Serine/arginine-rich Genes Research Articles

Evidence Supporting a Role of Alternative Splicing Participates in Melon (Cucumis melo L.) Fruit Ripening.

One key post-transcriptional modification mechanism that dynamically controls a number of physiological processes in plants is alternative splicing (AS). However, the functional impacts of AS on fruit ripening remain unclear. In this research, we used RNA-seq data from climacteric (VED, Harukei 3) and non-climacteric (PI, PS) melon cultivars to explore alternative splicing (AS) in immature and mature fruit. The results revealed dramatic changes in differential AS genes (DAG) between the young and mature fruit stages, particularly in genes involved in fruit development/ripening, carotenoid and capsaicinoid biosynthesis, and starch and sucrose metabolism. Serine/arginine-rich (SR) family proteins are known as important splicing factors in AS events. From the melon genome, a total of 17 SR members were discovered in this study. These genes could be classified into eight distinct subfamilies based on gene structure and conserved motifs. Promoter analysis detected various cis-acting regulatory elements involved in hormone pathways and fruit development. Interestingly, these SR genes exhibited specific expression patterns in reproductive organs such as flowers and ovaries. Additionally, concurrent with the increase in AS levels in ripening fruit, the transcripts of these SR genes were activated during fruit maturation in both climacteric and non-climacteric melon varieties. We also found that most SR genes were under selection during domestication. These results represent a novel finding of increased AS levels and SR gene expression during fruit ripening, indicating that alternative splicing may play a role in fruit maturation.

Transcription and splicing variations of SR genes accompany with genome-wide accumulation of long-introns in pine

Most of mRNAs in Eukaryote were matured after the removal of introns in their pre-mRNA transcripts. Serine/arginine-rich (SR) proteins are a group of splicing regulators regulating the splicing processes globally. Expressions of SR proteins themselves were extensively regulated, at both transcription and splicing levels, under different environmental conditions, specially heat stress conditions. The pine genome is characterized by super-long and easily methylated introns in a large number of genes that derived from the extensive accumulation of transposons (TEs). Here, we identified and analyzed the phylogenetic characteristics of 24 SR proteins and their encoding genes from the pine genome. Then we explored transcription and pre-mRNA splicing expression patterns of SR genes in P. massoniana seedlings under normal and heat stress temperature conditions. Our results showed that the transcription patterns of SR genes in pine exhibited significant changes compared to other plant species, and these changes were not strictly correlated with the intron length and DNA methylation intensity of the SR genes. Interestingly, none of the long introns of SR genes underwent alternative splicing (AS) in our experiment. Furthermore, the intensity of AS regulation may be related to the potential DNA methylation intensity of SR genes. Taken together, this study explores for the first time the characteristics of significant variations in the transcription and splicing patterns of SR proteins in a plant species with an over-accumulation of super-long introns.

SCR106 splicing factor modulates abiotic stress responses by maintaining RNA splicing in rice.

Plants employ sophisticated molecular machinery to fine-tune their responses to growth, developmental, and stress cues. Gene expression influences plant cellular responses through regulatory processes such as transcription and splicing. Pre-mRNA is alternatively spliced to increase the genome coding potential and further regulate expression. Serine/arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Several studies have reported SR protein genes in the rice genome, subdivided into six subfamilies based on their domain structures. Here, we identified a new splicing factor in rice with an RNA recognition motif (RRM) and SR-dipeptides, which is related to the SR proteins, subfamily SC. OsSCR106 regulates pre-mRNA splicing under abiotic stress conditions. It localizes to the nuclear speckles, a major site for pre-mRNA splicing in the cell. The loss-of-function scr106 mutant is hypersensitive to salt, abscisic acid, and low-temperature stress, and harbors a developmental abnormality indicated by the shorter length of the shoot and root. The hypersensitivity to stress phenotype was rescued by complementation using OsSCR106 fused behind its endogenous promoter. Global gene expression and genome-wide splicing analysis in wild-type and scr106 seedlings revealed that OsSCR106 regulates its targets, presumably through regulating the alternative 3'-splice site. Under salt stress conditions, we identified multiple splice isoforms regulated by OsSCR106. Collectively, our results suggest that OsSCR106 is an important splicing factor that plays a crucial role in accurate pre-mRNA splicing and regulates abiotic stress responses in plants.

