Transcriptome And Proteome Of Photoperiod-sensitive Male Sterile Rice Nongken58S

Photoperiod-sensitive Male Sterile Rice Nongken58S have been found since1973. Three genes controlling Photoperiod-sensitive Male Sterile (pms) have been mapped. Those are designated as pmsl, pms2, pms3, and located on chromosome7, chromosome3and chromosome12respectively. Unfortunately, none of them has been cloned due to difficulty determine the male sterility phenotype in the mapping populations. Previous researches of Photoperiod-sensitive Male Sterility (PMS) have been focused on biochemistry, physiology and cytology. However, the transcriptome and proteome were not investigated. In this work, the transcriptome and proteome of Nongken58S were analyzed by using rice microarray and isobaric Tags for Relative and Absolute Quantization (iTRAQ). The main results as follows:A, to figure out the transcriptional difference between long-day (LD) and short-day (SD) in Nongken58S, we compared the transcriptome of leaf from Nongken58S between SD and LD conditions at the photoperiod-sensitive transformation stages. The results showed that73genes were differentially expressed between SD and LD at the glumes primordium differentiation stage. Five genes were downregulated under LD with Fold Change (FC)>5, while only one gene upregulated under SD with FC≥5;128genes were differentially expressed between SD and LD conditions at the pistil&stamen primordium forming stage. Out of22genes were downregulated under LD with FC≥5, and only five genes upregulated under SD with FC≥5. Those results suggested that gene expression was downregulated under LD at the second photoperiod of Nongken58S. The facts that the gene numbers of differentially expressed under LD were increased with the rice inflorescence development proceeding suggested that gene repression and activation under LD has accumulative effects. It was consistence with accumulative effects of the pollen abortion under LD.B, in order to figure out the regulatory pathways of those genes involved, we used GO annotation and KEGG pathway software to analyze those differentially expressed genes. We found that those genes were falled into circadian rhythm and flowering controlling pathways. The expression level of RICE PSEUDORESPONSE REGULATOR1(OsPRR1), RICE PSEUDORESPONSE REGULATOR37(OsPRR37), RICE GIGANTEA (OsGI) and RICE LATE ELONGATED HYPOCOTYL (OsLHY) involved in circadian rhythm were significantly changed under LD; The expression fold changes of EARLY HEADING DATE1(Ehd1), HEADING DATE3a (Hd3a), RICE FLOWERING LOCUS T1(RFT1), OsMADS BOX1(OsMADS1) involved in flowering pathway were observed between LD and SD, indicating those genes might involved in fertility transformation of Nongken58S.C, to understand the circadian rhythms of the genes involved in fertility transformation, we analyzed circadian rhythm of the genes involved in the circadian rhythm and flowering controlling pathways by quantitative PCR technique. The results showed that the rhythmic patterns of OsPRRl, OsPRR37, OsGI and OsLHY in Nongken58S were significantly different from Nongken58N under LD. It suggested that those genes could participate in fertility transformation at the second photoperiod.D, we found that the expression level of Ehd2, Ehd1, Hd3a, RFT1and OsMADS1were downregulated under LD, while no difference in circadian rhythmic patterns between Nongken58S and Nongken58N were observed. However both OsGI and Hd1were not inhibited under LD and appears the similar circadian rhythm pattern. And circadian rhythm pattern of Hd1and OsGI were significantly different between Nongken58S and Nongken58N. It strongly suggested that Hd1and OsGI could participate in the fertility transformation, speculating that OsGI is located on the upstream of Hd1.E, the expression level of a transcriptional factor belongs to Dof type zinc finger (OsDof) in Nongken58S is significantly different from Nongken58N and it showed similar pattern with OsLHY based on the microarray data. The quantitative PCR analysis revealed that the circadian rhythmic pattern of OsDof in Nongken58S was different from Nongken58N. These results suggested that OsDof could involve in the fertility transformation and might be on the downstream of OsLHY.F, no difference of circadian rhythmic patterns of photoreceptors, such as phytochromes and crytochromes, were found between Nongken58S and Nongken58N. It suggested that photoreceptors of phytochromes and crytochromes could not directly affect fertility transformation of Nongken58S under LD condition.G., to find out whether the difference of PHYA, PHYB and CRY1b in spatial specificity existed between Nongken58S and Nongken58N under LD and SD, we detected the expression patterns of those genes in inflorescence by tissue in situ hybridization. The results showed that PHYA, PHYB and CRY1b mRNA were enriched in shoot apical meristem of inflorescence primordium, but no tissue specificity difference were observed between Nongken58S and Nongken58N under LD and SD conditions.H, to figure out whether there were differences on translational level during fertility transformation, we compared the leaf proteome between Nongken58S and Nongken58N under LD at the photoperiod-sensitive stages by iTRAQ. Of75proteins were found to differentially express at the glume primordium differentiation stage, while102genes were found to differentially express at the pistil&stamen primordium forming stage. Of33proteins expression level in Nongken58S were higher than that of Nongken58N at the glume primordium differentiation stage, while the number of genes differentially expressed were increased up to62in Nongken58S at the pistil&stamen primordium forming stage. This implies that the inhibition effect in Nongken58S under LD was weaker than that of Nongken58N. These results were consistence with the transcriptomic analysis.I, the proteomic results indicated that proteins involved in photosynthesis in Nongken58S were affected under LD. Amounts of13proteins in Nongken58S were lower than that of in Nongken58N at the glume primordium differentiation stage, while amounts of other13proteins in Nongken58S were higher than that of in Nongken58N at the pistil&stamen primordium forming stage. Those proteins are photosynthesis related suggested that the chloroplast in Nongken58S could be abnormal function. This results were consistence to the previous studies of the abnormal structure of chloroplasts in Nongken58S under LD.J, we found six proteins related to translational machinery were downregulated in Nongken58S, while11proteins translation related were inhibited at the pistil&stamen primordium forming stage. It implies that the proteins related to translational machinery in Nongken58S were affected under LD condition.Summarily, based on the analysis of transcriptome and expression the circadian rhythm patterns of those genes, a regulatory model of PMS was proposed. In this model, the fertility transformation of Nongken58S under LD was simultaneously regulated by both circadian rhythm and flowering control pathways. In the circadian rhythm signal transduction pathway, the signal transduction was passed to the downstream regulators, such as OsDof could be via circadian rhythm genes OsPRR1, OsPRR37, OsGI and OsLHY, and then the signals was passed to the other downstream regulators and finally caused the pollen abortion. In flowering control pathway, the light signal from LD firstly activated OsG1, and then it is via activation of Hdl gene expression to pass the signal to next unknown genes that control the pollen development to induce the pollen abortion.