The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1) is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.
The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1) is an important regulator of seed dormancy and controls the timing of seedling emergence. The amount of DOG1 protein in mature seeds correlates with their dormancy level. It has been demonstrated that DOG1 is an important contributor to natural variation for seed dormancy and Arabidopsis accessions vary in their DOG1 levels. In this study we showed that the DOG1 gene produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants lack dormancy and do not accumulate DOG1 protein. Simultaneous expression of two or more DOG1 transcript variants encoding different isoforms, however, leads to the accumulation of DOG1 protein and increased seed dormancy. DOG1 protein can bind to itself and Arabidopsis seeds that contain a DOG1 variant unable to bind show reduced seed dormancy. We observed variation for DOG1 binding efficiency between Arabidopsis accessions, indicating a role for DOG1 self-binding in natural variation for seed dormancy.
Citation: Nakabayashi K, Bartsch M, Ding J, Soppe WJJ (2015) Seed Dormancy in Arabidopsis Requires Self-Binding Ability of DOG1 Protein and the Presence of Multiple Isoforms Generated by Alternative Splicing. PLoS Genet 11(12): e1005737. https://doi.org/10.1371/journal.pgen.1005737
Editor: Steven Penfield, University of York, UNITED KINGDOM
Received: August 21, 2015; Accepted: November 23, 2015; Published: December 18, 2015
Copyright: © 2015 Nakabayashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Max Planck Society and a Deutsche Forschungsgemeinschaft (http://www.dfg.de) SFB572 grant (WJJS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Alternative splicing has an important role in the post-transcriptional regulation of higher eukaryotes, but it was long believed to be of minor significance in plants. During the last years consecutive reports demonstrated a steadily increasing percentage of alternatively spliced genes in plants. At the beginning of this century only 1.5% of the Arabidopsis thaliana genes were estimated being alternatively spliced . Within one decade this fraction went up to 61% . Alternative splicing can lead to different outcomes and produces transcripts that code for proteins with altered or lost function. It can also lead to tissue specific transcripts or affect mRNA stability and turnover via nonsense-mediated decay [3,4]. The regulation and function of alternative splicing in plants is still largely unexplored but several examples have demonstrated its functional importance in various processes like photosynthesis, defence responses, the circadian clock, hormone signalling, flowering time, and metabolism [5,6,7,8]. A few examples have shown a role of alternative splicing during seed development and germination. The central regulator of seed maturation, ABSCISIC ACID INSENSITIVE 3 (ABI3), has been cloned in Arabidopsis over 20 years ago , but it was only recently found that this gene is alternatively spliced in a developmentally regulated fashion . The PHYTOCHROME INTERACTING FACTOR 6 gene is also alternatively spliced during seed development and one of its two splice forms, PIF6-β influences germination potential .
The timing of seed germination determines the seasonal environmental conditions that a plant encounters during its life and thereby its growth and reproductive success. Seed germination is regulated by dormancy, which is defined as the incapacity of a viable seed to germinate under favourable conditions . Seed dormancy is induced during seed maturation and released by dry storage of seeds (after-ripening) or imbibition at low temperatures (stratification) , and is regulated by environmental and endogenous factors. Extensive research on seed dormancy in several plant species, including Arabidopsis, has revealed the requirement of the plant hormone abscisic acid (ABA) to induce dormancy during seed maturation, whereas gibberellins (GAs) are required for germination [13,14]. In addition, mutant analyses identified a number of seed dormancy regulators. Apart from factors involved in hormone metabolism and seed maturation, these included several chromatin modifiers and transcriptional regulators .
The Arabidopsis gene DELAY OF GERMINATION 1 (DOG1) is a master regulator of seed dormancy acting independent of ABA. DOG1 was first identified as a major Quantitative Trait Locus (QTL) for seed dormancy . Mutations in the DOG1 gene lead to a complete absence of dormancy. DOG1 shows a seed-specific expression pattern and encodes a protein with unknown function. Its transcript and protein abundances in freshly harvested seeds highly correlate with dormancy levels [17,18]. This correlation has been observed under both lab and natural conditions. Environmental conditions that enhance seed dormancy, such as low temperatures during seed maturation, are associated with enhanced DOG1 transcript levels [18,19,20]. Arabidopsis accessions from the south of Europe are in general more dormant and show higher DOG1 transcript levels compared to northern accessions . Interestingly, DOG1 transcript levels also showed the highest correlation among a set of seed dormancy genes with changes in dormancy in buried seeds in the field . During after-ripening DOG1 protein remains stable but loses its activity due to unknown post-translational modifications. Therefore, DOG1 is likely to be part of a timing mechanism for the release of seed dormancy . The DOG1 protein belongs to a small family in Arabidopsis that is conserved in plants. Several DOG1 homologues have been shown to be functionally conserved and are able to enhance seed dormancy in Lepidium sativum  and Triticum aestivum .
We are only starting to understand the regulation of DOG1 at the protein level and its transcriptional regulation remains to be further investigated. DOG1 transcript levels are enhanced by the TFIIS transcription elongation factor [24,25] and by histone monoubiquitination . DOG1 is alternatively spliced and four splicing variants encoding three different isoforms have been reported . It has recently been shown that the spliceosome disassembly factor NTR1 is required for proper transcript levels and splicing of DOG1 .
We have identified a fifth splicing variant of DOG1 that constitutes the majority of its transcripts. Here we show that the accumulation of DOG1 protein requires alternative splicing because single DOG1 protein isoforms are not able to accumulate efficiently in the seed. The DOG1 protein can bind to itself and this self-binding is required for full DOG1 function. Variation in self-binding ability of DOG1 exists in nature and contributes to variation in seed dormancy levels between Arabidopsis accessions.
Quantification of DOG1 transcripts during seed maturation
The Arabidopsis DOG1 gene contains three exons. Its second intron is alternatively spliced and shows both alternative 3’ and alternative 5’ splice site selection, leading to four different transcripts DOG1-α, DOG1-β, DOG1-γ, and DOG1-δ . We quantified the individual transcripts using specific primers. Comparison of total DOG1 transcript levels with the combined levels of the four individual splicing forms indicated that the majority of DOG1 transcript does not exist out of these four forms. Detailed analysis of the DOG1 transcripts by 3’-RACE revealed the presence of a fifth splicing form encoding the same protein as DOG1-β and DOG1-γ, which was designated as DOG1-ε. This splicing form misses the complete third exon (Fig 1A).
