
Hutchinson
Ibore
Jawahir
Jiang
Johnson
Jones
Kalra
Keller
Kephart
Kilgore
Kirchner
Kruse
Kutchan
LaMontagne
Larue
Leach
Leuchtman
Li
Elizabeth
Matt
Joyce
Lingxiao
Malia
Kerrigan
Monique
John
Kevin
Aleel
Katherine
Adam
Eric
Natalie
Elizabeth
Antje
Veronica
Jeremy
Nicholas
Matthew
Rosalyn
Hayley
Jessica
Sarah
Martha
Vanessica
Lingyan
Eden
Cody
Isha
Caroline
Anthony
Matthew
Nicholas
Colin
Toni
Erica
Clayton
Kristen
Daniel
Maoyin
Washington University in St. Louis
Kansas State University
Chicago State University
Bowling Green State University
Donald Danforth Plant Science Center
Donald Danforth Plant Science Center
Chicago State University
University Of Toledo
Ohio University
University of Illinois
Northwest Missouri State University
Northern Illinois University
Washington University in St. Louis
University of Missouri - St. Louis
Washington University in St. Louis
University Of Missouri-Columbia
Southern Illinois University Edwardsville
Ohio University
University Of Illinois at Urbana-Champaign
Indiana University
Truman State University
Ohio University
Iowa State University
Southern Illinois University Edwardsville
Iowa State University
University of Missouri St. Louis
University Of Missouri - Columbia
University Of Missouri - Columbia
University Of Illinois at Urbana-Champaign
Miami University, Oxford, Ohio
University Of Illinois at Urbana-Champaign
Northern Illinois University
Donald Danforth Plant Science Center
Northern Illinois University
Ohio University
Donald Danforth Plant Science Center
University Of Missouri - Columbia
Monsanto Company
Biological Sciences, University of Missouri
University Of Missouri
Donald Danforth Plant Science Center
52
Li
Liu
Lopez
Lu
Luesse
Marchal
Markham
McSteen
Morales
Morriss
Nusinow
Osika
Paciorek
Pandey
Pandurangan
Peck
Peng
Petrik
Pikaard
Pugh
Rahaman
Rajapaksha
Randall
Reem
Riggs
Robison
Ross
Sagwan-Barkdoll
Sahi
Schreier
Scott
Shah
Sharma
Shurzinske
South
Sternberger
Strader
Studer
SU
Subramanian
Tang
Ying
Helen
Edgar
Xiaolong
Darron
Melissa
Jonathan
Paula
Elisa
Stephanie
Dmitri
Megan
Tomasz
Sona
Sudhakar
Scott
Shuming
Deborah
Craig
Josiah
MD Mizanur
Nadeesha
Steve
Nathan
Kara
Jennifer
Jared
Laxmi
Shivendra
Spencer
Michael Walter
Dilip
Nilesh
Amanda
Paul
Anne
Lucia
Anthony
YUAN
Senthil
Fang
University Of Missouri St. Louis
University Of Illinois at Urbana-Champaign
Ohio State University
Saint Louis University
Southern Illinois University Edwardsville
University of Iowa
University of Nebraska-Lincoln
University Of Missouri
Southern Illinois University Edwardsville
Iowa State University
Danforth Plant Science Center
Ohio University
Monsanto
Donald Danforth Plant Science Center
Western University, Canada
University of Missouri Columbia
University of Missouri - St. Louis
Illinois State University
HHM, Indiana University
Ohio University
North Dakota State University
Saint Louis University Department of Biology
Indiana University Purdue University Indianapolis
Iowa State University
University Of Missouri
IUPUI
Southern Illinois University Edwardsville
Southern Illinois University Carbondale
Western Kentucky University
South Dakota State University
University Of Toledo
Donald Danforth Plant Science Center
Western Kentucky University
University of Missouri - St. Louis
USDA-ARS University of Illinois
Ohio University
Washington University in St Louis
The Donald Danforth Plant Science Center
University of Missouri - St. Louis
South Dakota State University
University Of Kentucky
53
Taylor
Tian
Tsogtbaatar
Valenta
Vega-Sanchez
Veley
Verslues
Vierling
Voothuluru
Wang
Williams
Wilson
Winters-Rozema
Wu
Wyatt
Wyman
Yang
Youngstrom
Zhang
Zhu
Zolman
Isaiah
Lu
Enkhtuul
Joy
Miguel
Kira
Paul
Michael
Priya
Geliang
Jordyn
Margaret
Elizabeth
Xiaoyun
Sarah
Aaron
Xiaodong
Christopher
Yingying
Dennis
Bethany
University of Missouri
Ohio University
The Ohio State University
University Of Missouri-St. Louis/DDPSC
Monsanto Company
Washington University in Saint Louis
Academia Sinica
Southern Illinois University Edwardsville
University Of Missouri
University of Missouri - St. Louis
Truman State University
Washington University in Saint Louis
East Central College
Monsanto Company
Ohio University
Spring Arbor University
University Of Nebraska-Lincoln
University of Iowa
University of Missouri - St. Louis
University Of Missouri-Columbia
University of Missouri - St. Louis
54
NOTES
NOTES
NOTES
NOTES
On behalf of The Donald Danforth Plant Science Center, we thank you for
attending the American Society of Plant Biologists 2015 Midwestern Section
Annual Meeting. We hope you have enjoyed your visit.
The Donald Danforth Plant Science Center
St. Louis, MO