Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity
2009; Springer Nature; Volume: 28; Issue: 15 Linguagem: Inglês
10.1038/emboj.2009.170
ISSN1460-2075
AutoresUrszula Piskurewicz, Veronika Turečková, Éric Lacombe, Luis Lopez‐Molina,
Tópico(s)Plant Molecular Biology Research
ResumoArticle25 June 2009free access Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity Urszula Piskurewicz Urszula Piskurewicz Département de Biologie Végétale, Université de Genève, Genève 4, Switzerland Search for more papers by this author Veronika Turečková Veronika Turečková Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Olomouc, Czech Republic Search for more papers by this author Eric Lacombe Eric Lacombe Biochimie & Physiologie Moléculaire des Plantes, Unité Mixte de Recherche CNRS/INRA, Université Montpellier II, SupAgro, Montpellier Cedex 1, France Search for more papers by this author Luis Lopez-Molina Corresponding Author Luis Lopez-Molina Département de Biologie Végétale, Université de Genève, Genève 4, Switzerland Search for more papers by this author Urszula Piskurewicz Urszula Piskurewicz Département de Biologie Végétale, Université de Genève, Genève 4, Switzerland Search for more papers by this author Veronika Turečková Veronika Turečková Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Olomouc, Czech Republic Search for more papers by this author Eric Lacombe Eric Lacombe Biochimie & Physiologie Moléculaire des Plantes, Unité Mixte de Recherche CNRS/INRA, Université Montpellier II, SupAgro, Montpellier Cedex 1, France Search for more papers by this author Luis Lopez-Molina Corresponding Author Luis Lopez-Molina Département de Biologie Végétale, Université de Genève, Genève 4, Switzerland Search for more papers by this author Author Information Urszula Piskurewicz1, Veronika Turečková2, Eric Lacombe3 and Luis Lopez-Molina 1 1Département de Biologie Végétale, Université de Genève, Genève 4, Switzerland 2Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Olomouc, Czech Republic 3Biochimie & Physiologie Moléculaire des Plantes, Unité Mixte de Recherche CNRS/INRA, Université Montpellier II, SupAgro, Montpellier Cedex 1, France *Corresponding author. Département de Biologie Végétale, Université de Genève, 30, quai Ernest-Ansermet—Sciences III, 1211 Genève 4, Switzerland. Tel.: +41 22 379 3206; Fax: +41 22 379 3205; E-mail: [email protected] The EMBO Journal (2009)28:2259-2271https://doi.org/10.1038/emboj.2009.170 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Under the canopy, far-red (FR) light represses seed germination by inactivating phytochrome photoreceptors. This elicits a decrease in gibberellins (GA) levels and an increase in abscisic acid (ABA) levels. GA promotes germination by enhancing the proteasome-mediated destruction of DELLA repressors. ABA prevents germination by stimulating the expression of ABI repressors. How phytochromes elicit changes in hormone levels or how GA- and ABA-dependent signals are coordinated to repress germination remains poorly understood. We show that repression of germination by FR light involves stabilized DELLA factors GAI, RGA and RGL2 that stimulate endogenous ABA synthesis. In turn, ABA blocks germination through the transcription factor ABI3. The role of PIL5, a basic helix-loop-helix transcription factor stimulating GAI and RGA expression, is significant, provided GA synthesis is high enough; otherwise, high GAI and RGA protein levels persist to block germination. Under white light, GAI and RGA driven by the RGL2 promoter can substitute for RGL2 to promote ABA synthesis and repress germination, consistent with the recent findings with RGL2. The three DELLA factors inhibit testa rupture whereas ABI3 blocks endosperm rupture. Introduction In Arabidopsis, the mature seed consists of a protective outer layer of dead tissue, the testa, under which the endosperm, a single layer of cells, surrounds the embryo (Debeaujon et al, 2000). Arabidopsis seed germination chronologically involves testa rupture followed by concomitant endosperm rupture and embryonic axis (i.e. radicle) protrusion, the latter being the usual definition of germination (Kucera et al, 2005; Muller et al, 2006). When seeds are non-dormant, as in this