Overexpression of ABI5 Binding Proteins Suppresses Inhibition of Germination Due to Overaccumulation of DELLA Proteins

Abscisic acid (ABA) and gibberellic acid (GA) antagonistically regulate many aspects of plant growth, including seed dormancy and germination. The effects of these hormones are mediated by a complex network of positive and negative regulators of transcription. The DELLA family of proteins repress GA response, and can promote an ABA response via interactions with numerous regulators, including the ABA-insensitive (ABI) transcription factors. The AFP family of ABI5 binding proteins are repressors of the ABA response. This study tested the hypothesis that the AFPs also interact antagonistically with DELLA proteins. Members of these protein families interacted weakly in yeast two-hybrid and bimolecular fluorescence complementation studies. Overexpression of AFPs in sleepy1, a mutant that over-accumulates DELLA proteins, suppressed DELLA-induced overaccumulation of storage proteins, hyperdormancy and hypersensitivity to ABA, but did not alter the dwarf phenotype of the mutant. The interaction appeared to reflect additive effects of the AFPs and DELLAs, consistent with action in convergent pathways.


Introduction
Seed germination is regulated by diverse environmental signals, including light, temperature, and water availability (reviewed in [1]). Many of these signals affect the balance between germination inhibition by abscisic acid (ABA) and promotion by gibberellic acid (GA) [2][3][4]. Following many years of studies with exogenously applied hormones, genetic evidence for intrinsic control by this balance was provided by the isolation of non-germinating dwarf mutants that were deficient in GA synthesis [5], and germinating revertants that had additional mutations disrupting ABA synthesis [6]. In addition to control by the relative concentrations of ABA and GA, the abundance and activity of numerous signaling intermediates affect the sensitivity to these hormones.
The ABA-insensitive loci ABI1, ABI2, ABI3, ABI4, and ABI5, major regulators in ABA response at this stage, were identified by screens for ABA-resistant germination (reviewed in [7]). The initial abi1-1 and abi2-1 mutants had dominant negative mutations in members of a clade of protein phosphatases (PP2Cs) later shown to be a central part of the ABA core signaling pathway. Subsequent screens for ABA-hypersensitive germination (AHG) identified loss of function mutations in additional members of this clade, reinforcing the interpretation that these were negative regulators of ABA response. In contrast, ABI3, ABI4, and ABI5 encode transcription factors that both activate ABA-induced genes and repress ABA-downregulated genes [8][9][10][11].
Screens for defects in GA response focused on plants with defects in regulating elongation, either GA insensitive dwarfs (gai and gid) [12,13] or displayed constitutive response to GA (spindly) [14]. The initial gai mutant also had a dominant negative mutation [12],

Direct Interactions between AFPs and DELLA Proteins
We initially tested for possible direct interactions between AFPs and DELLA proteins by yeast two hybrid and bimolecular fluorescence complementation (split YFP) assays, including ABI5 as a positive control (Figures 1 and S1). We focused on the DELLA proteins with similarly significant expression in dry or imbibing seeds, i.e., all except RGL1 ( Figure S2). For the yeast two hybrid assays, the DELLAs were presented as GAL4 activation domain (AD)-fusions, the AFPs were fused to the GAL4 binding domain (BD), and ABI5 was present in both AD-and BD-fusions. Both AFP1 and AFP2 interacted strongly with RGA1 and RGA2/GAI, and AFP1 also interacted to a lesser extent with RGL2 and RGL3. As documented previously [20,30], ABI5 interacted directly with the AFPs and the DELLAs, especially RGA1 and RGA2. The DELLA protein interactions with the AFPs were further mapped to the GRAS domain of RGA2, but this was much weaker than the interaction with the full-length protein. Although ABI5 appeared to interact with both the GRAS and DELLA domains of RGA2, these interactions were again weaker than those with full-length RGA2. present in both AD-and BD-fusions. Both AFP1 and AFP2 interacted strongly with RGA1 and RGA2/GAI, and AFP1 also interacted to a lesser extent with RGL2 and RGL3. As documented previously [20,30], ABI5 interacted directly with the AFPs and the DELLAs, especially RGA1 and RGA2. The DELLA protein interactions with the AFPs were further mapped to the GRAS domain of RGA2, but this was much weaker than the interaction with the full-length protein. Although ABI5 appeared to interact with both the GRAS and DELLA domains of RGA2, these interactions were again weaker than those with fulllength RGA2. The split YFP assays also repeated strong interactions between ABI5 and the AFPs, but weaker interactions between DELLA fusions and either ABI5 or the AFPs ( Figure S1). Surprisingly, although RGL2 did not interact with AFP2 in the yeast two-hybrid assays, this combination interacted quite strongly in the split YFP assay.