(A) Schematic representation of transcript variants and protein isoforms. The genomic structure is presented at the top. Exons are shown as boxes, and introns, the partial promoter and downstream regions as lines; asterisk represents STOP codon. (B) qRT-PCR analysis of DOG1 splicing variants during seed maturation of NIL DOG1. The mRNA level of each DOG1 variant was normalised to the ACT8 mRNA level. Error bars represent the S.E.M. of at least three biological replicates. DAP, days after pollination. (C) The relative ratios of the DOG1 splicing variants during seed maturation, calculated from the data in (B). The right panel is a magnified image of the bottom of the graph in the left. For clarity, the epsilon variant is not shown in the right panel. Error bars represent S.E.M. DAP, days after pollination. The data normalised to the second reference gene HBT for (B) and (C) are shown in S1 Fig. (D) Subcellular localisation of DOG1 isoforms fused to YFP transiently expressed in Nicotiana benthamiana leaves. YFP fluorescence (i–iii), merged image of YFP fluorescence, autofluorescence of chlorophyll, and transmission (iv–vi). Scale bars = 20 μm.
The abundance of the five DOG1 splicing variants was followed during seed maturation. As shown previously [17,18], DOG1 expression peaks in the middle of the seed maturation phase and is reduced in fully mature dry seeds. The DOG1-ε transcript represents about 90–95% of the DOG1 transcripts at the measured time-points (Fig 1B and S1A Fig). The ratio between the different splicing forms is fairly constant (Fig 1C and S1B Fig), although DOG1-α is relatively more abundant at the beginning of seed maturation. DOG1-δ is very low abundant, but increases at the end of seed maturation (Fig 1C and S1B Fig).
The alternative splicing of DOG1 only affects the C-terminal part of the protein. DOG1-β is the smallest isoform (consisting of 278 amino acids in the Landsberg erecta (Ler) accession) and shares nearly the complete protein sequence with DOG1-α and DOG1-δ, apart from its last nine amino acids. The DOG1-α and DOG1-δ proteins are longer, respectively 292 and 303 amino acids in Ler, and share their last 24 amino acids with each other (Fig 1A).
All three DOG1 isoforms are located in the nucleus
The cellular localisation of the three DOG1 isoforms was analysed by transient expression of their N-terminal yellow fluorescent protein (YFP) fusion proteins in Nicotiana benthamiana leaves. All three fusion proteins were mainly detected in the nucleus similar as previously shown in transgenic Arabidopsis seeds containing YFP fused with the DOG1 genomic fragment (Fig 1D) . These results were confirmed by transformation of Arabidopsis protoplasts with C-terminal YFP fusion proteins of the three DOG1 isoforms (S2 Fig). The presence of all three DOG1 protein isoforms in the nucleus suggests that they are able to meet each other.
The accumulation of DOG1 protein requires the presence of multiple isoforms
The Near Isogenic Line DOG1 (NIL DOG1) contains an introgression of a strong DOG1 allele from the Cape Verde Islands (Cvi) accession in the Ler background and has a high level of seed dormancy. In contrast, the dog1-1 mutant (in NIL DOG1 background) does not produce full-length DOG1 protein and is nondormant (Fig 2A) . The function of the three DOG1 protein isoforms was studied by complementation of the dog1-1 mutant with single DOG1 isoforms driven by the native DOG1_Cvi promoter. Transgenic plants with single insertion events were obtained. None of these showed convincing functional complementation as their seed dormancy level was comparable to that of the dog1-1 mutant. Only introduction of DOG1-β caused a very weak restoration of dormancy (Fig 2B). Therefore, single DOG1 isoforms appeared largely non-functional when expressed from a native DOG1 promoter.
Germination profiles of control lines (A), single (B), double (C) and triple (D) pDOG1_Cvi:DOG1-α, DOG1-β, and DOG1-δ transformants in dog1-1 after different periods of dry storage. Error bars represent S.E.M. of at least three biological replicates. w, week. DOG1 overall mRNA (E) and protein (F) levels in transgenic lines. (E) DOG1 mRNA level was normalised to ACT8 mRNA level. (F) The top panel shows DOG1 protein, and the bottom panel a nonspecific band around 60 kD that is used as loading control (LC) . An asterisk on the left of the top panel shows molecular mass marker around 36 kD. The dog1-1 mutant produces only truncated protein and serves as a negative control. Individual line names were indicated at the bottom for (E) and (F). The line colours beneath the transgenic line names correspond to the line colours in (A-D).
Subsequently, transgenic plants with single DOG1 isoforms were mutually crossed and F3 or F4 plants homozygous for two isoforms were selected for all possible combinations. Transgenic lines containing two DOG1 isoforms showed enhanced dormancy compared to lines containing a single DOG1 isoform. Although dormancy levels of these double transformants showed variation, we observed the tendency that plants containing a combination of DOG1-β with DOG1-α or DOG1-δ had higher dormancy levels than plants containing a combination of DOG1-α and DOG1-δ, which germinated already 70–80% directly after harvest (Fig 2C). The dormancy level of double transgenic plants containing DOG1-β as one of the two splicing forms was similar to that of the low dormant Ler accession but much lower than NIL DOG1, which is the wild-type background of the dog1-1 mutant. Finally, we obtained transgenic plants that contain all three DOG1 isoforms after further crossing and selection. Seeds of all these triple transgenic plants showed dormancy restoration and none of them had similar low dormancy levels as the double transgenic lines containing α and δ (Fig 2D).
