The Role Of Brassinosteroid And HsfA1a In The Regulation Of Pollen Development And Heat Tolerance In Tomato

Tomato is one of the most widely cultivated vegetable crops in the world,which is also an important vegetable crop in facility cultivation in China.Recently,with the continuous expansion of facility production scale in our country,as a vegetable crop favored by the majority of consumers,tomato is also increasing its cultivation area.Efficient pollination and fertilization are critical for tomato fruit setting and development to ensure fruit yield and quality.A large number of studies have shown that the male fertility of plants is of great significance to crop breeding,hybrid seed production and gene drift regulation,thus,the development of anther and pollen and the tolerance to abiotic stress are of great importance to tomato production.As an important plant hormone,Brassinosteroids(BRs)have a high content in pollen.Although the effects of BR on plant growth and development and the regulation of Hsfs on plant response to stress have been reported,there are still many questions about the regulation mechanisms of BR and Hsfs on tomato pollen development and stress tolerance.In our study,tomato(Solanum lycopersicum L)was used as materials.We studied the effect of BR on tomato pollen development and elucidated the regulatory mechanism of BR-mediated ROS on tapetal degradation during pollen development by cytology,molecular biology,biochemistry and genetic techniques.We studied the role of HsfA1a on tomato pollen resistance to high temperature.The main results are as follows:1.We have illuminated that BR synthesis and signaling improved tomato pollen fertility and seed yield.In BR synthetic gene DWF mutant d^im and BR signaling transcription factor BZR1 mutant bzr1-1,the proportion of abnormal pollen was increases,pollen vitality and germination rate were reduced and the average seed number of each fruit was decreased compared with wild type.However,DWF:OE2 and BZR1:OE2 plants had the opposite results.Self-and cross-pollination among these materials in different levels of BR showed the consistent tend with the pollen fertility.Pollen germination ability of BR mutant d^im and bzr1-12 on selfor cross-pollinated stigmas was weak.Furthermore,average seed number in each fruit was less compared with wild type.On the contrary,pollen of BR overexpression plants DWF:OE2 and BZR1:OE2 had strong gennination capacity on stigmas,and fruit seed setting was also higher than those plants pollinated with WT pollen.These results suggested that endogenous BR level and BZR1-mediated BR signal transduction promoted seed setting in tomato fruits by improving pollen quality.2.We found that ROS was involved in BRs facilitating tapetal cells PCD and tapetal layer degradation.It was showen that the absence of DWF or BZR1 led to tapetal cells,while overexpression of DWF or BZR1 resulted in proper premature degradation of tapetal cells by histochemical staining analysis.These phenotypes were proved to be associated with the timing changes of tapetal PCD by TUNEL assay.We detected the transcription level of eight tomato homologous genes of Arabidopsis tapetum and pollen development genetic network by qRT-PCR and found that the gene expression were decrease in d^im and bzr1-1 mutant anthers,while significantly increased in DWF and BZR1 overexpression plants.H2DCF-DA and NBT staining showed that during pollen development,reactive oxygen species(ROS)accumulation was lower in the tapetum of d^im and bzrl-1 mutant anthers,but higher in DWF and BZR1 overexpression plants.We then analyzed the expression of genes encoding NADPH OXIDASE by qRT-PCR and found that RBOH1(RESPIRATORY BURST OXIDASE HOMOLOG 1)was specifically expressed in stamens and roots.Moreover,RBOH1 transcription level in d^im and bzrl-1 mutant stamens was reduced during pollen development,oppositely,RBOH1 expression in DWFand BZR1 overexpression plants were raised compared with wild type.The results suggested that BR promoted tapetal PCD and degradation of tapetal cells by inducing ROS during pollen development in tomato.3.We clarified that BRs induces ROS production by activating RBOH1 gene expression to promotes tapetum degradation.We proved that BZR1 regulated RBOH1 gene expression by binding to the promoter of RBOH1 by yeast one hybrid and ChIP-qPCR analysis.To further confirm the important role of RBOH1 in tomato pollen development,we constructed RBOH1 RNA interference tomato plants as the experimental material.It was shown that ROS produced by RBOH1 could induce tapetal PCD and tapetal layer degradation,reduced transcription level of RBOH1 led to a significant decrease of ROS accumulation in the tapetum.RBOH1 RNA interference in DWF and BZR1 overexpression plants inhibited the enhancement of pollen viability and germination ability.Similarly,the seed yield in these RBOH1 defective plants fruits was also decreased compared with those of wild type.These results demonstrated that BRs induced ROS production by directly regulating the transcription of RBOH1 to promote tapetal cells degradation during tomato pollen development.4.HsfAla improve tomato pollen heat tolerance by inducing HSPs protein accumulation and autophagy genes expression.It was shown that HsfAla overexpression increased pollen heat tolerance,while HsfAl a mutant showed weakened pollen resistance to high temperature by testing pollen fertility under heat stress.The gene expressions of HsfA2,HsfA3,HSP70 and HSP90 in tomato anthers were significantly up-regulated by HsfAla overexpression under high temperature stress,but inhibited in HsfAla knockout plant anthers.In addition,HsfA1a overexpression promoted the accumulation of HSP70 and HSP90 proteins in anthers undergoing heat stress,oppositely,these proteins accumulation was inhibited in HsfA1 a knockout plant anthers.Furthmore,it was showen that the expressions of ATG18f,ATG18g and ATG18h were significantly up-regulated after heat stress in anthers of plants overexpressing HsfA1a however,inhibited in HsfA1a knockout plants.These results suggested that HsfA1a enhance the heat tolerance of tomato pollen by inducing HSPs and regulating autophagy.