Function Analysis Of Serine/Arginine-rich (SR) Proteins In Phosphorus Homeostasis In Rice

Phosphorus plays an important role in the growth and development of plants as an essential macroelement.However,the amount of phosphorus that can be used by plants is very limited in the soil.Therefore,it is of great significance to research the mechanism of phosphorus deficiency response in plants for cultivating crops with high phosphorus use efficiency.Alternative splicing plays an important role in plant growth,development and stress response,as a post-transcriptional regulatory mechanism.At present,the research on the regulation of plant phosphorus deficiency response mainly focus on transcriptional regulation,and no studies have been reported from the perspective of alternative splicing.The SR proteins are the important splicing factors that regulate the alternative splicing process in the organism and is related to the process of plant stress response.However,up to now,the mechanism of SR in plant stress resistance is still unclear.Most of the studies on SR function of plants are based on Arabidopsis thaliana,and there are no related reports in crops(or crops such as rice).This study mainly focused on the function of SR proteins in phosphorus homeostasis in rice.There are 22 members in rice SR family.The results of alternative splicing analysis of SR genes in rice under phosphorus deficiency indicated that most members of SR family existed differental alternative splicing under phosphorus deficiency condition.We collected 11 T-DNA insertion mutants of the SR genes,and found that there were 10 SR mutants with significantly higher total phosphorus content in shoots under phosphorus deficiency than those in wild type by analyzing the changes of total P content in these mutants.Among them,ossr40,osscl57,osrs2z36 and ossr33 were the four mutants with the largest difference,which were 46.1%,43.3%,49.5%and 48.9%higher than the wild type respectively.We selected two mutants,ossr40 and osscl57,whose changes in total phosphorus content are larger for further phenotypic analysis.The results showed that ossr40 and osscl57 showed inhibition of growth and brown spot on the tip of old leaves under the condition of sufficient phosphorus(normal phosphorus and high phosphorus),while the growth of two mutants was better than wild type under phosphorus deficiency.The inorganic phosphorus(Pi)content of ossr40 and osscl57 increased from the latest to the oldest leaf,and the most phosphorus was accumulated in the oldest leaf.The results of the time-course experiments showed that the accumulation rate of Pi in root and leaves of ossr40 and osscl57 was faster than that of wild type,and it was obvious that the mobilization of phosphorus in the oldest leaf was inhibited.These results indicate that the SR family is involved in the phosphorus deficiency response in rice.The mutations in SR40 and SCL57 affect the uptake of phosphorus and the reassignment of phosphorus from old leaves to new leaves.The results of alternative splicing analysis of SCL25 showed that SCL25 has 6 transcripts in rice.Among them,SCL25.1 is the major transcript of SCL25,which has the highest expression level and can produce a complete SR protein with a full RRM domain and SR-rich region.Intron retention events occurred in the remaining 5 transcripts.SCL25.2,SCL25.3,SCL25.4,and SCL25.5 introduced a premature termination codon,resulting in the loss of some RRM domains and the the entire SR-rich region.SCL25.6 lacks some of the SR-rich regions in the 3’-end region due to frameshifts.The results of the subcellular localization show that SCL25.1 is a nuclear localization protein,like most of the splicing factors.Tissue localization results showed that SCL25 was expressed in column of roots,rhizomes and leaves.Knocking out SCL25 had no effect on Pi content in roots,but the contents of phosphorus in old leaves were significantly higher than those in wild type under both phosphorus-deficient and phosphorus-sufficient conditions,and tips of the oldest Leaf appear the spot of phosphorus poisoning under high Pi conditions.The results of time-course experiments with different concentrations of Pi treatment showed that the deletion of SCL25 resulted in the obstruction of phosphorus reassignment from old leaf to new leaf in rice.Either overexpression of SCL25.1 or SCL25.2 in rice resulted in an increase of Pi content in the old leaves.Compared with SCL25.2,overexpression of SCL25.1 had a greater effect on Pi content in rice.These results indicate that SCL25 is involved in the maintenance of phosphorus homeostasis in rice,and SCL25.1 plays an important role in this process,but other transcripts of SCL25 may also participate in this regulation process.In order to further understand the regulatory mechanism of SCL25,we performed a high-throughput transcriptome sequencing of rice SCL25 mutant(osscl25-1)and the wild type rice,and we compared the alternative splicing events between them.Four major types of alternative splicing events were detected in the mutant osscl25-1 and wild-type rice genomes,IR,A3SS,A5SS and ES respectively,among them,IR events account for the largest proportion.The mutation SCL25 did not affect the number of splicing events in the rice genome,but resulted in many different alternative splicing events,and we found that there is a small overlap between differential splicing and differentially expressed genes.The results of functional clustering showed that these genes were significantly enriched in several biological metabolic processes related to phosphorus metabolism,such as’phosphorus-containing compounds metabolism’ and‘phosphorus metabolism’.Most of the reported key genes regulating the metabolism of phosphorus metabolism in rice did not show any differences in alternative splicing or differential expression,and we found that only PHR3 had a differential A3SS events.These results indicated that SCL25 may directly participate in the regulation of phosphorus metabolism in rice through regulating the splicing or expression of PHR3 and other genes related to phosphorus metabolism,and this is likely to be a new regulatory pathway besides the known phosphorus regulation network.The exact mechanism remains to be further studied.In addition,some of the differential alternative splicing genes were significantly enriched in the ferrous ion transport pathway and stress-related processes,and we also observed that the SCL25 mutant has a low temperature sensitive phenotype,indicating that SCL25 may also be involved in the regulation of iron homeostasis and low temperature stress response process.In summary,SR40,SCL57,SCL25 and other SR proteins play an important role in the regulation of rice phosphorus homeostasis.