Arabidopsis DET1 Represses Photomorphogenesis in Part by Negatively Regulating DELLA Protein Abundance in Darkness

Transcription

1 Arabidopsis DET1 Represses Photomorphogenesis in Part by Negatively Regulating DELLA Protein Abundance in Darkness Kunlun Li 1, Zhaoxu Gao 1, Hang He 1, William Terzaghi 2, Liu-Min Fan 1, Xing Wang Deng 1,3, * and Haodong Chen 1, * 1 Peking-Yale Joint Center for Plant Molecular Genetics and Agro-biotechnology, State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, College of Life Sciences, Peking University, Beijing , China 2 Department of Biology, Wilkes University, Wilkes-Barre, PA 18766, USA 3 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT , USA *Correspondence: Xing Wang Deng (xingwang.deng@yale.edu), Haodong Chen (chenhaodong@pku.edu.cn) Research Article ABSTRACT Arabidopsis De-etiolated 1 (DET1) is one of the key repressors that maintain the etiolated state of seedlings in darkness. The plant hormone gibberellic acid (GA) also participates in this process, and plants deficient in GA synthesis or signaling show a partially de-etiolated phenotype in darkness. However, how DET1 and the GA pathway work in concert in repressing photomorphogenesis remains largely unknown. In this study, we found that the abundance of DELLA proteins in det1-1 was increased in comparison with that in the wildtype plants. Mutation in DET1 changed the sensitivity of hypocotyl elongation of mutant seedlings to GA and paclobutrazol (PAC), an inhibitor of GA synthesis. However, we did not find obvious differences between det1-1 and wild-type plants with regard to the bioactive GA content or the GA signaling upstream of DELLAs. Genetic data showed that removal of several DELLA proteins suppressed the det1-1 mutant phenotype more obviously than GA treatment, indicating that DET1 can regulate DELLA proteins via some other mechanisms. In addition, a large-scale transcriptomic analysis revealed that DET1 and DELLAs play antagonistic roles in regulating expression of photosynthetic and cell elongation-related genes in etiolated seedlings. Taken together, our results show that DET1 represses photomorphogenesis in darkness in part by reducing the abundance of DELLA proteins. Key words: DET1, DELLA, gibberellic acid (GA), photomorphogenesis, Arabidopsis Li K., Gao Z., He H., Terzaghi W., Fan L.-M., Deng X.W., and Chen H. (2015). Arabidopsis DET1 Represses Photomorphogenesis in Part by Negatively Regulating DELLA Protein Abundance in Darkness. Mol. Plant. 8, INTRODUCTION Light is one of the most important environmental factors regulating all stages of plant growth and development. Under light conditions, seedlings undergo photomorphogenesis, characterized by open and expanded cotyledons and short hypocotyls. By contrast, in the dark seedlings go through skotomorphogenesis, whereby seedlings exhibit long hypocotyls, closed cotyledons, undifferentiated chloroplasts, and apical hooks (vonarnim and Deng, 1996). After germination in the soil, the skotomorphogenic pattern is extremely important for protecting the shoot apical meristem and cotyledons as they push through the soil until they reach the appropriate light conditions. The CONSTITUTIVE PHOTOMORPHOGENIC/DE-ETIOLATED/ FUSCA (COP/DET/FUS) proteins are essential negative regulators that maintain the skotomorphogenic state in the dark. Seedlings of mutants lacking these factors usually exhibit photomorphogenic phenotypes in darkness, or accumulate high anthocyanin levels in the seeds (Schwechheimer and Deng, 2000). COP/DET/FUS proteins mainly form three protein complexes, the COP1-SPA1 complex, the COP9 signalosome, and the CDD complex, all of which are involved in the ubiquitin-mediated proteolytic degradation of photomorphogenisis-promoting factors (Yanagawa et al., 2004; Wei et al., 2008; Zhu et al., 2008). Among these three complexes, COP1 was the first cloned and characterized COP/ DET/FUS locus (Deng et al., 1991; Deng et al., 1992). In darkness, COP1 acts as an E3 ubiquitin ligase and mediates Published by the Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS , , April 2015 ª The Author 2015.