Comprehensive study of serine/arginine-rich (SR) gene family in rice: characterization, evolution and expression analysis.

As important regulators of alternative splicing (AS) events, serine/arginine (SR)-rich proteins play indispensable roles in the growth and development of organisms. Until now, the study of SR genes has been lacking in plants. In the current study, we performed genome-wide analysis on the SR gene family in rice. A total of 24 OsSR genes were phylogenetically classified into seven groups, corresponding to seven subfamilies. The OsSR genes' structures, distribution of conserved domains, and protein tertiary structure of OsSR were conserved within each subfamily. The synteny analysis revealed that segmental duplication events were critical for the expansion of OsSR gene family. Moreover, interspecific synteny revealed the distribution of orthologous SR gene pairs between rice and Arabidopsis, sorghum, wheat, and maize. Among all OsSR genes, 14 genes exhibited NAGNAG acceptors, and only four OsSR genes had AS events on the NAGNAG acceptors. Furthermore, the distinct tissue-specific expression patterns of OsSR genes showed that these genes may function in different developmental stages in rice. The AS patterns on the same OsSR gene were variable among the root, stem, leaf, and grains at different filling stages, and some isoforms could only be detected in one or a few of tested tissues. Meanwhile, our results showed that the expression of some OsSR genes changed dramatically under ABA, GA, salt, drought, cold or heat treatment, which were related to the wide distribution of corresponding cis-elements in their promoter regions, suggesting their specific roles in stress and hormone response. This research facilitates our understanding of SR gene family in rice and provides clues for further exploration of the function of OsSR genes.

Insights into sweet potato SR proteins: from evolution to species-specific expression and alternative splicing.

SR proteins from sweet potato have conserved functional domains and similar gene structures as that of Arabidopsis and rice in general. However, expression patterns and alternative splicing regulations of SR genes from different species have changed under stresses. Novel alternative splicing regulations were found in sweet potato SR genes. Serine/arginine-rich (SR) proteins play important roles in plant development and stress response by regulating the pre-mRNA splicing process. However, SR proteins have not been identified so far from an important crop sweet potato. Through bioinformatics analysis, our study identified 24 SR proteins from sweet potato, with comprehensively analyzing of protein characteristics, gene structure, chromosome localization, and cis-acting elements in promotors. Salt, heat, and mimic drought stresses triggered extensive but different expressional regulations on sweet potato SR genes. Interestingly, heat stress caused the most active disturbances in both gene transcription and pre-mRNA alternative splicing (AS). Tissue and species-specific transcriptional and pre-mRNA AS regulations in response to stresses were found in sweet potato, in comparison with Arabidopsis and rice. Moreover, novel patterns of pre-mRNA alternative splicing were found in SR proteins from sweet potato. Our study provided an insight into similarities and differences of SR proteins in different plant species from gene sequences to gene structures and stress responses, indicating SR proteins may regulate their downstream genes differently between different species and tissues by varied transcriptional and pre-mRNA AS regulations.

A Comprehensive Identification and Function Analysis of Serine/Arginine-Rich (SR) Proteins in Cotton (Gossypium spp.).