study, imbibition by water is sufficient to trigger germination. Under normal conditions, the process can be completed within 48–72 h after seed imbibition (Piskurewicz et al, 2008). Germination is under tight control by the environment, being affected by light quality, temperature and water potential. Environmental factors eventually determine the relative levels of the phytohormones gibberellins (GA) and abscisic acid (ABA) that have an antagonistic and important function in the control of seed germination (Olszewski et al, 2002; Nambara and Marion-Poll, 2005). Conditions favourable for germination lead shortly after seed imbibition to an increase of GA levels (Ogawa et al, 2003), which is essential for germination to occur. In parallel, the levels of ABA drop rapidly after dry seed imbibition, and thereafter the role of ABA becomes facultative: a sudden osmotic stress or direct ABA application effectively blocks endosperm rupture and its effect on testa rupture is significantly lesser (Muller et al, 2006; Piskurewicz et al, 2008). Importantly, exogenous GA does not stimulate endosperm rupture in ABA-treated seeds, suggesting that ABA acts downstream of GA for the control of endosperm rupture (Muller et al, 2006). The ABA-dependent growth arrest occurs only within a limited time window of about 48 h after seed imbibition (Lopez-Molina et al, 2001). ABA or osmotic stress stimulates the de novo accumulation of embryonic transcription factors ABI3 and ABI5, which are both necessary to repress germination (Lopez-Molina et al, 2001, 2002). Similarly, as during seed maturation, ABI5 expression is positively regulated by ABI3 and both transcription factors ensure the de novo induction of LEA gene expression, thus maintaining the embryonic nature of the arrested embryo (Parcy et al, 1994; Finkelstein and Lynch, 2000; Lopez-Molina and Chua, 2000). GA promotes seed germination by enhancing the destruction of the DELLA repressor proteins through the 26S-proteasome machinery. DELLA genes are predicted to encode GRAS-family transcription factors, although no DNA-binding activity has been characterized so far. Rather, it has been proposed that they influence transcription by interacting with other DNA-binding transcription factors (Zentella et al, 2007). In the proposed model, GA binds to Arabidopsis GID1-like receptors, so as to enhance DELLA protein interaction with the F-box protein SLY1, thus facilitating DELLA ubiquitination and subsequent degradation (Sun and Gubler, 2004; Feng et al, 2008). There are five DELLA genes in the Arabidopsis genome: RGA, GAI, RGL1, RGL2 and RGL3. Even though all DELLA genes are expressed during seed germination, only RGL2, GAI and RGA have been shown to have a function to repress germination (Lee et al, 2002; Tyler et al, 2004; Penfield et al, 2006). Light quality affects seed germination by invoking changes in GA and ABA levels (Seo et al, 2006). This notably involves phytochrome B (phyB) and PIL5, a basic helix-loop-helix (bHLH) transcription factor indirectly modulating GA and ABA levels and directly stimulating RGA and GAI gene transcription (Bae and Choi, 2008; Seo et al, 2009). phyB is a protein photoreceptor with a covalently attached light-sensitive chromophore, whose activity is mainly set by the intensity ratio of red light to far-red (FR) light. An elevated ratio of red light to FR light (R conditions) leads to the active state of phyB (Pfr state). When active, phyB triggers the proteasome-mediated destruction of PIL5 through a process in which phyB and PIL5 interact (Oh et al, 2004, 2006). As a result, GA synthesis occurs normally and GAI and RGA transcription is low (Oh et al, 2007). In contrast, under the canopy, a low ratio of red to FR light (FR conditions) will inactivate phyB (Pr state) (Chen et al, 2004). When inactive, phyB no longer interacts with PIL5 and this prevents PIL5 destruction by the 26S-proteasome machinery. As a result, PIL5 accumulates and represses seed germination. Under this model, PIL5-dependent repression involves (1) GAI and RGA, (2) SOMNUS (SOM), a CCCH-type zinc-finger protein also repressing germination and (3) regulation of ABA and GA metabolic genes (Kim et al, 2008; Seo et al, 2009). PIL5 activates the transcription