Genetic Interactions between AFPs and DELLA Proteins
Our previous studies showed that AFP2 overexpression results in extreme ABA resistance of seeds and consequently a loss of dormancy. In contrast, sleepy (sly1) mutants lack the F-box protein that ubiquitinates DELLA proteins, such that the DELLAs are hyperstable in these mutants, GA response is repressed and the sly1 seeds are hyperdormant. To determine whether the apparent physical interactions between AFP2 and DELLAs result in epistatic or additive effects on germination potential, we overexpressed AFP2 in the sly1-2 background. The YFP-AFP2 transgene was crossed into sly1 from a previously characterized line in the wild-type background [32]. Although AFP1 overexpression was The split YFP assays also repeated strong interactions between ABI5 and the AFPs, but weaker interactions between DELLA fusions and either ABI5 or the AFPs ( Figure S1). Surprisingly, although RGL2 did not interact with AFP2 in the yeast two-hybrid assays, this combination interacted quite strongly in the split YFP assay.

Genetic Interactions between AFPs and DELLA Proteins
Our previous studies showed that AFP2 overexpression results in extreme ABA resistance of seeds and consequently a loss of dormancy. In contrast, sleepy (sly1) mutants lack the F-box protein that ubiquitinates DELLA proteins, such that the DELLAs are hyperstable in these mutants, GA response is repressed and the sly1 seeds are hyperdormant. To determine whether the apparent physical interactions between AFP2 and DELLAs result in epistatic or additive effects on germination potential, we overexpressed AFP2 in the sly1-2 background. The YFP-AFP2 transgene was crossed into sly1 from a previously characterized line in the wild-type background [32]. Although AFP1 overexpression was less effective than AFP2 for promoting extreme ABA resistance [32], we also tested the effects of AFP1 overexpression in the sly1 mutant because the strongest interactions observed in the yeast two-hybrid assay were between AFP1 and the DELLAs. The YFP-AFP1 overexpression transgene from sly1 line #3 was backcrossed into the wild-type background. As in the wild-type background, all sly1 lines with good seedling viability were hemizygous for the transgene, such that 25% of these seed populations lacked the transgene. Overexpression of either AFP was sufficient to reduce dormancy and permit some ABA-resistant germination in this background (Figure 2A). In both backgrounds, YFP-AFP2 overexpression promoted cotyledon expansion and greening of most germinated seeds ( Figure 2B,C). However, the fraction capable of germinating, especially in the presence of ABA, was greatly reduced in the sly1 background; a similar percentage germination was achieved for YFP-AFP2 seeds in the wild-type background exposed to 50 µM ABA as for those in the sly1 background on hormone-free medium. In contrast, the YFP-AFP1 overexpression transgene from sly1 line #3 permitted germination on media containing at least five-fold higher ABA concentrations in the wild-type background (similar responses to 50 µM vs. 10 µM ABA in wild-type vs. sly1 backgrounds), but had more limited effects on promoting ABA-resistant greening. An independent sly1, YFP-AFP1/+ line (#4) had stronger effects in promoting both germination and post-germinative growth in the presence of ABA, but still less than the weaker transgene in the wild-type background. less effective than AFP2 for promoting extreme ABA resistance [32], we also tested the effects of AFP1 overexpression in the sly1 mutant because the strongest interactions observed in the yeast two-hybrid assay were between AFP1 and the DELLAs. The YFP-AFP1 overexpression transgene from sly1 line #3 was backcrossed into the wild-type background. As in the wild-type background, all sly1 lines with good seedling viability were hemizygous for the transgene, such that 25% of these seed populations lacked the transgene. Overexpression of either