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/lp/ou_press/arabidopsis-aspartic-protease-aspg1-affects-seed-dormancy-seed-9DuGQpnkEJ
- Publisher
- Oxford University Press
- Copyright
- © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
- ISSN
- 0032-0781
- eISSN
- 1471-9053
- DOI
- 10.1093/pcp/pcy070
- pmid
- 29648652
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- See Article on Publisher Site
Abstract
Abstract Seed storage proteins (SSPs) provide free amino acids and energy for the process of seed germination. Although degradation of SSPs by the aspartic proteases isolated from seeds has been documented in vitro, there is still no genetic evidence for involvement of aspartic proteases in seed germination. Here we report that the aspartic protease ASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1) plays an important role in the process of dormancy, viability and germination of Arabidopsis seeds. We show that aspg1-1 mutants have enhanced seed dormancy and reduced seed viability. A significant increase in expression of DELLA genes which act as repressors in the gibberellic acid signal transduction pathway were detected in aspg1-1 during seed germination. Seed germination of aspg1-1 mutants was more sensitive to treatment with paclobutrazol (PAC; a gibberellic acid biosynthesis inhibitor). In contrast, seed germination of ASPG1 overexpression (OE) transgenic lines showed resistant to PAC. The degradation of SSPs in germinating seeds was severely impaired in aspg1-1 mutants. Moreover, the development of aspg1-1 young seedlings was arrested when grown on the nutrient-free medium. Thus ASPG1 is important for seed dormancy, seed longevity and seed germination, and its function is associated with degradation of SSPs and regulation of gibberellic acid signaling in Arabidopsis. Introduction Seed dormancy controls the distribution of germination in space and time through preventing germination of mature seeds during unsuitable ecological conditions (Née et al. 2017a). The primary seed dormancy is generally initiated during seed maturation and it reaches a high level in freshly harvested seeds. A dormant seed may gradually lose its dormancy in a subsequent period of dry seed storage (so-called after-ripening) and eventually enter into a non-dormant state (Graeber et al. 2012). In winter annual species such as Arabidopsis thaliana, seed dormancy can be broken by stratification (Baskin and Baskin 2004). With release from dormancy, seed germination begins with water uptake (imbibition) and ends with radicle protrusion through the testa and endosperm (seed coat) (Han and Yang 2015). In Arabidopsis, the essential role of the endosperm in inhibiting the germination of a dormant seed has been documented in reports (Lee et al. 2010, Lee and Lopez-Molina 2013). To explore the influence of seed coat on germination, a ‘seed coat bedding assay’ has been used for monitoring the growth of embryos cultured on a layer of endosperm tissue with the testa attached. Results from the ‘seed coat bedding assay’ demonstrated that the biosynthesized ABA which is released from the endosperm of dormant seeds could block embryonic growth (Lee et al. 2010, Kang et al. 2015). Although the testa and endosperm play a significant role in maintaining seed dormancy, the cause of dormancy is mainly determined by the inherent characteristics of embryos (Finch-Savage and Leubner-Metzger 2006). Gibberellic acid and ABA are two major hormones antagonistically controlling seed dormancy and germination (Gubler et al. 2005, Graeber et al. 2012, Shu et al. 2016). ABA accumulates abundantly and is maintained at a high level in dormant seeds. However, ABA contents in seeds gradually decrease and drop to a low level during germination. An increased endogenous level of ABA in plants overexpressing NCED6 (9-CIS-EPOXYCAROTENOID DIOXYGENASE 6), a gene encoding a rate-limiting enzyme in ABA biosynthesis, results in enhanced seed dormancy in Arabidopsis (Martínez-Andújar et al. 2011). Seeds of the nced mutants germinate faster than those of the wild type (Frey et al. 2012). ABA signaling plays a crucial role in controlling seed dormancy. PP2C (PROTEIN PHOSPHATASE 2C), ABI1 (ABA INSENSITIVE 1) and ABI2 (ABA INSENSITIVE 2) are major repressors of ABA signaling. ABI1 and ABI2 interact with and inactivate SnRK2 (SNF1-RELATED KINASE 2), a positive regulator of ABA response. In the presence of ABA, the action of ABI1 and ABI2 is inhibited after binding to the ABA receptor PYR/PYL (PYRABACTIN RESISTANCE/PYRABACTIN-LIKE). The seeds of dominant-negative abi1-1 and abi2-1 mutants showed reduced dormancy, because their mutated ABI1 and ABI2 proteins failed to interact with PYR/PYL receptors (Park et al. 2009). In contrast to ABA, gibberellic acid contents in seeds start to build up during imbibition and stratification (Rodríguez-Gacio et al. 2009, Weitbrecht et al. 2011, Arc et al. 2013). With the gibberellic acid level increased, dormancy breaks down and seed germination is initiated (Holdsworth et al. 2008). Two gibberellic acid-deficient mutants, ga1 and ga2, both have an enhanced seed dormancy phenotype. Exogenously applied gibberellic acid is able to improve seed germination in ga1 and ga2 mutants (Lee et al. 2002). The DELLA proteins such as RGA (REPRESSOR OF ga1-3) and GAI (GIBBERELLIC ACID INSENSITIVE) are negative regulators in the gibberellic acid signal transduction pathway. Mutations in other repressors of gibberellic acid signaling, such as RGL2 (RGA-LIKE2) and SPY (SPINDLY), can rescue the non-germination phenotype of ga1 (Jacobsen and Olszewski 1993, Lee et al. 2002). A group of dormancy-specific genes have been identified in studies and their mutants have a strong dormant phenotype. Amongst these genes, the function of DOG1 (DELAY OF GERMINATION 1) is best characterized (Bentsink et al. 2006). DOG1 regulates seed dormancy via ABA-dependent and ABA-independent pathways (Nakabayashi et al. 2012, Graeber et al. 2014, Huo et al. 2016, Née et al. 2017b). The transcription of DOG1 can be affected by HUB1/RDO4 (HISTONE MONOUBIQUITINATION1/REDUCED DORMANCY4) and RDO2 (REDUCED DORMANCY2), two positive players in the regulation of seed dormancy (Liu et al. 2007, 2011). DOG18/RDO5 (DELAY OF GERMINATION 18/REDUCED DORMANCY 5), a protein phosphatase 2C, positively regulates seed dormancy through suppressing expression of RNA-binding proteins such as APUM (ARABIDOPSIS PUMILIO) (Xiang et al. 2014, 2016). In seed maturation ABI3, LEC1 (LEAFY COTYLEDON 1), LEC2 (LEAFY COTYLEDON 2) and FUS3 (FUSCA 3) are four central regulators of seed dormancy establishment (Stone et al. 2001, Chiu et al. 2012, Ding et al. 2014). During seed development and maturation, plants accumulate and store seed storage proteins (SSPs), which will subsequently be mobilized for providing free amino acids and nutrients to seed germination and seedling growth. In Arabidopsis, the major SSPs are comprised of 12S globulins and 2S albumins (Tan-Wilson and Wilson 2012). Most of the identified proteolytic enzymes degrading SSPs in seed germination are cysteine proteases; others such as serine, aspartic and metalloproteases are also reported (Tan-Wilson and Wilson 2012). Plants often ensure early initiation of SSP mobilization by depositing active proteases during seed maturation (Wang et al. 2014). Many studies have implied that aspartic proteases in dry seeds might be responsible for the first step of SSP mobilization (Belozersky et al. 1989, Dunaevsky et al. 1989, Capocchi et al. 2000, Jones 2005). Aspartic proteases are a group of proteolytic enzymes which use an activated water molecule bound to two aspartate residues for catalyzing their peptide substrates. Aspartic proteases contain two highly conserved aspartates in the active sites and are optimally active in an acidic environment (Simões and Faro 2004). From studies in animals, aspartic proteases are found to control a wide range of biological functions and processes, including cell