Seed dormancy highly correlates with DOG1 transcript and protein levels in freshly harvested seeds . Therefore, transcript and protein levels were measured in the single, double and triple transgenic plants. Freshly harvested seeds of the transgenic lines showed some variation in their DOG1 expression but were mostly in a similar range as those of NIL DOG1 (Fig 2E). This was to be expected because DOG1 is transcribed from the same DOG1_Cvi promoter in NIL DOG1 and the transgenic lines. The transcript levels of the transgenes are expected to be slightly lower than the observed DOG1 transcript levels, which include a low amount of transcript from the dog1 mutant gene. Taken this into account, transcript levels in the transgenic lines still did not correlate with dormancy levels. Several of the transgenic lines with single DOG1 isoforms showed high DOG1 expression levels, comparable to NIL DOG1, but were non-dormant. In contrast, dormancy correlated with DOG1 protein accumulation in these transgenic seeds (Fig 2B–2F). DOG1 protein was scarcely detectable in the transgenic lines containing single DOG1 isoforms, in accordance with their lack of seed dormancy. The double and triple transgenic lines accumulated varying levels of DOG1 protein that were mostly in the same range as those found in seeds of the Ler accession, but much lower compared to the wild-type NIL DOG1. Interestingly, the double transgenic lines accumulated slightly higher levels of DOG1 protein than the triple, although they were less dormant. This suggests that the presence of three isoforms gives higher DOG1 activity in comparison to two isoforms. In the double and triple transformants that contain both DOG1-β and DOG1-α or DOG1-δ protein, two bands could be detected. In the non-transgenic controls, however, only the faster migrating band is visible, which corresponds to the DOG1-β protein. This is most likely due to the different ratio of the DOG1 transcript variants between controls and transgenic lines. In the double and triple transformants the different DOG1 transcript variants are expressed at similar levels, whereas in the wild type the transcripts that encode the DOG1-β protein are most abundant (Fig 1C). As DOG1 protein accumulates to higher levels in NIL DOG1 compared to the double and triple transgenic lines despite their comparable transcript levels, the ratio between the isoforms probably influences DOG1 protein accumulation.
Single DOG1 isoforms can accumulate when highly overexpressed
We were interested whether an increase in the transcript level of single DOG1 transcripts could lead to complementation of the dog1 mutant. Three constructs with DOG1-α, DOG1-β, and DOG1-δ driven by the constitutive 35S promoter were separately transformed into dog1-1 plants. About 10 to 40% of the obtained independent transformants for all three constructs showed high levels of seed dormancy (Fig 3A). Interestingly, the DOG1-β isoform was most effective in dormancy induction, followed by DOG1-δ. DOG1-α showed relatively lower dormancy levels. Overall, this experiment demonstrated that every DOG1 isoform is biochemically functional to induce seed dormancy.
(A) Germination profiles of p35S:DOG1-α, DOG1-β, and DOG1-δ transformants in dog1-1 after different periods of dry storage. Error bars represent S.E.M. of at least three biological replicates. w, week. Germination profiles of the control lines are shown in Fig 2A. DOG1 overall mRNA (B) and protein (C) levels in overexpression transformant lines. (B) DOG1 mRNA levels were normalised to ACT8 mRNA level. The top left graph shows the values for the controls and non-complementing transformant lines. (C) Details are described in the legend of Fig 2F.
Both complementing and non-complementing lines for all three constructs were selected for further analysis. Comparison of DOG1 transcript levels between these lines showed a more than 100-fold difference. High levels of DOG1 transcript were only detected in lines with high dormancy levels (Fig 3B). Consistent with the transcript levels, DOG1 protein could only be detected in the dormant transformants where it accumulated to even higher levels than in the NIL DOG1 control (Fig 3C). Taken together with the results from the complementation experiments using the native DOG1 promoter (Fig 2A–2F), these data suggest that all the single DOG1 isoforms are functional but unstable in the cell and can only accumulate when they have very high transcript levels.
Self-binding of DOG1 enhances seed dormancy
Overlapping nuclear localisation and instability of single DOG1 isoforms have prompted us to test mutual binding abilities of the DOG1 isoforms in a yeast two-hybrid experiment. All three DOG1 isoforms were able to bind to themselves and to each other in any combination (Fig 4A). These results were confirmed using the bimolecular fluorescence complementation assay based on split YFP. As shown in Fig 4B, YFP fluorescence could be observed in the nuclei of Arabidopsis embryo cells containing two transgenes consisting of the N- and C- terminal halves of YFP fused to different DOG1 splicing forms. Fluorescence was not detected in the controls.
(A) Interaction between DOG1 isoforms detected with the yeast two-hybrid assay. BD, fusion with GAL4-DNA binding domain; AD, fusion with GAL4- activation domain; -LW, dropout media without leucine and tryptophan; -LWH, dropout media without leucine, tryptophan and histidine; -, empty vector without DOG1 cDNA. (B) Interaction between DOG1 isoforms in planta detected with the split YFP system. Restored YFP fluorescence was observed by confocal microscopy in the embryo of 1-h imbibed seeds. Bar = 10 μm. α/β means the combination of N-terminal half YFP-DOG1 alpha fusion and C-terminal half YFP-DOG1 beta fusion. GUS protein was used as a negative control. (C) Interaction between the substituted mutant dog1 proteins and wild-type DOG1 delta protein. Details are described in the legend of Fig 4A. E13A is shown as a representative of a substitution that does not affect DOG1 binding abilities. (D) Germination profiles upon harvest of transgenic lines with alanine-substitutions. The values are the average of several independent lines for Y16A and E13A in dog1-1. Error bars represent S.E.M. of at least three biological replicates. (E) DOG1 protein accumulation in Y16A transgenic lines. Lines 1 to 7 represent independent transgenic lines. Details are described in the legend of Fig 2F.
A series of truncated DOG1 proteins was prepared to identify the region required for self-binding. A yeast two-hybrid assay between these truncated DOG1 proteins and full-length DOG1-δ identified a region of 10 amino acids whose absence makes the protein incapable of binding. Further alanine-scanning experiments in this region revealed that a single replacement, a tyrosine (the 16th amino acid of DOG1_Ler) with alanine, strongly reduced self-binding (Fig 4C), whereas the other seven obtained substitution mutants did not show altered self-binding abilities.