2 DET1 Negatively Regulates DELLA Proteins the degradation of several photomorphogenesis-promoting transcription factors, including ELONGATED HYPOCOTYL 5 (HY5), LONG AFTER FAR-RED LIGHT 1 (LAF1), LONG HYPOCOTYL IN FAR RED (HFR1), LIGHT-REGULATED ZINC FINGER1/SALT TOLERANCE HOMOLOG3 (STH3), CONSTANS-LIKE3 (COL3), and GATA2 (a GATA-type transcription factor) (Osterlund et al., 2000a, 2000b; Seo et al., 2003; Duek et al., 2004; Jang et al., 2005; Yang et al., 2005; Datta et al., 2006, 2008; Luo et al., 2010). In the light, photoreceptors promote the nuclear export of COP1 and abrogate its repressive activity, resulting in the accumulation of photomorphogenesis-promoting proteins and the light-grown phenotypes (Osterlund and Deng, 1998). Arabidopsis DET1 was originally isolated by the de-etiolated phenotype of its loss-of-function mutants in darkness (Chory et al., 1989). DET1 was demonstrated to interact with DDB1 to regulate Arabidopsis photomorphogenesis (Schroeder et al., 2002). Furthermore, DET1 has been shown to interact with DAMAGED DNA BINDING PROTEIN 1 (DDB1) and COP10 to form the CDD complex, which enhances the activity of Ubconjugating enzymes (Yanagawa et al., 2004; Lau and Deng, 2009). The CDD complex physically associates with CULLLIN4 (CUL4) to form an E3 ligase that degrades light signal-promoting transcription factors to repress photomorphogenesis in darkness (Wertz et al., 2004; Chen et al., 2006, 2010; Lin and Wang, 2007; Nixdorf and Hoecker, 2010). In addition, DET1 is necessary for CUL4-DDB1-mediated proteolytic degradation of the UV-lesion recognition factor DDB2, to maintain genome integrity under UV stress (Castells et al., 2011). Experiments and tests in living plant cells showed that Arabidopsis DET1 interacted with the nonacetylated amino-terminal tails of histone H2B in the context of nucleosomes (Benvenuto et al., 2002), which indicated that DET1 may be involved in chromatin regulation and hence may regulate transcription. Recently, DET1 has also been suggested to function as a transcriptional co-repressor and interact physically with two MYB transcription factors, CCA1 and LHY, to decrease the transcription of targets such as TOC1, a key regulator of the circadian clock (Lau et al., 2011). The plant hormone GA is also involved in the regulation of seedling morphogenesis. For instance, Arabidopsis or pea (Pisum sativum) plants with defects in bioactive GA synthesis or GA signaling showed light-grown characteristics when grown in darkness. All of these partial photomorphogenic phenotypes in darkness could be suppressed by the lack of DELLA proteins, the negative regulators in GA signaling (Reed et al., 1996; Alabadí et al., 2004; Achard et al., 2007). Hence, light and GA have antagonistic effects on regulating seedling morphogenesis (Alabadí et al., 2004). In Arabidopsis, the GA receptor GA INSENSITIVE DWARF1 (GID1) perceives the GA signal. Binding of bioactive GA to the C-terminal domain of GID1 induces a conformational change in its N-terminal extension (N-Ex) to create a hydrophobic surface for DELLA binding, which in turn promotes the recognition of DELLAs by the SCF SLY1 complex (Murase et al., 2008; Shimada et al., 2008). DELLA proteins are then poly-ubiquitinated and subsequently degraded via the 26S-proteasome pathway (Dill et al., 2004; Fu et al., 2004). Hence, DELLAs are central negative regulators that repress GA signaling and restrain plant growth (Sun, 2008). The Arabidopsis genome encodes five DELLAs, namely GAI, RGA, RGL1, RGL2, and RGL3, which share both distinct and overlapping functions in the regulation of plant development (Peng et al., 1997; Dill and Sun, 2001; Silverstone et al., 2001; Lee et al., 2002; Wen and Chang, 2002). Among the five DELLA family members, GAI and RGA were demonstrated to be the main repressors regulating etiolated growth (Alabadí et al., 2004). Several signaling integrators have been identified as to connect light and GA pathways. GA has been shown to induce HY5 protein accumulation by modulating the activity of COP1 (Alabadí et al., 2008). Phytochrome-interacting factors (PIFs), another group of major repressors of photomorphogenesis, also integrate light and GA signaling to regulate seedling morphogenesis. DELLAs can directly interact with the DNA-recognition domains (basic helix-loop-helix domain) of PIF3 and PIF4, and prevent them from binding to their target gene promoters, leading to the abrogation of PIF3/4-mediated light control of hypocotyl elongation (de Lucas