As one of the most important factors in alternative splicing (AS) events, serine/arginine-rich (SR) proteins not only participate in the growth and development of plants but also play pivotal roles in abiotic stresses. However, the research about SR proteins in cotton is still lacking. In this study, we performed an extensive comparative analysis of SR proteins and determined their phylogeny in the plant lineage. A total of 169 SR family members were identified from four Gossypium species, and these genes could be divided into eight distinct subfamilies. The domain, motif distribution and gene structure of cotton SR proteins are conserved within each subfamily. The expansion of SR genes is mainly contributed by WGD and allopolyploidization events in cotton. The selection pressure analysis showed that all the paralogous gene pairs were under purifying selection pressure. Many cis-elements responding to abiotic stress and phytohormones were identified in the upstream sequences of the GhSR genes. Expression profiling suggested that some GhSR genes may involve in the pathways of plant resistance to abiotic stresses. The WGCNA analysis showed that GhSCL-8 co-expressed with many abiotic responding related genes in a salt-responding network. The Y2H assays showed that GhSCL-8 could interact with GhSRs in other subfamilies. The subcellular location analysis showed that GhSCL-8 is expressed in the nucleus. The further VIGS assays showed that the silencing of GhSCL-8 could decrease salt tolerance in cotton. These results expand our knowledge of the evolution of the SR gene family in plants, and they will also contribute to the elucidation of the biological functions of SR genes in the future.

Alternative splicing analysis provides insight into the domestication and improvement of pear (Pyrus pyrifolia) fruit

Alternative splicing (AS) plays a vital role in response to environmental stress. However, the functional impacts of AS on fruit quality in domestication and improvement remain unknown. In this study, we analyzed AS in fruit using RNA-seq of wild, landrace, and improved pear accessions. A total of new 237,085 transcripts were mapped to 29,254 genes. Based on these assembled transcripts, 191,060 AS events for 15,324 genes were identified, of which retained intron (RI) was the most frequent AS type, accounting for approximately 46.96% of AS events. Furthermore, 847 and 841 differential AS genes (DASGs) were identified during domestication and improvement, respectively. According to gene ontology (GO) enrichment analysis, the DASGs exclusively during domestication affected glycosylation and carbohydrate metabolism, while the DASGs exclusively during improvement were enriched in response to chemical stimulus. We found little overlap between DASGs and differentially expressed genes (DEGs) in both the domestication and improvement processes, which indicated that AS was another gene regulatory mechanism for fruit development. Eight serine/arginine-rich (SR) genes related to AS regulation were differentially alternatively spliced during domestication or improvement, which indicated that SR genes might play regulatory roles in AS during the two processes in pear. Combined with gene annotation and previously identified quantitative trait loci (QTLs), 91 candidate DASGs related to fruit traits, including genes involved in sugar metabolism, acid metabolism, stone cell formation, and fruit firmness, were screened out during the investigation of domestication and improvement. Among them, Pectin acetylesterase 8 (PbrPAE8) produced PbrPAE8_1 and PbrPAE8_2 transcripts through AS, which was validated by qRT-PCR. The significant difference of AS ratio between landrace and improved groups were correlated with fruit firmness variability in these two populations. Our analysis is the first to reveal that AS variation is related to fruit quality during domestication and improvement of pear. Taken together, our study enriches the understanding of AS and its potential role in pear fruit improvement, thus providing useful references for other fruit tree crops.

Genome-Wide Characterization of Serine/Arginine-Rich Gene Family and Its Genetic Effects on Agronomic Traits of Brassica napus

Serine/arginine-rich (SR) proteins are indispensable factors for RNA splicing, and they play important roles in development and abiotic stress responses. However, little information on SR genes in Brassica napus is available. In this study, 59 SR genes were identified and classified into seven subfamilies: SR, SCL, RS2Z, RSZ, RS, SR45, and SC. In each subfamily, the genes showed relatively conserved structures and motifs, but displayed distinct expression patterns in different tissues and under abiotic stress, which might be caused by the varied cis-acting regulatory elements among them. Transcriptome datasets from Pacbio/Illumina platforms showed that alternative splicing of SR genes was widespread in B. napus and the majority of paralogous gene pairs displayed different splicing patterns. Protein-protein interaction analysis indicated that SR proteins were involved in the regulation of the whole lifecycle of mRNA, from synthesis to decay. Moreover, the association mapping analysis suggested that 12 SR genes were candidate genes for regulating specific agronomic traits, which indicated that SR genes could affect the development and hence influence the important agronomic traits of B. napus. In summary, this study provided elaborate information on SR genes in B. napus, which will aid further functional studies and genetic improvement of agronomic traits in B. napus.

