| The day-temperature that is suitable for the growth of tomato(Solarium lycopersicum L.) is20-25℃and the night-temperature is16-20℃. If the normal tomato varieties exposed to the temperature less than15℃, all including the seeds germination, pollination and fertilization, and ripening will be affected. When seeds suffered30℃, the seeds germination rate, potential and index are all decreased. Tomato reproductive development is significantly affected in higher than34℃, while the plants suffered physiological disorders to thermal death in higher than40℃. High temperature influences microsporogenesis, which is directly related to pollination and fertilization process and the final fruits and seeds quality and yield.In this study, cytological morphology studies were performed in the microsporogenesis and anther development in Micro-Tom tomato when suffered continuous high temperature of35℃/30℃for7d. Using Affymetrix Tomato Genome Array technology, we compared the transcriptome differences of anthers (tetrad and binucleate stage, respectively) that after heat stress, and screening the righr heat stress response genes expressed in pollen for temporal and spatial expression analysis. We used heat-tolerance tomato BL1175and heat-sensitive tomato LA2006suffered to heat stress of35℃/30℃to study the spatial and temporal expression characteristics of the right genes in them. This study aimed to clarify the heat stress effects on the cytology of the tomato microspore development, to reveal the expression profile of effects of high temperature stress at different developmental stages of tomato microspores, to obtain genes spatial and temporal expression characteristics that screened from genechips which expressed significant difference in pollen and responsed to heat stress. We attempted to explore the molecular mechanisms of the tomato heat stress response and heat tolerance, and then provide a theoretical basis for the cultivation of new varieties of heat-resistant tomato.The major study results are as following:1) Through morphological observation, DAPI staining and paraffin section, the’Micro-Tom’ tomato microsporogenesis process was divided into5stages carried out under normal temperature. Our study found that35℃/30℃high temperature stress not only led to pollen abnormal or abortion, pollen decrease in the number, and low pollen viability and germination capacity, but also led to the tapetum, connective tissue, endothecium, epidermis, circular cell cluster and other anther cell structures abnormal. We determined the most sensitive periods of the tomato microsporogenesis process to high temperature stress were tetrad stage and binucleate stage.2) Using gene chip analysis, we compared the transcriptome differences of Micro-Tom tomato anthers (tetrad and binucleate stage, respectively) that grew at high temperature (35℃/30℃) and normal temperature (25℃/20℃). A large number of right genes were obtained from the tomato anther transcription information in gene chips. The functional classification of differentially expressed genes mainly contained abiotic stress response, transcriptional regulation, cell construction, signal transduction, carbohydrate metabolism, lipid metabolism and other categories, but each group of genes in the two stages had some differences.3) By RT-PCR analysis, the expression characteristics of SIMAPKs in microspore tetrad stage and binucleate stage of tomato anther treated with heat stress were identified. The experimental results showed that genes of SIMAPK1, SIMAPK3, SIMAPK7, SIMAPK12and SIMAPK16were up-regulated during tomato microspore development process in high temperature stress, but the peak of the genes expression appeared at different HS length.4) We screened16genes of tomato microspore development from the gene chips related and responsed to high temperature. The genes spatial and temporal expression analysis showed that, LeARG2, TFHS1and WRKY4were significantly up-regulated expression in the tetrad stage of microspore development; HSP70, TAGL12, SIMAPK3, SIMAPK7, MAP3Ka were significantly up-regulated expression in the period of binucleate stage; GPAT, TMP and Frk1were significantly down-regulated expression in the tetrad stage; EXPA5was significantly down-regulated expression in the binucleate stage; Rnale was significantly up-regulated expression in the tetrad stage, while significantly down-regulated in the binucleate stage; Cel5was down-regulated expression in the tetrad stage, while significantly up-regulated expression in the binucleate stage; LOC543824(alternative oxidase1a gene) was up-regulated expression in these two stages; and MAPK12was slightly decreased expression in the tetrad stage, while its expression increased slightly in the binucleate stage, but none were significantly.5) Suffered to35℃/30℃heat stress, the heat-tolerance tomato ’BL1175’ had a stronger cell membrane thermostability, higher SOD activity&pollen viability&germination rate, when compared with the heat-sensitive tomato ’LA2006’. QRT-PCR analysis of the LeARG2, FRK1, MAPK3and MAPK7genes of ’BL1175’ and ’LA2006’ anthers in the tetrad stage was carried out. The results showed that the four heat stress related genes play important roles in thermotolerance of tomato. Tomatoes with different genotypes had large differences in response to high temperature stress. The fact that the heat-sensitive tomato has lower level of polyamines and lower pollen germination rate than the heat-tolerance one, under high temperature, was verified on the molecular level. |
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