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. 2021 Jan 13;185(4):1697–1707. doi: 10.1093/plphys/kiaa114

The gibberellin signaling negative regulator RGA-LIKE3 promotes seed storage protein accumulation

Yilong Hu 1,#, Limeng Zhou 1,2,#, Yuhua Yang 1, Wenbin Zhang 1,2, Zhonghui Chen 1,2, Xiaoming Li 1, Qian Qian 1, Fanjiang Kong 3, Yuge Li 1, Xu Liu 1, Xingliang Hou 1,✉,3
PMCID: PMC8133674  PMID: 33793917

Abstract

Seed storage protein (SSP) acts as one of the main components of seed storage reserves, of which accumulation is tightly mediated by a sophisticated regulatory network. However, whether and how gibberellin (GA) signaling is involved in this important biological event is not fully understood. Here, we show that SSP content in Arabidopsis (Arabidopsis thaliana) is significantly reduced by GA and increased in the GA biosynthesis triple mutant ga3ox1/3/4. Further investigation shows that the DELLA protein RGA-LIKE3 (RGL3), a negative regulator of GA signaling, is important for SSP accumulation. In rgl3 and 35S:RGL3-HA, the expression of SSP genes is down- and upregulated, respectively, compared with that in the wild-type. RGL3 interacts with ABSCISIC ACID INSENSITIVE3 (ABI3), a critical transcription factor for seed developmental processes governing SSP accumulation, both in vivo and in vitro, thus greatly promoting the transcriptional activating ability of ABI3 on SSP genes. In addition, genetic evidence shows that RGL3 and ABI3 regulate SSP accumulation in an interdependent manner. Therefore, we reveal a function of RGL3, a little studied DELLA member, as a coactivator of ABI3 to promote SSP biosynthesis during seed maturation stage. This finding advances the understanding of mechanisms in GA-mediated seed storage reserve accumulation.

Two regulators of the plant hormones GA and ABA signaling interdependently modulate seed storage protein accumulation.

Introduction

Seed storage reserves are crucial for seed longevity and plant propagation by providing protection for seeds detached from mother plants and nutrition for germination prior to seedling establishment (D'Hooghe et al., 2014; Nguyen et al., 2015). Meanwhile, they contribute to half of the world’s intake of dietary proteins, oils and starch, and green renewable carbon resources for humans (Fatihi et al., 2016). In the model plant Arabidopsis (Arabidopsis thaliana), storage compound accumulation initiates at 7 d after pollination (DAP) when embryo growth occurs at the expense of the endosperm that progressively degenerates thereafter (Baud et al., 2008). Oil and seed storage protein (SSP) are predominant storage reserves in Arabidopsis, taking up about 40% and 30% of the dry seed weight. Specifically, SSP classes in Arabidopsis mainly include 12S and 2S proteins, which are encoded by CRUCIFERIN A1 (CRA1), CRUCIFERIN 2 (CRU2/CRB), and CRU3, and 2S SEED STORAGE PROTEIN 1 (2S1), 2S2, 2S3, 2S4, and 2S5 genes, respectively (Kroj et al., 2003).

Past decades have witnessed a great progress in the seed storage compound biosynthesis mechanisms by the identification of several master regulators of seed maturation, namely, FUSCA3 (FUS3), ABSCISIC ACID INSENSITIVE3 (ABI3), LEAFY COTYLEDON 1 (LEC1), and LEC2 (Parcy et al., 1997; Baud et al., 2007). Mutants of these genes share similar phenotypes, such as shortages in seed storage reserve accumulation and reduced desiccation tolerance. LEC1 belongs to the Nuclear Factor Y-B family, while FUS3, ABI3, and LEC2 are B3-domain transcription factors (TFs; Suzuki and McCarty, 2008; Jo et al., 2019). FUS3, ABI3, and LEC2 directly regulate downstream genes via binding to the RY-box (CATGCA) at the promoter regions (Santos-Mendoza et al., 2008; Baud et al., 2016). Among these transcriptional regulators, ABI3 is the most critical for the regulation of SSP accumulation (Koornneef et al., 1989; Kagaya et al., 2005a), and chromatin immunoprecipitation (ChIP)-on-chip assay has identified numerous SSP biosynthetic genes as the targets of ABI3 (Monke et al., 2012).

Gibberellins (GAs) impose great effects on various aspects of plant development during the whole lifespan, notably in seed germination and flowering (Sun, 2008; Yamaguchi, 2008). Besides flowering, GA is also involved in other reproductive growth procedures, such as petal and stamen elongation (Cheng et al., 2004), anther development (Wang et al., 2020), pollen development (Plackett et al., 2014), and ovule development (Gomez et al., 2016). Recently, GA was revealed to promote embryo development by releasing the inhibition of DELLA on LEC1 (Hu et al., 2018). GA also plays a negative role in seed oil accumulation (Chen et al., 2012); however, its function in SSP accumulation and related molecular mechanisms are still not clear and thus worth an investigation.