We were interested whether self-binding is necessary for DOG1 function. To answer this question, a complementation experiment was carried out using two constructs. The first contained the genomic region of the DOG1 gene from Ler and the second was identical except for the replacement of tyrosine 16 by alanine (Y16A). Both constructs were transformed into the dog1-1 mutant and transformants with single insertion events were selected. As shown previously , the genomic DOG1 clone complemented the dog1 mutant and seeds from the transformants were dormant. In contrast, the Y16A clone showed very weak complementation and 65% of the seeds germinated directly after harvest (Fig 4D and S3 Fig). Therefore, DOG1 protein requires self-binding to induce seed dormancy, although lack of binding ability does not abolish its function completely. A control experiment showed that replacement of glutamic acid at position 13 with alanine, an amino acid that is not affecting self-binding, did not cause a reduced complementation of the dog1 mutant (Fig 4D and S3 Fig).
Several independent Y16A transgenic lines were further analysed for DOG1 protein accumulation. Interestingly, DOG1 protein could be detected in all of these lines at similar levels as in the dormant NIL DOG1 control (Fig 4E). This indicated that the weak complementation of the Y16A lines was not due to reduced accumulation or instability of the Y16A mutant protein. Because the Y16A-DOG1 protein cannot bind to itself, we concluded that self-binding of DOG1 does not influence its protein accumulation but is required for its full function.
Differences in DOG1 self-binding ability contribute to natural variation for seed dormancy
DOG1 was originally identified as a major QTL underlying natural variation in dormancy between the Arabidopsis accessions Ler and Cvi. Later on, the DOG1 dormancy QTL was identified in several additional recombinant inbred line populations . This suggests that DOG1 is a major contributor to natural variation for seed dormancy in Arabidopsis. DOG1 alleles from different accessions show sequence polymorphisms in both promoter and coding regions. A correlation was found between dormancy levels and DOG1 transcript levels among different genotypes . Accordingly, it is likely that polymorphisms in the promoters of different accessions cause variation in DOG1 strength between accessions.
Surprisingly, a comparison of DOG1 transcript and dormancy levels in the low dormant accessions Ler and Col, and the dormant line NIL DOG1 (which contains the Cvi allele of DOG1) showed that this correlation was absent in Col, which has relatively high DOG1 transcript levels (Fig 5A and 5B). The DOG1 protein levels of these three genotypes correlated with their transcript levels (Fig 5B) and the Col seeds showed low dormancy despite having high levels of DOG1 protein. This lack of correlation might be caused by a reduced function of the DOG1_Col protein. Therefore, the self-binding ability of DOG1_Col was analysed. As shown in Fig 5C, DOG1_Col showed significantly reduced self-binding in a yeast two-hybrid assay. A sequence comparison of the predicted DOG1 proteins of Ler, Col and Cvi showed a polymorphism within AA13-16 (Fig 5D). In Ler and Cvi this region contains the amino acids ECCY, which are replaced by DSY in Col. Interestingly, the tyrosine at AA16 that is required for self-binding is present in all three accessions. Nevertheless, the observed amino acid changes are likely to affect self-binding because the Col DOG1 protein is not able to bind to itself (Fig 5C). To test the influence of this polymorphism on seed dormancy, two constructs containing different DOG1 alleles were introduced into the dog1-2 mutant (in Col background) for complementation. One of the constructs contained the wild-type DOG1_Col allele, including a 2.2 kb fragment upstream of the START codon and 1.1 kb downstream of the STOP codon. The other construct coded for a modified Col DOG1 protein in which the amino acids DSY at AA13-16 were replaced by ECCY. Transgenic plants containing single introgression events were selected for both constructs and their seed dormancy levels were assessed by following their germination rate during extended seed storage. Similar to the DOG1 complementation lines previously obtained , independent transgenic lines with the same construct showed varying degrees of dormancy (S3 Fig). However, plants with the construct encoding the ECCY Col DOG1 protein showed enhanced dormancy levels compared to plants containing the wild-type Col DOG1 construct (Fig 5E and S4 Fig). Therefore, we assume that the polymorphism at AA13-16 contributes to the low seed dormancy of Col by reducing the self-binding ability of the DOG1 protein.
(A) Germination profiles of Col, Ler and NIL DOG1 after different periods of dry storage. Pink triangle, Col; green open diamond, Ler; and blue square, NIL DOG1. Error bars represent S.E.M. of at least three biological replicates. w, week. (B) DOG1 overall mRNA and protein levels in Ler, NIL DOG1 and Col. Details are described in the legend of Fig 2E and 2F. Numbers below the blot indicate relative DOG1 protein levels, normalized to the loading control. Error bars represent S.E.M. of at least three biological replicates. The data for Ler and NIL DOG1 were taken from  (www.plantcell.org): Copyright American Society of Plant Biologists. (C) Yeast two-hybrid binding assay of the beta isoforms of DOG1_Col and controls. Details are described in the legend of Fig 4A. (D) Alignment of the first 20 amino acid residues of DOG1 of Ler, Cvi and Col. The first M is the starting methionine. Polymorphic residues are shaded in grey, and the asterisk represents the 16th tyrosine_Ler that is required for self-binding. (E) The ECCY substitution of DOG1_Col shows enhanced dormancy. Germination profiles upon harvest of transgenic lines with ECCY substitution and controls. Col WT represents 14 independent lines of dog1-2 transformed with wild-type Col_DOG1, Col ECCY represents 13 independent lines of dog1-2 transformed with the ECCY variant of Col_DOG1. Error bars represent S.E.M.
Natural variation for DOG1 self-binding ability was further explored by analysing DOG1 in 58 accessions. A protein sequence comparison identified three main haplotypes for the AA13-16 region, which were named DSY (Col-type), DRY (Sei0-type) and ECCY (Ler/Cvi-type) (S5 Fig). Several accessions from each haplotype were analysed for their DOG1 binding ability using a yeast two-hybrid assay. As expected, the haplotype DSY showed very weak self-binding, which was also the case for the DRY haplotype. In contrast, the ECCY accessions showed strong DOG1 self-binding (Fig 6A). We further studied the DOG1 haplotypes by analysis of their dormancy level and DOG1 protein accumulation in fresh seeds of representative accessions of the three groups (Fig 6B and 6C). By combining DOG1 protein levels and haplotype, we could explain a major part of the dormancy levels of these accessions. The DSY accessions had relatively low dormancy levels except for Sha, Daejoen, Kondara and Kas1 that all showed high DOG1 protein levels. Most of the ECCY accessions had high dormancy levels while having low to medium high DOG1 protein levels. The ECCY accession Ler showed low dormancy, but this accession had very low DOG1 protein levels (Fig 5B). Overall, these data demonstrated that not only expression levels of DOG1 but also differences in self-binding ability affect the strength of DOG1 function in dormancy induction of Arabidopsis natural accessions. We propose that combining the analysis of DOG1 protein levels in freshly harvested seeds with amino acid composition at the 13–16 AA region can lead to an improved prediction of seed dormancy levels in Arabidopsis accessions.