et al., 2008; Feng et al., 2008). It has also been suggested that phytochromes can inhibit GA biosynthesis during the deetiolation process by regulating the mrna levels of GA metabolic genes (Reid et al., 2002; Alabadí et al., 2008). Further studies in pea showed that LONG1 (the pea ortholog of Arabidopsis HY5), which acts downstream of LIP1 (the pea ortholog of Arabidopsis COP1), mediated the light regulation of bioactive GA synthesis and stimulated photomorphogenic development (Weller et al., 2009). Although several interactions between light and GA signals have been found, the relationship between COP/DET/FUS proteins and the GA pathway has not been fully established. In particular, the way in which the key repressor of photomorphogenesis DET1 is involved in the interaction between light and GA signaling remains unclear. Here, we show that DET1 negatively regulates the abundance of DELLA proteins, which partially explains how DET1 represses photomorphogenesis in the dark. RESULTS DET1 Suppresses the Accumulation of DELLA Proteins in Darkness Light and GA are believed to play antagonistic roles in regulating photomorphogenesis (Alabadí et al., 2004). Consistent with previous studies (Chory et al., 1989; Alabadí et al., 2004), both dark-grown wild-type (Col) seedlings in the presence of PAC (a GA biosynthesis inhibitor) and det1-1 mutants exhibited short and thick hypocotyls, opened cotyledons, and disappearance of the apical hook (Figure 1A and 1B). Furthermore, rgad17 gain-of-function mutants also showed partial photomorphogenesis phenotype in darkness (Figure 1A and 1B). These data suggested that DELLAs are involved in the regulation of skotomorphogenic development in the dark. The similarities in phenotypes between det1-1 mutants and mutations or treatments that caused accumulation of DELLA proteins encouraged us to investigate whether DET1 and DELLA proteins could regulate each other s abundance. Immunoblot analysis showed that GA or PAC treatments had no effect on both the protein levels of endogenous DET1 (Figure 1C), and transgenic Myc-DET1 driven by the 35S promoter (Figure 1D). In contrast, RGA protein levels were significantly higher in det1-1 mutants (Figure 1E). These data showed 8, , April 2015 ª The Author

3 DET1 Negatively Regulates DELLA Proteins Figure 1. DET1 Negatively Regulates DELLA Protein Abundance in the Dark. (A) Images of 4-day-old dark-grown seedlings. For PAC treatment, wild-type (Col) seedlings were grown on medium supplemented with 0 mm (Mock) or 0.5 mm PAC in the dark for 4 days. (B) Hypocotyl lengths of seedlings shown in (A). Mean values ± SD were calculated from 30 seedlings. (C) DET1 protein levels in 4-day-old darkgrown wild-type (Col) seedlings treated with GA or PAC. The medium was supplemented with 10 mm GA 3,0,or1mM PAC, respectively. Total proteins were analyzed by Western blots using anti-det1. A non-specific band was used as a control. (D) Myc-DET1 protein levels in 4-day-old darkgrown 35S:Myc-DET1 seedlings. The medium was supplemented with 10 mm GA 3,0,or1mM PAC, respectively. Total proteins were analyzed by Western blots using anti-myc. A non-specific band was used as a control. (E) RGA protein levels in rga-24, wild-type (Col), and det1-1 seedlings. Total proteins were extracted from 4-day-old dark-grown seedlings and then analyzed by Western blots using anti-rga and anti-rpt5 antibodies. RPT5 was used as a control. (F) DELLA genes transcript levels in wild-type (Col) and det1-1 mutants quantified by qrt PCR. Total RNAs were extracted from 4-day-old dark-grown seedlings. PP2A served as the internal control. Means values and SD were calculated from three independent replicates. that DET1 negatively regulated DELLA protein levels while DELLAs had no effect on DET1 protein levels. This indicated that DELLAs might act downstream of DET1 in regulating photomorphogenesis. To explore whether DET1 modulates DELLA protein abundance at the transcriptional or posttranscriptional levels, we examined the levels of mrnas encoding five DELLA proteins, GAI, RGA, RGL1, RGL2, and RGL3. The transcription levels of these DELLA genes were lower in det1-1 compared with wild-type (Figure 1F), whereas the abundance of the representative DELLA protein RGA was higher. Taken together, these data indicate that DET1 may posttranscriptionally lower DELLA protein levels in darkness. Mutation of DET1 Affects the Sensitivity of Plants to GA Since more DELLA proteins accumulated in det1-1 compared with wild-type (Figure 