growth, cell death, protein turnover and immune defense (Brik and Wong 2003). The knowledge of the functions of aspartic proteases in plants, however, remains limited. In our previous report, we showed that the aspartic protease ASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1, At3G18490) confers drought avoidance in Arabidopsis (Yao et al. 2012). Overexpressing ASPG1 results in enhanced ABA sensitivity in guard cells and reduced water loss in ASPG1-overexpression (ASPG1-OE) transgenic plants (Yao et al. 2012). In this study, we further explored the function of ASPG1 in Arabidopsis development. Seed dormancy and seed germination were altered in the loss-of-function mutant aspg1-1. When no exogenous nutrient was supplied, severely impaired seedling growth and delayed mobilization of SSPs was detected in aspg1-1 mutants. We suggest that ASPG1 affects seed dormancy, seed longevity and seed germination in Arabidopsis. Results ASPG1 is involved in seed dormancy of Arabidopsis To investigate the role of ASPG1 in Arabidopsis in depth, we generated RNA interference (RNAi) transgenic plants to knock down the expression of ASPG1 in the wild-type Columbia (Col-0) background. The reduction of ASPG1 expression was quantitatively analyzed and the result showed that most RNAi transgenic lines were efficient in silencing ASPG1 expression. In particular, the expression level of ASPG1 in RNAi line #3 (RNAi3) and line #5 (RNAi5) was decreased >94% in comparison with that in Col (Supplementary Fig. S1). Because in our previous study no significant phenotypes were observed when analyzing the growing seedlings and adult plants of knockout aspg1 mutants (Yao et al. 2012), we turned our attention to phenotypic analysis of the mutants during seed development and seed germination. Occasionally we found that aspg1-1 seeds germinated much more slowly than Col seeds when their siliques naturally fell onto the surface of the soil. This finding prompted us to investigate the effect of ASPG1 on seed dormancy. We examined the germination phenotype of developing seeds which were still embedded in green siliques of aspg1-1 and RNAi plants. The detached green siliques were sterilized and placed on the water–agarose plates, and then the germination rates were quantified on the 10th day of growth (Fig. 1A). A germination rate >50% was scored with Col or overexpressing ASPG1 transgenic lines (OE1 and OE2). However, a significant reduction in the germination rate was measured in aspg1-1 mutants and RNAi lines (Fig. 1B). To confirm the role of ASPG1 in seed germination, we generated and analyzed the complementation lines (Com), i.e. the lines expressing ASPG1 coding sequences controlled by its promoter in the aspg1-1 background. The retarded germination phenotype of aspg1-1 seeds was rescued in the Com1 and Com2 lines (Fig. 1A, B). Fig. 1 View largeDownload slide The dormancy in seeds of aspg1-1 mutants and RNAi lines was enhanced. (A and B) Seeds in the developing siliques of aspg1-1 and RNAi lines had lower germination rates than those in Col. (A) Immature long-green siliques (15 d after pollination) of Col, OE1, OE2, Com1, Com2, aspg1-1, RNAi3 and RNAi5 were grown on water–agarose plates for 10 d. Scale bar = 3 mm. (B) Quantitative analysis of germination rates of seeds in siliques. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). (C–F) Mature seeds of aspg1-1 showed deep dormancy, which could be rescued by GA3 (1.0 μM) or stratification. (C) Freshly harvested seeds (0 d of dry storage) were germinated for 5 d on water–agarose plates containing 1.0 μM GA3 or ethanol (Mock). Before incubation, seeds were treated with or without stratification. Scale bar = 2 mm. (D) Germination rates of after-ripening seeds were analyzed. Seeds which were stored at 22°C for the indicated time (day) were grown on water–agarose plates for 5 d. (E and F) GA3 or stratification treatment could rescue the deep dormancy phenotype of aspg1-1 mutants and RNAi lines. Data represent the mean ± SD of three biological replicates (n >100 for each experiment). Fig. 1 View largeDownload slide The dormancy in seeds of aspg1-1 mutants and RNAi lines was enhanced. (A and B) Seeds in the developing siliques of aspg1-1 and RNAi lines had lower germination rates than those in Col. (A) Immature long-green siliques (15 d after pollination) of Col, OE1, OE2, Com1, Com2, aspg1-1, RNAi3 and RNAi5 were grown on water–agarose plates for 10 d. Scale bar = 3 mm. (B) Quantitative analysis of germination rates of seeds in siliques. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). (C–F) Mature seeds of aspg1-1 showed deep dormancy, which could be rescued by GA3 (1.0 μM) or stratification. (C) Freshly harvested seeds (0 d of dry storage) were germinated for 5 d on water–agarose plates containing 1.0 μM GA3 or ethanol (Mock). Before incubation, seeds were treated with or without stratification. Scale bar = 2 mm. (D) Germination rates of after-ripening seeds were analyzed. Seeds which were stored at 22°C for the indicated time (day) were grown on water–agarose plates for 5 d. (E and F) GA3 or stratification treatment could rescue the deep dormancy phenotype of aspg1-1 mutants and RNAi lines. Data represent the mean ± SD of three biological replicates (n >100 for each experiment). To monitor the state of dormancy in mature seeds of various genetic backgrounds, freshly harvested seeds from opened siliques were grown on water–agarose plates for 5 d and their germination rates were measured. A germination rate as high as 90% was scored with Col, OE1, OE2, Com1 and Com2; a germination rate <35% was measured with aspg1-1, RNAi3 and RNAi5 (Fig. 1C, D). Based on these results, we speculated that seeds of aspg1-1 and the RNAi lines (RNAi3 and RNAi5) might have deeper dormancy. Arabidopsis seed dormancy can be released after storage at room temperature (22°C) for weeks (Graeber et al. 2012). We compared the germination phenotypes of dry stored seeds in different genetic backgrounds. Freshly harvested seeds of aspg1-1 and RNAi lines could lose their dormancy by being stored in desiccated conditions at room temperature (dry storage). When stored dry for 2 d, the germination of aspg1-1 seeds was markedly improved and the germination rate reached 52%. In contrast, the germination rate of freshly harvested (0 d of dry storage) seeds of aspg1-1 was only 29%. When aspg1-1 seeds were stored dry for 7 d, nearly 90% of them were able to germinate (Fig. 1D;Supplementary Fig. S2). A similar phenotype was observed with the germinations of RNAi lines. In addition to dry storage, exogenous gibberellic acid application is also efficient in breaking seed dormancy (Peng et al. 1999, Rodríguez-Gacio et al. 2009). To test the effect of gibberellic acid on seed germination of aspg1-1 and RNAi lines, we sowed seeds on water–agarose plates containing GA3 (1.0 μM). At day 5, >90% germination of aspg1-1 mutants and RNAi lines was observed on the plates containing GA3 (Fig. 1C, E;Supplementary Fig. S2). Thus GA3 was evidently helpful for overcoming deep dormancy in seeds of aspg1-1 mutants and RNAi lines. Because stratification can promote seed germination (Nordborg and Bergelson 1999, Shu et al. 2013), we analyzed the germination phenotype of freshly harvested seeds under the condition of stratification. All tested seeds were stratified in the darkness at 4°C for 2 d prior to incubating on the water–agarose plates. On day 5 of incubation, up to 72% germination was observed with the freshly harvested and then stratified seeds of aspg1-1 mutants. In contrast, the germination rate of non-stratified (Mock) aspg1-1 seeds was as low as 29% (Fig. 1C, F;Supplementary Fig. S2). A similar result was obtained when examining seed germination of RNAi lines. Taken together, dry storage, application of exogenous GA3 and stratification are all efficient treatments to overcome deep dormancy of freshly harvested seeds of aspg1-1 mutants and RNAi lines. Furthermore, we analyzed the