Mechanisms And Functions Of Autophagy In Growth And Development And Stress Response In Tomato

Due to the simplicity of horticultural infrastructure and the poorly ability to withstand natural disasters,the vegetables often encounter environmental stresses,such as extreme temperature and salt stress,which seriously restrict the yield and quality of vegetables in our country.Therefore,exploring the ways to relieve and overcome those environmental stresses to ensure the adequate vegetable production,improved quality and economic benefit has a significance in both science and realism.Autophagy plays a critical role in growth and development and response to environmental stresses.It is an evolutionary conserved recycling process in eukaryotes whereby intracellular components are engulfed by autophagosomes,which are subsequently transferred to the vacuoles for further degradation and reuse.At present,the study of autophagy in plants is primarily focused on the identification of autophagy related components and their respective functions in Arabidopsis.However,our understanding of the regulation of autophagy in plants is scanty,especially in horticultural plants.This paper uses tomato(Solanum lycopersicum)as material to obtain the tor mutant plants using CRISPR/Cas9 technology and characterize its functions in fruit development.In addition,we identify the interaction between MAPK12 and Atg6 using yeast two-hybride system and bimolecular fluorescence complementation.And then,we study the role of MAPK12 mediated autophagy in pollen developmen.Furthermore,we analyze the mechanism of brassinosteroids(BRs)signal inducing autophagy by using different BR levels,BZR1 silencing and overexpressing plants.We also study the function of BR-induced autophagy under nitrogen starvation.At last,we study the role of heat shock transcription factor Ala regulated autophagy under drought stress.The main results are as follows:1.We have found that TOR negatively regulated autophagy and fruit yield in tomato.We identified one TOR gene in tomato,the homologous of TOR gene in Arabidopsis,through the amino acid sequence alignment and the similarity and identity were 86.83%and 79.36%,respectively.Tomato TOR gene encodes 2470 amino acids,which have the conserved FAT,FRB,kinase domain and FATC domain.Tomato tor mutant did not display embryo lethality.The number of autophagosomes in tor mutants were higher than that in wild-type(WT)plants under normal condations.In addition,tor mutants increased light saturated CO2 assimilation rate,fruit setting rate,fruit number per plant and fruit yield.Taken together,TOR negatively regulated autophagy and fruit yield in tomato.2.We have studied the role of MAPK12 interaction with Atg6 regulated autophagy in pollen development.Tomato MAPK12 and ATG6 highly expressed in stamen,and MAPK12 interacted with Atg6 to involve in the formation of autophagosomes.The pollen viability and pollen germination rate in mapk12 and atg6 mutants were significantly lower than that of WT plants.The tapetum of mapk12 and atg6 mutants began to degrade at tetrads periods and most of the tapetum were degraded absolutely at vacuolated period.Furthermore,the tapetum of WT plants exhibited a number of autophagic bodies at vacuolated period,which were compromised in mapk12 and atg6 mutants.WT plants accumulated reactive oxygen species(ROS)at early uninucleate period,but were appeared at tetrads period in mapk12 and atg6 mutants,which resulted in earlier tapetum degradation,pollen deformity,inhibiting pollen viability and germination,decreased fruit seeds number and fruit weight per fruit.These results indicate that MAPK12 interaction with Atg6 mediates the formation of autophagosomes,and maintains the dynamic balance of ROS in tapetum cells during pollen development to regulate the degradation of tapetum,leading to affect the development of pollen and fruit.3.We have studied that brassinosteroid signal induced autophagy through transcriptional regulation of ATG and RBOH1 genes by BZR1 and plays a critical role under nitrogen starvation in tomato.The formation of autophagosomes is triggered by enhanced levels of brassinosteroid(BR),either through endogenous manipulation or exogenous application.Furthermore,tomato transcription factor brassinazole resistant 1(BZR1),a positive regulator of the BR signaling pathway,is involved in BR-induced autophagy.Treatment with BR enhanced dephosphorylated(active)form of BZR1,transcripts of autophagy-related genes(ATGs)and the formation of autophagosomes in BZR1-overexpressing plants,while silencing of BZR1 totally compromised the effect of exogenous BR in plants.In addition,BR-activated BZR1-induced autophagy was associated with the expression of respiratory burst oxidase homolog 1(RBOH1)and production of hydrogen peroxide(H2O2)in plants.ChIP-qPCR analysis revealed that BZR1 bound to promoters of four ATGs and RBOH1 genes.BR-induced H2O2 production was compromised in RBOH1 silencing plants but not in ATGs silencing plants,while BR-induced formation of autophagosomes was decreased both in ATGs and RBOH1 silencing plants.Moreover,the resistance to nitrogen starvation was compromised in BZR1 silencing plants,but was enhanced in BZR1-overexpressing plants.Taken together,our results suggest that BZR1-dependent BR signaling upregulates ATGs expression and promotes H2O2 production,while BR-induced H2O2 acts as a positive feedforward signal towards regulation of autophagy in tomato plants.Whilst,BR signal induced autophagy plays a vital role under nitrogen starvation in tomato.4.We have demonstrated that tomato transcription factor HsfAla played a critical role in drought tolerance by activating ATGs and inducing autophagy.The expression of HsfAla was induced by drought stress.Virus-induced HsfAla gene silencing reduced while its overexpression increased plant drought tolerance based on both symptoms and membrane integrity.HsfAla-silenced plants were more sensitive to endogenous ABA mediated stomatal closure,while its overexpression lines were resistant under drought stress,indicating that phytohormone ABA did not play a major role in HsfAla-induced drought tolerance.On the other hand,HsfAla-silenced plants increased while its overexpressing plants decreased the levels of insoluble proteins which were highly ubiquitinated under drought stress.Furthermore,drought stress induced numerous ATGs expression and autophagosome formation in wild-type plants.The expression of ATG10 and ATG18f,and the formation of autophagosomes were compromised in HsfAla-silenced plants but were enhanced in HsfAla-overexpressing plants.Both electrophoretic mobility shift assay(EMSA)and chromatin immunoprecipitation coupled with qPCR(ChIP-qPCR)analysis revealed that HsfAla bound to the promoters of ATG10 and ATG18f gene.Silencing of ATG10 and ATG18f reduced HsfAla-induced drought tolerance and autophagosome formation in HsfAla-overexpressing plants.These results demonstrate that HsfAla induces drought tolerance by activating ATG genes and inducing autophagy,which may promote plant survival by degrading ubiquitinated protein aggregates under drought stress.