| Tomato, one of the most important vegetable crops, is widely cultivated around the world. Tomato is the highly self-pollinated crop and the cross-pollination rate does not exceed2%. In hot seasons, the stigma of the flowers often extends out of the anther cone and causes the unsuccessful self-pollination, and finally markedly lowers fruit set.In this study, the phenotypic characterization of tomato on the growth of stamen and pistil as well as the stigma exsertion was observed in tomato flower buds when exposed to continuous heat stress; Histological analysis of heat stress in filament and style of tomato at binucleate pollen stage was performed; The influence of plant hormones on the heat-induced exserted stigma was evaluated by exogenous spraying of indole-3-acetic acid (IAA), gibberellin (GA3), jasmonic acid (JA),2,4-epibrassinolide (EBR) and paclobutrazol (PAC) with different concentrations on the tomato flower buds, meanwhile, the changes of expression levels of some phytohormone-related genes were detected as exposed to exogenous GA3and paclobutrazol (PAC) under heat stress conditions using quantitative real-time PCR (qRT-PCR); Finally, using RNA-seq transcriptome sequencing technology, we compared the changes of transcriptome pattern in tomato stamen and pistil before and after stigma exsertion after heat stress, GO analysis and pathway analysis were carried out in our study for preliminary clarify the regulation model of the heat stress influence on tomato stigma exsertion. Our results will provide the theoretical basis to enchance fruit set under high-temperature in tomato.The major results are as follows:(1) Tomato variety’Micro-Tom’was exposed to day/night temperatures of25℃/20℃,30℃/25℃,35℃/30℃, and40℃/35℃to clarify the heat stress influence on tomato stigma exsertion. The results indicated that, tomato under heat stress of30℃/25℃and35℃/30℃developed the flowers with protruding stigma, the number of flowers with stigma exsertion was more than40%, and the flowers has the1mm extension of stigma tube. Under temperature of35℃/30℃, the flowers exhibited abnormally smaller and the pollen vitality was significantly reduced. Under temperature of30℃/25℃, the pollen vitality was not significantly influenced. When grown under40℃/35℃, the growth of flower buds was inhibited and couldn’t develop to opening flowers. Histological analysis of high-temperature injury in tomato filament and style found that the number of anther cells was deceased significantly, the cells of filament in longitudinal sections were shorter than the control’s, but not in developing style cells.(2) The effect of exogenous spraying of IAA, GA3, JA, EBR and paclobutrazol (PAC) with different concentrations on the tomato flower buds was evaluated under heat stress conditions. The results showed that spraying GA3under heat stress accelerated the tomato stigma exsertion. The percentage of stigma-exserted flowers was increased, as well as the extent of stigma exsertion, whereas the inhibitor of GA biosynthesis, PAC can rescue the extent of stigma exsertion to some degree. What’s more, continuous GA3application on the flower buds could significantly increase tomato fruit set. Furthermore, JA treatment showed similar effects on the percentage of stigma exserted flowers with GA3treatment, but it had no significant effects on the extent of stigma exsertion. The effect of IAA and EBR treatment was not obvious. These results suggested that GA and JA may participate in the signal transduction process of heat induced tomato stigma exsertion.(3) Using quantitative real-time PCR (qRT-PCR), the expression analysis of phytohormone-related genes including IAA, GA and ABA was carried out in the heat-induced stigma exsertion process. All the genes response to heat stress significantly especially the genes related to IAA and GA. In pistil, a IAA biosynthesis gene SlToFZY2and a GA biosynthesis gene SlGA20ox2were up-regulated, whereas the gene SIDELLA, a repressor in the GA signaling pathway was down-regulated. SlGA3oxl, expected to be under the GA negative feedback regulation, was down-regulated. However, in stamen, SlToFZY2was down-regulated while SlGA20ox2was up-regulated. Meanwhile, the expression changes of some important genes involving IAA, GA, and ABA signaling pathway were evaluated as exposed to exogenous GA3and PAC under heat stress conditions. The results showed that the relative expression levels of the GA biosynthesis genes were differentially changed in pistil and stamen. In pistil, SlGA20ox2was up-regulated when treated with GA3under heat stress, whereas SlDELLA and SlGA3ox1, were down-regulated. In contrast, in stamen, the expression of SlGA3oxl was down-regulated when treated with GA3under heat stress. Moreover, SIPIF1-1and SIPIF4were significantly down-regulated in pistil whereas there was no significant change in stamen.(4) Based on the transcriptome profile analysis using RNA sequencing, a total of5839genes were found to be differently expressed in stamen and pistil of tomato when treated with heat stress after2d or12d. Among these,5320genes were heat-responsive in stamen, the others from pistil, while190genes were found to be differently expressed in both stamen and pistil. In addition, before the stigma exsertion,74genes were identified to be differentially expressed in pistil and2044genes in stamen. After the stigma of heat-treated flowers appeared to exsert at12d,445genes were identified to be differentially expressed in pistil and3276genes in stamen. It showed that the gene expression profiling was significantly different between stamen and pistil when exposed to heat stress. Moreover, the validity of different expressed genes detected by the RNA sequencing was examined by qRT-PCR.(5) Gene ontology enrichment analysis and biochemical pathways were performed in this study. The heat-responsive genes of2d and12d at the stamen and pistil were mainly involved in stress response, transcriptional regulation, cell construction and signal transduction. After the stigma exsertion, the hormone related biochemical pathways affected by heat stress in stamen mainly were cytokinins-O-glucoside biosynthesis, Jasmonic acid biosynthesis and gibberellin biosynthesis. In contrast, the hormone related biochemical pathways were not markedly changed in pistil. Heat stress for12d had a significant effect on the gene expression pattern in stamen, a large number of cell division-and cell wall-related genes were down-regulated, whereas they had no significant changes in pistil. Transcription factors also played an important role in heat stress response in tomato stamen and pistil. |
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