AFP was sufficient to reduce dormancy and permit some ABA-resistant germination in this background ( Figure 2A). In both backgrounds, YFP-AFP2 overexpression promoted cotyledon expansion and greening of most germinated seeds ( Figure 2B,C). However, the fraction capable of germinating, especially in the presence of ABA, was greatly reduced in the sly1 background; a similar percentage germination was achieved for YFP-AFP2 seeds in the wild-type background exposed to 50 μM ABA as for those in the sly1 background on hormone-free medium. In contrast, the YFP-AFP1 overexpression transgene from sly1 line #3 permitted germination on media containing at least five-fold higher ABA concentrations in the wild-type background (similar responses to 50 M vs. 10 M ABA in wild-type vs. sly1 backgrounds), but had more limited effects on promoting ABA-resistant greening. An independent sly1, YFP-AFP1/+ line (#4) had stronger effects in promoting both germination and post-germinative growth in the presence of ABA, but still less than the weaker transgene in the wild-type background. During seed development, the ABI transcription factors mediate ABA-promoted accumulation of storage proteins and lipids. Recently, RGL3 was shown to be the major During seed development, the ABI transcription factors mediate ABA-promoted accumulation of storage proteins and lipids. Recently, RGL3 was shown to be the major DELLA protein expressed in maturing seeds, where it promotes the expression of the cruciferin and At2S families of storage proteins [35]. Consistent with this, we found a slightly increased accumulation of these proteins in the sly1 mutant background (Figure 3  In contrast to the effects on seeds, both classes of sly1, 35S-YFP-AFP lines displayed the dwarf stature, slow growth, loss of apical dominance, and poor fertility of the sly1 parental line, although YFP-AFP2 overexpression slightly suppressed lateral branching in the sly1 background (Figures 4 and S3). Fertility was delayed the most in the primary inflorescence, with lateral shoots producing much fewer sterile flowers. Although more variable, the YFP-AFP1 transgenes enhanced the sterility in the primary inflorescence of the sly1 parental line but did not affect fertility in a wild-type background.  In contrast to the effects on seeds, both classes of sly1, 35S-YFP-AFP lines displayed the dwarf stature, slow growth, loss of apical dominance, and poor fertility of the sly1 parental line, although YFP-AFP2 overexpression slightly suppressed lateral branching in the sly1 background (Figures 4 and S3). Fertility was delayed the most in the primary inflorescence, with lateral shoots producing much fewer sterile flowers. Although more variable, the YFP-AFP1 transgenes enhanced the sterility in the primary inflorescence of the sly1 parental line but did not affect fertility in a wild-type background. DELLA protein expressed in maturing seeds, where it promotes the expression of the cruciferin and At2S families of storage proteins [35]. Consistent with this, we found a slightly increased accumulation of these proteins in the sly1 mutant background (Figure 3, lane 7). Transgenic lines overexpressing either AFP reduced accumulation of the storage proteins ( Figure 3, lanes 2 and 3), with those in the sly1 background again showing an intermediate phenotype (Figure 3, lanes 4-6). In contrast to the effects on seeds, both classes of sly1, 35S-YFP-AFP lines displayed the dwarf stature, slow growth, loss of apical dominance, and poor fertility of the sly1 parental line, although YFP-AFP2 overexpression slightly suppressed lateral branching in the sly1 background (Figures 4 and S3). Fertility was delayed the most in the primary inflorescence, with lateral shoots producing much fewer sterile flowers. Although more variable, the YFP-AFP1 transgenes enhanced the sterility in the primary inflorescence of the sly1 parental line but did not affect fertility in a wild-type background.