As the critical repressors in GA signaling, DELLA proteins are targeted to degradation in a 26S proteasome-dependent manner in the presence of GA (Dill et al., 2001; McGinnis et al., 2003; Gao et al., 2008). There are mainly two working models for DELLAs in the regulation of gene expression. One is that DELLAs interact with TFs or regulatory proteins to impair their binding affinity to the downstream genes, which is the principal molecular mechanism for the crosstalk between GA and some plant hormones, such as auxin, brassinolide, and ethylene (Daviere and Achard, 2016). The other one is that DELLAs interact with TFs to coactivate downstream gene expression (Marín-de la Rosa et al., 2015; Liu et al., 2016). In Arabidopsis, there are five DELLA proteins, GIBBERELLIN-INSENSITIVE (GAI), REPRESSOR OF GA1-3 (RGA), RGA-LIKE1 (RGL1), RGL2, and RGL3, among which GAI and RGA are two major GA repressors during plant development, while RGL3 is less studied compared with other DELLAs (Sun, 2008). RGL3 is commonly regarded to be involved in abiotic stresses, such as drought, cold stress, and salt stress (Colebrook et al., 2014; Shi et al., 2017; Zhou et al., 2017), and biotic stresses (Wild et al., 2012; Li et al., 2019), while it plays relatively a minor role in plant development compared to other DELLAs (Piskurewicz and Lopez-Molina, 2009; Bai et al., 2014). In this study, we unveiled the molecular mechanism of RGL3 interacting with ABI3 in the regulation of SSP accumulation, providing new insight into the function of RGL3 in seed development.

Results

GA negatively regulates SSP accumulation through RGL3

To investigate the role of GA in SSP accumulation, we determined the SSP content in Col-0 Arabidopsis treated with or without GA3 from 6 DAP when seeds are ready for storage compound accumulation (to avoid side effects of GA on earlier stages) and GA-deficient mutants. GA-treated seeds accumulated significantly less SSP (Figure 1A). Consistently, SSP accumulation increased in the triple mutant of GA biosynthetic genes ga3ox1/3/4 (Figure 1B). These results indicate that GA may negatively regulate SSP biosynthesis. We then explored the involvement of the GA signaling repressors DELLAs in this event by examining their transcription levels during seed development. Among the five DELLAs, RGL3 exhibited an obviously increasing trend after 11 DAP, the time point for embryo enlargement accomplishment and SSP rapid accumulation, and had the most abundant transcripts at late stages (Figure 1C). In contrast, the expression of RGL3 was rather limited and much lower than RGA and GAI in seedlings and rosette leaves (Supplemental Supplemental Figure S1, A and B). These results were consistent with the data in the Arabidopsis electronic Fluorescent Pictograph browser, which further confirmed the specific RGL3 expression pattern in seeds (Supplemental Figure S1C). Moreover, RGL3 protein in rgl3 pRGL3:RGL3-6HA seeds also showed a markedly increasing pattern during seed maturation stages (Figure 1D). Among five della single mutants, strikingly, rgl3 showed the least storage protein accumulation, although SSPs also decreased to different degrees in other della seeds (Figure 1E). Thus, we chose RGL3 as the representative to investigate the role of DELLA proteins in SSP biosynthesis. To further confirm the function of RGL3 in SSP accumulation, we detected SSP content in RGL3 complementation and overexpression transgenic lines. As expected, pRGL3:RGL3-6HA completely rescued the phenotype of lower SSP in rgl3, while 35S:RGL3-6HA accumulated more SSPs (Figure 1F). These results together highlight the important function of RGL3 in modulating SSP accumulation.

Figure 1.

Figure 1

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GA regulates SSP accumulation in Arabidopsis. A, SSP contents in Col-0 mature seeds treated with or without 100 µM GA3. Values were calculated by weight percentage (w/w), the same as below. Data represent means ± sd of three biological replicates. Asterisks indicate significant differences in GA treatment compared with Mock (P < 0.05, by Student’s t test). B, SSP contents in Col-0, ga3ox1/4, and ga3ox1/3/4. Data represent means ± sd of three biological replicates, and letters above the bars indicate significant difference between groups (P <0.05, by one-way ANOVA). C, RT-qPCR analysis of five DELLA genes expression in Col-0 during seed development. Data represent means ± sd of three biological replicates. UBQ10 was amplified as an internal control. D, RGL3 protein level in rgl3 pRGL3:RGL3-6HA during seed development. Actin protein was used as the loading control. E and F, SSP contents in mature seeds of Col-0, della single mutants, rgl3 pRGL3-RGL3-6HA, and 35S:RGL3-6HA. Data represent means ± sd of three biological replicates, and letters above the bars indicate significant difference between groups (P < 0.05, by one-way ANOVA).