1), and DELLAs had been shown to be the key repressors in the GA signaling pathway (Sun, 2008), we postulated that det1-1 mutants would exhibit altered responses to GAtreatments. We therefore compared the responses of dark-grown ga1-3 (a GA-deficient mutant), det1-1, and wild-type plants to exogenous GA 3. Dark-grown ga1-3 and det1-1 seedlings had shorter hypocotyls than their wild-type counterparts before treatment, but after treated with GA 3 of different concentrations, the hypocotyl lengths of ga1-3 and det1-1 seedlings dramatically 624 8, , April 2015 ª The Author increased to a comparable level to those of the wild-type (Figure 2A and 2B). Since GA 3 treatment was less effective for induction of hypocotyl elongation in det1-1 than in ga1-3 seedlings, abnormality of GA pathway might be partially responsible for the short hypocotyls of dark-grown det1-1 mutant seedlings. In addition, the hypocotyl lengths of det1-1 mutant seedlings decreased less than Col after treatment with PAC (Figure 2C and 2D). The differences in sensitivity to GA or PAC treatments between det1-1 and wild-type correlated well with their DELLA protein levels. DET1 Can Regulate DELLA Proteins by Mechanisms Other Than GA Pathway Our data showed that more DELLA proteins accumulated in det1-1 than in wild-type (Figure 1). There are three possible ways in which DET1 may regulate DELLA protein levels: first, by regulating GA synthesis; second, by regulating GA signaling upstream of DELLA proteins; and third, by regulating DELLA proteins via mechanisms other than GA pathway. First, we checked whether DET1 affects GA synthesis and bioactive GA content. Although more than 100 GAs have been identified in plants, relatively few, such as GA 1 and GA 4, have been shown to function as bioactive hormones, while GA 12 is considered to be

4 DET1 Negatively Regulates DELLA Proteins Figure 2. Mutation of DET1 Changes the Sensitivity of Seedling Hypocotyls Elongation to GA and PAC. (A) Hypocotyl lengths of det1-1, ga1-3, and their wild-type counterparts (Col and Ler) seedlings grown on medium containing various GA concentrations. Seedlings were grown on medium supplemented with 0, 2, 5, or 10 mm GA 3 in the dark for 7 days. (B) Relative hypocotyl lengths of the seedlings in (A). (C) Hypocotyl lengths of wild-type (Col) and det1-1 seedlings growing on medium containing PAC with various concentrations. Seedlings were grown on medium supplemented with 0, 0.01, or 0.1 mm PAC in the dark for 7 days. (D) Relative hypocotyllengths ofthe seedlings in (C). Means ± SD were obtained from 20 independent plants. In (B) and (D), the hypocotyl lengths of the seedlings grown on the medium without GA 3 or PAC were set to 1. the common precursor of all GAs in plants (Yamaguchi, 2008). Successive steps from GA 12 to GA 4 require the activity of GA 20-oxidases (GA20ox) and GA 3-oxidases (GA3ox), whereas GA2-oxidases (GA2ox) catalyze the oxidation and deactivation of the bioactive GAs (Spray et al., 1996; Thomas et al., 1999; Itoh et al., 2001). We therefore tested the effect of DET1 on the regulation of the transcript levels of representative GA synthetic (GA20ox and GA3ox) and metabolic genes (GA2ox). In darkgrown det1-1 seedlings, GA20ox1, GA3ox1, and GA3ox4 transcript levels were much lower than wild-type whereas GA2ox1, GA2ox6, and GA2ox7 transcript levels were higher than wildtype (Supplemental Figure 1A and 1B). These results indicate that the biosynthesis of bioactive GA might be repressed in det1-1 seedlings. However, no obvious differences were observed in the levels of bioactive GA (GA 1 and GA 4 ) in Col and det1-1 mutant seedlings (Supplemental Figure 2). seeds removing all DELLA and DET1 proteins, and what we were able to obtain were det1-1 gai-t6 rgl1-1, det1-1 rga-t2 rgl1-1, and det1-1 gai-t6 rga-t2 rgl1-1 mutants. We then compared the phenotypes of these triple and quadruple mutants with that of det1-1 in the dark. As shown in Figure 4, det1-1 mutant We therefore checked for defects in GA signaling that may affect DELLA protein levels in det1-1. Figure 2 clearly showed that GA or PAC treatment altered det1-1 hypocotyl lengths, which indicated that the GA signaling pathway still worked in the det1-1 mutant background. To further test this result, we treated det1-1 and wild-type seedlings with GA 3 for varying times, and measured the degradation of the DELLA protein RGA (which is normally degraded in response to GA 3 ). We found that RGA degraded quickly in both det1-1 and wild-type (Figure 3A and 3B). These data indicated that DET1 had no obvious effects on GA signaling upstream of DELLA proteins. Taking these results together, we speculated that DET1 can regulate DELLA protein levels via mechanisms other than the GA pathway. Absence of DELLA Proteins Partially Suppresses det1-1 Mutant Phenotypes To test the importance of the accumulation of DELLA proteins in det1-1 for the regulation of photomorphogenesis, we crossed della mutants into the det1-1 background and checked their phenotypes. Due to fertility problems, it was hard to obtain mutant Figure 3. DELLA Degradation Mediated by GA Signaling Is Normal in det1-1. (A) RGA protein levels in 4-day-old dark-grown wild-type (Col) and det1-1 seedlings. Seedlings grown on regular medium were treated with 10 mm GA 3 for the indicated times. Total proteins were then extracted and analyzed by Western blots using anti-rga and anti-rpt5 antibodies. RPT5 was used as a control. (B) Relative RGA protein levels in (A). RGA and RPT5 protein levels were quantified using Image J software. RGA protein levels were normalized to RPT5, and the value of the starting point was set to 1. Means values and SE were calculated from two independent replicates. 8, , April 2015 ª The Author

5 DET1 Negatively Regulates DELLA Proteins Figure 4. Mutations of DELLA Genes Suppress the det1 Mutant Phenotype in the Dark. (A) Representatives of 4-day-old dark-grown wild-type (Ler and Col), della, det1-1, det1-1 gai-t6 rgl1-1, det1-1 rga-t2 rgl1-1, and det1-1 gai-t6 rga-t2 rgl1-1 seedlings. (B) Hypocotyl lengths of the seedlings shown in (A). Means ± SD were obtained from 30 independent plants. Statistical significance was determined using Student s t tests between the various crossed mutants and det1-1. ***P < seedlings exhibited short hypocotyls and open cotyledons in darkness, and det1-1 gai-t6 rgl1-1, det1-1 rga-t2 rgl1-1 had significantly (P < 0.001) longer hypocotyls than det1-1. Furthermore, the lack of GAI, RGA and RGL1 together had an even stronger effect on suppressing the det1-1 phenotype as det1-1 gai-t6 rga-t2 rgl1-1 mutants showed much longer hypocotyls than det1-1 in the dark. Therefore, GAI, RGA and RGL1 may function together in repressing hypocotyl growth in det1-1 mutants in darkness. However, the absence of GAI, RGA, and RGL1 did not completely suppress the short hypocotyls of the det1-1 mutant. Therefore, the other two DELLA members RGL2 and RGL3, and possibly other factors, are also likely involved in repressing det1-1 hypocotyl growth in the dark. Taken together, these genetic data strongly support the conclusion that DELLA proteins play important roles downstream of DET1 in the regulation of seedling development. DET1 Regulates Transcriptomic Changes during Seedling De-Etiolation in Part through DELLAs Our biochemical and genetic data all showed that DET1 represses photomorphogenesis partially via DELLA proteins (Figures 1, 2 and 4). Since det1-1 mutants over-accumulated DELLA proteins, det1-1 and plants overexpressing DELLA proteins may coregulate some downstream genes. We previously analyzed the transcriptomes of dark-grown det1-1 and Col seedlings by RNA-seq and identified 3740 differentially expressed genes (>2.0-fold, P % 0.05) (Dong et al., 2014). Also, microarray data comparing dark-grown GA-deficient ga1-3 mutants with its wildtype counterpart Ler are available (Cheminant et al., 2011), and ga1-3 is a typical mutant with over-accumulated DELLA proteins. To evaluate the relationship between DET1 and DELLA proteins in regulating target genes, we compared the expression of the , , April 2015 ª The Author Figure 5. DET1 Regulates Target Genes Partially through DELLAs in Darkness. (A) Venn diagram of overlapping genes regulated by det1-1 and ga1-3. (B) Heat map of genes co-regulated by det1-1 and ga1-3. Genes that were differentially expressed in both of these two mutants compared with their wild-type counterparts were analyzed. The bar represents the log2 of fold change. (C) Percentages of genes co-regulated by det1-1 and ga1-3: coupregulated, co-downregulated, or in other patterns. genes regulated by DET1 with that of the 1139 genes regulated by the over-accumulation of DELLA proteins. A total of 338 genes showed differential expression in both det1-1 and ga1-3 mutants compared with wild-type (Figure 5A and Supplemental Table 1). The cluster analysis and the heat map revealed that these coregulated genes showed highly similar expression patterns (Figure 5B). Among these 338 genes, 