germination phenotype of seeds which were dry stored for a longer time (such as for 1 week, 2 weeks and 6 months) under the condition without stratification (No stratification) or with stratification (Stratification) (Supplementary Fig. S3). Although the delay in seed germination persisted in aspg1-1 mutants and RNAi lines, dry storage was obviously beneficial for recovering their seed germination (Supplementary Fig. S3). ASPG1 promotes embryonic growth Arabidopsis embryos are embedded in the surrounding seed coat which consists of a single cell layer of endosperm and an outer layer of dead tissue, the testa. Embryos isolated from some dormant seeds of Arabidopsis mutants are actually not dormant (coat-enhanced dormancy), while in other species the embryo itself is dormant (Bewley 1997, Piskurewicz et al. 2016). Thus we sought to find out whether the seed coat or an embryo itself is responsible for the altered seed dormancy in aspg1-1 mutants. Seed coats of freshly harvested seeds from Col, aspg1-1 and OE2 were carefully removed and then the embryos were isolated. The dissected embryos were incubated on water–agarose plates. The radicle length of each growing embryo was measured and analyzed statistically. Initially, there was no difference in the average radicle length of embryos among aspg1-1, Col and OE2 (Fig. 2A, B). After growing for 3 d, a difference in radicle growth was observed. The average radicle length of Col embryos was 0.76 mm and the average radicle length of aspg1-1 embryos was only 0.61 mm (Fig. 2A, B). A seed coat bedding assay is useful to identify active components controlling germination from endosperm or embryo (Lee et al. 2010). To examine the influence of aspg1-1 seed coat (testa and endosperm) on dormancy, we dissected embryos from freshly harvested seeds of Col, OE2 and aspg1-1, and then placed them on a layer of aspg1-1 seed coats. In contrast to aspg1-1 embryos, the faster radicle growth in Col and OE2 embryos was scored (Supplementary Fig. S4). Thus we can conclude that the embryos rather than the seed coats may have the main responsibility for the delayed radicle growth of aspg1-1 seeds. Fig. 2 View largeDownload slide Retarded embryonic growth shown in aspg1-1. (A and B) Embryonic growth of aspg1-1 was delayed. Embryos were dissected from freshly harvested seeds of Col, aspg1-1 and OE2 and grown on water–agarose plates. HAI: hours after incubation. Scale bar = 0.3 mm. (B) The length of growing radicles was measured. Data represent the mean ± SD of three biological replicates (n >60 for each experiment). (C) The embryonic growth was inhibited by pepstatin A (0.2 μM). DMSO was added to the control plates (Mock). DAI: days after incubation. Scale bar = 0.5 mm. Fig. 2 View largeDownload slide Retarded embryonic growth shown in aspg1-1. (A and B) Embryonic growth of aspg1-1 was delayed. Embryos were dissected from freshly harvested seeds of Col, aspg1-1 and OE2 and grown on water–agarose plates. HAI: hours after incubation. Scale bar = 0.3 mm. (B) The length of growing radicles was measured. Data represent the mean ± SD of three biological replicates (n >60 for each experiment). (C) The embryonic growth was inhibited by pepstatin A (0.2 μM). DMSO was added to the control plates (Mock). DAI: days after incubation. Scale bar = 0.5 mm. To verify the impact of aspartic proteases including ASPG1 on the growth of embryos, we carried out a pharmacological assay using pepstatin A, a potent inhibitor that specifically blocks aspartic protease activity (Kulkarni and Rao 2009, Yao et al. 2012). To exclude the interference of seed coats with limiting pepstatin A uptake, we conducted this assay with the seed coats removed. Embryos isolated from Col seeds were sown on water–agarose plates containing pepstatin A (0.2 μM) and their growth was completely blocked (Fig. 2C). This result indicated that the role of aspartic protease in facilitating the growth of dissected embryos should not be ignored. Seed longevity was affected in aspg1-1 mutants Seed longevity and seed dormancy are two major characteristics determining seed quality. Seed longevity is defined as seed viability after dry storage. Arabidopsis seeds may lose their germination ability completely after a few years of dry storage (Rajjou and Debeaujon 2008, Nguyen et al. 2012). To explore the impact of ASPG1 on seed longevity, we compared the phenotype of seed aging and seedling growth in Col, aspg1-1 and OE lines. To examine natural seed aging, all tested seeds were stored at 22°C in open and dry air for 6 months or 1 year, or even longer, and then they were incubated on water–agarose plates for 7 d. The phenotype of seed germination and abnormal seedlings was quantitatively analyzed. The seeds of Col, aspg1-1 and OE stored for 6 months were well germinated on water–agarose plates (Fig. 3A, B). When stored for 1 year, nearly 100% of seeds of Col and OE2 germinated. However, about 80% of aspg1-1 seeds could germinate (Fig. 3A, B). As the storage time extended, the viability of aspg1-1 seeds declined much faster than that of Col and OE2 seeds (Fig. 3A, B). Additionally, we scored a higher rate of abnormal seedlings in aspg1-1 than in Col and OE lines (Supplementary Table S1). To confirm that aspg1-1 mutants had a higher proportion of dead seeds, we further tested the viability of non-germinated seeds using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Seeds cannot be stained when they are dead. We found that the majority of non-germinated seeds had lost their viability (Supplementary Fig. S5A; Supplementary Table S1). The controlled deterioration test (CDT) system is able to accelerate seed aging by increasing the temperature and relative humidity of the seed storage environment (Tesnier et al. 2002, Nguyen et al. 2015). We then used the CDT system to analyze the germination phenotype of aspg1-1, RNAi3 and RNAi5 seeds. First, we treated seeds with 80% relative humidity at 40°C for weeks; next, the germination phenotype was monitored weekly. When kept in the CDT system for 1 week, the germination rates of aspg1-1, RNAi3 and RNAi5 seeds dropped to 60% or even less; however, the germination rate of Col seeds was mainained at 80% (Fig. 3C;Supplementary Fig. S5B). As the duration of CDT treatment increased, the germination rates in aspg1-1 mutants and RNAi lines were distinctly decreased. When kept in the CDT system for 2 weeks, seed germination of aspg1-1 mutants and RNAi lines was nearly undetectable (Fig. 3C;Supplementary Fig. S5). These results together demonstrated that seeds of aspg1-1 mutants and RNAi lines were susceptible to artificial aging treatment, suggesting the significance of ASPG1 for maintaining seed longevity. Fig. 3 View largeDownload slide Seed longevity was affected in aspg1-1. (A and B) Seed viability of aspg1-1 was lost much more rapidly than that of Col. Seeds stored at 22°C for the indicated time (years) were germinated on water–agarose plates. (A) Photos were taken on day 7 of growth. Scale bar = 0.5 cm. (B) Seed germination rate was quantified on day 7 of growth. Data represent the mean ± SD of biological replicates (n >100 for each experiment). (C) Seeds of aspg1-1, RNAi3 and RNAi5 were more sensitive to artificial aging treatment (controlled deterioration test, CDT). Seeds were stored in 80% relative humidity at 40°C for the indicated time (weeks) and then they were sown on water–agarose plates. The germination rates were measured on day 7 of growth. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). Fig. 3 View largeDownload slide Seed longevity was affected in aspg1-1. (A and B) Seed viability of aspg1-1 was lost much more rapidly than that of Col. Seeds stored at 22°C for the indicated time (years) were germinated on water–agarose plates. (A) Photos were taken on day 7 of growth. Scale bar = 0.5 cm. (B) Seed germination rate was quantified on day 7 of growth. Data represent the mean ± SD of biological replicates (n >100 for each experiment). (C) Seeds of aspg1-1, RNAi3 and RNAi5 were more sensitive to artificial aging treatment (controlled deterioration test, CDT). Seeds were stored in 80% relative humidity at 40°C for the indicated time (weeks) and then they were sown on water–agarose plates. The germination rates were measured on day 7 of growth. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). ASPG1 is preferentially expressed in developing organs and germinating seeds To better understand the function of ASPG1, we examined the expression profiles of ASPG1 in developing organs and germinating seeds in the transgenic lines harboring the plasmid ASPG1pro-GUS. Previously we have reported the predominant guard cell expression of ASPG1pro-GUS (Yao et al. 2012). However, we wanted to investigate more details of the expression of ASPG1, especially its role in seed development and germination. The results showed, consistent with our previous report (Yao et al. 2012), that β-glucuronidase (GUS) signal was observed in seedlings, but also that abundant GUS signal was also detected in the early stage of floral buds and flowers (Fig. 4A), developing siliques (Fig. 4B) as well as germinating seeds (Fig. 4C). Although we did not detect the GUS signal in mature embryos of ASPG1pro-GUS seeds, we observed it in the cotyledons of seeds imbibed for 24 h (Fig. 4C). In addition, supply of exogenous GA3 or treatment by stratification prior to imbibition could significantly induce expression of ASPG1pro-GUS in embryos (Fig. 4C). Similar expression patterns of ASPG1 in Col were consistently obtained using quantitative analysis. The results demonstrated that ASPG1 expression was detectable mainly in developing organs such as the inflorescences and siliques at the fifth day of pollination (Fig. 4D). The expression level of ASPG1 in germinating seeds was 5.0-fold higher than that in non-imbibed mature seeds (Fig. 4D). In addition, the expression level of ASPG1 in mature seeds which were stratified or treated with GA3 (1.0 μM) was 3.0-fold higher than that in non-treated seeds (Fig. 4D). These results were in fact consistent with the annotated public microarray data (The BAR, http://bar.utoronto.ca/; Winter et al. 2007, Bassel et al. 2008) (Supplementary Fig. S6). When we analyzed the microarray data, we noticed that among aspartic protease family members, ASPG1 is one of the major genes which are highly expressed during seed development and seed germination (Supplementary Fig. S7; Supplementary Table S4). Thus the expression characteristic of ASPG1 implies its functional specificity in seed development and germination. Fig. 4 View largeDownload slide The tissue-specific expression profile of ASPG1pro-GUS was analyzed. (A) The expression of ASPG1pro-GUS was detectable in inflorescences (A1, scale bar = 1 mm); younger floral buds (A2, scale bar = 0.5 mm); flowers at stage 10, 12 and 14 (A3–A5, scale bar = 0.5 mm); and anthers at stage 8 to stage 14 (A6–A12, scale bar = 50 μm). (B) A stronger ASPG1pro-GUS signal shown in younger siliques. DAP: day after pollination (B1–B2, scale bars = 0.5 mm; B3, 20 μm). (C) Application of GA3 or stratification treatment could increase the expression level of ASPG1pro-GUS in embryos imbibed for 24 h. C1, embryo before imbibition; C2, embryo imbibed for 24 h on a water–agarose plate; C3, embryo imbibed for 24 h on a water–agarose plate containing 1.0 μM GA3; C4, embryo stratified for 2 d before imbibition. Scale bar = 20 μm. (D) The expression level of ASPG1 in corresponding Arabidopsis tissues was quantitatively measured. Fig. 4 View largeDownload slide The tissue-specific expression profile of ASPG1pro-GUS was analyzed. (A) The expression of ASPG1pro-GUS was detectable in inflorescences (A1, scale bar = 1 mm); younger floral buds (A2, scale bar = 0.5 mm); flowers at stage 10, 12 and 14 (A3–A5, scale bar = 0.5 mm); and anthers at stage 8 to stage 14 (A6–A12, scale bar = 50 μm). (B) A stronger ASPG1pro-GUS signal shown in younger siliques. DAP: day after pollination (B1–B2, scale bars = 0.5 mm; B3, 20 μm). (C) Application of GA3 or stratification treatment could increase the expression level of ASPG1pro-GUS in embryos imbibed for 24 h. C1, embryo before imbibition; C2, embryo imbibed for 24 h on a water–agarose plate; C3, embryo imbibed for 24 h on a water–agarose plate containing 1.0 μM GA3; C4, embryo stratified for 2 d before imbibition. Scale bar = 20 μm. (D) The expression level of ASPG1 in corresponding Arabidopsis tissues was quantitatively measured. Genes involved in the regulation of dormancy and the gibberellic acid signaling pathway were differentially modulated in aspg1-1 seeds To explore further the function of ASPG1 in seed dormancy and germination, we analyzed the impact of the mutation of ASPG1 on expression of key regulators of dormancy and germination in dry and imbibed seeds. The results demonstrated that expression levels of a set of key positive regulators of seed dormancy, including DOG1, DOG18, RDO2 and RDO4, were obviously increased in aspg1-1 dry seeds. In a similar fashion, expression of four key positive transcriptional regulators, i.e. LEC1, LEC2, FUS3 and ABI3, was significantly enhanced in aspg1-1 dry seeds (Fig. 5). DOG1 is a key regulator of seed dormancy in Arabidopsis and other species. Deep dormancy of Arabidopsis seeds is associated with a high level of DOG1 transcription (Huo et al. 2016). The expression level of DOG1 in aspg1-1 dry seeds was already 2.5-fold higher than that in dry seeds of Col and OE2. Strikingly, after imbibition, the difference in the DOG1 expression level between aspg1-1 and Col was reduced to various degrees (Fig. 5). Similar results were obtained when analyzing expression of other positive regulators of seed dormancy, i.e. DOG18, RDO2, RDO4, ABI3, FUS3, LEC1 and LEC2 (Fig. 5). It is widely recognized that ABA and gibberellic acid are primary hormones that antagonistically regulate seed dormancy and germination (Shu et al. 2016). Thus we compared expression of ABA and gibberellic acid biosynthesis genes as well as their signaling components in aspg1-1 and in Col seeds during imbibition. Two ABA biosynthesis genes, ABA1 and ABA2, showed a similar expression level in seeds of aspg1-1 and Col. The expression levels of another two ABA biosynthesis genes, NCED6 and NCED9, were at least 6.0-fold higher in aspg1-1 dry seeds than in Col dry seeds. However, the expression levels of NCED6 and NCED9 in imbibed seeds of aspg1-1 were not very different from those in imbibed Col seeds (Fig. 5). We measured the endogenous ABA contents in dry seeds of aspg1-1 and Col, and we found that ABA contents in dry seeds of aspg1-1 and Col were not significantly different (Supplementary Fig. S8). For ABA signaling components, we compared expression levels of ABI4, ABI5, ABF3 and RD29B genes in seeds of aspg1-1 and Col; no obvious differences were detected (Fig. 5). Additionally, we analyzed expression levels of gibberellic acid biosynthesis genes such as KAO1, KAO2, GA20ox1 and GA3ox1 in seeds of Col, aspg1-1 and OE2. No significant difference were found in expression of KAO1 and KAO2 genes between Col and aspg1-1. However, clearly decreased expression levels of GA20ox1 and GA3ox1 were detected in aspg1-1. We also analyzed expression of gibberellic acid signaling repressor genes including GAI, RGA, RGL2 and RGL3 (encoding DELLA transcriptional regulators). Compared with OE2, a higher expression level of GAI, RGA, RGL2 and RGL3 was detected in aspg1-1 seeds (Fig. 5). Considering that DELLA proteins are important for repressing testa rupture during seed germination (Piskurewicz and Lopez-Molina 2009), our data implied that ASPG1 function might be executed through altering the transcription of a set of key regulators including gibberellic acid signaling components in seeds. Fig. 5 View largeDownload slide The expression levels of a set of genes in dry and imbibed seeds were analyzed. The expression levels of dormancy-related key genes, gibberellic acid biosynthesis genes