AFP, DELLA and ABI5 Protein Accumulation
ABA-resistant germination can result from decreased levels of inhibitors such as ABI5 or DELLA proteins or increased levels of proteins that antagonize their function. To determine whether the altered extent of germination was associated with changes in the levels of these proteins, we used immunoblots to compare their accumulation in seeds and following stratification in the presence or absence of added 1 µM ABA, a concentration sufficient to delay but not block germination ( Figure 5). In populations segregating a YFP-AFP transgene in the sly1 background, ABA-resistant germination was limited to individuals accumulating the fusion protein ( Figure S4A).

AFP, DELLA and ABI5 Protein Accumulation
ABA-resistant germination can result from decreased levels of inhibitors such as ABI5 or DELLA proteins or increased levels of proteins that antagonize their function. To determine whether the altered extent of germination was associated with changes in the levels of these proteins, we used immunoblots to compare their accumulation in seeds and following stratification in the presence or absence of added 1 M ABA, a concentration sufficient to delay but not block germination ( Figure 5). In populations segregating a YFP-AFP transgene in the sly1 background, ABA-resistant germination was limited to individuals accumulating the fusion protein ( Figure S4A). YFP-AFP1 levels are relatively constant in seeds and seedlings, but are below detection in rosette leaves of older plants (Figures 5A and S4BC). The YFP-AFP2 fusions accumulate as germination proceeds and show a slight shift toward higher mobility, likely reflecting changes in post-translational modifications, but are also not detected in older plants ( Figure 5B). Levels of the YFP-AFP fusions also varied among independent transformants, but generally paralleled the degree of ABA resistance in a given background ( Figure S4C,D). However, similar amounts of the fusion protein were far less effective in promoting germination in sly1 mutants compared to the wild-type background. For example, the YFP-AFP2 fusion protein accumulated to at least three-fold higher levels in the wild-type background than the same construct in the sly1 background ( Figures 5B and S5) YFP-AFP1 levels are relatively constant in seeds and seedlings, but are below detection in rosette leaves of older plants ( Figures 5A and S4B,C). The YFP-AFP2 fusions accumulate as germination proceeds and show a slight shift toward higher mobility, likely reflecting changes in post-translational modifications, but are also not detected in older plants ( Figure 5B). Levels of the YFP-AFP fusions also varied among independent transformants, but generally paralleled the degree of ABA resistance in a given background ( Figure S4C,D). However, similar amounts of the fusion protein were far less effective in promoting germination in sly1 mutants compared to the wild-type background. For example, the YFP-AFP2 fusion protein accumulated to at least three-fold higher levels in the wild-type background than the same construct in the sly1 background ( Figures 5B and S5) and permitted comparable germination on at least 50-fold higher ABA concentrations. Similarly, although the YFP-AFP1 fusion protein accumulated to similar levels in sly1 and wild-type backgrounds, the transgene permitted a similar degree of germination on 1 µM ABA in the sly background as on 30 µM ABA in the wild-type background (Figure 2). Differences in YFP-AFP2 protein accumulation did not reflect different transcript levels ( Figure S5), suggesting the possibility of post-transcriptional control.
The DELLA protein RGA1 is barely detectable in wild-type seeds and seedlings ( Figure 5B). In contrast, RGA1 accumulates substantially post-imbibition in the sly1 background, consistent with the roughly ten-fold increase in transcript levels reported for incubation in the presence of either water or ABA, and accumulates to even higher levels in rosette leaves ( Figure 5 and Figure S4B). RGA1 levels are not substantially altered by the presence of either YFP-AFP transgene.
As previously reported, ABI5 levels increase post-imbibition when exposed to ABA and decrease during germination [29]. In the present study, they are mostly reflective of germination status, increasing during delayed germination of ABA-treated wild-type seeds, but showing only a slight, further delayed increase in sly1 mutant seeds, which are not germinating at all ( Figure 5 and Figure S6). In lines carrying the transgenes, germination proceeds more rapidly and by 3d post-stratification, seeds exposed to ABA are greening and ABI5 levels are not substantially higher than those in dry seeds. Similar to AFP2, ABI5 undergoes a shift in mobility in seeds incubated on media containing ABA, in this case, toward higher apparent molecular weight forms ( Figure 5). ABI5 is fully degraded in all genotypes that have germinated on hormone-free media.