182 (54%) were upregulated in both, 101 (30%) were downregulated in both, and 55 (16%) showed other regulation patterns (Figure 5C and Supplemental Table 1). Therefore, our data support the conclusion that DET1 may affect expression of a group of genes to repress photomorphogenesis, and this is partially mediated through their negative regulation by DELLA proteins. DET1 Regulates Photosynthesis and Cell Elongation Genes Partially via DELLA Proteins to Repress Photomorphogenesis To identify the biological processes in which the genes coregulated by DET1 and DELLA proteins participated, we performed gene ontology (GO) analysis and functional clustering using the functional annotation of DAVID (Huang et al., 2009). Interestingly, as illustrated in Figure 6A and Supplemental Table 2, GO analysis of the overlapping genes suggested that the first and second abundant clusters were related to photosynthesis and response to light stimulus, which is consistent with the observation that both det1-1 and ga1-3 mutants exhibit light-grown phenotypes in darkness (Chory et al., 1989; Cheminant et al., 2011). This GO analysis result

6 DET1 Negatively Regulates DELLA Proteins Figure 6. DET1 and DELLAs Antagonistically Regulate Photosynthesis and Cell Elongation in Darkness. (A) Functional categories of genes co-regulated by det1-1 and ga1-3. y-axis represents the enrichment scores (ES). (B and C) qrt PCR analysis of several major genes involved in photosynthesis and chlorophyll biosynthesis (B) and cell elongation (C). Total RNAs were extracted from 4-day-old dark-grown seedlings, and PP2A served as the internal control. indicates that two of the major correlated functions of DET1 and GA are to regulate photosynthesis and light signal transduction. For further confirmation, quantitative Real Time PCR (qrt PCR) was used to measure the expression levels of the four photosynthetic and chlorophyll biosynthetic genes PSAE1, HEMA1, HEMA3, and GUN5. We found that PAC treatment and mutation of DET1 enhanced the expression of all four genes (Figure 6B). Considering that both DET1 and GA (or DELLAs) regulate hypocotyl elongation, transcription levels of representative genes regulating cell elongation were also checked. Compared with the controls, the expression levels of EXP, PRE, and XTH genes were all dramatically reduced in the det1-1 mutant and wild-type treated with PAC (Figure 6C). Among the genes tested in Figure 6B and 6C, only GUN5, PSAE1, and XTH19 are in the list of co-regulated genes in Figure 6A, which indicates that far more genes are co-regulated by DET1 and DELLA than those identified in our transcriptomic comparison. One of the major reasons for this is that the ga1-3 transcriptomic data were obtained using microarrays, which cannot identify differentially expressed genes as well as RNAseq. Taken together, these data indicate that DET1 may regulate the expression of photosynthetic and cell elongation genes to repress photomorphogenesis by negatively regulating DELLA proteins in the dark. DISCUSSION DET1 Negatively Regulates DELLA Protein Levels Our study clearly showed that DELLA protein abundance increased in det1-1 mutants (Figure 1). It is of interest to resolve how DET1 regulates DELLA protein abundance. Previous study showed that the light-induced reduction in bioactive GA levels is evident in Arabidopsis, barley, and pea, which indicated that GAs play negative regulatory roles in de-etiolation (Symons et al., 2008). In addition, both COP1 and DET1 have been demonstrated to negatively regulate HY5 protein levels in the dark (Osterlund et al., 2000a, 2000b). LONG1, the pea ortholog of Arabidopsis HY5, can mediate the light regulation of bioactive GA synthesis and stimulated photomorphogenic development (Weller et al., 2009). These clues indicated that GA synthesis might be altered in det1-1, which further regulates the DELLA protein level. However, our data showed that although the transcription levels of several GA synthetic and metabolic genes changed in det1-1, the bioactive GA content (GA 1 and GA 4 )did not show obvious changes (Supplemental Figures 1 and 2). GA treatment experiments showed that DELLA degradation in det1-1 was comparable to that in the wild-type plants (Figure 3). Combining these results with the genetic data showing that removal of DELLA proteins suppresses the det1-1 mutant phenotype more obviously than GA treatment (Figures 2 and 4), we speculate that DET1 can regulate DELLA through mechanisms other than the GA