of GAI, RGA and SOM genes by directly binding to their promoter sequences. In parallel, both PIL5 and SOMNUS are proposed to indirectly lower GA levels and elevate ABA levels by modulating the expression of GA and ABA metabolic genes in an unknown manner. In turn, lower GA levels further increase DELLA protein stability, thus enhancing RGA-, GAI- and RGL2-dependent repression, whereas higher ABA levels repress seed germination by stimulating ABI3 and ABI5 expression. Some key aspects of the model described above have not been addressed. First, ga1/pil5 (and ga1/som) double mutants cannot germinate under FR conditions, suggesting that additional unidentified repressive activities occur when GA synthesis is severely prevented (by the ga1 mutation). Second, the role of ABA and ABA-response factors to repress germination has not been directly investigated. Third, the nature of the PIL5- and SOM-dependent regulation of the expression of genes involved in hormone metabolism is unclear (Oh et al, 2007; Kim et al, 2008). Indeed, the observed changes in the expression of genes involved in GA and ABA metabolism in pil5 or som mutants may be interpreted as secondary effects resulting from pil5 and som seed germination under FR conditions. Finally, the notion that the balance of GA and ABA levels determines the germination potential of the Arabidopsis seed lacks precision in terms of their effects on development. Thus, even though the observation that GA and ABA levels are inversely correlated strongly points to a regulatory crosstalk between their metabolic pathways, this does not necessarily imply that each hormone regulates germination through symmetrically inversed mechanisms. Indeed, in a recent report, we showed that RGL2 and ABI5 have distinct developmental functions under white light that are consistent with how GA and ABA differently influence testa and endosperm rupture. In response to low GA levels, a stabilized RGL2 efficiently blocks testa rupture and increases endogenous ABA levels. ABA in turn stimulates ABI5 expression and product activity to repress endosperm rupture (Piskurewicz et al, 2008). This points to the general notion that GA promotes testa rupture and limits ABA levels, thus ensuring low expression and activity of the ABA-response factors repressing endosperm rupture. Here, we report that this view can be extended to the phyB-dependent control of seed germination. We show that under FR conditions, low GA levels allow an overaccumulation of GAI, RGA and RGL2, which block testa rupture and promote an increase in the levels of ABA, ultimately responsible to prevent endosperm rupture. This may involve DELLA-dependent stimulation of the expression of XERICO, encoding an RING-H2 factor promoting ABA synthesis in an unknown manner. ABA-dependent repression under FR conditions is orchestrated by ABI3. We show that the role of PIL5 to influence GAI and RGA protein levels is limited to FR conditions in which GA levels are not drastically reduced. As a result, in pil5 seeds under low GA conditions, GAI and RGA levels increase sufficiently to block testa rupture and promote higher endogenous ABA levels. Finally, we show that GAI and RGA can complement rgl2 mutants when both are under the control of RGL2 promoter sequences, further confirming their role to repress testa rupture and to stimulate endogenous ABA synthesis during germination. Results RGL2, GAI and RGA are the main GA-response repressors of testa and endosperm rupture under FR light Under phyB-dependent repression of seed germination, a pulse of FR light (FR conditions) ensures a complete blockade of seed germination in darkness unless followed by a pulse of red light (R conditions, phyB is active). FR conditions (i.e. phyB is inactive) are associated with lower GA levels and higher ABA levels, whereas the converse (high GA, low ABA) occurs under R conditions (Seo et al, 2006). The relative role of each hormone as well as each GA- or ABA-response factor to repress seed germination remains unclear. Earlier reports have suggested that in conditions other than white light illumination, RGL2 is no longer the main DELLA factor repressing seed germination in response to low GA levels (Cao et al, 2005). Indeed, contrarily