Accepted Manuscript
Cautionary Notes on the Usage of abi1-2 and abi1-3 Mutants of Arabidopsis ABI1 for
Functional Studies
Yaorong Wu, Yaqiong Li, Yongchang Liu, Qi Xie
PII:
S1674-2052(14)00022-7
DOI:
10.1016/j.molp.2014.10.007
Reference:
MOLP 21
To appear in:
MOLECULAR PLANT
Received Date: 26 August 2014
Revised Date:
26 September 2014
Accepted Date: 3 October 2014
Please cite this article as: Wu Y., Li Y., Liu Y., and Xie Q. (2014). Cautionary Notes on the Usage
of abi1-2 and abi1-3 Mutants of Arabidopsis ABI1 for Functional Studies. Mol. Plant. doi: 10.1016/
j.molp.2014.10.007.
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LETTER TO THE EDITOR
Cautionary Notes on the Usage of abi1-2 and abi1-3 Mutants of Arabidopsis
Yaorong Wu1, Yaqiong Li1, Yongchang Liu2 and Qi Xie1
State Key Laboratory of Plant Genomics, National Center for Plant Gene Research,
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ABI1 for Functional Studies
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
No.1 West Beichen Road, Chaoyang District, Beijing 100101, China
College of Agriculture/Key Laboratory of Oasis Ecology, Agriculture of BINTUAN,
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Shihezi University, Shihezi 832003, China
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Running title: abi1-2 and abi1-3 are not true null mutants
To whom correspondence should be addressed:
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Qi XIE
State Key Laboratory of Plant Genomics
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Institute of Genetics and Developmental Biology
Chinese Academy of Sciences
No.1 West Beichen Road
Beijing 100101, China
TEL:
86-10-64806619
FAX:
86-10-64806619
E-mail: [email protected]
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Dear Editor,
Abscisic acid (ABA) is a key hormone involved in plant growth, development, as
well as stresses adaptation. Recently, a core ABA signal pathway was proposed, in
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which the ABA receptor proteins (PYR/PYL/RCAR) bind and inhibit type 2C protein
phosphatases (PP2Cs) in the presence of ABA, which in turn releases the SnRK2s
kinases, and the phosphorylated SnRK2s accumulate and subsequently phosphorylate
the ABA-responsive element binding factors (Melcher et al., 2009; Cutler et al.,
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2010).
ABI1, belonging to cluster A of the PP2C family, is an early-step negative
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regulator in the ABA signaling pathway. The dominant ABA-insensitive mutant allele
abi1-1 is impaired in ABA-related mechanisms (Koornneef et al., 1989; Leung et al.,
1994; Meyer et al., 1994; Allen et al., 1999). Saez et al. described two T-DNA
insertion mutants of ABI1: abi1-2 (SALK_072009) and abi1-3 (SALK_076309). Both
abi1-2 and abi1-3 are recessive loss-of-function alleles of ABI1, and exhibit enhanced
drought avoidance and ABA sensitivity both in seed and vegetative tissues (Saez et al.,
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2006). Since then, abi1-2 and abi1-3 have been widely used in many laboratories
(Mishra et al., 2006; Ludwikow et al., 2009; Rubio et al., 2009; Nishimura et al.,
2010).
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Microarray was performed to identify the genes differentially expressed in the
abi1-3 and wild type seedlings upon to ABA or auxin treatment. Other than no
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transcripts of ABI1 (At4g26080), transcripts of the adjacent gene, MKK1
(AT4g26070), were also not detected in abi1-3 with or without ABA and auxin
treatment. RT-PCR analysis confirmed the absence of full-length coding sequence
(CDS) of both ABI1 and MKK1 in abi1-3 under the same physiological conditions,
but MKK1 transcripts could be detected in the abi1-2 mutant (Figure 1A). This
indicates that abi1-3 has a different effect on the gene expression of MKK1 from that
of abi1-2. MKK1 is located adjacent to ABI1 on the genome in a tail-to-tail manner
(Figure 1B). It drew our attention because MKK1 was reported to be involved in the
ABA signaling pathway (Xing et al., 2007). To demonstrate that the loss of expression
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of MKK1 was not a secondary effect of the T-DNA in ABI1, we searched the
transcripts and genomic DNA fragments that covered the MKK1 gene. The transcripts
amplified with the primers P3 and P4 were present but genomic DNA were absent in
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the P1/P2 region (Figures 1B and 1C). This suggested that there was another T-DNA
insertion in SALK_076309.
