Mechanism Of Autophagy And Alternative Splicing In Regulating Plant Acquired Thermotolerance

High temperature will cause protein denaturation,organelle damage,enzyme inactivation and increase of ROS content in plants,all of which will cause the imbalance of plant homeostasis and ultimately cause severe heat damage to plants.On the one hand,denatured proteins caused by heat stress can be renatured and reused by heat shock proteins（HSPs）,and on the other hand,proteins that cannot be renatured can be degraded by 26S proteasome or autophagy.Heat shock proteins are easily induced by heat.They are mainly involved in biological processes such as the folding and processing of new proteins,the maturation of immature proteins,and the renaturation and degradation of denatured proteins.They play an important role in enhancing the heat resistance of plants.FES1 is an important auxiliary molecular chaperone of HSP70,it acts as a nucleotide conversion factor（NEF）to assist HSP70 in normal function by mediating the conversion of ATP and ADP.Autophagy is an efficient pathway to clear denatured protein aggregates and damaged organelles in vivo,which plays an important role in maintaining cellular homeostasis in plants under stress.According to different autophagy substrates,autophagy is divided into non-selective autophagy and selective autophagy.Part 1:Molecular mechanism of autophagy in regulating plant acquired thermotolerance.Studies have shown that Arabidopsis autophagy mutants have different phenotypes in basal heat tolerance and heat stress memory.This means that autophagy plays a completely different role in different types of heat stress and the function of autophagy in acquired heat resistance is unclear.The Arabidopsis fes1a mutant is a very good material with acquired thermotolerance deficiency.In this study,a series of autophagy genes were knocked out in the context of fes1a to explore the regulation of autophagy in plant acquired thermotolerance.We knocked out ATG2,ATG5,ATG7,ATG11,and ATG18a genes involved in non-selective autophagy,respectively,in the context of fes1a,and found that blocking the non-selective autophagy pathway significantly enhanced the acquired thermotolerance of fes1a.At the same time,we found that the atg7 fes1a mutant became as hypersensitive to heat as fes1a after replenishing the ATG7 gene,suggesting that non-selective autophagy is involved in the regulation of fes1a acquired heat resistance.In addition,we knocked out NBR1,DSK2,ATI1,and ATI3A genes involved in selective autophagy on the basis of fes1a,and found that blocking the known selective autophagy pathway did not enhance the acquired thermotolerance of fes1a.Western blot results showed that the content of HSPs protein was significantly increased after heat shock in WT,fes1a,atg7 fes1a and atg7.However,compared with WT and fes1a,the change trend of HSPs in atg7 fes1a and atg7 mutants before and after heat shock was not significantly different,indicating that blocking non-selective autophagy-induced fes1a increased heat resistance is not directly related to HSPs.The proteome sequencing results of atg7 fes1a and fes1a mutants before and after heat shock showed that blocking the non-selective autophagy pathway did not cause the accumulation of HSPs,but caused the accumulation of a large number of peroxisome-related proteins.These proteins were mainly involved in lipid oxidation,fatty acid beta-oxidation,lipid modification,organic catabolic process,and other biological processes.In addition,Catalase,APX3 and DHAR1 located in the peroxisome,these ROS scavengers were also significantly enriched,which is conducive to faster ROS scavenging and lessening of ROS damage to the membrane,thereby enhancing the resistance of fes1a.Western blot results showed that blocking the non-selective autophagy pathway before and after heat shock caused the accumulation of CAT2and catalase,which further confirmed the results of proteome sequencing.Meanwhile,fluorescently labeled peroxisome GFP-SKL transgenic lines showed significantly more peroxisomes in atg7 fes1a GFP-SKL root tips and leaf epidermis than fes1a GFP-SKL after both room temperature and heat shock.This indicates that blocking the non-selective autophagy pathway has caused the accumulation of peroxisomes before heat shock,and the accumulated peroxisomes during heat shock play a positive role in plant heat resistance.Heat stress causes an increase in plant ROS content,and peroxisomes can effectively scavenge excess ROS.We found that there was no significant difference in the content of H₂O₂and O^2-in each material before heat shock,but the contents of H₂O₂ and O^2-in atg7 fes1a and atg7 were significantly lower than those in fes1a after heat shock.This indicates that the increase of peroxisomes caused by blocking non-selective autophagy effectively removes the accumulated ROS under heat stress and enhances the acquired thermotolerance of fes1a to a certain extent.Studies have shown that overexpression of the ABCD1（ATP-binding cassette D1）gene in Arabidopsis