Discussion
ABA and GA act antagonistically throughout plant development. Plant responses to these conflicting signals are mediated through a combination of changing hormone levels and sensitivities. Levels of these hormones are controlled by environmental signals, e.g., light and temperature, and cross-regulation of hormone metabolism, while sensitivity reflects relative activities of a complex network of positive and negative regulators of response (reviewed in [2,36]). In the current work, we have focused on the interaction between two sets of negative regulators, each of which have been characterized as signaling hubs due to their extensive interactions with other signaling proteins: the DELLA repressors of GA response [37] and the AFP repressors of ABA response [32]. During seed maturation, RGA2 and RGL3 expression in seeds is promoted by ABI5 ( Figure S2). In mature seeds RGA1, RGA2, and RGL2 transcripts increase following imbibition with or without ABA ( Figure S2) and RGL2 is subject to proteasomal degradation when GA is present, thereby permitting germination [25]. AFP1 and AFP2 transcripts also increase following imbibition, but are induced by ABA, high glucose and salt stress over several days, potentially acting in a feedback loop to gradually release seeds from ABA-inhibition of germination [30].
Physical interactions between subsets of the DELLA and AFP families were detected in yeast and BiFC assays in N. benthamiana. Although not all interactions were observed in both assays, it is not unusual to see discrepancies between their results [38]. The interactions in yeast were dependent on the GRAS domain, previously shown to be important for interactions with over 150 transcriptional regulators that can have either positive or negative effects on gene expression [39,40]. The stronger interactions observed with the full-length DELLAs might reflect contributions of additional domains not tested individually in these constructs or effects on the conformation of the GRAS domain.
Because both DELLAs and AFPs comprise families with partially redundant functions that can mask the effects of loss of function mutations, we chose to analyze interactions between the gain of function lines that over-accumulate the respective repressors. Although the use of the sly1 background does not readily distinguish between DELLAs relevant for different developmental effects, previous studies have identified RGL2 as the major regulator of seed dormancy and germination [24], but whose function is enhanced by RGA1, RGA2/GAI, and RGL1 [15]. RGL2 acts in an ABI5-dependent manner both indirectly by increasing ABI5 expression [26,27] and directly through complex formation [20,28]. RGL2 and several ABI transcription factors all repress AtGASA6, thereby inhibiting the promotion of cell wall loosening needed for cell expansion in germination [41]. RGL2 also forms a complex with DNA BINDING1 ZINC FINGER6 (DOF6) that directly activates the expression of GATA12, another transcription factor implicated in maintaining primary seed dormancy [42]. The Circadian clock protein REVEILLE(RVE)1 also promotes dormancy by interactions with RGL2 that stabilize it, blocking its degradation by SLY1-mediated targeting to the COP9 signalosome [43,44]. However, in after-ripened sly1 seeds, loss of RGL2 is not required to permit germination [45].
In the present study, overexpression of either AFP1 or AFP2 was sufficient to release dormancy and promote some degree of ABA-resistant germination in the sly1 background despite no ability to degrade DELLA proteins. Furthermore, ABI5 was maintained at similar levels in transgenic seeds germinating on a low concentration of ABA, regardless of whether in the wild-type or sly1 background. This indicates that AFP-induced germination does not require loss of either the DELLAs or ABI5, and further suggests that the AFPs are acting by inhibiting the function of ABI5 and/or the DELLAs, but does not distinguish between direct and indirect inhibition. Furthermore, YFP-AFP2 appears to be subject to post-transcriptional regulation resulting in reduced accumulation in the sly1 background. Although the mechanism is not known in this case, the YFP-AFP2 fusion is subject to proteasomal degradation [34].
While the balance between ABA and GA signaling shifts toward GA response in controlling the transition from seed to seedling at maturity, ABA signaling predominates in maturing seeds. The maize VP1 transcription factor, the ortholog to Arabidopsis ABI3, has a dual role as a positive regulator of ABA response in maturing seeds and repressor of GA response post-imbibition [8]. DELLA proteins, although initially characterized as repressors of GA signaling, can also function as positive regulators of ABA synthesis via the promotion of XERICO expression [26,46]. Increased ABA levels further promote ABA response by induction of transcription factors, including ABI5 (reviewed in [7]). During seed maturation, when GA levels are low, RGL3 is highly expressed and interacts directly with ABI3 to promote seed storage protein accumulation; overexpression of RGL3 leads to slight increases in storage protein content [35]. The sly1 mutant has a similarly mild increase in storage proteins. In contrast, overexpression of either AFP substantially reduced storage protein accumulation, consistent with recent proteomic studies of the AFP2 overexpression line [34]; the combination of excess AFP and DELLAs was again intermediate.
All of the DELLA proteins are expressed in flowers and siliques, and genetic evidence indicates that RGA1, RGL1, and RGL2 all regulate floral development [25]. Some aspects of DELLA regulation promote fertility: RGA1 and RGA2/GAI function is required for the production of viable pollen [47] and RGL2 positively regulate ovule number and fertility in Arabidopsis [48]. However, DELLA proteins repress cell elongation in stamens, contributing to male sterility in GA-deficient mutants [49]. Whereas loss of function for RGA1 or RGA2/GAI can suppress the defects in stem elongation, apical dominance, and flowering time in sly1 mutants, this is not sufficient to restore fertility [18]. Although YFP-AFP2 overexpression slightly reduced branching in the sly1 background, AFP overexpression generally failed to rescue the sly1 mutant's growth defects.
In summary, although the mechanism is not yet clear, the AFP transgenes and the over-accumulated DELLAs exert additive antagonistic effects on seed development and germination. The minimal effects of the YFP-AFP transgenes on vegetative growth and fertility are consistent with the strong expression of RGA1 and failure to detect the YFP-AFP fusions at later stages in growth, e.g., rosette leaves. These results show a stage-specific antagonism between these two classes of signaling hubs.