(A) Interaction of DOG1 beta forms with themselves in different accessions by yeast two-hybrid assay. cDNAs for beta DOG1 were cloned from several accessions of each DOG1 haplotype. Acc, accession; -LW, dropout media without leucine and tryptophan; -LWH, dropout media without leucine, tryptophan and histidine. (B) Germination profiles of representative accessions of the three DOG1 haplotypes after different periods of dry storage. Error bars represent S.E.M. of at least three biological replicates. (C) DOG1 protein accumulation in accessions. Details are described in the legend of Fig 2F. Numbers below the blot indicate relative DOG1 protein levels, normalised to the loading control.
The timing of seed germination determines successful plant establishment. Seed dormancy is a major factor controlling germination potential and has a complex regulation involving several independent pathways [15,28]. The molecular mechanisms that regulate seed dormancy are only beginning to be understood. DOG1 has been identified as a key dormancy gene in Arabidopsis [17,18]. Seed dormancy levels are well correlated with DOG1 protein levels in freshly matured seeds. The DOG1 protein undergoes modification during dry seed storage, which is paralleled by loss of dormancy . A better understanding of seed dormancy requires a thorough knowledge of the regulation of DOG1. Our present study demonstrated that the accumulation of DOG1 protein is influenced by alternative splicing of the DOG1 gene. In addition, we have shown that DOG1 protein function is enhanced by its self-binding.
Alternative splicing is widespread in plants, but relatively few studies have been performed to study its regulatory action in specific plant processes. The main consequences of alternative splicing are alteration or loss of protein function, tissue specificity, and changed mRNA stability and turnover via nonsense mediated decay [3,4]. Here, we demonstrated that the production of several protein isoforms by alternative splicing stimulates the accumulation of DOG1 protein. Five DOG1 splicing variants were identified that are translated into three protein isoforms, DOG1-α, DOG1-β and DOG1-δ. These isoforms could not complement the dog1 mutant when they were expressed from the endogenous DOG1 promoter and DOG1 protein accumulation required the presence of at least two of these isoforms. However, overexpression experiments showed that the isoforms had small differences in their functionality with DOG1-β being the most effective, followed by DOG1-δ and DOG1-α (Fig 3). The individual DOG1 splicing variants and protein isoforms showed a high variation in abundance in wild-type plants. The transcripts encoding the DOG1-β isoform were about 20 times more abundant in mature seeds compared to the transcripts encoding the other two protein isoforms. In accordance, in wild-type seeds DOG1-β showed the highest accumulation and the other isoforms could not be clearly detected on immunoblots. However, our analyses with transgenic plants suggested that the DOG1-β isoform could not sufficiently accumulate in the absence of additional isoforms.
The mechanism that underlies the requirement of multiple isoforms for DOG1 protein to accumulate still needs further characterisation, but we have identified several of its characteristics. First, it is likely that the mechanism depends on active protein degradation because overexpression of single DOG1 isoforms by the 35S promoter can lead to the accumulation of DOG1 protein. We assume that the active DOG1 degradation mechanism is not able to deal with high amounts of single DOG1 isoforms that are continuously translated from abundantly present transcripts. Secondly, the ratio between isoforms appears to be important because equal amounts of splicing forms in the (double and triple) transgenic plants lead to a low level of protein accumulation. The natural ratio between DOG1 isoforms, in which DOG1-β is much more abundant than the other isoforms, leads to high levels of protein accumulation.
DOG1 is an essential gene for seed dormancy and its protein abundance is correlated with dormancy levels. Therefore, the regulation of DOG1 protein accumulation by alternative splicing could be part of a mechanism to fine-tune seed dormancy. We previously showed that DOG1 protein abundance does not decrease during the last part of the seed maturation phase, although DOG1 transcript levels are strongly reduced . Interestingly, the DOG1-δ transcript variant becomes relatively more abundant at the end of seed maturation in comparison to the variants encoding DOG1-β (Fig 1C). This altered ratio might enhance DOG1 protein accumulation and could explain the persistence of DOG1 protein at this time point, despite a general reduction in DOG1 transcript levels. Identification of the factors controlling alternative splicing of DOG1 can lead to a better understanding of its regulation and give new insights in seed dormancy. We have previously identified a splicing factor, SUPPRESSOR OF ABI3 (SUA), which functions during seed maturation and regulates alternative splicing of ABI3 . We analysed DOG1 splicing in the sua mutant, but did not observe any difference compared to wild-type plants. Therefore, other splicing factors than SUA regulate DOG1 alternative splicing. One of these has recently been identified as the spliceosome disassembly factor AtNTR1. The atntr1 mutants have altered transcript levels and splicing of DOG1 as well as reduced dormancy .
Our observation that the accumulation of DOG1 protein requires the presence of multiple isoforms inspired us to analyse whether these isoforms can bind to each other. Indeed, we observed self-binding of DOG1 but contrary to our expectation self-binding was not required for protein accumulation because DOG1 still accumulated in a modified version of DOG1 that is unable to bind to itself (Fig 4). However, this modified version of DOG1 had a strongly reduced function as evidenced by the low dormancy level of seeds that were derived from transgenic plants containing DOG1 that is unable to bind to itself. This observation suggests that DOG1 acts in a protein complex that comprises of at least a homodimer.