RGL3 regulates SSP gene expression in developing seeds

SSP content is tightly controlled at the transcriptional level (Kroj et al., 2003; Kagaya et al., 2005a). Ten to 16 DAP is the key period for SSP rapid accumulation (Baud et al., 2008). We hence chose this period for SSP gene expression analysis. The results showed that GA treatment led to a significant decrease in the expression of 2S1, 2S2, 2S3, CRA, CRB, and CRU3 in Col-0 compared with the mock (Figure 2A), suggesting that GA may regulate SSP essentially at the transcriptional level. DELLA proteins are subject to destabilization in the presence of GA (Sun, 2008; Li et al., 2019). Thus, we questioned whether GA-mediated transient decrease of SSP transcripts was accompanied by a reduction of RGL3 protein accumulation. To this end, we examined the RGL3 protein abundance in rgl3 pRGL3:RGL3-6HA and found that it was greatly degraded by GA treatment and stabilized by paclobutrazol (PAC), a GA biosynthesis inhibitor, in seeds (Figure 2B), suggesting that GA may regulate SSP gene expression depending on the degradation of RGL3.

Figure 2.

Figure 2

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SSP genes are positively regulated by RGL3. A, RT-qPCR analysis of SSP gene expression in 12 DAP seeds treated with 100 µM GA3 or mock for 3 h. Data represent means ± sd of three biological replicates. Asterisks indicate significant differences between groups (P < 0.05, by Student’s t test). B, RGL3 protein analysis in 12 DAP rgl3 pRGL3-6HA seeds treated with 100 μM PAC and 100 μM GA3 for 6 h. Col-0 was used as the negative control. Coomassie bright blue stain was used as the loading control. C, RT-qPCR analysis of SSP gene expression in Col-0, rgl3, and 35S:RGL3-6HA at 8, 12, and 16 DAP. Data represent means ± sd of three biological replicates. UBQ10 was amplified as an internal control.

To probe whether SSP genes were regulated by RGL3, we checked SSP gene expression in Col-0, rgl3, and 35S:RGL3-6HA. The analysis showed that the expression levels of 2S1, 2S2, 2S3, CRA1, CRB, and CRU3 were generally downregulated in rgl3 and upregulated in 35S:RGL3-6HA, respectively, in comparison to those in the wild-type (Figure 2C). It is worthy to note that the difference in these transcripts occurs in 12 and 16 DAPs, but not in 8 DAP, which is consistent with the observation that RGL3 began its rapid accumulation after 11–12 DAP (Figure 1, C and D). These data together support that RGL3 contributes to SSP accumulation via regulating SSP genes.

RGL3 interacts with ABI3

We next questioned how RGL3 regulates SSP gene expression. Bioinformatic analysis revealed that RGL3 is co-expressed with ABI3, the critical factor gene for seed storage reserve biosynthesis (Marín-de la Rosa et al., 2014). We thus examined the expression of ABI3 and RGL3 during seed development. ABI3 and RGL3 shared a similar expression pattern. Both genes maintained at a relative low expression level at early stages and displayed an increasing trend at seed maturation stages (Supplemental Figure S2A), implying that RGL3 may work with ABI3 in seed maturation. A previous study has reported the interaction of ABI3 with two DELLAs, RGA and GAI, in seed germination under high temperature (Lim et al., 2013). To investigate the regulatory mechanism of RGL3 in SSP accumulation, we tested the possible protein interaction between RGL3 and ABI3. Since ABI3 transformation caused yeast cell death, yeast two-hybrid assay failed to determine their interaction (Supplemental Figure S3). Instead, pull-down assay using prokaryotically expressed His-RGL3 and GST-ABI3 demonstrated that RGL3 was co-precipitated by GST-ABI3, but not by  glutathione S-transferase (GST; Figure 3A), indicating the in vitro physical interaction between ABI3 and RGL3 proteins. Bimolecular fluorescence complementation (BiFC) analysis was next conducted in Arabidopsis mesophyll protoplast to examine their in vivo interaction. The fluorescence by ABI3-EYFPN together with RGL3-EYFPC was localized in the cell nuclei (Figure 3B). The co-immunoprecipitation assay using protein extracts from 11 to 16 DAP siliques (pRGL3:RGL3-6HA/35S:ABI3-FLAG) showed that RGL3 can be immunoprecipitated by ABI3 (Figure 3C). Taken together, these data evidence the direct interaction between RGL3 and ABI3 proteins in plants.

Figure 3.