and signaling components were altered in aspg1-1 seeds. Seeds stored for 2 weeks which were imbibed for the indicated number of hours were used in this experiment. The SAR1 expression level was used as the internal control. Data represent the mean ± SD of three biological replicates. Fig. 5 View largeDownload slide The expression levels of a set of genes in dry and imbibed seeds were analyzed. The expression levels of dormancy-related key genes, gibberellic acid biosynthesis genes and signaling components were altered in aspg1-1 seeds. Seeds stored for 2 weeks which were imbibed for the indicated number of hours were used in this experiment. The SAR1 expression level was used as the internal control. Data represent the mean ± SD of three biological replicates. aspg1-1 seeds were sensitive to PAC treatment Because the transcription of gibberellic acid signaling repressors and gibberellic acid biosynthesis genes was differentially modulated in aspg1-1 seeds during germination (Fig. 5), we thus explored the germination phenotypes of aspg1-1 mutants and RNAi lines under treatment with GA3 and PAC (a gibberellic acid biosynthesis inhibitor). We observed that the germination of aspg1-1 seeds stored for 2 weeks was completely inhibited in the presence of PAC (5.0 μM), whereas 86% of seed germination was scored with Col on day 7 of growth (Fig. 6). We noticed that the inhibitory influence of PAC on germinations of aspg1-1 and RNAi lines was dose dependent (Supplementary Fig. S9). Compared with Col, the influence of PAC on germination of OE lines was evidently less severe (Fig. 6; Supplementary Fig. S9). Interestingly, the addition of GA3 (10.0 μM) to the PAC-containing plates could successfully rescue the inhibited germination of aspg1-1 and RNAi lines (Fig. 6A, E). Fig. 6 View largeDownload slide Phenotypes of seed germination were analyzed in the tested lines under treatments by PAC and GA3. (A) Seeds stored for 2 weeks were sown on water–agarose plates containing PAC (5.0 μM), GA3 (10.0 μM) or PAC (5.0 μM) + GA3 (10.0 μM). The same volume of solvent (ethanol) was added to the control plates. The plates were then placed in the growth chamber. The pictures were taken on day 5 of germination. Scale bars= 0.5 mm. (B–E) The emerged radicles were quantified at the indicated time points (DAI: days after incubation). Data are means ± SDs of three biological replicates (n >100 for each experiment). Fig. 6 View largeDownload slide Phenotypes of seed germination were analyzed in the tested lines under treatments by PAC and GA3. (A) Seeds stored for 2 weeks were sown on water–agarose plates containing PAC (5.0 μM), GA3 (10.0 μM) or PAC (5.0 μM) + GA3 (10.0 μM). The same volume of solvent (ethanol) was added to the control plates. The plates were then placed in the growth chamber. The pictures were taken on day 5 of germination. Scale bars= 0.5 mm. (B–E) The emerged radicles were quantified at the indicated time points (DAI: days after incubation). Data are means ± SDs of three biological replicates (n >100 for each experiment). ASPG1 promotes break down of seed storage protein Degradation of SSPs is a crucial physiological process during seed germination and early seedling establishment. It is believed that approximately 84% of total proteins in Arabidopsis seeds are present in the form of legumin-type 12S globulin and napin-type 2S albumin storage proteins (Gruis et al. 2002, Higashi et al. 2006). Some aspartic proteases are required for proteolytic processing or degradation of SSPs (Otegui et al. 2006, Tamura et al. 2007). We sought to examine the involvement of ASPG1 in the degradation of SSPs during seed germination. Total proteins extracted from germinations of seeds stored for 2 weeks were analyzed using one-dimensional SDS–PAGE. Profiles of seed proteins from Col, aspg1-1 and OE2 are presented in Fig. 7. There were no obvious differences in the contents of SSPs between Col and aspg1-1 seeds before imbibition (Fig. 7; Supplementary Fig. S10). In general, SSPs start to be broken down to provide resources for synthesis of new proteins after imbibition (Tan-Wilson and Wilson 2012). In our analysis, we found that the amount of legumin-type SSPs in germinating seeds of aspg1-1 was retained, compared with that in germinating seeds of Col and OE2. The most obvious change, in terms of SSP type in germinating aspg1-1 seeds, were polypeptides with molecular masses of 25, 32 and 40 kDa (Fig. 7). Thus the degradation of legumin-type 12S globulin protein in aspg1-1 seeds was probably altered. We also found that the amount of new peptides with a molecular mass of 78 kDa in germinating seeds of aspg1-1 was reduced. This suggested that the biosynthesis of new peptides in germinating seeds of aspg1-1 might be affected. Fig. 7 View largeDownload slide The seed storage proteins were analyzed. Total proteins were extracted from germinating seeds of Col, aspg1-1 and OE2. An equal number of seeds were imbibed on water–agarose plates for 0, 24 and 48 h (HAI: hours after imbibition). Arrowheads indicate the differences shown in the SDS–polyacrylamide gel. Fig. 7 View largeDownload slide The seed storage proteins were analyzed. Total proteins were extracted from germinating seeds of Col, aspg1-1 and OE2. An equal number of seeds were imbibed on water–agarose plates for 0, 24 and 48 h (HAI: hours after imbibition). Arrowheads indicate the differences shown in the SDS–polyacrylamide gel. ASPG1 is important for early seedling growth As mobilization and degradation of SSPs is vital for establishment and growth of young seedlings, we examined the growth phenotype of aspg1-1 young seedlings when their own storage reserves were the only nutrient sources. Seeds stored for 2 weeks were sown on water–agarose plates (without any supply of exogenous nutrients) and Murashige and Skoog (MS) plates, respectively. Results showed that seedlings of aspg1-1 and RNAi lines growing on MS plates were all normal (Fig. 8). Seedlings of aspg1-1 and RNAi lines growing on water–agarose plates for 1 week appeared normal (Fig. 8A). However, when young seedlings of aspg1-1, RNAi3 and RNAi5 were continuously grown on water–agarose plates for 3 weeks, their leaves became pale or even transparent, and their survival rates were significantly decreased (Fig. 8B). Therefore, we measured the total Chl (chlorophyll) content in the seedlings and found that the Chl content in seedlings of aspg1-1, RNAi3 and RNAi5 was drastically decreased (Fig. 8C). Overall, our results demonstrated that by consuming their own reserve, the ability of aspg1-1 mutants and RNAi lines to survive was obviously reduced. Fig. 8 View largeDownload slide Decrease in growth ability shown in aspg1-1 mutants and RNAi lines. (A) The seedling survival ability was obviously declined in aspg1-1 mutants and RNAi lines when they were grown on water–agarose plates for 3 weeks. However, all tested seeds grew similarly on MS plates. Photos were taken in the first and third week of growth. Scale bars = 0.5 cm. (B) The survival rate was measured in the third week of growth. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). (C) The Chl content was measured in the third week of growth. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). Fig. 8 View largeDownload slide Decrease in growth ability shown in aspg1-1 mutants and RNAi lines. (A) The seedling survival ability was obviously declined in aspg1-1 mutants and RNAi lines when they were grown on water–agarose plates for 3 weeks. However, all tested seeds grew similarly on MS plates. Photos were taken in the first and third week of growth. Scale bars = 0.5 cm. (B) The survival rate was measured in the third week of growth. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). (C) The Chl content was measured in the third week of growth. Data represent the mean ± SD of three biological replicates (n >100 for each experiment, **P < 0.01). Discussion Seed development and seed germination are two crucial stages in the life cycle of plants. Seed dormancy is induced during seed development, and then is completely broken during seed germination. Lots of studies have sought to decipher the complex regulatory network that controls seed dormancy and germination. ABA and gibberellic acid are two major plant hormones in establishing and releasing seed dormancy. During seed development and maturation, ABA accumulates and induces seed dormancy. At the same time, seeds start to build up and store SSPs and proteases which are required for seed germination and seedling growth (Tan-Wilson and Wilson 2012, Shu et al. 2016). However, our knowledge about the role of SSPs and proteases in mature seeds during the process of dormancy and germination is limited. ASPG1 plays a negative role in seed dormancy but promotes seed germination The ASPG1 expression pattern that was annotated from the public microarray data (Supplementary Figs. S6, S7) and the ASPG1pro-GUS distribution characteristics (Fig. 4) suggested the impact of ASPG1 on seed development, maturation and germination. To explore the function of ASPG1, we first analyzed the germination phenotype of the mutants and OE lines. We found that loss of ASPG1 function could increase the level of seed dormancy (Fig. 1; Supplementary Fig. S2), and the deep dormancy phenotype of freshly harvested seeds from aspg1-1 and RNAi plants could be rescued by treatments such as dry storage, application of exogenous GA3 and stratification (Fig. 1; Supplementary Fig. S2). Although dry storage and the stratification process could improve the germination of aspg1-1 seeds on water–agarose plates, their germination always progressed more slowly than that of Col (Fig. 1; Supplementary Fig. S3). Thus, the role of ASPG1 in seed germination was implicated. Seed dormancy can be imposed by the embryo or the seed coat (endosperm and testa), or a combination of both factors to some extent (Debeaujon et al. 2000). Previous research showed that ABA can be released from endosperm and in turn can inhibit seed germination (Lee et al. 2012, Lee and Lopez-Molina 2013). In our case, both endosperm and testa, isolated from freshly harvested aspg1-1 seeds, had a limited influence on embryonic growth (Fig. 2; Supplementary Fig. S4). In fact, the delayed embryonic growth of aspg1-1 mutants might be caused mainly by the embryos themselves, not by the endosperm. It has been reported that the aspartic protease inhibitor VrAPI, purified from the seeds of Vigna radiate, can inhibit seed germination by controlling the activity of endogenous aspartic proteases (Kulkarni and Rao 2009). In agreement with their finding, we observed that the growth of Col embryos was completely inhibited when they were placed on plates containing the inhibitor pepstatin A (Fig. 2). These results suggested the importance of activities of aspartic proteases including ASPG1 in seed germination. In addition, we found that both radicle extension and cotyledon greening were facilitated in the growth of dissected embryos of OE lines (Fig. 2). Hence, the role of ASPG1 in promoting seed germination should not be ignored. Maintaining dormancy and longevity are two crucial traits for a seed. Seed dormancy and longevity have been thought to be positively correlated in plants (Nguyen et al. 2012, Sano et al. 2016). This theory is mainly based on the performance of those mutants possessing a low level of dormancy, such as lec1, abi3, green seed mutant (enhancer of abi3-1), tt (TRANSPARENT TESTA), ats (ABERRANT TESTA) and dog1 (Debeaujon et al. 2000, Clerkx et al. 2003, Bentsink et al. 2006, Sugliani et al. 2009). All these mutants have a reduced dormancy level that is associated with reduced seed longevity. However, a study discussing natural variations for seed longevity reveals a negative correlation between dormancy and longevity (Nguyen et al. 2012). The transgenic plants in the Ler (Landsberg erecta) background carrying the DOG1 allele of Cvi (Cape Verde Islands) exhibit increased seed dormancy and reduced seed longevity (Nguyen et al. 2012). Therefore, DOG1 may not only control seed dormancy but also influence seed longevity (Nguyen et al. 2012, Nguyen et al. 2015). In this study, we showed that loss of ASPG1 function enhanced seed dormancy and decreased seed viability (Figs. 1, 3). The reduction of seed longevity and increase in seed dormancy in aspg1-1 mutants may be attributed to a higher expression level of DOG1 in aspg1-1 dry seeds. Taken together, ASPG1 may play a negative role in the control of seed dormancy and act as a positive factor in seed longevity and germination. ASPG1-modulated seed dormancy and germination is associated with gibberellic acid signaling Seed dormancy is induced during seed maturation. Mutants of seed maturation regulators such as ABI3, FUS3, LEC1 and LEC2 have poor seed quality with a decreased dormancy level (Stone et al. 2001, Holdsworth et al. 2008, Chiu et al. 2012, Ding et al. 2014). In our analysis, we found that genes such as ABI3, FUS3, LEC1 and LEC2 were all highly expressed in aspg1-1 dry seeds (Fig. 5). Their higher expression levels were correlated to the delayed germination phenotype of aspg1-1 mutants (Supplementary Fig. S3). In addition, the expression of DOG1, as well as other positive regulators of dormancy including DOG18, RDO2 and RDO4, was significantly higher in aspg1-1 dry seeds (Fig. 5). DOG1 and DOG18 protein levels in freshly harvested seeds are associated with the seed dormancy level (Nakabayashi et al. 2012, Xiang et al. 2014). Hence, a higher level of DOG18, RDO2 and RDO4 expression in aspg1-1 seeds may explain their enhanced dormancy phenotype. Numerous studies suggest that accumulation of aspartic proteases during seed development is significant for initiating seed germination (Elpidina et al. 1990, Marttila et al. 1995, Müntz et al. 2001). We speculate that disruption of ASPG1 function alters expression of seed dormancy genes such as DOG1, RDO2 and RDO4, thus producing deeper dormancy in aspg1-1 seeds and, in turn, affecting seed germination of aspg1-1 mutants. ABA is required to induce and maintain seed dormancy during seed maturation (Shu et al. 2016). Although the expression levels of ABA biosynthesis genes NCED6 and NECD9 were increased in aspg1-1 dry seeds, no difference in expression of ABA-responsive genes were observed between seeds of aspg1-1 and Col dry stored for 2 weeks (Fig. 5). We did not detect any obvious difference in endogenous ABA concentration between aspg1-1 and Col dry seeds (Supplementary Fig. S8). RDO5 regulates seed dormancy through an ABA-independent pathway (Xiang et al. 2014). The rdo5 mutant has a strongly reduced seed dormancy phenotype. Mutation in RDO5 neither affects ABA level in dry and imbibed seeds nor ABA sensitivity during seed germination. It will be interesting to investigate further whether or not ASPG1 regulates seed dormancy through an ABA-dependent pathway. In contrast to ABA, gibberellic acid signaling was severely impaired in aspg1-1 during germination. DELLA proteins are negative regulators of gibberellic acid responses (Yoshida et al. 2014). During seed germination, the expression levels of DELLA components were significantly increased in aspg1-1 but only slightly reduced in OE2 (Fig. 5). Among these DELLA components, the most significant change was observed in the expression of RGL2, a main negative regulator of seed germination (Cao et al. 2005, Yoshida et al. 2014). Hence, loss of ASPG1 function could produce repression of gibberellic acid signaling during seed germination. Mutants containing a higher level of active gibberellic acid or robust gibberellic acid signaling may have stronger resistance to PAC (Zhang et al. 2011, Shu et al. 2013). Indeed, we found that overexpression of ASPG1 led to a higher level of resistance to PAC, while aspg1-1 and RNAi lines were more sensitive to PAC (Fig. 6; Supplementary Fig. S9). Furthermore, an exogenous supply of GA3 could recover the impaired germination phenotype