pathway. Although our measurements did not find an obvious difference in GA content between det1-1 and wild-type, we cannot exclude the involvement of GA synthesis in the regulation of DELLA by DET1. Since the GA content levels tested were quite low in our study, it was possible that the measurement failed to detect the difference between det1-1 and wild-type plants. Also, previous study collected expanding stem material for measurement of GA content in pea (Weller et al., 2009), while we used the whole darkgrown seedlings, which could be another reason why we did not find a difference in GA content. In addition, reduction in bioactive GA levels during de-etiolation was transitory in both barley and Arabidopsis seedlings (Symons et al., 2008), and the plants used in our study were grown and harvested in continuous darkness. Accordingly, it remains unclear as to how DET1 negatively 8, , April 2015 ª The Author

7 DET1 Negatively Regulates DELLA Proteins METHODS Figure 7. A Model of How DET1 Represses Photomorphogenesis in Darkness. PIFs are a group of transcription factors repressing photomorphogenesis in the dark (Shin et al., 2009). DET1 positively regulates PIF abundance mainly by stabilizing them in darkness (Dong et al., 2014). DELLAs have been shown to interact with PIFs and to inhibit their binding to targets (de Lucas et al., 2008; Feng et al., 2008). This study shows that DET1 can negatively regulate DELLA protein levels to repress photomorphogenesis in the dark. DET1 may thus positively regulate PIFs to repress photomorphogenesis in darkness in two ways: by positively regulating the abundance of PIFs and by negatively regulating the abundance of DELLA proteins to release PIFs. The arrow represents activation; lines with flat ends represent inhibition; solid lines mean that the mechanisms are relatively clear; the dashed line means that the mechanism is unclear. regulates DELLA protein levels via mechanisms other than the GA pathway. DET1 has been shown to form a CUL4-CDD E3 ligase (Yanagawa et al., 2004; Chen et al., 2006), which may negatively regulate the amount of target proteins. However, the mechanism of how DET1 negatively regulates DELLA proteins needs further tests. DET1 Positively Regulates PIFs to Repress Photomorphogenesis in the Dark As the first locus shown to repress photomorphogenesis in darkness, DET1 has been studied for decades (Chory et al., 1989). However, the molecular mechanism of how DET1 represses photomorphogenesis remains largely unknown. Recently, DET1 has been demonstrated to positively regulate the abundance of PIF transcription factor proteins to repress photomorphogenesis in the dark (Dong et al., 2014). Previous research has clearly shown that DELLAs may interact with PIF3 and PIF4 and inhibit their binding to the target genes (de Lucas et al., 2008; Feng et al., 2008). Here, we found that DET1 negatively regulated the accumulation of DELLA proteins. So in det1-1 mutants, the over-accumulated DELLA proteins may further inhibit the remaining PIFs functions to promote photomorphogenesis. Together, DET1 may positively regulate PIF protein levels and enhance their transcriptional activation activity via the removal of the inhibition of DELLAs to repress photomorphogenesis in the dark (Figure 7) , , April 2015 ª The Author Plant Materials and Growth Conditions The wild-type Arabidopsis accessions used in this study were Col-0 and Ler. Mutants and transgenic lines previously described are as follows: det1-1 (Chory et al., 1989), rga-24, rgad17, ga1-3, and della (gai-t6 rgat2 rgl1-1 rgl2-1 rgl3-1) (Dill et al., 2004; Fu et al., 2004), and 35S:Myc- DET1 (Schroeder et al., 2002). det1-1 gai-t6 rgl1-1, det1-1 rga-t2 rgl1-1 and det1-1 gai-t6 rga-t2 rgl1-1 mutants were generated by crossing det1-1 and della pentuple mutants. Genotyping of the det1-1, gai-t6, rgat2, rgl1-1, and rgl2-1 alleles was performed as described previously (Peng et al., 1997; Dill and Sun, 2001; Silverstone et al., 2001; Lee et al., 2002; Dong et al., 2014). All seeds were surface-sterilized with 15% NaClO for about 5 min, rinsed with sterile water at least five times, and then sown on MS medium containing 1% sugar. The seeds were then vernalized at 4 C for 4 days in the dark, and germination was induced by 3 h continuous white light, after which the plates were kept in the conditions indicated in the text. For experiments including ga1-3, all lines were treated with GA 3 (10 mm) for 4 days at 4 C and then washed five times with distilled