Yeast Two-Hybrid Constructs and Assays
Full-length ORF cDNAs for RGA1 (AT2G01570), RGA2 (AT1G14920), and RGL3 (AT5G17490), (U13937, U14047, and U60167, respectively) [50] were obtained from the ABRC and an RGL2 (AT3G03450) cDNA was constructed by PCR of the coding sequence from genomic DNA, adding BglII sites to the ends using primers described in Table S1, then subcloned into the BamHI site of pUNI51. Subclones encompassing either the DELLA or GRAS domains of RGA2 and RGL2 were constructed by PCR using the full-length cDNAs as templates and primers as indicated (Table S1), and ligated into pUNI51. The RGA2 DELLA domain clone included codons 1-121, the RGA2 GRAS domain clone included codons 115-533, RGL2 DELLA domain clone encoded amino acids 1-145, and the RGL2 GRAS domain included codons 139-547. Fusions between the GAL4 activation domain (AD) and these DELLA clones were constructed by recombination with pACT2lox, catalyzed by GST-CRE as described in [51]. The GAL4 binding domain (BD) fusions to the AFPs and both AD-and BD-fusions for ABI5 have been described previously [32]. Interactions were tested by matings between pairs of haploid lines carrying BD-and AD-fusions, replicaplated onto selective media to test for activation of the HIS3 and ADE2 reporter genes, as described in [32].

Plant Materials and Transgenes
Arabidopsis plants were grown in pots in growth chambers under continuous light at 22 • C. SLY1 loss of function lines was described in [16]; we used a line carrying the sly1-2 allele that had been backcrossed three times into the Col-0 background. The 35S:YFP:AFP fusions and split YFP fusions for ABI5, AFP1 and AFP2 were described in [32]. The 35S:YFP:AFP2 line used was #4A2. Agrobacterium tumefaciens-mediated direct transformation of sly1-2 mutants was performed by the floral dip method [52], followed by the selection of BASTA-resistant seedlings. Essentially all germinating seeds were transgenic, reflecting suppression of the strong dormancy of sly1 mutants. Progeny continued to segregate BASTA-resistance over multiple generations. A 35S:YFP:AFP1 transgene was backcrossed from the sly1 background into Col-0 and F1s screened for BASTA resistance; within the F2 population, germination is limited to approximately 94% of seeds that have a wild-type SLY1 allele and/or the transgene.
Split YFP fusions for RGA, RGA2, and RGL2 were constructed using the Gateway compatible pSITE-cEYFP-C1 (Acc# GU734652) vector and PCR products with attL ends added as described in [53], using primers described in Table S1, following manufacturer's instructions for LR Clonase reactions (Invitrogen). BiFC assays were conducted as described in [32].

Plant Growth Conditions
Germination assays testing ABA sensitivity of age-matched seeds were performed on minimal nutrient media supplemented with ABA at concentrations over the range from 0-50 µM, as described in [32]. Following sterilization and plating, plates were incubated 3d at 4 • C; this was sufficient to break residual dormancy for all except the hyperdormant sly1 parental line. Accumulation of fusion proteins was assayed by immunoblots of seeds or seedlings harvested after 3-4d incubation on Germination Medium (GM: 0.5 × MS salts and vitamins, 1% sucrose) with or without 1 µM ABA or 8 µg/mL BASTA, solidified with 0.7% agar.
For analysis of adult plant development, seedlings were transferred to soil and photographed at weekly intervals. The number of shoots was scored for individual mature plants (n = 11 −22), and the number of sterile pods on the main inflorescence and at least 4 lateral branches of each plant was counted.

Transcript Analyses
RNA was extracted, as described in [32], then incubated with RQ1 DNase (Promega, Madison, WI, USA) and RNAsin for 15 min at room temperature. The reaction was stopped by the addition of EGTA (1.8 mM final), then RNA was purified over Zymo-Clean columns (Zymo Resesarch, Irvine, CA, USA) according to the manufacturer's instructions. Approximately 0.5 ug of RNA was used as a template for cDNA reactions using MMLV reverse transcriptase or GoScript (Promega, Madison, WI, USA) and a 10:1 mix of random hexamers and oligo dT as primers. cDNA concentrations were assayed by qRT-PCR using EvaGreen Master Mix (Midwest Scientific, Valley Park, MO, USA) or Forget-Me-Not EvaGreen Master Mix (Biotium, Fremont, CA, USA) in an iQ5 cycler (BioRad, Hercules, CA, USA) according to the manufacturer's instructions. Primers used for normalizing were selected for uniform expression in seeds and seedlings grown in a variety of conditions [54]. Reactions with both primer sets were quantified relative to a standard curve spanning the range of concentrations present in all samples, as described in [55].