Figure 3

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ABI3 and RGL3 interact in vitro and in vivo. A, In vitro pull-down assay showing direct interaction between His-RGL3 and GST-ABI3 recombinant proteins. Arrows and triangles indicate GST-ABI3 and GST protein, respectively. B, BiFC showing the interaction between ABI3 and RGL3 in Arabidopsis protoplasts. EYFP, the fluorescence of enhanced yellow fluorescent protein; mCherry was used as the nucleus indicator; Merge, merge of EYFP, mCherry, and chloroplast auto-fluorescence; C, Co-immunoprecipitation analysis of the interaction between RGL3 and ABI3 in plants.

RGL3 acts as a coactivator to promote transcriptional activity of ABI3 on target genes

To further dissect the regulatory relationship between RGL3 and ABI3, we examined the expression of ABI3 in rgl3 and 35S:RGL3-6HA transgenic seeds. RGL3 had no effect on ABI3 at transcriptional level (Supplemental Figure S2B). Furthermore, we crossed 35S:ABI3-FLAG into rgl3 background and found that RGL3 had no effect on ABI3-FLAG protein stability either (Figure 4B). Nevertheless, ChIP assay revealed that the binding of ABI3 to the 2S1, 2S3, and CRU3 promoters was significantly attenuated in rgl3 background (Figure 4, A and C). These results suggest that RGL3 may enhance ABI3 binding affinity to target gene promoters. Though DELLA proteins have no DNA binding domain identified, they associate with DNA via other TFs (Daviere and Achard, 2016; Liu et al., 2016). We wondered whether RGL3 could also associate with SSP gene promoters. ChIP assay using rgl3 pRGL3:RGL3-6HA showed that RGL3 was enriched at promoter regions of target genes as well (Supplemental Figure S4). We next examined whether RGL3 association with DNA was dependent on ABI3. Since ABI3 slightly repressed RGL3 expression (Supplemental Figure S2C), we crossed 35S:RGL3-6HA into abi3 background to exclude the transcriptional effect (Supplemental Figure S2D). ChIP assay showed that the association of RGL3 with SSP gene promoters was greatly reduced in the absence of ABI3 even if RGL3 protein abundance was unaffected (Figure 4, D and E), indicating that ABI3 is required for the binding of RGL3 to DNA.

Figure 4.

Figure 4

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RGL3 facilitates ABI3 transcriptional activation on downstream genes. A, Schematic diagram of 2S1, 2S3, and CRU3 for ChIP-qPCR analysis. Black boxes indicate exons, white boxes indicate untranslated regions, and short vertical lines indicate RY-boxes. P1–P3 indicate fragments for ChIP-qPCR amplification. B, Quantification of ABI3-FLAG proteins in nuclear extracts or immunoprecipitated fractions of 35S:ABI3-FLAG plants in the wild-type or rgl3 background. Twelve DAP siliques were harvested for immunoblot analysis. C, ChIP analysis of ABI3-FLAG binding to target genes. The plants used were described in (B). Asterisks indicate significant difference of DNA enrichment in rgl3 compared with Col-0 background (P <0.05, by Student’s t test). D, Quantification of RGL3-HA proteins in nuclear extracts or immunoprecipitated fractions of 35S:RGL3-6HA plants in the wild-type or abi3 background. Twelve DAP siliques were harvested for immunoblot analysis. E, ChIP analysis of RGL3-HA binding to target genes. The plant materials used were described in (D). Asterisks indicate significant difference of DNA enrichment in abi3 compared with Col-0 background (P <0.05, by Student’s t test). F, Plasmid constructs for effectors and reporter in dual-luciferase reporter assay. G, RGL3 enhances ABI3 transcriptional activation in the 2S1 gene. REN was used as an internal control. LUC/REN represented the transcriptional activity of the 2S1 promoter. Data represent mean ± sd of three biological replicates, and letters above the bars indicate significant difference (P < 0.05, by one-way ANOVA).

To examine the RGL3 effect on ABI3 transcriptional activation ability, we performed a dual-luciferase (LUC) reporter assay using 35S:RGL3 and 35S:ABI3 as effectors and Promoter2S1:LUC as reporter (Figure 4F) since 2S1 was significantly repressed in rgl3 (Figure 2C) and is directly regulated by ABI3 (Lara et al., 2003). The ABI3-effector alone could activate the 2S1 expression, while the individual RGL3-effector had minor effects on 2S1 promotion. Strikingly, co-expression of RGL3 and ABI3 resulted in a dramatic increase in LUC signals (Figure 4G), indicating that RGL3 promotes the activation of ABI3 on downstream genes.

Taken together, these findings strongly support that RGL3 acts as a coactivator of ABI3 to regulate SSP genes via enhancing the ABI3 binding to DNA.