water before sterilization. In experiments with PAC treatments, seeds were transferred to control or PAC plates after light treatment for germination, then plates were kept in darkness for the indicated number of days. GA 3 and PAC Treatment For hypocotyl elongation and qrt PCR experiments, Arabidopsis seeds were sown on MS medium containing various concentrations of GA 3 or PAC. For immunoblotting experiments, 4-day-old seedlings were kept immersed in 10 mmga 3 (dissolved in ethanol) or ethanol alone for the indicated times before plant tissues were harvested. Manipulation of seedlings in darkness was performed under dim green safelight. Quantification of Gibberellins For GA content analysis, 1 g (fresh weight) of Col and det1-1 seedlings grown in darkness for 4 days were harvested and immediately frozen in liquid nitrogen and stored at 80 C until GA extraction. The endogenous GAs contents were measured by nano-lc-esi-q-tof-ms analysis as described previously by Lvjiankeruixin Technology Co. Ltd. (Chen et al., 2011). All measurements were conducted three times. Protein Extraction and Western Blots Materials were harvested at the indicated times and ground into fine powders in liquid nitrogen. Total proteins for analysis in this study were extracted by homogenizing seedlings using denaturing buffer (100 M NaH 2 PO 4, 10 mm Tris HCl ph 8.0, 8 M urea), and the extracts were centrifuged at rpm for 10 min at 4 C. Protein concentrations in the supernatants were quantified by the Bradford assay. Aliquots of denatured total proteins were resolved on 8% SDS PAGE gels and transferred to polyvinylidene fluoride membranes. Antibodies against DET1, RGA, and PRT5 were used in the tests. Quantitative RT PCR Total RNA was extracted from 4-day-old seedlings grown in darkness using the RNeasy plant minikit (Qiagen). Reverse transcription was performed using the SuperScript II first-strand cdna synthesis system (Invitrogen) according to the manufacturer s instructions. qrt PCR analysis was performed using SYBR Green PCR Master Mix (Applied Biosystems) with a Bio-Rad CFX96 real-time PCR detection system. Each experiment was repeated with three independent samples, and RT PCR reactions were performed in three technical replicates for each sample. The primers are listed in Supplemental Table 3. Hypocotyl Length Measurements After the indicated times of growth and treatment, seedlings were laid horizontally on the agar plates and digital pictures were taken. Then hypocotyl lengths were measured using Image J software. The relative hypocotyl

8 DET1 Negatively Regulates DELLA Proteins lengths were presented as the percentage of the hypocotyl lengths under GA 3 or PAC treatments relative to those given the Mock treatment. Bioinformatics Analyses Our RNA-seq data were processed previously and the differentially expressed genes were identified by the filtering condition: the log2 value larger than 1 or less than 1 and statistically significant P value of % The ga1-3 data were screened by the same criteria as in the original paper (Cheminant et al., 2011). Cluster3.0 and Treeview software were used for the heat-map analysis. Functional classification was performed via the DAVID functional annotation clustering tool ( ncifcrf.gov/home.jsp). The functional clusters enrichment analysis was calculated by comparing the selected genes with the whole Arabidopsis genome, and the highest classification stringency was chosen for clustering. The significantly enriched biological processes were chosen for our analysis. SUPPLEMENTAL INFORMATION Supplemental Information is available at Online. FUNDING This work was supported by grants to H.C. from the National Natural Science Foundation of China ( ), the National Program on Key Basic Research Project of China (973 Program: 2011CB100101), the National High Technology Research and Development Program of China (863 Program: 2012AA10A304), the Ministry of Agriculture of China (948 Program: 2011-G2B), and State Key Laboratory of Protein and Plant Gene Research; and grants to X.W.D. from the National Natural Science Foundation of China ( , U ), the National Program on Key Basic Research Project of China (973 Program: 2012CB910900), Peking-Tsinghua Center for Life Sciences, and State Key Laboratory of Protein and Plant Gene Research. ACKNOWLEDGMENTS We thank Renbo Yu, Jie Dong, and other laboratory members for their constructive discussion and help. No conflict of interest declared. 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