to what is observed under white light conditions, ga1/rgl2 double mutants can no longer germinate in darkness. In contrast, ga1/rgl2/gai/rga mutants can germinate in the dark, which shows that RGL2, GAI and RGA redundantly repress germination in the absence of light. However, darkness is a rather ill-defined treatment from the perspective of phytochrome-dependent repression of seed germination. Indeed, varied seed germination responses may occur in darkness depending on the seed batch, including germination of the entire seed population (Cao et al, 2005; Supplementary Figure 1). This is most likely because of the presence of different pools of active phyB in imbibed seeds that could be inherited from embryogenesis or from the experimental manipulations performed in the presence of light before seed imbibition. Therefore, seed germination in ga1/rgl2/gai/rga could still involve active phyB, so that the relative contribution of phyB and DELLA factors remains unclear. Here, we explore the role of RGL2, GAI and RGA together with ABA to repress seed germination under conditions in which phyB is inactive (i.e. FR conditions). A 5-min FR irradiation treatment is sufficient to inactivate phyB and ensure repression of germination in darkness (Supplementary Figure 1). We first examined testa and endosperm rupture events in ga1, ga1/rgl2 and ga1/rgl2/gai/rga seeds under FR conditions, a situation that had not been yet explored. Under white light conditions, testa and endosperm rupture could be observed in ga1/rgl2 and ga1/rgl2/gai/rga seeds, but not in ga1 seeds, consistent with the earlier results (Figure 1A and B). In contrast, under FR conditions, testa rupture and endosperm rupture could only be observed in ga1/rgl2/gai/rga seeds (Figure 1A and B). FR conditions were associated with higher GAI and RGA protein contents in ga1 and ga1/rgl2, consistent with the earlier reports (Supplementary Figure 2). This further supports the notion that GAI and RGA, together with RGL2, become key GA-response repressors of testa and endosperm rupture when the phytochrome is inactive. Figure 1.RGL2, GAI and RGA redundantly repress testa rupture under FR conditions. (A) Representative pictures showing ga1-3, ga1-3/rgl2-13 and ga1-3/rga-t2/gai-t6/rgl2-1 seeds 30 h and 5 days after imbibition under white light conditions or far-red (FR) light conditions. Picture showing testa rupture in ga1-3/rgl2-13 seed is taken 72 h after imbibition. Arrows indicate testa rupture events. (B) Histogram shows percentage of testa rupture events 5 days after imbibition of WT (Col), ga1-3, ga1-3/rgl2-13 and ga1-3/rgat2/gait6/rgl2-1 seeds under white (white) or FR light conditions (in three independent seed batches (n=150–300) testa rupture percentage is always either 0 or 100% depending on the genotype and the light conditions as shown in the histogram). Download figure Download PowerPoint GAI, RGA and RGL2 are necessary to increase endogenous ABA levels under FR conditions Earlier reports have suggested that GAI and RGA may stimulate endogenous ABA synthesis in seedlings (Zentella et al, 2007). In addition, we showed earlier that RGL2-dependent repression of seed germination in white light involves stimulation of endogenous ABA synthesis (Piskurewicz et al, 2008). We thus hypothesized that under FR conditions, GAI and RGA, together with RGL2, are necessary to repress germination also by stimulating ABA synthesis. To address this possibility, we monitored endogenous ABA levels as well as ABI5 accumulation in protein blots, as it is a convenient marker for changes in endogenous ABA levels on seed imbibition and before seedling establishment (Piskurewicz et al, 2008). ABA levels, high in dry seeds, dropped markedly after imbibition under white light conditions in all ga1, ga1/rgl2 and ga1/rgl2/gai/rga seeds, consistent with the earlier results (Figure 2A) (Piskurewicz et al, 2008). Thereafter, ABA levels remained 5–10-fold higher in ga1 relative to ga1/rgl2 or ga1/rgl2/gai/rga seeds, consistent with the earlier results (Figure 2A) (Piskurewicz et al, 2008). As expected, ABI5 protein levels were high in ga1 seeds, but rapidly decayed in ga1/rgl2 or ga1/rgl2/gai/rga seeds under white light conditions (Figure 2C). In