can cause abnormal peroxisome function and lead to an increase in ROS content in plants.We found that the atg7 fes1a ABCD1-OE overexpressing line became as heat sensitive as fes1a again.This indicates that peroxisomes with normal physiological functions are very important for enhancing the acquired thermotolerance of fes1a.Peroxisomes can convert indolebutyric acid（IBA）to indoleacetic acid（IAA）throughβ-oxidation.By introducing DR5pro:GFP to fluorescently label IAA,we found that the content of IAA in the root tip before and after heat shock was as follows:atg7 DR5pro:GFP>atg7fes1a DR5pro:GFP>DR5pro:GFP>fes1a DR5pro:GFP.Therefore,the above results suggest that blocking non-selective autophagy leads to massive accumulation of peroxisomes,and the accumulated peroxisomes cause an increase in IAA content.In addition,we exogenously applied IAA or naphthalene acetic acid（NAA）to fes1a,atg2 fes1a,and atg7 fes1a,and found that exogenous application of IAA or NAA could significantly increase the acquired thermotolerance of fes1a.In conclusion,we found that blocking non-selective autophagy significantly enhanced the acquired thermotolerance of fes1a,but blocking some selective autophagy had no effect.At the same time,the accumulation of peroxisomes caused by blocking non-selective autophagy is the main reason for the enhanced acquired thermotolerance of fes1a.The accumulated peroxisomes on the one hand facilitate the rapid elimination of ROS and on the other hand produce more auxin,which enhances the acquired thermotolerance of fes1a.In addition,we also found that exogenous application of IAA or NAA was beneficial to enhance the acquired thermotolerance ability of fes1a.Part 2:Molecular mechanism of SR proteins and their protein kinases SRPKs in regulating plant acquired heat toleranceAlternative splicing is a process in which a splicing complex converts the same precursor m RNA（pre-m RNA）into a variety of different types of mature m RNA through different splicing methods.It is a ubiquitous post-transcriptional regulatory mechanism in eukaryotes.The splicing complex is a large RNA-protein complex whose assembly and function depend on SR proteins and SRPKs.There are 19 SR proteins and five SRPKs proteins in Arabidopsis.Plants can increase the complexity of the proteome through alternative splicing,thereby enhancing plant stress resistance to a certain extent.Studies have shown that heat stress can induce alternative splicing in plants,and alternative splicing plays an important role in plants coping with heat stress.The current research on the involvement of SR genes in the regulation of plant heat stress is mainly based on bioinformatics evidence.Some SR genes in Arabidopsis were found to be induced by heat at the transcriptional level,but genetic evidence was lacking,so the specific mechanism of SR genes involved in plant heat stress is still unclear,and there are fewer related studies on plant SRPKs.Whether the deletion of SR or SPRKs genes can cause plant heat sensitivity and how alternative splicing involving SR and SRPKs genes regulates the acquired heat tolerance of plants needs further study.Based on bioinformatics data,we drew a heat map of the gene expression of SR genes in Arabidopsis after heat shock,and found that SR30,SCL33 and SC35 genes were all induced by heat,with SR30 being the most significant.However,RNA-seq sequencing results before and after WT heat shock showed that only SCL33 and SR30 were significantly up-regulated among the 19 SR genes in Arabidopsis.We further examined the acquired heat tolerance of mutants in the partially related SR and SRPKs genes,and found that scl30,scl33,sr30,sc35,sc35-scl,srpk2a 2b 2c and the srpk1a 1b 2a 2b 2c mutants all exhibited distinct heat-sensitive phenotypes,whereas the sr45,rsz21,and srpk1a 1b mutants were heat-insensitive.It indicated that SCL30,SCL33,SR30,SC35 and SRPK2 genes in Arabidopsis may be involved in the regulation of plant acquired heat thermotolerance.We detected HSPs in sc35-scl,srpk2a 2b 2c and srpk1a 1b 2a 2b 2c thermosensitive mutants by Western blot,We found that the protein levels of HSPs in the two mutants before and after heat shock were not different from WT,indicating that the heat-sensitive phenotypes of sc35-scl,srpk2a 2b 2c and srpk1a 1b 2a 2b 2c were not related to the expression levels of HSPs.However,the content of catalase in srpk2a 2b 2c was significantly lower than that in WT before and after heat shock.It may be that the deletion of the SRPK2 gene family caused the synthesis of catalase to decrease,resulting in its mutants being unable to be effectively eliminated ROS under heat stress.In conclusion,based on bioinformatics analysis,we found that SR30,SCL33 and SC35genes were all induced by heat,with SR30 being the most significant.In addition,mutants such as scl30,scl33,sr30,sc35,sc35-scl,srpk2a 2b 2c and srpk1a 1b 2a 2b 2c all exhibited distinct heat-sensitive phenotypes,genetically demonstrating that alternative splicing is involved in plant acquired thermotolerance regulation.