Function And Mechanism Of Arabidopsis Callose Synthase PMR4 In Regulating Plant Senescence

In Arabidopsis,there are 12 genes encoding callose synthase,also known as glucan synthase-like(GSL,GSL1-GSL12)proteins.Among them,GSL5 induces the deposition of callose in plants under stress,and the pmr4 mutations enhance the resistance of plants to powdery mildew,so it is also called PMR4(powdery mildew resistance 4).In our study,we have discovered that pmr4 mutants also have a premature senescence phenotype,indicating that PMR4 also plays a role in the regulation of plant senescence.In this study,transcriptome profiling was used to identify the downstream genes of PMR4 and explorexplore their roles its mechanism of participating in PMR4-regulated plant senescence;The senescence suppressors of pmr4(spm)were obtained from chemically mutagenized pmr4 mutants,and their molecular genetics was systematically studied;The relationship between the changes of cell wall components and premature senescence of plants was studied by means of Fourier transform infrared spectroscopy(FTIR).The main research results are as follows:1.Transcriptome analysis of PMR4 downstream genes and their roles in plant senescenceThe transcriptome profiling results of pmr4 mutant showed that the expression of three genes in pmr4 mutant was significantly down regulated: TED6,TED7 and XTH31.TED6 and TED7 are related to the formation of plant secondary wall and the differentiation of vascular elements;XTH31 affects plant cell expansion.In order to determine whether the down-regulation of three genes plays a critical role in the premature senescence phenotype of pmr4 mutant,the overexpression vectors of three genes were constructed respectively,and the transgenic plants TED6/pmr4,TED7/pmr4 and XTH31/pmr4 were screened.Compared with wild-type Col-0 and pmr4 mutants,the senescence phenotypes of the three overexpressed transgenic plants were significantly recovered.2.Isolation and analysis of senescence suppressors of pmr4 mutant(spm)Two suppressors spm4-4 and spm5-1 with a normal senescence phenotype were obtained from EMS-mutagenized pmr4 mutant,The suppressors were analyzed by whole genome re-sequencing of bulked segregation DNA samples.The spm4-4suppressor gene was found to encode FLD,which regulates plant flowering time.FLD overexpression vector was constructed and transformferred into spm4-4revertant mutant to restore the early senescence phenotype.q RT-PCR results showed that the relative expression of FLD in the FLD/spm4-4 T1 lines with early senescence phenotype was higher than those in wild-type Col-0,pmr4 mutants,spm4-4 revertant mutants and the FLD/spm4-4 T1 lines with normal senescence phenotype.These results indicate that FLD influences early senescence of the pmr4 mutants.The spm5-1 suppressor gene is PAD4,which is involved in salicylic acid signal transduction.Comparing the senescence phenotypes of sid2/pmr4,pad4/pmr4,Col-0and pmr4 showed that the senescence of pad4/pmr4 double mutants senescenced normally,while sid2/pmr4 double mutants still senescenced early,indicating that PAD4 regulates plant senescence in a manner independent of salicylic acid.3.Measurement of cell wall contents by FTIRThe dried leaf samples were detected by FTIR-ATR method.The results showed that the plants with normal senescence phenotype had the highest content of cellulose and other cell wall polysaccharides,as well as protein,nucleic acid,and lipid at 6weeks,while the mutants with early senescence phenotype had the highest content of these substances at 4 weeks.These results suggest that the senescence of normal senescent plants begins in about 6 weeks,while the premature senescent plants start senescence in 4 weeks or even earlier.The results showed that the contents of protein,nucleic acid,lipid and cellulose in pad4/pmr4 mutant were higher than those in Col-0,while Col-0 was significantly higher than that in pmr4 mutant,indicating that PAD4 mutation can delay the senescence of pmr4 mutant.The normal senescence of TED7/pmr4 was associated with increased contents of these substances,indicating that PMR4 and TED7 interact to jointly affect plant senescence.Compared with pmr4 mutant,the content of several substances in spm4-4 revertant mutant greatly increased and even exceeded that of Col-0.The early senescence of transgene FLD/spm4-4plants was associated with reduced content of several substances.We propose that FLD regulates plant senescence by affecting the process of plant growth and development.