The plant-specific SCL30a SR protein regulates ABA-dependent seed traits and salt stress tolerance during germination

SR (serine/arginine-rich) proteins are conserved RNA-binding proteins best known as key regulators of splicing, which have also been implicated in other steps of gene expression. Despite mounting evidence for their role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain elusive. Here we show that the plant-specific SCL30a SR protein negatively regulates abscisic acid (ABA) signaling to control important seed traits and salt stress responses during germination in Arabidopsis. The SCL30a gene is upregulated during seed imbibition and germination, and its loss of function results in smaller seeds displaying enhanced dormancy and elevated expression of ABA-responsive genes as well as of genes repressed during the germination process. Moreover, the knockout mutant is hypersensitive to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA sensitivity and enhanced tolerance to salt stress during seed germination. An ABA biosynthesis inhibitor rescues the mutant’s enhanced sensitivity to stress, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the SR protein positively regulates stress tolerance during seed germination by reducing sensitivity to the phytohormone. Our results reveal a new key player in ABA-mediated control of early development and stress response, and underscore the role of plant SR proteins as important regulators of the ABA signaling pathway. Author Summary Seed germination is a critical step in plant development determining the transition to aerial growth and exposure to a more challenging environment. As such, seeds have evolved mechanisms that prevent germination under adverse conditions, thereby increasing the chances of plant survival. As a general regulator of plant development and a key mediator of stress responses, the hormone abscisic acid (ABA) promotes a prolonged non-germinating state called dormancy, influences seed size and represses germination under environmental stress. Here, we show that an RNA-binding protein, SCL30a, controls seed size, dormancy, germination and tolerance to high salinity in the model plant Arabidopsis thaliana. Loss of SCL30a gene function results in smaller and more dormant seeds with reduced ability to germinate in a high-salt environment; by contrast, SCL30a overexpression produces larger seeds that germinate faster under salt stress. Using a large-scale gene expression analysis, we identify the ABA hormonal pathway as a putative target of SCL30a. We then use genetic and pharmacological tools to unequivocally demonstrate that the uncovered biological functions of SCL30a are achieved through modulation of the ABA pathway. Our study reveals a novel regulator of key seed traits and has biotechnological implications for crop improvement under adverse environments.

Introduction 206 only about one third of that of wild-type seeds (Fig 2B). The germination rate of stratified 207 scl30a-1 mutant seeds was slightly lower, exhibiting a significant delay when compared to 208 wild-type seeds (Fig 2C). 209 The seed phenotypes of the scl30a-1 mutant prompted us to analyze the expression of the 210 ABI3 and ABI5 genes, two major transcriptional regulators controlling seed development, 211 dormancy and germination [37,38]. RT-qPCR analyses of germinating seeds showed that the 212 expression of ABI5, and to a lesser extent also ABI3, is significantly increased in the scl30a-1 213 mutant (Fig 2D). In agreement, the expression of Em1, Em6, and LEA4-5, three downstream 214 targets of the ABI3 and ABI5 transcription factors [39,40], was also upregulated in scl30a-1, 215 even to a larger extent (Fig 2D). 216 These findings indicate that the SCL30a SR protein plays an in vivo role in embryonic 217 tissues, where it affects seed size, dormancy and germination, and controls the expression of 218 key genes regulating seed development and germination.  (Table 1). Although of low magnitude (ΔPSI < 25 in all cases), the RNA-seq alternative 232 splicing changes were confirmed in the four events selected for validation by RT-PCR, using 233 wild-type and scl30a-1 RNA samples independent from those analyzed by RNA-seq (S2 Fig). 234 Interestingly, all of the seven differentially-regulated IR events showed lower inclusion levels 235 in the scl30a-1 mutant, suggesting that SCL30a negatively regulates splicing of these introns. 260 Our RNA-seq analysis revealed 382 genes whose expression was significantly changed by at 261 least two-fold in the scl30a-1 mutant. Among these, 315 displayed higher transcript levels than 262 the wild type, whereas 67 were downregulated in the scl30a-1 mutant (S2 Table).

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Given the seed and germination phenotypes of the scl30a-1 mutant (see Fig 2), we then 264 asked whether the genes whose expression was affected by the SCL30a protein were 265 transcriptionally regulated during the seed germination process. To address this question, we 291 in the mutant (Fig 2D and S3 Table), while 49 % (33 genes) of the genes 301 downregulated in the scl30a-1 mutant were repressed by ABA (Fig 4D and S6 Table). We then  2). In contrast to what was observed for the scl30a-1 mutant, imbibed seeds 317 from the SCL30a-overexpressing plants were significantly (10%) larger than those from wild-318 type plants ( Fig 5B and S3B Fig). Furthermore, stratified SCL30a-overexpressing seeds 319 germinated slightly faster under control conditions than wild-type seeds (Fig 5C).

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The differential expression of ABA-related genes observed in the scl30a-1 mutant prompted 321 us to analyze ABA response of the different genotypes during germination. We found that the  343 NaCl on the germination of wild-type seeds (Fig 6A). Most importantly, the presence of 344 fluridone rescued the salt stress hypersensitive phenotype of the scl30a-1 mutant, which 345 germinated at rates similar to the wild type in NaCl (Fig 6A). This result indicates that the 346 mutant's salt stress germination phenotype depends on endogenous ABA production.  358 We then assessed the seed size and dormancy of the different genotypes. Both the aba2-1 359 and the abi4-101 mutations suppressed the reduced size displayed by scl30a-1 seeds, with the 360 area of scl30a-1aba2-1 imbibed seeds being even significantly larger than those of the wild 361 type, as previously reported for the aba2-1 mutant [8] (Fig 6C). Regarding seed dormancy, the 362 double mutants again showed strikingly similar phenotypes to those induced by single 363 mutations in the ABA2 and ABI4 genes that, in agreement with early reports [6,57], conferred 364 strongly reduced and normal dormancy, respectively ( Fig 6D). Therefore, both ABA2 and ABI4 365 are epistatic to the SCL30a gene, indicating that the seed/germination roles of the encoded SR 366 protein are fully dependent on a functional ABA pathway.

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The above findings raised the question of whether changes in SCL30a levels affect ABA 368 biosynthesis or sensing/signaling of the stress hormone. To address this issue, we measured the 369 endogenous ABA content of wild-type, scl30a-1 mutant and SCL30a-overexpressing seeds 370 germinated in control conditions or under high salinity stress.  478 Seed imbibition (Fig 1, 2A, 5B and 6C) was always performed at 4 ºC (equivalent to 479 stratification). After 2-3 weeks, seedlings were transferred to soil in individual pots.

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PCR-based genotyping of the SALK_041849 line (obtained from NASC) with primers 481 specific for SCL30a and the left border of the T-DNA (S7 Table)

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The area of dry and imbibed seeds was measured using the ImageJ software 508 (http://rsbweb.nih.gov/ij). To determine seed weight, six groups of 1000 dry seeds were 509 weighed using an Acculab ALC-80.4 (Sartorius) analytical balance.

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For compositional analysis, dry seeds were bulk harvested by genotype and homogenized.