Identification and Regulation of Tomato Serine/Arginine-Rich Proteins Under High Temperatures.

Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.

Overexpression of SCL30A from cassava (Manihot esculenta) negatively regulates salt tolerance in Arabidopsis.

Soil salinity is a significant threat to sustainable agricultural production. Plants must adjust their developmental and physiological processes to deal with environmental salt conditions. We previously identified 18 serine-arginine-rich (SR) proteins from cassava (Manihot esculenta Crantz) that play pivotal roles in alternative splicing when encountering the external stress condition. However, functional characterisation of SR proteins is less reported in cassava, which is an important staple crop in the world. In the current study, we found that the expression of cassava spliceosomal component 35-like 30A (MeSCL30A) was significantly induced in response to drought and salt stress. The MeSCL30A overexpressing lines were also obtained in Arabidopsis thaliana L., which flowered earlier when compared with Col-0. Moreover, the MeSCL30A overexpressing lines were hypersensitive to salt and drought stress with lower germination and greening rate in comparison to Col-0. Importantly, soil-grown overexpression lines exhibited salt sensitivity through modulating the reactive oxygen species homeostasis and negatively regulating the gene expression that involved in ionic stress pathway. Therefore, these findings refined the SR protein-coding genes and provided novel insights for enhancing the resistance to environmental stress in plant.

Genome-wide identification and expression analysis of the SR gene family in longan (Dimocarpus longan Lour.)

Longan (Dimocarpus longan Lour.) is an important commercial fruit tree in southern China. The embryogenesis of longan affects the quality and yield of fruit. A large number of alternative splicing events occurs during somatic embryogenesis (SE), which is regulated by serine/arginine-rich (SR) proteins. However, the functions of SR proteins in longan are poorly understood. In this study, 21 Dlo-SR gene family members belonging to six subfamilies were identified, among which Dlo-RSZ20a, Dlo-SR30, Dlo-SR17, Dlo-SR53 and Dlo-SR32 were localized in the nucleus, Dlo-RSZ20b, Dlo-RSZ20c, Dlo-RSZ20d, Dlo-SC18, Dlo-RS2Z29, Dlo-SCL41, and Dlo-SR33 were localized in chloroplasts, and Dlo-RS43, Dlo-SC33, Dlo-SC37, Dlo-RS2Z33, Dlo-RS2Z16, Dlo-RS2Z24, Dlo-SCL43, Dlo-SR112, and Dlo-SR59 were localized in the nucleus and chloroplasts. The Dlo-SR genes exhibited differential expression patterns in different tissues of longan. The transcript levels of Dlo-RSZ20a, Dlo-SC18, Dlo-RS2Z29, DLo-SR59, Dlo-SR53, and Dlo-SR17 were low in all analyzed tissues, whereas Dlo-RS43, Dlo-RS2Z16, Dlo-RS2Z24, and Dlo-SR30 were highly expressed in all tissues. To clarify their function during SE, the transcript levels of Dlo-SR genes were analyzed at different four stages of SE, comprising non-embryonic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic culture (ICpEC) and globular embryo (GE). Interestingly, the transcript levels of Dlo-RS2Z29 and Dlo-SR112 were increased in embryogenic cells compared with the NEC stage, whereas transcript levels of Dlo-RSZ20a, Dlo-RS43, Dlo-SC37, and Dlo-RS2Z16 were especially increased at the GE stage compared with the other stages. Alternative splicing events of Dlo-SR mRNA precursors (pre-mRNAs) was detected during SE, with totals of 41, 29, 35, and 44 events detected during NEC, EC, ICpEC, and GE respectively. Protein–protein interaction analysis showed that SR proteins were capable of interaction with each other. The results indicate that the alternative splicing of Dlo-SR pre-mRNAs occurs during SE and that Dlo-SR proteins may interact to regulate embryogenesis of longan.