RGL3 and ABI3 function interdependently in SSP accumulation

To investigate the genetic relationship between RGL3 and ABI3, rgl3 35S:ABI3-FLAG, and abi3 35SS:RGL3-HA plants were established for genetic analysis. As expected, more SSPs accumulated in 35S:ABI3-FLAG than Col, which was slightly, but significantly reduced by the loss of RGL3 (Figure 5A). In line with the previous reports (Koornneef et al., 1989; Kagaya et al., 2005a; Monke et al., 2012), SSPs were reduced in the abi3 mutant (Figure 5B). Notably, loss of ABI3 function greatly repressed the SSP accumulation in the 35S:RGL3-6HA transgenic line (Figure 5B). In addition, we examined SSP gene (2S1 and CRU3) expression in 10 and 12 DAP seeds of these plant materials. Consistent with the above observations, rgl3 mutation repressed the upregulation of SSP genes in 35S:ABI3-FLAG (Figure 5C). Likewise, the upregulation of SSP genes in 35S:RGL3-6HA was also repressed by loss of ABI3 function (Figure 5C). The rgl3 abi3 double mutant was then generated to further elucidate their genetic relationship. Both SSP content and SSP gene transcripts decreased in the rgl3 abi3 double mutant compared with those in single mutants and the wild-type (Supplemental Figure S5, A and B). Therefore, the genetic evidence strongly suggests that RGL3 and ABI3 function interdependently to regulate SSP genes for SSP accumulation.

Figure 5.

Figure 5

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RGL3 and ABI3 function interdependently to regulate SSP accumulation. A and B, SSP contents in mature seeds of Col-0, rgl3, rgl3 ABI3-OE, ABI3-OE, abi3, abi3 RGL3-OE, and RGL3-OE. Data represent means ± sd of three biological replicates. C, RT-qPCR analysis of 2S1 and CRU3 expression in various plant seeds at 10 and 12 DAP. Data represent means ± sd of three biological replicates. Letters above the bars indicate significant difference between groups (P < 0.05, by one-way ANOVA).

Discussion

Studies have demonstrated that GA closely regulates various processes related to seed development, including ovule development (Gomez et al., 2016), embryogenesis (Hu et al., 2018), and seed oil synthesis (Chen et al., 2012), etc. However, the relationship between GA and SSP accumulation is not clear. Here, we reveal that GA negatively regulates the synthesis of SSPs. GA signaling repressor RGL3 can facilitate ABI3 transcriptional activation via protein–protein interaction, thus promoting the expression of SSP synthesis genes, and ultimately contributing to SSP accumulation.

GA content and bioactivity are dynamically regulated during embryogenesis (Hu et al., 2018). The content of bioactive GA types GA1, GA3, and GA4 reaches a peak at 9 DAP of seeds, which plays an important role in late embryo development (Kanno et al., 2010; Hu et al., 2018). Nine DAP is the critical period for the completion of embryonic morphogenesis and the start of rapid nutrient accumulation, thus the dramatic decline of bioactive GAs after this time point (Hu et al., 2018) suggests that, in contrast to its positive role in late embryogenesis, GA may play a negative role during seed maturation. This is finely verified with the changes in SSP phenotype and associated gene expression caused by increased (exogenous GA application) or reduced (in GA biosynthesis mutants) GA content. Furthermore, both previous reports and our results have shown that GA negatively regulates seed oil accumulation in Arabidopsis (Chen et al., 2012; Supplemental Figure S6), which supports the negative role of GA in regulation of storage substance accumulation in Arabidopsis seeds. Collectively, these findings reveal that the precise spatio-temporal control of bioactive GAs content is crucial for normal seed development.

DELLAs are critical repressors in GA signaling pathway. Among five DELLAs in Arabidopsis, RGL3 has very limited contribution to plant growth but can rapidly respond to stresses (Wild et al., 2012; Shi et al., 2017). Both results from a public microarray database and our  reverse transcriptionquantitative PCR (RT-qPCR) analysis showed that RGL3 is prominently expressed during seed development and after seed stratification while the other four DELLA genes are expressed in various plant tissues (Winter et al., 2007). High transcriptional level of RGL3 at seed maturation stage might be caused by the dehydration process in maturing seeds, comparably to the inducible mode of RGL3 by drought (Colebrook et al., 2014). Moreover, our study showed that RGL3 regulates SSP synthesis. It is worth noting that the time when SSP gene expression level starts to be induced is in line with the increasing expression pattern of RGL3, but not the other DELLAs (Figures 1, C and 2, C; Supplemental Figure S2A). The della single mutants (except rgl1) and dellaq (rga gai rgl1 rgl2) mutant presented a slight but significant SPP accumulation reduction compared with the wild-type (Figure 1E;Supplemental Figure S7), suggesting a possible role of other DELLAs in SSP biosynthesis. However, the SSP content in the dellap mutant (rga gai rgl1 rgl2 rgl3) was only slightly lower than that in rgl3 (Supplemental Figure S7). Thus, these results support that RGL3 is the most important DELLA protein involved in regulation of the SSP accumulation.