To identify additional T-DNA insertions in the SALK_076309 line, primer LBb1
was combined with either P1 or P2, and only the LBb1/P2 pair would detect the
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expected DNA fragment. The sequence of the T-DNA flanking region showed that
another T-DNA insert was localized 677 nucleotides downstream from the ATG start
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codon in the MKK1 gene (Figures 1B and 1C). Furthermore, the expected size of
fragment can be amplified with primers P1 and P5 (Figure 1B). Sequence of LBb1/P2
and P1/P5 PCR fragments showed that this T-DNA maintains no deletions (Details to
see Supplementary data 1A and 1B). This also excludes the possibility of the T-DNA
rearrangement from the one inserted into ABI1 gene near this location. This T-DNA
insertion line was subsequently termed mkk1-2 because mkk1 has been reported
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previously (Xing et al., 2007). Thus, in addition to the previous identified insertion in
ABI1 in this mutant, a second T-DNA insertion in the same direction was found
(Figure 1B). This SALK_076309 line was renamed abi1-3mkk1-2.
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Mutations in ABI1 and MKK1 resulting from two T-DNA insertions are closely
linked, therefore successful segregation could not be achieved through repeated
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backcrossing. Subsequently, genomic MKK1 complementation was carried out to
obtain a single abi1-null mutant in abi1-3mkk1-2 background. Two independent
complementation lines without any visible phenotypes were selected for further
analysis based on the similar MKK1 expression level to that of the wild type plants
(Supplemental Figure 1A). The growth was compared between the wild type,
abi1-3mkk1-2, and transgenic plants in Murashige and Skoog (MS) medium with or
without ABA, in addition with Ler and abi1-1 plants as control. The seeds of different
genotype plants were germinated on medium containing 0.5 µM or 1 µM ABA, and
differences were observed at both germination and postgermination stages. As
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previously described, the abi1-3mkk1-2 plants were more sensitive to ABA than wild
type plants, and abi1-1 plants were ABA-insensitive. Seeds of both transgenic plants,
C1.2 and C16.2, germinated much later than that of wild type and abi1-3mkk1-2
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(Supplemental Figure 1B and 1C). And 49.16% of wild type cotyledon and 26.67% of
abi1-3mkk1-2 cotyledon were green, while the green cotyledon ratio of two
abi1-3mkk1-2::gMKK1 transgenic lines were 15.83% and 15% respectively, so the
abi1-3mkk1-2::gMKK1 transgenic plants were the most sensitive to ABA among the
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plants compared (Figures 1D and 1E). This indicates that mkk1 obscures the sensitive
phenotype of abi1-3.
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As SALK_076309 harbors two T-DNA insertions and both genes affect ABA
signaling, then we analyzed the other widely used ABI1-null mutant, abi1-2. RT-PCR
with primers selected to amplify the product covering the entire open reading frame
gave a weak band, and an enhanced product was produced when the PCR cycles were
increased (Figure 1A). A RNA gel blot assay was performed to determine whether
partial transcripts of ABI1 were present in abi1-2, and abi1-3mkk1-2 was used as a
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control. Surprisingly, as shown in Figure 1F, partial and weaker transcripts of ABI1
were present in these two mutants, especially in the seedlings treated with ABA. A
shorter band was detected in abi1-2, particularly where high induction had taken place
during the ABA treatment (Figure 1F). To demonstrate that at least some of the
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remaining short version of the ABI1 transcripts remained functional, we firstly
sequenced RT-PCR product and found it covered the intact ORF of ABI1 gene
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(Supplemental data 1C). Since no ABI1 antibody is available, next we analyzed the
expression of several genes differently affected in our microarray data. Figure 1F
shows that there was almost no difference in the expression levels of GH3.2 and
GH3.3 in the wild type and abi1-2 mutant treated with or without ABA and IAA;
however, GH3.2 and GH3.3 expression was greatly reduced in the abi1-3mkk1-2
mutant treated with IAA (Figure 1F). This suggests that GH3.2 and GH3.3 expression
may be dependent on either MKK1 or ABI1. To rule out the possibility that GH3.2 and
GH3.3 expression is dependent on MKK1, a RNA gel blot was carried out to analyze
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the new single MKK1 mutant, mkk1-3 (SALK_027645). As shown in Figure 1G, the
null mutant of MKK1 had no effect on the induction of GH3.2 in the plants treated
with ABA or IAA. The expression of GH3.2 and GH3.3 were checked in wild type
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and abi1-3mkk1-2 plants using quantitative RT-PCR with the primers specific to
GH3.2 and GH3.3 respectively, and the similar results were observed as detected by
Northern blot (Supplemental Figure 1D). This indicated that the induction of the
expression of GH3 genes is ABI1-dependent in the IAA-treated plants. The above
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results showed that while the abi1-2 mutant is a knockdown mutant, partial functional
transcripts of ABI1 remained.