DOG1 has originally been identified as a seed dormancy QTL and the DOG1 locus shows sequence variation in both promoter and coding region among accessions . It was also shown that variation in DOG1 expression contributes to variation in seed dormancy levels between Arabidopsis accessions . In this work, we have shown additional natural variation at AA13-16 for DOG1 self-binding. Variation in self-binding ability could contribute to variation in DOG1 function and thereby seed dormancy. As DOG1 is a conserved gene, combining the variation in DOG1 expression levels with the variation in DOG1 self-binding has the potential to be developed into a marker to predict dormancy levels of crop seeds. However, these two factors are still not enough to fully explain the variation in seed dormancy levels between Arabidopsis accessions. Although DOG1 is a major dormancy QTL in Arabidopsis, ten other dormancy QTLs have been identified and variation in these QTLs should also be considered. For instance, the high dormancy levels of the Sha and Kondara accessions that have weak non-self-binding DOG1 alleles might be explained by their strong DOG6 alleles .
In this and previous studies we have observed a strong negative correlation between DOG1 protein levels and germination potential with two important exceptions. Firstly, after-ripened seeds can germinate in the presence of high levels of DOG1 protein . Secondly, we showed in the present work that seeds containing DOG1 that is not able to bind to itself can germinate. We speculate that the lack of a negative correlation between DOG1 protein accumulation and germination potential could have the same cause in both cases. After-ripening has been shown to be associated with DOG1 protein modifications. These protein modifications might prevent or reduce DOG1 self-binding and thereby its function.
Plant materials and growth conditions
Arabidopsis NIL DOG1_Cvi is a near isogenic line that contains the DOG1 allele from Cvi in a Ler background . The mutant dog1-1 has been obtained in the NIL DOG1 background , dog1-2 in Col . Arabidopsis accessions used in this study are listed in S1 Table. The double and triple homozygous transgenic lines were selected after crossings using PCR to confirm their homozygosity. All plants were sown on soil and grown in a growth chamber with 16 h- light/8 h-dark cycle (22°C/16°C), or in a greenhouse where the temperature was maintained close to 23°C, 16 h of light was provided daily. Six weeks vernalisation was applied to the late-flowering accessions to promote flowering. Freshly harvested seeds were immediately used for experiments or stored under constant conditions (21°C, 50% humidity, in the dark) for after-ripening treatment.
About 50 seeds were plated onto a filter paper moistened with demineralized water in Petri dishes and incubated in an alternating condition (12 h light/12 h dark, 25°C/20°C cycle). Radicle emergence was scored after three days, since dog1-1 mutant and after-ripened seeds of other accessions fully germinate within this period. Each germination test was done in at least three replicates from independent plants.
RNA extraction and quantitative RT-PCR
Total RNA was extracted from developing Arabidopsis siliques as described previously . Quantitative RT-PCR was performed as described previously , except for the annealing temperature, which was 64°C for splicing variant-specific primer sets. Sequences of the primers used for qRT-PCR are listed in S2 Table. The expression value for each gene was quantified using a standard curve with a serial dilution of plasmid of known concentration, and they were normalised to the value of ACT8 (At1g49240) or HBT (At2g20000) genes. At least three biological replicates were analysed.
To identify additional splicing variants, 3’-RACE was performed using RNA extracted from Ler siliques at 16 days after pollination (DAP) following the standard protocol of 3’-Full RACE Core Set (TAKARA) using the oligo-dT-adapter primer, adapter primer and DOG1 primer 5’-GGATTCTATCTCCGGTACAAGGA- 3’.
Protein extraction and immunoblot analysis
Seed protein extraction and immunoblot analysis were performed as described previously using peptide antibody against DOG1 .
Construction of transgenic lines
All the binary constructs were prepared using the Gateway technology (Invitrogen). A 5.07 kb fragment of Col genomic DNA corresponding to the Ler fragment  including a 2.22 kb region upstream of the DOG1 start codon, the DOG1 coding region and 1.03 kb downstream of the stop codon, as well as cDNA fragments of each splicing variant from Cvi were cloned into pENTR/D-TOPO vector. Entry clones carrying Y16A and E13A mutations in DOG1_Ler or the ECCY mutation in DOG1_Col were generated by site-directed mutagenesis based on the sequences of DOG1_Ler using the QuickChange II XL site-directed mutagenesis kit (Stratagene). The resultant genomic entry clones were converted into pGWB1  by LR reaction. For isoform specific complementation, each variant cDNA fragment was cloned under the DOG1 promoter_Cvi into the pGreen backbone. For fluorescent protein fusion constructs, each variant cDNA was cloned into 2x Pro 35S: YFP vectors, pENSG-YFP (N-terminal fusion) and pEXSG-YFP (C-terminal fusion), and split YFP vectors, pBatTL-B-sYFPn and pBatTL-B-sYFPc  via LR reaction. All the binary constructs were introduced by electroporation into Agrobacterium tumefaciens strains GV3101 or GV3101 carrying the helper plasmid pMP90RK  or pSoup , which were subsequently used to transform Ler, dog1-1 or dog1-2 plants by floral dipping . All the transgenic lines were first selected based on their antibiotics resistance, their homozygosity was further confirmed by PCR-based genotyping.
Confocal microscopy analysis
Subcellular localisation of each DOG1 isoform was analysed using binary constructs with single variant cDNA from Cvi fused to YFP at their N-terminus or C-terminus and cloned under the CaMV 35S promoter. Transiently expressed fusion proteins were observed in Nicotiana benthamiana leaves as described  or in Arabidopsis thaliana protoplasts (from Col) as described .
For BiFC assays, embryos from 1 h-imbibed seeds of the double homozygous transformants were dissected from testa/endosperm, and restored YFP fluorescence was analysed. Observations were performed with either Zeiss LSM510 or LSM700 confocal laser scanning microscope system using 514 nm lasers for excitation with 63x oil-immersion objective. The images were analysed using the LSM5 software or ZEN imaging software (Zeiss, Germany).