Previous studies have reported that LEC1, the key regulator of late embryogenesis, regulates the expression of SSP genes, and loss-of-function of DELLA genes causes the upregulation of SSP genes at the early stage of seed maturation (8–9 DAP; Hu et al., 2018). The misregulation of SSP genes in della developing embryos results from the release of LEC1 activity (Hu et al., 2018). However, LEC1 transcripts and proteins are primarily restricted in 5–11 DAP of embryos (Hu et al., 2018), while SSPs mainly accumulate at the seed maturation stage (11–19 DAP) during which DELLA and ABI3 proteins are highly expressed (Figure 1, D and Supplemental Figure 2, A; Le et al., 2010). LEC1 and ABI3 function largely at different seed developmental stages, even though LEC1 acts as a key TF to initiate the expression of ABI3 (Kagaya et al., 2005b). Thus, it is suggested that DELLAs regulate the expression of seed development genes to modulate late embryogenesis via DELLA–LEC1 interaction, and subsequently induce SSP genes to promote seed storage compound accumulation via interacting with ABI3.

Plant hormones GA and ABA function antagonistically in plant growth and stress resistance. Their crosstalk generally occurs in hormone biosynthesis and signaling pathways (Liu and Hou, 2018). During seed germination, high temperature results in the accumulation of ABI3 and DELLA proteins RGA and GAI that facilitates their binding to the promoter of germination repressor genes, thus germination is restrained (Lim et al., 2013). Similarly, during seed maturation, bioactive GA content decreases while ABA biosynthesis increases (Yan and Chen, 2017), resulting in ABI3 and RGL3 accumulation. We reveal that RGL3 interacts with ABI3 and acts as a coactivator to strengthen ABI3 transcriptional activation activity, thus promoting SSP synthesis. Our findings suggest that the DELLA-ABI3 TF coactivation mode is likely to be universal in GA and ABA crosstalk in answer to different environmental hints and internal developmental signals.

Genetic engineering is one of the main approaches in breeding. Through recognizing and manipulating TFs, seed traits like color, size, nutrient content, and storage product type and proportion could possibly be modified according to human wishes. However, it sometimes can lead to unexpected phenotypes since many genes function in diverse development or tissues. Due to less undesirable side effects in plant development, the RGL3-type DELLA gene might be a promising target for crop breeding with high SSP in the future.

Materials and methods

Plant materials and growth conditions

All Arabidopsis (A. thaliana) plants used in this study were in Col-0 background. rga-28 (SALK_089146), gai (SAIL_587_C02), rgl1 (SALK_136162), rgl2 (SALK_124231; Hu et al., 2018), rgl3-5 (SALK_082546; Wild et al., 2012), abi3-8 (Nambara et al., 2002), dellaq (Hu et al., 2018), dellap (Park et al., 2017), ga3ox1/ga3ox4 and ga3ox1/ga3ox3/ga3ox4 (Hu et al., 2008), pRGL3:RGL3-6HA (Li et al., 2019), and 35S:ABI3-FLAG (Yang et al., 2019) have been reported previously. Seeds were incubated at 4°C for 3 d and then grown in full-spectrum LED light (about 11k lux) at 22°C under long day (16-h light/8-h dark) conditions.

Plasmid construction and plant transformation

For the 35S:RGL3-6HA construct, the RGL3 coding sequence was cloned into pGreen-OE6HA vector (Liu et al. 2016). Primers used for plasmid construction are listed in Supplemental Table S1. 35S:RGL3-6HA transgenic lines were obtained through Agrobacterium tumefaciens-mediated transformation using the floral dip method in Col-0 wild-type and a screening with 1/500 10% (w/v) basta (Ingbio, Lot: CB26213210).

SSP measurement

Mature seeds from the same batch of plants were collected and dried in 37°C for 7 d to eliminate the impact of seed water content. Seeds were precisely weighed for 20.00 mg (METTLER TOLEDO, XSE105DU) and finely ground in an electronic homogenizer (Dinghaoyuan, TL 2010S) with three cycles of 25 s in operation and 5 s in pause. One milliliter SSP extraction buffer (50 mM  2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES; pH = 7.5); 5 mM MgCl2; 5 mM dithiothreitol (DTT); 1 mM phenylmethylsulfonyl fluoride (PMSF); 1 mM ethylene diamine tetraacetic acid (EDTA); 10% (v/v) Ethylene glycol) was added to the ground seed powder. After centrifugation at 13,200 rpm and 4°C for 10 min twice, the supernatant of each sample was transferred to a new tube and then used for storage protein measurements according to a previously reported method (Bradford, 1976; Boyondtime Protein Assay Kit, P0017B). The remaining insoluble proteins were recovered by 1 M sodium hydroxide and measured as described above. In both assays,  bovine serum albumin (BSA) was used for calibration.

