Functional Study Of Peroxidase In Arabidopsis Thaliana

Peroxisomes are single-membrane-bound organelles found in most eukaryotes.Peroxisomes play important roles in fatty acid?-oxidation,degradation of branched-chain amino acids,and ROS detoxification.There are two main types of proteins in peroxisomes:membrane proteins and matrix proteins.Matrix proteins usually carry targeting sequence signals PTS1 or PTS2.In addition to enzymes involved in various metabolic reactions,Arabidopsis peroxisomes have a variety of proteases involved in protein degradation.It has been reported that there may be nine proteases in peroxisomes,which are encoded by AT5G47040(LON2),AT1G28320(DEG15),AT2G41790(PXM16),AT2G18080,AT2G35615,AT3G57810,AT4G14570(AARE),AT4G36195 and AT4G20310 genes,respectively.Except for LON2,DEG15,PXM16,AT2G18080 and AT2G35615,little is known about the functions and mechanisms of other proteases.In this study,AT3G57810,AT4G14570,AT4G36195and AT4G20310 were selected as the research objects to explore the possible roles of several proteases in the process of protein quantity control of peroxisomes.The main work includes the following aspects:1.Subcellular localization of proteasesThe vector that expressed the fusion protein of full-length or partial sequence of four proteases and enhanced yellow fluorescent protein(EYFP)were constructed and their subcellular localization was analyzed by transforming onion epidermal cells.The results showed that AT3G57810 was located in peroxisome,mitochondria and chloroplast;AT4G14570(752-820 aa)was located in peroxisome;AT4G36195 was located in endoplasmic reticulum;AT4G20310 and AT4G14570(468-820aa)were both located in nucleus and cytoplasm.The main purpose of this project is to study the function of protease in peroxisome,so further experiments are focused on the peroxisome-targeted AT3G57810 gene.2.Tissue-specific expression and functional analysis of AT3G578102.1 Tissue-specific expression of AT3G57810The expression vector of Pro _AT3G57810::GUS was constructed and transformed into Arabidopsis thaliana to detect its tissue-specific expression.The results showed that the expression level AT3G57810 was higher in stem leaves,flowers and horns than in rosette leaves.2.2 Construction of at3g57810 mutant and functional analysis of AT3G57810In order to study the function of the AT3G57810 gene,two homozygous mutants of the AT3G57810 gene,at3g57810-1 and at3g57810-2,were obtained using the CRISPR-Cas9 technologhy,but their editing sites were different,at3g57810-1 lost 2bases at 28 bp after the start codon,and at3g57810-2 lost 7 bases at 23 bp after the start codon.The phenotypes under various treatment conditions were observed and analyzed.2.2.1 Phenotypic observation of at3g57810 mutant under normal culture conditionsUnder normal conditions,the phenotypes of the mutants at3g57810-1 and at3g57810-2 were not significantly different from the wild type during the whole growth stage of the plant.2.2.2 Phenotypic observation of at3g57810 mutant under heat and salt stressExpression analysis made by the Arabidopsis e BF Browser shows thatthe expression level of the AT3G57810 gene was significantly increased by heat induction within 3 hours.In this experiment,the mutant seedlings germinated and grown on MS medium for 8 days were subjected to heat shock treatment,and the phenotype was observed after 3 days.The results showed that the at3g57810 mutant grew well,there was no significant difference in phenotype compared with the wild type.The seedlings of at3g57810 mutant germinated and grown on MS medium for 6days and were then transferred to MS medium containing 0,100,150 and 200 m M Na Cl for 5 days.The results showed that at3g57810 mutant grew well and had no significant difference from wild type.2.2.3 Response of at3g57810 mutant to IBA stress and dependence on sucroseIn order to observe whether AT3G57810 is involved in the?