林讚標臺灣大學:植物科學研究所林伯昊Lin, Po-HaoPo-HaoLin2010-05-112018-07-062010-05-112018-07-062009U0001-1408200912464300http://ntur.lib.ntu.edu.tw//handle/246246/181969Under the threat of global warming and population explosion, improving crop yield under abiotic stress in order to secure future food supply is important for the survival of humankind. In order to achieve such goal, we need a more thorough understanding of all aspects of abiotic stress. I believe that the apoptosis-like programmed cell death (AL-PCD) pathway maybe an aspect that have been largely overlooked, despite some publication suggesting its involvement in abiotic stress. Vacuolar processing enzyme (VPE) is a functional homolog of caspase-1 that is found in plants and participates in plant AL-PCD by facilitating vacuolar collapse. Arabidopsis thaliana consists of four VPE genes, and one of the member δVPE is involved in activating AL-PCD pathway in maturing silique seed coat to reduce the thickness of seed coat. The bioinformatic tools suggested that δVPE expression was low in vegetative tissue, and no abiotic stress elevated the expression of the gene. Two mutant alleles, δvpe-4 and δvpe-5, were selected, with T-DNA disrupting the upstream sequence of δVPE for the former allele, and at the second exon of δVPE for the later allele. Both mutants were considered as knockout mutant. The AL-PCD sensitivity of δvpe-4 was examined using Fumonosin B1 (FB1), an AL-PCD-inducing agent, and the δVPE mutants were also tested against various abiotic stresses. The results indicated that δvpe-4 mutant was more sensitive to FB1 than that of WT. Yet, both mutant δvpe-4 and δvpe-5 were more tolerant to drought stress. Since δVPE mutant was more prone to AL-PCD, the drought resistance phenotype cannot be attributed to improving survival of individual cells under drought. The crosstalk between δVPE and abiotic stress needs further dissection, and one possible mechanism is that it changes the AL-PCD pattern under the stress. This will be further investigated through the examination of AL-PCD patterns under abiotic stress by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. If a shift in TUNEL pattern was observed under such stress, then we might be able to attribute the difference in abiotic stress tolerance to these changes. The results might indicate that Arabidopsis actively sacrifices certain cell under abiotic stress, and the pattern contributes to overall fitness of the plant under the stress.審定書...................................................................................................................................................i謝......................................................................................................................................................iiBBREVIATION...............................................................................................................................iii文摘要............................................................................................................................................ivBSTRACT........................................................................................................................................vNTRODUCTION...............................................................................................................................1Programmed cell death and apoptosis in animals......................................................................2Apoptosis-like PCD in plants.....................................................................................................4Vacuolar processing enzymes.....................................................................................................6Understand the connection between AL-PCD and abiotic stress ..............................................7ATERIALS and METHODS...........................................................................................................9Plant materials and growth condition ...............................................................................10DNA extraction, PCR, and sequencing....................................................................................10RNA extraction and RT-PCR....................................................................................................11FB1 treatment and relative ion leakage measurement..............................................................11Conserved protein residue analysis...........................................................................................12Bioinformatic gene expression analysis...................................................................................12Drought treatment.....................................................................................................................12Salt treatment............................................................................................................................13ESULTS..........................................................................................................................................14Mutant alleles of δVPE ............................................................................................................15Mutant δvpe-4 showed higher susceptibility toward FB1.........................................................15Bioinformatic analysis..............................................................................................................16Stress treatment of δVPE mutants.............................................................................................16ISCUSSION...................................................................................................................................18Mutant δvpe-4 might be more sensitive to FB1........................................................................19Mutant showed drought resistant phenotype............................................................................20VPE and the evolution of AL-PCD..........................................................................................21AL-PCD needs more detailed categorization...........................................................................23IGURES..........................................................................................................................................25EFERENCES................................................................................................................................34PPENDIX.......................................................................................................................................39application/pdf65540470 bytesapplication/pdfen-US細胞凋亡非生物逆境VPEapoptosis-like programmed cell deathabiotic stresshttp://ntur.lib.ntu.edu.tw/bitstream/246246/181969/1/ntu-98-R96b42009-1.pdf