Molecular Mechanisms Underlying How Arabidopsis SRPK And AFC Family Proteins Regulate Plant Development,ABA And Salt Stress

The alternative splicing of precursor mRNA is a crucial step of post-transcriptional regulation,which can produce different transcripts,thereby increasing protein diversity and regulating various physiological processes in plants.The SR protein family is an important group of splicing factors that regulate splicing by identifying and binding splicing sites and recruiting active spliceosomes.In mammalian and yeast cells,the localization and function of SR proteins are influenced by the phosphorylation of their RS domain.However,little is known about how phosphorylation of SR protein affects plant growth and development as well as response to abiotic stress by regulating gene transcription and alternative splicing.In this study,Arabidopsis thaliana was used as plant material to study the biological function and mechanism of two families of SR protein kinases SRPK and AFC families.The functional deletion mutants of SRPK or AFC family were taken as the research object by comprehensive use of molecular,cellular,genetic and biochemical experimental techniques.The mechanism of SRPK and AFC in responses to growth and development,ABA and salt stress was analyzed,which pave the way for future agricultural production and plant alternative splicing.The main findings and conclusions are summarized as follows:1.The SRPKⅡ family of Arabidopsis thaliana srpkii-1 mutant exhibits developmental defects such as late flowering and fruit order disorder,as well as ABA and salt-sensitive phenotypes of delayed seed germination,suggesting that the SRPKⅡ family of Arabidopsis thaliana plays an important role in plant development,ABA and salt stress response.2.The SRPKⅡ family proteins are located in the nucleus and cytoplasm.They are widely distributed in various tissues.In addition,the expression of SRPKⅡ genes is induced by exogenous ABA and salt signals,indicating that the SRPKⅡ genes have a broad spectrum of functional sites in plants and play an important functional role in various physiological processes.3.The SRPKⅡ family protein regulates the phosphorylation of specific SR proteins(including SR45 and SC35)and affect the localization of SR proteins in cytoplasm and nucleus,indicating that SRPKⅡ proteins affect the function of SR proteins by mediating their subcellular localizations.4.At the genetic level,it was confirmed that the Arabidopsis SRPKⅡ family regulates plant growth and development by influencing SR45 phosphorylation and responds to ABA and salt stress,indicating that SRPKⅡ family mediated SR45 phosphorylation plays an important role in plant growth and development and response to ABA and salt stress.5.Alternative splicing of many ABA-responsive genes,especially intron retention events(RI),such as HAB1.2/HAB1.1(RI)ratio,was significantly higher in the srpkii-1 mutant than in the wild type.Overexpression of HAB1.1 in srpkii-1 partially rescues for the ABA-sensitive phenotype,suggesting that the SRPKⅡ family proteins regulate alternative splicing of HAB1 in response to ABA signal transduction.In addition,overexpression of SR45(S/D)in srpkii-1partially rescues for the ABA-sensitive phenotype,suggesting that the SR45 phosphorylation state regulated by the SRPKⅡ family proteins respond to ABA signal transduction,thereby regulating seed germination and early seedling development.6.The ABA-sensitive phenotype of srpkii-1 was rescued by knocking out ABI4 or ABI5,the positive regulator of ABA transduction,indicating that Arabidopsis SRPKⅡ family regulates ABA signal transduction through the HAB1-ABI4/5 module.7.Compared with the srpkii mutant,the srpki/ii quinqual mutant showed no more obvious growth and ABA-sensitive phenotype,but showed salt-sensitive phenotype with delayed seed germination,indicating that both SRPK family proteins of Arabidopsis respond to salt stress,but only the SRPKⅡ family protein plays a key role in plant growth and development and ABA response.8.Arabidopsis AFC protein family functionally deficient mutant afc123 has no growth and developmental deficiency phenotype,but has ABA and salt-sensitive phenotype of delayed seed germination,indicating that AFC family does not play an important role in normal plant growth and development,but responds to ABA and salt stress.9.srpki/ii afc2 and srpki/ii afc3 mutants showed no further growth and developmental defect phenotype compared with srpki/ii mutants,but showed more obvious ABA-sensitive phenotype with delayed seed germination than srpkii mutants,indicating that SRPK family co-regulates ABA signal transduction with AFC2 or AFC3.In summary,our results suggest that the SRPKⅡ family of Arabidopsis thaliana regulates the phosphorylation status of the SR protein and regulates the expression and alternative splicing of FLC,thereby controlling flowering time.Arabidopsis SRPKⅡ family regulates seed germination and early seedling development by mediating the phosphorylation of SR45,regulating alternative splicing of ABA signal response gene HAB1,and regulating ABA signal transduction by HAB1-ABI4/5 model.The Arabidopsis SRPKⅡ family responds to salt stress by mediating the phosphorylation of SR45.These findings further indicate the importance of the Arabidopsis SRPKⅡ family in plant growth and development.ABA and salt stress signaling pathways.Enabl plants to respond rapidly to the changing surrounding environment.Our study provided evidence on how plant alternative splicing regulated by SR protein kinase and might contribute to future agricultural production against abiotic stresses.