-oxidation process of fatty acids,the at3g57810 mutant was subjected to IBA stress and sucrose dependence experiments.LON2 is also a protease in peroxisomes and is involved in processes such as autophagy of peroxisomes.In this experiment,the mutant lon2 was used as a control.Appropriate IBA concentration can induce lateral roots in Arabidopsis seedlings.Deletion of LON2 can cause defects in peroxisome IBA metabolism,making lon2insensitive to IBA.Under the treatment condition of 10?M IBA,the lon2 mutant had no lateral root production.The mutants at3g57810-1,at3g57810-2 and wild type had lateral roots,there was no significant difference in growth.Loss of LON2 inhibits?-oxidation of fatty acids,thereby inhibiting plant growth.The seeds of the at3g57810 mutant were grown on MS+1%sucrose and MS-sucrose medium for 8 days respectively to observe the mutant's dependence on sucrose.The results showed that under the condition of sucrose,lon2 was smaller,at3g57810-1 and at3g57810-2 had no obvious phenotypic difference with wild type growth.Under the condition of no sucrose,the growth of lon2 was inhibited,and there was no significant phenotypic difference between at3g57810-1,at3g57810-2and wild type.The results indicate that AT3G57810 may not be involved in fatty acid?-oxidation.2.2.4 Detection and analysis of protein expression level of at3g57810 mutantLON2 was used as a control for this experiment.LON2 is involved in the degradation of peroxisome proteins.Studies have shown that LON2 affects the conversion of PMDH(peroxidase malate dehydrogenase)from precursor to mature protein,while has no significant effect on the key enzymes of glyoxylate cycle,ICL(isocitrate lyase)and MLS(malate synthase)Both AT3G57810 and LON2 are peroxisome proteases.In order to explore whether there is a relationship between them,the seedlings grown for 4,6 and 8 days were used as experimental materials to detect changes of the expression levels of ICL,MLS and PMDH.The results showed that the degradation trends of ICL and MLS in lon2,at3g57810 mutants and wild-type seedlings were not significantly different.The PMDH precursors in lon2 accumulated significantly.But there was no accumulation of PMDH precursors in at3g57810mutants and wild type seedlings.The results indicate that AT3G57810 does not affect the normal degradation of ICL and MLS,nor does it affect the maturation transition of PMDH precursors.3.Screening for proteases involved in peroxisome protein degradation by transcriptomics analysisThe results show that AT3G57810 does not participate in the degradation of ICL and MLS,so we continue to screen proteases that may be involved in the degradation of ICL and MLS.The 4,6 and 8 d grown Col-0 seedlings were sequenced for RNA-Seq,and the differentially expressed genes were analyzed.A total of 45 genes encoding proteases with increasing expression levels from 4 to 8 days were screened.The proteases were also screened base on the PTS signal.Five proteases with PTS signal sequence,AT3G57460,AT3G57470,AT2G03200,AT4G12910,and AT4G36195,were selected.In the early stage of this experiment,AT4G36195 was located on the endoplasmic reticulum,AT2G03200 and AT4G12910 were localized in the cytoplasm and nucleus.They may not directly participate in the degradation of peroxisome-related proteins.Further research is needed to understand whether the AT3G57460 and AT3G57470 protease participate in the degradation of ICL and MLS.4.NBR1 cannot block peroxisome autophagy induced in lon2 mutantThe study found that LON2 protease can participate in the peroxisome autophagy process,the deletion of LON2 enhances pexophagy,and the deletion of the key autophagy related genes ATG2,ATG3 and ATG7 can block lon2-induced pexophagy.In this process,pexophagy receptors in Arabidopsis thaliana have not been identified.In animals,NBR1 is a peroxisome autophagy receptor,as a homologous protein in plants,whether Arabidopsis NBR1 is also a peroxisome autophagy receptor has not been studied.Therefore,in this experiment,NBR1 was knocked out on the background of the lon2 mutant,and the expression levels of ICL,MLS,PMDH and Thiolase in the lon2 nbr1 double mutant were detected.The results showed that the degradation of ICL,MLS and Thiolase in lon2 nbr1 were not significantly different from that of lon2.The PMDH precursors in lon2 and lon2 nbr1mutants accumulated significantly,but there was no accumulation of PMDH precursors in wild type seedlings.The results indicated that the loss of NBR1 function did not block lon2-induced pexophagy.