Seed oil content measurement

The lipid extraction was performed as previously described with some modification (Li et al., 2006). Briefly, mature seeds were dried at 85°C for 2 d and cooled down to room temperature in a drying closet with silica gel. Seed oil was extracted with isopropanol and hexane. Then, 15% (w/v) aqueous sodium sulfate was added to result in phase separation. The upper phase was collected and the lower aqueous phase was re-extracted with 7/2 (v:v) hexane/isopropanol and collected as well. The combined lipid extracts were evaporated to constant weight under nitrogen. The oil content was calculated by dividing the weight of lipid extracted by the input seed weight.

GA treatment assay

Col-0 wild-type or pRGL3:RGL3-6HA flowers were marked after pollination (0 DAP). To apply GA for SSP content measurement, siliques at 6 DAP were directly treated with 100 µM GA3 mixed with 0.02% (v/v) Tween-20 once per day until 16 DAP. Mock group was treated with 0.02% (v/v) Tween-20 and 0.1% (v/v) ethanol. The treated seeds were used for SSP content measurement when matured. To apply GA for gene expression analysis, siliques at 12 DAP were mildly slit with a needle (Hu et al., 2018) and the slightly naked seeds in plants were treated with 100 µM GA3 for 3 h before collection.

Reverse transcription quantitative PCR

For SSP gene expression analysis, total RNA was extracted from 8, 10, 12, or 16 DAP of developing seeds and RT-qPCR was performed as described previously (Hu et al., 2018). Col-0 seeds were collected at 6–17 DAP to detect the DELLA gene expression patterns during seed development. Young siliques at 0–4 DAP and seeds at 5–14 DAP were collected to analyze the gene expression patterns of ABI3 and RGL3. UBQ10 (AT4G05320) was used as an internal control. Primers used are listed in Supplemental Table S1.

In vitro pull-down assay

The coding regions of ABI3 and RGL3 were cloned into the vectors pGEX-4T-1 (Pharmacia) and pQE30 (Qiagen), respectively. GST-ABI3 and His-RGL3 recombinant proteins were expressed in Escherichia coli Rosetta (DE3, Novagen). The soluble GST-ABI3 and His-RGL3 proteins were purified using Glutathione Sepharose Beads (Amersham Biosciences) and Ni-NTA agarose beads (Qiagen), respectively, according to the manufacturers’ instructions. For pull-down assay, 2 μg His-RGL3 fusion proteins were incubated with the Glutathione beads containing GST or GST-tagged ABI3 proteins at 4°C for 4 h. Proteins attached on beads were subsequently washed three times and then followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and detection with anti-His (AbM59012-18-PU, BGI) and anti-GST antibody (AB101-02, Tiangen).

BiFC assay

The coding regions of RGL3 and ABI3 were cloned into the pGreen binary vectors containing C- or N-terminal fusions of EYFP to generate 35S:RGL3-EYFPC and 35S:ABI3-EYFPN. Arabidopsis mesophyll protoplasts were prepared, transfected, and cultured as previously described (Liu et al., 2016). Mesophyll protoplasts were observed after 16-h culture using a confocal laser scanning microscope (Zeiss LSM 700) with a 488-nm excitation wavelength for enhanced yellow fluorescent protein (EYFP) and 587 nm for mCherry. Nuclear marker (Vir D2NLS-mCherry) was used to indicate the localization of the nucleus.

Co-immunoprecipitation assay

The 11–16 DAP pRGL3:RGL3-HA 35S:ABI3-FLAG siliques were treated with 100-μM PAC for 2 d. Siliques were ground into fine powder in liquid N2 and then homogenized in extraction buffer [50 mM Tris–Cl (pH = 7.5); 100 mM NaCl; 0.1% (v/v) NP40; 1 mM PMSF; 80 µM MG132; 1× complete protease inhibitor cocktail (Roche)], followed by 1 min sonification in ice. After centrifuged for 10 min at top speed twice, the supernatant was transferred to a new tube and then incubated with Protein G PLUS/Protein A-Agarose Suspension (IP10, CALBIOCHEN) plus either anti-FLAG or preimmune serum (IgG, I8765, Sigma) at 4°C overnight. After washing three times, the proteins bound to beads were resolved by 5× protein loading buffer and heated for 5 min at 100°C. Protein extracts were fractionated by SDS–PAGE and then detected by anti-FLAG (A3156, sigma) or anti-HA antibody (sc-7392, Santa Cruz).