Emerging Functions of Plant Serine/Arginine-Rich (SR) Proteins: Lessons from Animals

Serine/arginine-rich (SR) proteins are widely found in higher eukaryotes, including metazoans and plants. They form a major family of essential splicing factors that function in both constitutive splicing (CS) and alternative splicing (AS). Some SR proteins can also shuttle between the nucleus and cytoplasm and have roles beyond splicing. The activities of SR proteins are required for many living organisms to maintain normal growth and development. Although SR genes have been identified in numerous plants, they are less studied than those found in animals. The results of existing research suggest that plant SR proteins share many similar properties with their animal counterparts. However, plants generally have a higher number of SR proteins that display largely redundant functions under normal conditions but participate in specific stress responses. Moreover, many animal and plant SR genes are alternatively spliced and can be regulated by themselves or other SR proteins. These AS events, often coupled to nonsense-mediated decay (NMD), enable a mechanism for protein amount control under different conditions but sometimes also lead to translated protein isoforms with different functions. Here, we summarize the key findings of SR proteins in animals and plants and compare the essential characteristics of SR studies in these two research areas.

Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice.

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.

Genome-wide Characterization of Brassica rapa Genes Encoding Serine/arginine-rich Proteins: Expression and Alternative Splicing Events by Abiotic Stresses

Serine/arginine-rich (SR) gene family members can diversify the transcriptome and proteome of eukaryotes by facilitating the alternative splicing (AS) of precursor messenger RNAs. Herein, we investigated the evolutionary dynamics, AS patterns, and expression levels of the Brassica rapa SR (BrSR) gene family in young seedlings treated with abiotic stresses. A comparative genomic analysis identified 25 BrSR genes at 18 Arabidopsis loci and three BrSR-like genes at two Arabidopsis loci. Thirteen of these loci were singletons, while seven loci carried paralogs. All the duplicated pairs were determined to be under purifying selection pressure. The expansion of the BrSR gene family was found to be the result of segmental duplications only. Additionally, the expression levels of 78.6% (22 of 28) and the AS patterns of 60.7% (17 of 28) of the BrSR genes were altered in response to abiotic stresses. Among the analyzed abiotic stresses, oxidative, cold, and heat treatments induced the largest expression changes, while cold and heat stresses caused most AS events. Our findings provide insights into the evolutionary dynamics of BrSR genes following polyploidization events, and provide an important resource for future studies aimed at characterizing the specific function(s) of BrSR genes in plant growth, development, and defense.

Alternative splicing shapes transcriptome but not proteome diversity in Physcomitrella patens

Alternative splicing (AS) can significantly impact the transcriptome and proteome of a eukaryotic cell. Here, using transcriptome and proteome profiling data, we analyzed AS in two life forms of the model moss Physcomitrella patens, namely protonemata and gametophores, as well as in protoplasts. We identified 12 043 genes subject to alternative splicing and analyzed the extent to which AS contributes to proteome diversity. We could distinguish a few examples that unambiguously indicated the presence of two or more splice isoforms from the same locus at the proteomic level. Our results indicate that alternative isoforms have a small effect on proteome diversity. We also revealed that mRNAs and pre-mRNAs have thousands of complementary binding sites for long non-coding RNAs (lncRNAs) that may lead to potential interactions in transcriptome. This finding points to an additional level of gene expression and AS regulation by non-coding transcripts in Physcomitrella patens. Among the differentially expressed and spliced genes we found serine/arginine-rich (SR) genes, which are known to regulate AS in cells. We found that treatment with abscisic (ABA) and methyl jasmonic acids (MeJA) led to an isoform-specific response and suggested that ABA in gametophores and MeJA in protoplasts regulate AS and the transcription of SR genes.