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As to why ABA-induced similar size transcript, hybridization to ABI1 probe,
could be detected in abi1-3mkk1-2, we noticed that the T-DNA insertion was 550
nucleotides downstream from the ATG start codon in the ABI1 gene (Figure 1B), and
it might still produce partial ABI1 transcripts and continued transcription might stop in
the T-DNA region. To verify this, primer pairs covering different parts of the ABI1
gene were used for quantitative RT-PCR analysis. The results demonstrated that ABI1
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transcripts were present before the T-DNA insertion site in abi1-3mkk1-2
(Supplemental Figure 1C), but no ABI1 transcripts could be detected when we used
primers located after or spanning both sides of the insertion site (Supplemental Figure
1F and 1G). In addition, the 3’-RACE PCR, RT-PCR analysis and the sequence of
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product demonstrated that indeed the transcript appeared in Northern blot is a “fusion”
mRNA (Supplementary data 1D and 1E). The RT-PCR product (1500 nt) plus ABI1 5’
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un-translation region (458 nt) gives the similar size of the full length cDNA of ABI1
(2031 nt). So we see the similar size of a transcript appeared in abi1-3mkk1-2 (Figure
1F).
Based on all the data presented here, abi1-2 and abi1-3 are not true single locus
ABI1-null mutants. CDS of ABI1 in the abi1-2 mutant can be transcribed and that the
abi1-2 mutant is a knockdown mutant, and it is also evidenced by analysis of the
expression of downstream genes, such as GH3.2 and GH3.3. For the reason why we
could detected the RNA expression in abi1-2, which is different from the previous
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description (Saez et al., 2006), we think the enhanced PCR cycles and the treatment
condition enabled us to detect ABI1 expression in abi1-2. However, although no CDS
of ABI1 was present in the previous abi1-3 mutant, this mutant harbors a second
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mutation site in MKK1. Genomic MKK1 complementation of the abi1-3mkk1-2
mutant increased the sensitivity of the plants to ABA. So the ABA sensitivity of the
abi1-3mkk1-2 mutants is the combined result of both MKK1 and ABI1 mutations. The
abi1-3mkk1-2::gMKK1 transgenic plant line could be applied as an abi1-null mutant
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in the future. Because it is a transgenic line, when it is applied to cross with other
plants, additional MKK1 transgene detection by PCR in segregation lines would be
necessary when this line is used in genetic analysis. Therefore, studies using abi1-2
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and abi1-3mkk1-2 mutants should be evaluated carefully because abi1-2 is a
knockdown mutant, and the abi1-3mkk1-2 mutant contains two adjunct mutant genes,
in which one rather than the other might be responsible for alterations in the
molecular and related physiological phenotypes. abi1-3mkk1-2 should be an excellent
tool to understand the ABI1 and MKK1 co-regulating physiological process, including
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ABA-related phenotype.
FUNDING
This research was supported by grant NSFC 91317308/31030047/31270301 from the
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National Science Foundation of China and Qi Xie is supported by 973 Program
2011CB915402 from the National Basic Research Program of China.
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ACKNOWLEDGMENTS
We thank the Arabidopsis Biological Resource Center (ABRC) at The Ohio State
University for providing the T-DNA insertion lines.
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FIGURE LEGENDS
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Exp. Bot. 58, 2969-2981.
Figure 1. abi1-2 and abi1-3 (renamed abi1-3mkk1-2) are different sorts of alleles.