of aspg1-1 under PAC treatment (Fig. 6). These results are supportive of the suggestion that ASPG1 plays an important role in seed dormancy and seed germination; the perturbed seed dormancy and seed germination in aspg1-1 mutants are attributed to the repression of gibberellic acid signaling. The ubiquitin–proteasome system (UPS) plays a regulatory role in seed development and seed germination. Degradation of phytohormone-specific transcription factors such as RGL1 and RGL2 by the UPS is one of the mechanisms facilitating seed germination (Wang and Deng 2011). Recently, the UPS-independent degradation system in the regulation of hormonal signaling has been reported. DAG9 (DEGRADATION OF PERIPLASMIC PROTEINS 9), a serine protease, modulates the interaction of cytokinin and light signal via mediating the degradation of ARR4 (ARABIDOPSIS RESPONSE REGULATOR 4) (Chi et al. 2016). Acting as an aspartic protease, ASPG1 might play its role in modulation of gibberellic acid signaling, in which ASPG1 might execute its function through degradation of hormonal transcriptional regulators. A future study to confirm this hypothesis would enrich our understanding of the way in which ASPG1 regulates hormonal signaling. ASPG1 is involved in degradation of seed storage proteins during seed germination During seed germination, SSPs are degraded by proteases, which convert the insoluble storage proteins into soluble peptides and free amino acids. The free amino acids are mobilized to the embryonic axis for the support of growth of embryos and the initiation of seed germination (Shutov and Vaintraub 1987, Müntz et al. 2001). Numerous studies have proposed the possible involvement of aspartic proteases in processing SSPs (Hondt et al. 1993, Runeberg-Roos et al. 1994, Gruis et al. 2002, Otegui et al. 2006). We have reported the aspartic protease activity of ASPG1 in our previous study (Yao et al. 2012). Here, we suggest that ASPG1 is involved in degradation of SSPs during seed germination. The insignificant difference in the content of SSPs in aspg1-1, OE2 and Col (Fig. 7; Supplementary Fig. S10) indicates that loss of ASPG1 function may not be sufficient to affect accumulation of SSPs, and the functional redundancy among aspartic proteases in Arabidopsis may explain this phenomenon. Because ASPG1 is highly expressed in the earlier stage of seed development (Fig. 4), it may play its role in the process of SSP polypeptide precursors. However, little is known about the function of aspartic proteases on mobilization of SSPs during seed germination. Some aspartic proteases isolated and purified from dormant or germinating seeds of wheat (Belozersky et al. 1989, Capocchi et al. 2000, Tamura et al. 2007) and rye (Brijs et al. 1999) can digest their respective SSPs in vitro. However, there is no genetic evidence showing that aspartic proteases are directly involved in degradation of SSPs. In this study, we found that loss of function in ASPG1 could result in delayed degradation of SSPs during seed germination (Fig. 7). Also, the impaired breakdown of SSPs in aspg1-1 germination contributed to the reduced survival rate of its young seedlings grown in conditions without additional nutrient supplied (Fig. 8). According to our data, we propose that ASPG1 plays a positive role in degradation of SSPs during seed germination. Searching for the putative targets of thioredoxin (TRX) in Arabidopsis, the interaction between ASPG1 and AtTRX3 (THIOREDOXIN 3) has been detected in proteomic analysis (Marchand et al. 2004, Darabi and Seddigh 2015). Studies have attested to the fact that TRX protein can act as a signaling component in the early stage of seed germination. TRX facilitates mobilization of reserves in seeds by reducing SSPs, which enhances their solubility and susceptibility to proteolysis (Yano et al. 2001, Wong et al. 2004, Guo and Yin 2007, Shahpiri et al. 2008). In cells of germinating seeds, proteins such as TRX3 and some proteases are sorted and transported to the protein storage vacuoles (PSVs) after being synthesized in the endoplasmic reticulum (ER) (Onda et al. 2011, Gao et al. 2015). The ER localization of ASPG1 has been detected in mesophyll cells in a transient assay (Yao et al. 2012). To promote degradation of SSPs, ASPG1 may execute its function via association with TRX3 in PSVs of seed cells. Aspartic proteases are optimally active in an acidic environment (Simões and Faro 2004). During seed germination, the acidic condition in PSVs (He et al. 2007) may facilitate ASPG1 activity, promote degradation of SSPs and/or support other proteases such as aleurain (Gao et al. 2015) for breaking down SSPs. Overall, our findings in this study indicate that ASPG1 is involved in the control of seed quality and probably facilitates seed germination through promoting the degradation of SSPs. Future studies to confirm the interaction of ASPG1 and TRX3 and to address the involvement of ASPG1 in the degradation of SSPs will enrich our knowledge of the roles of aspartic proteases in seed germination. Materials and Methods Plant materials and plasmid construction All Arabidopsis plants used in this study were in the Col-0 background. The generation of transgenic plants overexpressing ASPG1 (At3G18490) and the source of the mutant aspg1-1 had been described previously (Yao et al. 2012). For generating the complementation lines, the plasmid pBI101-ASPG1pro-ASPG1 was constructed by cloning full-length ASPG1 genomic DNA (3,444 bp) with its upstream promoter (1,942 bp) into the pBI101 vector at SbfI/XmaI cloning sites. The plasmid pBI101-ASPG1pro-ASPG1 was then transformed into aspg1-1 plants using the floral dipping method (Clough and Bent 1998). The RNAi lines were generated by following the method described by Wesley et al. (2001). Two inverted 550 bp fragments of ASPG1 and an intron from the pKANNIBAL vector (CSIRO Plant industry) were cloned into the pBA002 binary vector (Kost et al. 1998) at the XhoI/SpeI cloning sites, leading to the construction of the plasmid pBA002-ASPG1-RNAi. Subsequently, this plasmid was transformed into Col-0 plants using the floral dipping method. All plants were grown under the same conditions (16 h light/8 h dark at 22°C). Primers used for plasmid construction are listed in Supplementary Table S2. Dormancy and germination assay For seed dormancy analysis, developing siliques at the long-green stage (15 d after pollination) were collected from the consistent position of the inflorescence of each plant. Once the siliques turned brown and opened, mature seeds were harvested and used for analyses. Harvested seeds were stored in the open air with approximately 45% relative humidity at 22°C. Surface-sterilized seeds were sown on water–agarose plates which are made from ddH2O plus 0.6% agarose at pH 5.7, and the seeds were treated with or without stratification at 4°C for 2 d (Barrero et al. 2010). Seeds were grown in a growth chamber under 16 h light/8 h dark at 22°C. Seeds with emerging radicles were defined as germinated. GA3 (G7645, Sigma-Aldrich), PAC (P687, Phytotech) or pepstatin A (P5318, Sigma-Aldrich) were added to the water–agarose medium as needed for the various treatments. To analyze the phenotype of seed dormancy and seed germination statistically, >100 seeds, which were harvested at the same time from three individual plants of each genotype, were used for each experiment. All assays were performed more than three times. RNA extraction and gene expression analysis To analyze the expression level of genes, quantitative reverse transcription–PCR (qRT–PCR) was used. Total RNA was extracted from dry and imbibed seeds using the RNAprep Pure Plant Kit (DP441, TIANGEN). The first-strand cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen). Then, the cDNAs were amplified using Advanced SYBR Green supermix (Bio-Rad) with the CFX connect real-time PCR detection system 185-5201 (Bio-Rad). Arabidopsis SECRETION-ASSOCIATED RAS SUPER FAMILY 1 (SAR1, At4g02080) was used