All three cDNA fragments corresponding to the alpha, beta and delta isoforms from Ler, and the beta isoforms from all other accessions were cloned into pENTR/D-TOPO (Invitrogen), and then recombined in the pACT2-gateway (GAL4 AD fusion) and pAS2-gateway (GAL4 BD fusion) vectors (modified from Clontech). Yeast two-hybrid assays were carried out in yeast strain PJ69-4A . Yeast transformation was performed using a LiAc/SS carrier DNA/PEG method as described . Co-transformed colonies were selected on synthetic dropout medium (SD) lacking Leu (L) and Trp (W). Interaction tests were performed on SD lacking L, W, and His (H) with 5 mM 3-aminotriazole.
Analysis of DOG1 genomic sequences in Arabidopsis accessions
DOG1 genomic sequences were collected either by Sanger sequencing of PCR-amplified genomic fragments or re-analysis of the publicly available next generation sequencing reads . The Cao’s dataset covered 80 accessions. Additionally, we included data from Col-0, Ler and Cvi .
The polymorphic data are available on Arabidopsis 1001 Genome browser (http://signal.salk.edu/atg1001/3.0/gebrowser.php), however the polymorphisms (SNPs and small INDELs) in the region of interest in Ler and Cvi (we obtained genomic and cDNA sequences by Sanger sequencing) were not correctly shown in the browser. In order to verify or discover new structural variations, the raw reads were assembled using a program (http://mandrake.mpipz.mpg.de:8081/cgi-bin/oscar.pl) and the contigs were constructed for the DOG1 genomic region in each accession. The obtained DOG1 sequences from accessions that successfully assembled into contigs were aligned to the Col-0 reference. The structural variations of those accessions were used for further analysis.
S1 Fig. Transcript analysis of DOG1 splicing variants during seed maturation.
(A) qRT-PCR analysis of DOG1 splicing variants during seed maturation of NIL DOG1. The mRNA level of each DOG1 variant was normalised to the HBT mRNA level. Error bars represent the S.E.M. of at least three biological replicates. (B) The relative ratios of the DOG1 splicing variants during seed maturation, calculated from the data in (A). The right panel is a magnified image of the bottom of the graph in the left. For clarity, the epsilon variant is not shown in the right panel. Error bars represent S.E.M.
S2 Fig. Subcellular localisation of transiently expressed DOG1 isoforms fused to YFP in Arabidopsis protoplasts.
Protoplasts were prepared from Arabidopsis young rosette leaves and transient transformation was performed by infiltration using the C-terminal YFP fusion constructs. YFP fluorescence (A–C), autofluorescence of chlorophyll (D-F), and merged image of YFP fluorescence, autofluorescence of chlorophyll, and transmission (G–I). Scale bars = 20 μm.
S3 Fig. Germination profiles of independent transformants of DOG1_Ler substitution lines after different periods of dry storage.
L WT, Ler WT construct; L Y16A, Ler Y16A substituted line; and L E13A, Ler E13A substituted line. Bars represent S.E.M. of at least three biological replicates. w, week. L WT data was taken from  (www.plantcell.org): Copyright American Society of Plant Biologists.
S4 Fig. Germination profiles of independent transformants of Col WT and ECCY substitution after different periods of dry storage.
Bars represent S.E.M. from at least three biological replicates. w, week.
S5 Fig. Natural variation in the primary amino acid sequence of exon1 of DOG1.
Genomic or cDNA sequences in the first exon of DOG1 were determined by either Sanger sequencing of amplified fragments or contig reconstruction from publicly available next generation sequencing (NGS) reads. Deduced amino acid sequences of the first exon are aligned based on the polymorphisms at the 13th E to 16th Y in Ler/Cvi, which is marked by the red open box. The first column shows a list with accessions and the top row the amino acid sequence of the Col accession. Three main haplotypes (DSY, DRY and ECCY) were distinguished based on polymorphisms at amino acids 13–16. Several accessions have missing dots in their sequences due to the ambiguous calls or poor coverage of the region with NGS reads. “.” = identical amino acid; “-” = absent amino acid.
S1 Table. Arabidopsis accessions used in this study.
We thank Christina Philipp and Mai Takahashi for technical assistance, Elmon Schmelzer for help with the confocal microscopy, and Regina Gentges for the propagation of plant material.
Conceived and designed the experiments: KN MB WJJS. Performed the experiments: KN MB. Analyzed the data: KN MB JD WJJS. Wrote the paper: KN WJJS.
- 1. Zhu M, Schlueter SD, Brendel V (2003) Refined annotation of the Arabidopsis genome by complete expressed sequence tag mapping. Plant Physiol 132: 469–484. pmid:12805580
- 2. Marquez Y, Brown JWS, Simpson C, Barta A, Kalyna M (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22: 1184–1195. pmid:22391557
- 3. McGlincy NJ, Smith CW (2008) Alternative splicing resulting in nonsense-mediated mRNA decay: what is the meaning of nonsense? Trends Biochem Sci 33: 385–393. pmid:18621535
- 4. Reddy ASN, Marquez Y, Kalyna M, Barta A (2013) Complexity of the alternative splicing landscape in plants. Plant Cell 25: 3657–3683. pmid:24179125
- 5. Carvalho RF, Feijão CV, Duque P (2012) On the physiological significance of alternative splicing events in higher plants. Protoplasma 250: 639–950. pmid:22961303
- 6. Reddy ASN (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu Rev Plant Biol 58: 267–294. pmid:17222076
- 7. Staiger D, Brown JWS (2013) Alternative splicing at the intersection of biological timing, development, and stress responses. Plant Cell 25: 3640–3656. pmid:24179132
- 8. Syed NH, Kalyna M, Marquez Y, Barta A, Brown JWS (2012) Alternative splicing in plants—coming of age. Trends Plant Sci 17: 616–623. pmid:22743067