(A) RT-PCR analysis demonstrating the absence of full-length coding sequence of
ABI1 and MKK1 in the abi1-3mkk1-2 mutant, but the full-length coding sequence of
ABI1 and MKK1 were present in abi1-2 mutant. PCR reactions were performed as
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described in the Methods section, and Actin1 was used as the control. ABI1:1327 bp,
MKK1:1065 bp. Actin1: 240 bp.
(B) Schematic diagram of ABI1 and MKK1 structure and primers for T-DNA
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diagnostic PCR and RT-PCR. Closed boxes denote exons; introns are represented by
lines between closed boxes. P1 and LP: Forward primers, P2 and RP: reverse primers,
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LBb1: primer for the T-DNA left border. P3 and P4 are RT-PCR primers. The primers
were listed in Supplemental Table 1. The arrow toward right stands for sense primer,
and the arrow toward left stands for antisense primer.
(C) Genomic DNA PCR and RT-PCR of ABI1 and MKK1 in the abi1-3mkk1-2 mutant.
DNA and RNA from a homozygous abi1-3mkk1-2 mutant were used. The primers
used were indicated on the left side of each lane. The sizes of PCR produces were the
following: LP+RP:1213 bp, RP+LBb1:573 bp, P1+P2:1150 bp, P3+P4:1143 bp,
P2+LBb1: 1143 bp.
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(D) Growth phenotype of wild type, abi1-3mkk1-2, and two abi1-3mkk1-2::gMKK1
transgenic lines grown on MS medium with or without 0.5 µM ABA for five days.
Bar=0.5 cm.
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(E) Phenotype analysis of wild type, abi1-3mkk1-2, and two abi1-3mkk1-2::gMKK1
transgenic lines grown on MS medium with or without 0.5 µM ABA. Results
(seedlings with green and expansive cotyledon) were scored at 5 days after plating.
The values are means±SD, n=40).
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(F) Expression of ABI1, GH3.2 and GH3.3 in wild type, abi1-3mkk1-2 and abi1-2
plants. Expression of ABI1, GH3.2, and GH3.3 in plants treated by 100 µM ABA and
2 µM IAA at different time courses. Total RNA was extracted from 2-week-old
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seedlings treated with ABA or IAA for 0, 2, and 6 h. Total RNA (10 µg) was loaded in
each lane and analyzed by RNA gel blots with gene-specific probes. The methylene
blue–stained total RNA gel was used as a loading control.
(G) Expression of MKK1 and GH3.2 in the wild type and mkk1-3 plants. Total RNA
was extracted from 2-week-old seedlings treated with ABA or IAA for 0 and 2 h.
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Total RNA (10 µg) was loaded in each lane and analyzed by RNA gel blots with
gene-specific probes. The methylene blue–stained total RNA gel was used as a
loading control.
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Supplemental Figure 1.
(A) Total RNA was extracted from 2-week-old seedlings, and quantitative RT-PCR
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was conducted to determine the expression of MKK1. Amplification of 18S
was used as the internal control. The ratio of MKK1/18S was set as “1” in Col0
wild-type plants.
(B-C) Quantitative analysis of germination of wild type, abi1-3mkk1-2, two
abi1-3mkk1-2::gMKK1 transgenic lines, Ler and abi1-1 grown on MS medium
with or without 0.5 µM ABA (B) or 1 µM ABA (C). Percentages are means
(n=40) of three repeats±SD.
(D-G) Total RNA was extracted from 2-week-old seedlings treated with ABA or IAA
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for 0 and 2 h. And Quantitative RT-PCR assay was conducted with different
pair of primers. Amplification of 18S was used as the internal control.
(D) Different expression of GH3.2 and GH3.3 in the wild type and abi1-3mkk1-2
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plants treated with IAA for 0 and 2 h.
(E) The expression of partial ABI1 amplified with primers located in front of T-DNA
insertion site of ABI1 in the wild type and mkk1-3 plants.
(F) The expression of partial ABI1 amplified with primers located on both side of
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T-DNA insertion site of ABI1 in the wild type and mkk1-3 plants. .
(G) The expression of partial ABI1 amplified with primers located behind T-DNA
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insertion site of ABI1 in the wild type and mkk1-3 plants.
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