contrast, endogenous ABA and ABI5 protein levels remained elevated in ga1 and ga1/rgl2 seeds under FR conditions (Figure 2B and C). In contrast, ga1/rgl2/gai/rga seeds maintained 5–10-fold lower endogenous ABA levels over time relative to ga1 and ga1/rgl2 and failed to maintain ABI5 protein levels (Figure 2B and C). Higher endogenous ABA levels were consistently associated with higher mRNA levels of NCED6, encoding a 9-cis-epoxycarotenoid dioxygenase involved in the first-step specific to ABA biosynthesis (Supplementary Figure 3) (Lefebvre et al, 2006). This is consistent with their ability to germinate and indicates that GAI and RGA, together with RGL2, are necessary to maintain high endogenous ABA levels under FR conditions. Figure 2.RGL2, GAI and RGA are necessary to promote endogenous ABA and ABI5 levels under FR conditions. (A) Time course of endogenous ABA levels in ga1-3, ga1-3/rgl2-13 and ga1-3/rga-t2/gai-t6/rgl2-1 seeds under white light conditions (normal MS medium). Units are pmol per gram of fresh weight. Error bars indicate s.d. (n=3). (B) Same as in (A) under far-red light conditions. (C) Protein gel blot analysis of a time course of ABI5 protein levels upon ga1-3, ga1-3/rgl2-13 and ga1-3/rga-t2/gai-t6/rgl2-1 seed imbibition under far-red (FR) or white (white) light conditions. Signals can be directly compared between different genetic backgrounds. Each lane contains proteins extracted from 5 mg of seeds stained with Ponceau S (Ponceau) before incubation with antibodies against ABI5. At each time point, the percentage of germination (i.e. endosperm rupture events) in the seed population material used in the protein blot is indicated. We measured endosperm rupture in three different seed batches (n=150–300): at 5 days endosperm rupture percentage is either 0 or 100% depending on the genotype and the light condition as shown in Figure 1. Download figure Download PowerPoint XERICO encodes a putative RING-H2 factor promoting ABA synthesis in an unknown manner (Ko et al, 2006; Zentella et al, 2007). We showed earlier that under white light conditions, RGL2 is necessary to elevate XERICO mRNA expression under low GA conditions (Piskurewicz et al, 2008). This suggested a possible mechanism accounting for the elevation of endogenous ABA levels after seed imbibition under low GA conditions. A similar mechanism, involving GAI and RGA, had been proposed earlier in seedlings, in which GAI and RGA bind XERICO promoter sequences and positively regulate its mRNA accumulation (Ko et al, 2006; Zentella et al, 2007). We, therefore, wished to explore whether GAI and RGA are necessary to promote XERICO mRNA accumulation under white and FR light conditions during seed germination and under low GA conditions. Supplementary Figure 4 shows that under white light conditions, XERICO mRNA expression was highest in ga1 seeds and markedly lower in ga1/rgl2 seeds, consistent with the earlier results using paclobutrazol (PAC)-treated wild type (WT) and rgl2 seeds, and reached its lowest levels in ga1/rgl2/gai/rga seeds (Piskurewicz et al, 2008). However, XERICO mRNA accumulation was comparable in ga1 and ga1/rgl2 seeds under FR light, but not in ga1/rgl2/gai/rga seeds, in which it was markedly lower (Supplementary Figure 4). Taken together, these data are further consistent with the notion that XERICO is a target gene of the DELLA factors RGL2, GAI and RGA to promote an elevation in endogenous ABA levels during seed germination. To discriminate between the role of DELLA factors to prevent testa rupture and their role to repress endosperm rupture by stimulating endogenous ABA levels, we treated ga1/rgl2/gai/rga with ABA under FR conditions. ABA-treated ga1/rgl2/gai/rga seeds ruptured their testa, but failed to rupture their endosperm (Figure 3A). As expected, this also correlated with higher ABI5 protein levels (Figure 3B, compare with Figure 2C). Similarly, treating ga1 seeds with both GA and ABA led to lower DELLA factor accumulation but maintained high ABI5 protein levels (Figure 4A), consistent with the earlier results (Zentella et al, 2007). Under these conditions, testa rupture was visible, but no endosperm rupture took place (Figure 4B). Figure 3.ABA blocks rupture of endosperm in