Insect herbivory elicits genome-wide alternative splicing responses in Nicotiana attenuata.

Changes in gene expression and alternative splicing (AS) are involved in many responses to abiotic and biotic stresses in eukaryotic organisms. In response to attack and oviposition by insect herbivores, plants elicit rapid changes in gene expression which are essential for the activation of plant defenses; however, the herbivory-induced changes in AS remain unstudied. Using mRNA sequencing, we performed a genome-wide analysis on tobacco hornworm (Manduca sexta) feeding-induced AS in both leaves and roots of Nicotiana attenuata. Feeding by M.sexta for 5h reduced total AS events by 7.3% in leaves but increased them in roots by 8.0% and significantly changed AS patterns in leaves and roots of existing AS genes. Feeding by M.sexta also resulted in increased (in roots) and decreased (in leaves) transcript levels of the serine/arginine-rich (SR) proteins that are involved in the AS machinery of plants and induced changes in SR gene expression that were jasmonic acid (JA)-independent in leaves but JA-dependent in roots. Changes in AS and gene expression elicited by M.sexta feeding were regulated independently in both tissues. This study provides genome-wide evidence that insect herbivory induces changes not only in the levels of gene expression but also in their splicing, which might contribute to defense against and/or tolerance of herbivory.

Differential recruitment of splice variants from SR pre-mRNAs to polysomes during development and in response to stresses.

We have previously shown that precursor mRNAs (pre-mRNAs) of serine/arginine-rich (SR) proteins are extensively alternatively spliced to generate approximately 100 distinct splice variants from 14 SR genes and that the splicing pattern of SR pre-mRNAs changes in different organs and in response to abiotic stresses. About half of the splice variants are potential targets of nonsense-mediated decay (NMD) and 25 splice forms were confirmed to be real NMD targets. However, it is not known whether (i) all splice variants are recruited to polysomes for translation; (ii) there is a preferential recruitment of specific splice isoforms to polysomes; and (iii) there is a differential recruitment of splice variants during development and in response to stresses. To address these questions, we analyzed the association of SR splice variants with polysomes from seedlings, different organs and seedlings exposed to heat and cold stress. In seedlings, about one-third of the splice variants (22 out of 72) are not recruited to polysomes. Among those associated with polysomes, the functional isoforms that code for full-length proteins and some candidate putative and confirmed NMD targets were identified. There was preferential recruitment of some splice forms over others. Predominant recruitment of functional isoforms along with a few NMD candidates was found in different organs. Furthermore, we observed differential recruitment of isoforms in different organs. Heat and cold stress enhanced or reduced recruitment of specific splice variants. Our studies reveal differential recruitment of SR splice variants to polysomes under normal conditions, during development and in response to stresses.

Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd2+ uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.

Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression.

Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses.

Discovery and Expression Analysis of Alternative Splicing Events Conserved among Plant SR Proteins

The high frequency of alternative splicing among the serine/arginine-rich (SR) family of proteins in plants has been linked to important roles in gene regulation during development and in response to environmental stress. In this article, we have searched and manually annotated all the SR proteins in the genomes of maize and sorghum. The experimental validation of gene structure by reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed, with few exceptions, that SR genes produced multiple isoforms of transcripts by alternative splicing. Despite sharing high structural similarity and conserved positions of the introns, the profile of alternative splicing diverged significantly between maize and sorghum for the vast majority of SR genes. These include many transcript isoforms discovered by RT-PCR and not represented in extant expressed sequence tag (EST) collection. However, we report the occurrence of various maize and sorghum SR mRNA isoforms that display evolutionary conservation of splicing events with their homologous SR genes in Arabidopsis and moss. Our data also indicate an important role of both 5' and 3' untranslated regions in the regulation of SR gene expression. These observations have potentially important implications for the processes of evolution and adaptation of plants to land.