| Photoperiod/temperature-sensitive male sterile rice has a second Photoperiod-sensitive stage. Previous studies indicated that the two Photoperiod-sensitive stages were not overlapped, and the second Photoperiod-sensitive stage was not well studied. Transcriptome analysis of Photoperiod and temperature-sensitive male sterile rice PeiAi64S under different temperature and light conditions can help to learn the gene regulation models and the crosstalking of different pathways during fertility transformation at the second Photoperiod-sensitive stage in PeiAi64S. Previous studies indicated that heat shock proteins might participate in the fertility transformation at the second Photoperiod-sensitive stage. We treated the PeiAi64S with different temperature and light conditions, and the transcriptome of them were analyzed by using rice microarray. We also investigated the relationship of differentially expressed heat shock transcription factors and heat shock proteins.Cellular protein homeostasis is important for cellular functions. Under physiological conditions, the demands for protein folding were consistent with the capacity of protein folding, result in cellular protein homeostasis. Under stressful conditions, unfolded and/or misfolded proteins accumulate in the ER, triggering an unfolded protein response (UPR). Heat shock also leaded to accumulation of unfolded proteins, triggering heat shock response (HSR). The heat shock response up-regulated the expression level of heat shock transcription factors, leading to rapid accumulation of heat shock proteins, which aid the folding and degradation of damaged proteins. We supposed that heat shock might lead to accumulation of unfolded protein, which induced both UPR and HSR. We investigated the effects of heat stress upon UPR in rice by detecting the expression levels of three kinds of UPR sensors and the chaperone BiP by quantitative real time polymerase chain reaction (qRT-PCR) experiments. We also investigated the relationship of heat shock factor and UPR sensors and the chaperone BiP using the transient assay. The main results as follows:A. Analysis Pollen Fertilities under different treatments using microscope. The results showed that the Pollens of PA64S were sterile under either high temperature and long-day light treatments or high temperature and short-day light treatments, indicating high temperature is major factor for male sterility in PA64S; the Pollens of PA64S were fertile under either low temperature and long-day light treatments or low temperature and short-day light treatments.B. We compared the transcriptome of the leaves from PA64S under different temperature and light conditions, and found that a set of the genes was differentially expressed. The results indicated that high temperature inhibited the expression of most of differentially expressed genes (about60%under short-day light conditions; about86.6%under long-day light conditions); and long-day light up-regulated about47.1%differentially expressed genes, down-regulated about52.9%differentially expressed genes.C. Pathways and function classifications of differentially expressed genes were further analyzed. We found that genes were downregulated by high temperature were mainly participated in Urea cycle and metabolism of amino groups, nitrogen metabolism, arginine and proline metabolism and glutamate metabolism. The results indicated that the expression levels of genes participated in thiamine metabolism was upregulated under long-day light and the expression levels of genes participated in circadian rhythm was downregulated. We found that different light treatments induced more differentially expressed genes, whose functions were more versatility.D. We compared the transcriptome of the leaves from PA64S with different temperature and light conditions, and found that the expression of a set of genes encoding HSFs and HSPs were inhibited under high temperature and long-day light conditions. We chose five HSPs (HSP90, HSP70, HSP201, HSP202, HSP203) and one HSF (HSF1) genes to further study using transient expression assay. The results showed that the HSF1inlvolved in the regulation of the expression of HSP90, HSP70, HSP201and HSP202genes. E. We also investigated the effects of heat stress and light stress upon HSR. The results showed that a HSFA2d gene had three transcripts. An alternatively spliced form of HSFA2d? was mainly induced by heat stress, and other two splicing forms, HSFA2d? and HSFA2d?, were mainly induced by long-day light stress.F. The cellular localizations of different transcripts of HSFA2d were studied by transient expression in onion bulb epidermal cells. We found that the HSFA2dI was localized in nucleolus either in normal condition or under heat stress, while HSFA2dII was localized in cytoplasm and nucleolus.G. We investigated the transcription activity of HSFA2dI and HSFA2dII by transient expression assay. The results suggested that the protein HSFA2dI functioned as a transcription activator while the protein HSFA2dII had no transcriptional activation activity.H. Our results showed that the expression levels of UPR sensors, IRE1, bZIP50, bZIP39, bZIP60, and BiP, a most abundant chaperone protein in the ER, were induced by heat shock. By contrast, no change of thoese genes were found under long-day light treatment, indicating long-day light did not invlove in UPR induction.I. We further investigated the relationship among heat shock factors, UPR sensors and a chaperone BiP by transient expression assay. The results indicated that protein HSFA2dI functions as a transcription activator to regulate expresion of BiP via binding to its HSE on the promoter region.Taking together, we found that temperature and light play important roles on fertility transformation at the second Photoperiod-sensitive stage in PeiAi64S. A set of the genes were differentially expressed under different temperature and light conditions, which affected the cellular process, the development and fertility decision of PeiAi64S. We also found that heat shock could induce both UPR and HSR. The signal transduction pathways of UPR and HSR were connected by heat shock factor HSFA2d. These results suggested that the signal transduction pathways were trigged by different environmental stresses. |
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