ga1/rga/gai/rgl2 seeds, but not that of testa. (A) Representative pictures show testa rupture events in ga1-3 and ga1-3/rga-t2/gai-t6/rgl2-1 seeds 5 days after imbibition under white (white) or far-red (FR) conditions in the presence of 3 μM ABA. Histogram shows percentage of testa and endosperm rupture events 5 days after seed imbibition. Arrows indicate testa rupture event (in three independent seed batches (n=150–300) testa rupture percentage is always either 0 or 100% depending on the genotype and the light conditions as shown in the histogram). (B) Protein gel blot analysis of ABI5 protein levels in ga1-3 and ga1-3/rga-t2/gai-t6sol;rgl2-1 seeds at the indicated times upon imbibition under far-red (FR) or white (W) light conditions in the presence of 3 μM ABA. Protein gel blot conditions as in Figure 2C. Download figure Download PowerPoint Figure 4.Exogenous GA promotes testa rupture by downregulating DELLA protein levels without overcoming ABA-dependent blockade of endosperm rupture. (A) Protein gel blot analysis of GAI, RGA, RGL2 and ABI5 protein levels in ga1-3 seeds at the indicated times upon imbibition under white (W) or far-red (FR) light conditions in the presence of 3 μM ABA without (ABA) or with 50 μM GA (ABA+GA). Each lane contains proteins extracted from 5 mg of seeds. (B) Representative pictures showing ga1-3 seeds after 5 days under white (white) or far-red (FR) light conditions in the presence of 3 μM ABA without (ABA) or with 50 μM GA (ABA+GA). Arrows indicate testa rupture event. Histogram shows percentage of testa and endosperm rupture events at this time point (in three independent seed batches (n=150–300) testa rupture percentage is always either 0 or 100% depending on the genotype and the light conditions as shown in the histogram). Download figure Download PowerPoint GAI and RGA can restore normal sensitivity to low GA in rgl2 mutants We suggested earlier that under white light conditions, RGL2 is the main factor repressing seed germination because it achieves the highest protein accumulation relative to other DELLA factors when GA levels are low. This may be due to the fact that ABA positively and strongly regulates RGL2 mRNA levels, which is not the case for GAI and RGA (Piskurewicz et al, 2008). Here, we provided genetic evidence that GAI and RGA are also necessary to elevate endogenous ABA levels under FR conditions. Thus, we reasoned that both GAI and RGA should be able to substitute RGL2's function under white light conditions, provided their expression is under the control of RGL2 promoter. To explore this possibility, we generated rgl2 mutant transgenic lines carrying a transgene containing either GAI or RGA coding sequences under the control of RGL2 promoter sequences (rgl2/pro-RGL2∷GAI and rgl2/pro-RGL2∷RGA). We examined seed germination responses to low GA levels in independent lines using PAC, which inhibits ent-kaurene oxidase, a key enzyme of the GA synthesis pathway. As expected, repression of testa and endosperm rupture could be observed in PAC-treated rgl2/pro-RGL2∷GAI and rgl2/pro-RGL2∷RGA transgenic lines (Figure 5A; Supplementary Figure 5B). RNA and protein blot analysis confirmed that this was associated with higher GAI or RGA mRNA and protein product levels relative to PAC-treated WT and rgl2 seeds (Figure 5B; Supplementary Figure 5A). In addition, rgl2/pro-RGL2∷GAI and rgl2/pro-RGL2∷RGA arrested seeds accumulated high ABI5 protein levels, unlike rgl2 mutant seeds (Figure 5C). This is consistent with the notion that sufficient GAI or RGA accumulation can increase endogenous ABA levels during seed germination even under white light conditions. Figure 5.When expressed under RGL2 promoter sequences, GAI and RGA can inhibit rgl2 mutant seed germination in response to low GA levels. (A) Pictures show rgl2-13 (Col) mutants transformed with pro-RGL2∷GAI and pro-RGL2∷RGA DNA constructs as well as rgl2-13 and WT (Col) plants 96 h upon imbibition under white light conditions in the presence of 15 μM PAC (concerning the use of PAC, see Supplementary Figure 10). Average percentage of testa rupture events measured 96 h on imbibition is indicated (see also Supplementary Figure 5B). In the absence of PAC, all genotypes germinated similarly (not shown). (B) Protein gel blot analysis of GAI and RGA protein levels in WT (Col), rgl2-13/pro-RGL2∷GAI, rgl2-13/pro-RGL2∷RGA and rgl2-13 seeds at the indicated time points upon seed imbibition under same conditions as in A; 10 μg of total protein is loaded per lane. The band indicated by a plus sign (+) is RGL2, which may be detected with anti-GAI antibody. At each time point, the percentage of germination (i.e. endosperm rupture events) in the seed population material used in the protein blot is indicated. (C) Same as in B, but ABI5 protein levels are monitored. Download figure Download PowerPoint Taken together, these data support the hypothesis that the DELLA factors RGL2, GAI and RGA collectively repress seed germination under FR conditions by (1) repressing testa rupture and (2) stimulating endogenous ABA synthesis. In turn, higher ABA levels prevent endosperm rupture by stimulating the expression and activity of ABA-response factors, such as ABI5. Absence of PIL5 does not prevent high GAI and RGA accumulation under low GA conditions PIL5 is a bHLH transcription factor preferentially interacting with the active Pfr form of the phytochrome, which facilitates its degradation by the 26S proteasome (Oh et al, 2004, 2006). Under FR conditions, PIL5 is stable and stimulates the transcription of GAI and RGA (Oh et al, 2007). pil5 mutants can germinate under FR conditions and this is associated with lower endogenous ABA levels. However, pil5 mutants cannot germinate in the absence of GA synthesis (as in a ga1 background), which is associated with increased endogenous ABA levels (Oh et al, 2006). This shows that PIL5 is no longer essential to repress seed germination under low GA levels. We wished to further clarify the role of PIL5 in the context of the repressive function of RGL2, GAI and RGA outlined above. Under FR conditions, pil5 mutants ruptured their testa before endosperm rupture (i.e. germination), unlike WT seeds (Figure 6A). Treating seeds with PAC prevented testa and endosperm rupture in both WT and pil5 seeds (Figure 6A). These observations are consistent with the earlier results (Oh et al, 2006). Figure 6.Absence of PIL5 does not prevent high GAI and RGA accumulation under low GA conditions. (A) Representative pictures show WT (Col) and pil5 seeds 24 h upon imbibition under far-red (FR) conditions in the absence (MS) or the presence of 5 μM PAC (PAC) (concerning the use of PAC, see Supplementary Figure 10). Arrows indicate testa rupture event. Histogram shows percentage of testa rupture events 5 days upon imbibition under white (white) or far-red (FR) conditions in the absence (MS) or the presence of 5 μM PAC (PAC) (in three independent seed batches (n=150–300) testa rupture percentage is always either 0 or 100% depending on the genotype and the light conditions as shown in the histogram). (B) Protein gel blot analysis of a time course of GAI, RGA and RGL2 protein levels in WT (Col) and pil5 upon imbibition under far-red (FR) conditions in the absence (MS) or the presence of 5 μM PAC (PAC); 10 μg of total protein is loaded per lane. At each time point, the percentage of germination (i.e. endosperm rupture events) in the seed population material used in the protein blot is indicated. Download figure Download PowerPoint Direct measurement of endogenous GAI and RGA protein levels in pil5 seeds under FR conditions was not reported earlier. Under FR conditions, we observed, as expected, that pil5 seeds had lower endogenous RGA and GAI protein levels relative to WT, although they could still be detected (Figure 6B). In contrast, PAC treatment led to higher RGA and GAI protein levels in both WT and pil5 seeds (Figure 6B). This may result from higher RGA and GAI protein stability as a result of lower GA levels. Critically, RGA and GAI protein levels in PAC-treated pil5 seeds were similar to those observed in WT seeds under FR conditions in the absence of PAC treatment (Figure 6B). This observation readily provides an explanation as to why preventing GA synthesis in pil5 mutants leads to blockade of testa rupture and higher endogenous ABA levels: sufficient GAI and RGA protein levels accumulate to stimulate endogenou
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