- 9. Giraudat J, Hauge BM, Valon C, Smalle J, Parcy F, Goodman HM (1992) Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4: 1251–1261. pmid:1359917
- 10. Sugliani M, Brambilla V, Clerkx EJM, Koornneef M, Soppe WJJ (2010) The conserved splicing factor SUA controls alternative splicing of the developmental regulator ABI3 in Arabidopsis. Plant Cell 22: 1936–1946. pmid:20525852
- 11. Penfield S, Josse EM, Halliday KJ (2010) A role for an alternative splice variant of PIF6 in the control of Arabidopsis primary seed dormancy. Plant Mol Biol 73: 89–95. pmid:19911288
- 12. Finch-Savage WE, Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytol 171: 501–523. pmid:16866955
- 13. Holdsworth MJ, Bentsink L, Soppe WJJ (2008) Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination. New Phytol 179: 33–54. pmid:18422904
- 14. Finkelstein R, Reeves W, Ariizumi T, Steber C (2008) Molecular aspects of seed dormancy. Annu Rev Plant Biol 59: 387–415. pmid:18257711
- 15. Graeber K, Nakabayashi K, Miatton E, Leubner-Metzger G, Soppe WJJ (2012) Molecular mechanisms of seed dormancy. Plant Cell Environ 35: 1769–1786. pmid:22620982
- 16. Alonso-Blanco C, Bentsink L, Hanhart CJ, Blankestijn-de Vries H, Koornneef M (2003) Analysis of natural allelic variation at seed dormancy loci of Arabidopsis thaliana. Genetics 164: 711–729. pmid:12807791
- 17. Bentsink L, Jowett J, Hanhart CJ, Koornneef M (2006) Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis. Proc Natl Acad Sci USA 103: 17042–17047. pmid:17065317
- 18. Nakabayashi K, Bartsch M, Xiang Y, Miatton E, Pellengahr S, Yano R, et al. (2012) The time required for dormancy release in Arabidopsis is determined by DELAY OF GERMINATION 1 protein levels in freshly harvested seeds. Plant Cell 24: 2826–2838. pmid:22829147
- 19. Chiang GCK, Bartsch M, Barua D, Nakabayashi K, Debieu M, Kronholm I, et al. (2011) DOG1 expression is predicted by the seed-maturation environment and contributes to geographical variation in germination in Arabidopsis thaliana. Mol Ecol 20: 3336–3349. pmid:21740475
- 20. Kendall SL, Hellwege A, Marriot P, Whalley C, Graham IA, Penfield S (2011) Induction of dormancy in Arabidopsis summer annuals requires parallel regulation of DOG1 and hormone metabolism by low temperature and CBF transcription factors. Plant Cell 23: 2568–2580. pmid:21803937
- 21. Footitt S, Douterelo-Soler I, Clay H, Finch-Savage WE (2011) Dormancy cycling in Arabidopsis seeds is controlled by seasonally distinct hormone-signaling pathways. Proc Natl Acad Sci USA 108: 20236–20241. pmid:22128331
- 22. Graeber K, Linkies A, Steinbrecher T, Mummenhoff K, Tarkowská D, Turečková V, et al. (2014) DELAY OF GERMINATION 1 mediates a conserved coat-dormancy mechanism for the temperature- and gibberellin-dependent control of seed germination. Proc Natl Acad Sci USA 111: E3571–E3580. pmid:25114251
- 23. Ashikawa I, Mori M, Nakamura S, Abe F (2014) A transgenic approach to controlling wheat seed dormancy level by using Triticeae DOG1-like genes. Transgenic Res 23: 621–629. pmid:24752830
- 24. Liu Y, Geyer R, van Zanten M, Carles A, Li Y, Hörold A, et al. (2011) Identification of the Arabidopsis REDUCED DORMANCY 2 gene uncovered a role for the Polymerase Associated Factor 1 Complex in seed dormancy. PLoS ONE 6: e22241. pmid:21799800
- 25. Mortensen SA, Sønderkær M, Lynggaard C, Grasser M, Nielsen KL, Grasser KD (2011) Reduced expression of the DOG1 gene in Arabidopsis mutant seeds lacking the transcript elongation factor TFIIS. FEBS Lett 585: 1929–1933. pmid:21569772
- 26. Liu Y, Koornneef M, Soppe WJJ (2007) The absence of histone H2B monoubiquitination in the Arabidopsis hub1 (rdo4) mutant reveals a role for chromatin remodeling in seed dormancy. Plant Cell 19: 433–444. pmid:17329563
- 27. Dolata J, Guo Y, Kołowerzo A, Smoliński D, Brzyżek G, Jarmołowski A, et al. (2015) NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis. EMBO J 34: 544–558. pmid:25568310
- 28. Bentsink L, Hanson J, Hanhart CJ, Blankestijn-de Vries H, Coltrane C, Keizer P, et al. (2010) Natural variation for seed dormancy in Arabidopsis is regulated by additive genetic and molecular pathways. Proc Natl Acad Sci USA 107: 4264–4269. pmid:20145108
- 29. Kushiro T, Okamoto M, Nakabayashi K, Yamagishi K, Kitamura S, Asami T, et al. (2004) The Arabidopsis cytochrome P450 CYP707A encodes ABA 8‘-hydroxylases: Key enzymes in ABA catabolism. EMBO J 23: 1647–1656. pmid:15044947
- 30. Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, et al. (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104: 34–41. pmid:17697981
- 31. Uhrig J, Mutondo M, Zimmermann I, Deeks MJ, Machesky LM, Thomas P, et al. (2007) The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis. Development 134: 967–977. pmid:17267444
- 32. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by novel type of Agrobacterium binary vector. Mol Gen Genet 204: 383–396.
- 33. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819–832. pmid:10890530
- 34. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743. pmid:10069079
- 35. Wu A-J, Andriotis VME, Durrant MC, Rathjen JP (2004) A patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase. Plant Cell 16: 2809–2821. pmid:15367718
- 36. James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for high efficient two-hybrid selection in yeast. Genetics 144: 1425–1436. pmid:8978031
- 37. Gietz RD, Triggs-Raine B, Robbins A, Graham KC, Wodds RA (1997) Identification of proteins that interact with a protein of interest: applications of the yeast-two-hybrid system. Mol Cell Biochem 172: 67–79. pmid:9278233
- 38. Cao J, Schneeberger K, Ossowski S, Günther T, Bender S, Fitz J, et al. (2011) Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nat Genet 43: 956–963. pmid:21874002
- 39. Wijnker E, James GV, Ding J, Becker F, Klasen JR, Rawat V, et al. (2013) The genomic landscape of meiotic crossovers and gene conversions in Arabidopsis thaliana. eLIFE 2: e01426. pmid:24347547