Total protein extraction and immunoblot assays

Arabidopsis mature green seeds from siliques were ground into fine powder in liquid N2 and then homogenized in extraction buffer [50 mM HEPES (pH 7.5); 150 mM NaCl, 1 mM EDTA; 10% (v/v) glycerol; 0.1% (v/v) TritonX 100; 1 mM PMSF; 1× complete protease inhibitor cocktail (Roche)]. After centrifugation for 10 min at top speed twice, the supernatant was transferred to a new tube with 5× protein loading buffer [250 mM Tris–Cl (pH 6.8), 10% (w/v) SDS, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, and 0.05% (w/v) bromophenol blue] and heated for 5 min at 100°C. Protein extracts were fractionated by 10% SDS–PAGE and immunoblot analyzed with anti-FLAG (A3156, sigma), anti-HA antibody (sc-7392, Santa Cruz), or anti-Actin (Novogen, Cat#NHT0049).

ChIP assay

ChIP assays were performed as previously described (Hu et al., 2018). Briefly, siliques at 12 DAP under various genetic backgrounds were collected and fixed with 1% (v/v) formaldehyde in vacuum for about 40 min. Chromatins were extracted and sonicated to generate DNA fragments ∼500 bp in length on average. The solubilized chromatins were immunoprecipitated by anti-HA plus Protein G PLUS agarose (16–201, Millipore) or anti-FLAG (A3156, sigma), and the co-immunoprecipitated DNA was recovered and analyzed by quantitative PCR. Relative enrichment fold was calculated by normalizing the amount of a target DNA fragment against that of a TUB8 (AT5G23860) genomic fragment, and then against respective input DNA samples. The enrichment of PP2A (AT1G13320) genomic fragment was used as a negative control. Primers used are listed in Supplemental Table S1.

Dual-luciferase reporter assay

To generate the p2S1:LUC reporter construct, ∼2 kb 2S1 promoter was cloned into the pGreenII 0800-LUC vector. The Renilla Luciferase (REN) gene under the control of 35S promoter in the pGreenII 0800-LUC vector was used as the internal control. The coding regions of ABI3 and RGL3 were cloned into the pGreenII 62-SK vector and used as effectors. All primers used for these constructs are listed in Supplemental Table S1. Arabidopsis mesophyll protoplasts were prepared, transfected, and cultured as previously described (Liu et al., 2016). The LUC and REN activities were measured using the Dual-Luciferase Reporter Assay System (Promega) under the manufacturers’ instructions. The LUC/REN ratio was presented with three biological replicates.

Accession numbers

Sequence data used in this study can be found in The Arabidopsis Information Resource (https://www.arabidopsis.org) under the following accession numbers: 2S1 (AT4G27140), 2S2 (AT4G27150) , 2S3 (AT4G27160), CRA1 (AT5G44120), CRB (AT1G03880), CRU3 (AT4G28520), RGA (AT2G01570), GAI (AT1G14920), RGL1 (AT1G66350), RGL2 (AT3G03450), RGL3(AT5G17490), and ABI3 (AT3G24650).

Supplemental data

The following supplemental materials are available.

Supplemental Figure S1. Expression pattern of DELLA genes in plants.

Supplemental Figure S2. Expression analysis of RGL3 and ABI3 in seeds with various genetic backgrounds.

Supplemental Figure S3. ABI3 transformation causes yeast cell death.

Supplemental Figure S4. ChIP analysis of RGL3 binding to the SSP gene promoters.

Supplemental Figure S5. Analyses of SSP content and relative gene expression in Col, rgl3, abi3, and rgl3 abi3.

Supplemental Figure S6. Oil content in Col-0 and della mutants.

Supplemental Figure S7. SSP content in Col-0 and della mutants.

Supplemental Table S1. Primers used in this study.

Supplementary Material

kiaa114_Supplementary_Data
Click here for additional data file. (567.4KB, docx)

Acknowledgments

We thank Dr Chuanyou Li for providing abi3-8 seeds, Dr Tai-Ping Sun for dellap seeds, and Dr Xiangyang Hu for 35S:ABI3-FLAG seeds.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31670319 and 31900212) and China Postdoctoral Science Foundation (2018M640835).

Conflict of interest statement. The authors declare no conflict of interest.

 

Y.H., L.Z., and X.H. designed and supervised the research. L.Z., Y.H., Y.Y., W.Z., X.L., Z.C., Q.Q., Y.L., and X.L performed the research. L.Z., Y.H., F.K., and X.H. analyzed the data. L.Z., Y.H., and X.H. wrote the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Xingliang Hou (houxl@scib.ac.cn).

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Associated Data

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Supplementary Materials

kiaa114_Supplementary_Data
Click here for additional data file. (567.4KB, docx)

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