as the internal control (Dekkers et al. 2012). The relative gene expression level was analyzed using the CFX manager software (Bio-Rad). Three biological repeats were carried out for each experiment. Each experiment was performed at least three times with similar results, and one representative result is shown. Primers used for qRT–PCR are listed in Supplementary Table S3. Embryo growth and seed coat bedding assays The seed coat bedding assay was modified from the protocol described by Lee and Lopez-Molina (2013). Seed coats and embryos were isolated from freshly harvested seeds which had been imbibed for 2 h. Fine syringe needles (0.45 × 28.5 mm, Hongda) were used for the dissection. In the process of the dissection, seeds were all placed on a wet Whatman 3MM paper. Isolated coatless embryos were transferred to water–agarose plates or laid on a layer of seed coats on the surface of water–agarose plates. The embryos were incubated in a growth chamber under 16 h light/8 h dark at 22°C. The images of embryo growth were acquired using a Nikon SMZ1500 stereomicroscope. The length of emerged radicles was measured using Image-Pro Plus software (http://www.mediacy.com/imageproplus, Media Cybernetics). Seeds from three individual plants of each genotype were harvested at the same time. At least 60 embryos which were isolated from seeds of each genotype were used for each experiment. This experiment was repeated three times in order to obtain statistical analytic data. Histochemical GUS assay The transgenic plants carrying plasmid ASPG1pro-GUS were generated as described previously (Yao et al. 2012). To determine the expression pattern of ASPG1pro-GUS, the embryos were dissected from seeds stored for 2 weeks which were treated with or without stratification; afterwards they were transferred to water–agarose plates for 0 or 24 h. For the gibberellic acid treatment, GA3 (1.0 μM) was added to the plates. The ethanol was added to the plates as for the controls (Mock). The GUS assay was performed following the method described by Jefferson et al. (1987). The GUS assay solution was made with 50 mM sodium phosphate buffer (pH 7.2), 0.2% Triton X-100, 5 mM ferrocyanide, 5 mM ferricyanide and 2 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid. The images were acquired using the Nikon SMZ1500 stereomicroscope. Analysis of seed storage proteins Total soluble proteins in dry and imbibed seeds were extracted following the protocol described by Chibani et al. (2006) with minor modifications. Seeds (100 mg) were homogenized into powder in liquid nitrogen. The powder was suspended in 1.2 ml of a thiourea/urea lysis buffer which comprised 7 M urea, 2 M thiourea, 4% CHAPS, 18 mM Tris–HCl (Trizma HCl), 14 mM Trizma Base, 215 μl of protease inhibitor cocktail complete Mini (Roche Diagnostics) from one tablet dissolved in 1.5 ml of sterilized water, 53 U ml–1 DNase I, 4.9 K U ml–1 RNase A and 0.2% (v/v) Triton X-100. After incubating for 10 min at 4°C, protein extracts were centrifuged (15,000×g for 10 min) at 4°C. The supernatants were subjected to a second centrifugation (15,000×g for 10 min) and stored at –20°C. The concentration of purified proteins was measured using the Bradford Protein Assay Kit (P0006C, Beyotime), and BSA (bovine serum albumin) was used as the standard. A 30 μg aliquot of protein of each sample was loaded for electrophoresis (140 V, 45 min) in the SDS–polyacrylamide gel (4–20%, ExpressPlus) (Genscript). The gel was stained using Coomassie Brilliant Blue R250 (ST031, Beyotime) for visualization. The image was taken using the Syngene Bio imaging system (InGenius, Syngene). Natural and artificial aging assay To evaluate seed longevity, we performed natural and artificial aging assays. For the natural aging assay, seeds harvested from three individual plants of each genotype were stored at room temperature for 0.5, 1.0, 2.0, 3.0 or 4.0 years before they were tested for viability in germination. For the artificial aging assay, the CDT was performed according to the protocol of Tesnier et al. (2002). Briefly, freshly harvested seeds were stored in the open air at room temperature for 2 weeks, and then they were transferred to an opened 1.5 ml Eppendorf tube and stored above a saturated NaCl solution in a closed desiccator (80% relative humidity and temperature at 40°C). After treatment for 0, 1, 2 or 3 weeks, the germination assay was performed. Seed longevity was determined on the basis of seed germination and seedling abnormality. Seedlings were judged as ‘abnormal’ if they were showing any malformation. Normally growing seedlings from germinations of the 0.5-year-old Col seeds were used as the control. The malformation phenotypes were categorized as: no cotyledons, asymmetrical cotyledons, narrow vitrified cotyledons, chlorotic or albino cotyledons, no cauline apex, no root, and short or elongated hypocotyls. To examine the viability of non-germinated seeds in the aging assay, the embryos of non-germinated seeds were isolated and incubated in 1% (w/v) aqueous solution of TTC (Sigma, T8877) at 30°C in the dark for 2 d, according to the method described by Wharton (1955). Viable seeds can be stained red whereas dead seeds cannot be stained. Measurements of ABA contents A 100 mg aliquot of seeds of Col and aspg1-1 stored for 2 weeks were used for the measurement of ABA contents. This experiment was carried out according to the method described in our previous report (Yao et al. 2012). Three biological replicates were conducted. Seedling survival and growth under no nutrient supply Seeds stored for 2 weeks were grown on water–agarose plates or on MS medium (Murashige and Skoog 1962) containing 1% sucrose and 0.6% agarose. Survival rates were calculated after 3 weeks of incubation. Seedlings were determined to be dying when their leaves turned pale or transparent. To evaluate the rate of survival of seedlings, the Chl contents were measured. All seedlings which were undergoing survival rate analysis were collected and used for determination of Chl contents. Total Chl was extracted in 85% acetone as described by Porra et al. (1989). The Chl content was determined at spectrophotometer settings of 639 and 645 nm (SpectraMax M2, Molecular Devices). All experiments were performed three times independently. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Nation Natural Science Foundation of China [grants to Y.W. (31270333 and 90817013)] and the Ministry of Science and Technology of China [the Major State Basic Research Program (2013CB126900)]. Acknowledgments We thank the members of the Wu Lab for their technical help and comments on this manuscript. We thank the ‘Large-scale Instrument and Equipment Sharing Foundation of Wuhan University’ for supporting the use of the instruments in the College of Life Sciences in Wuhan University. Disclosures The authors have no conflicts of interest to declare. References Arc E. , Sechet J. , Corbineau F. , Rajjou L. , Marion-Poll A. ( 2013 ) ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination . Front. Plant Sci. 4 : 63. Barrero J.M. , Millar A.A. , Griffiths J. , Czechowski T. , Scheible W.R. , Udvardi M. 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( 2011 ) SCARECROW-LIKE 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis . Proc. Natl. Acad. Sci. USA 108 : 2160 – 2165 . Abbreviations Abbreviations ABI ABA INSENSITIVE CDT controlled deterioration test Col Columbia Com complementation DOG DELAY OF GERMINATION FUS3 FUSCA3 GUS β-glucuronidase LEC LEAFY COTYLEDON MS Murashige and Skoog NCED 9-CIS-EPOXYCAROTENOID DIOXYGENASE OE overexpression PAC paclobutrazol PSV protein storage vacuole qRT–PCR quantitative reverse transcription–PCR RDO REDUCED DORMANCY RNAi RNA interference SSP seed storage protein TRX thioredoxin TTC 2,3,5-triphenyltetrazolium chloride UPS ubiquitin–proteasome system © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Plant and Cell Physiology – Oxford University Press
Published: Apr 10, 2018