Programmed Cell Death During Cotyledon Senescence In Cotton (Gossypium Hirsutum L.)

Cotton is the most important cash crop in the world. Premature senescence of cotton leads to decreased photosynthetic capacity, and consequently lowers yield and fiber properties. Programmed Cell Death (PCD) is a gene-regulated and initiative cell death, which was associated with biology development, biotic and abiotic stresses. Potentially, premature senescence of cotton was associated with abnormal PCD. In this dissertation, cotton (Gossypium hirsutum L.) cultivar Shan Nong No.6 was used, the PCD processes and senescence-associated genes (SAGs) were studied during cotyledon senescence. Physiological and biochemical changes of cotyledon during senescence in cotton were also studied. The main results of this paper were as follows.(1)Microscope morphological observation showed symptoms of cell death, decreased cell-to-cell adhesion, cell shrinkage, distortion and decreased the frequency of the nucleus-containing cells were found during cotyledon senescence in cotton. And the timing course of cell death in different regions of the cotton cotyledon was asynchronous. Cell death was initially detected at the cotyledon margin, and then expanded centrally. Intact cell with nuclei could be detected around the midrib vascular bundle in the fallen cotyledons seemed to be efficient nutrient remobilization.(2)The results of gel electrophoresis and TUNEL assay proved that the cell death of cotyledon in cotton was a PCD process. The onset of programmed cell death in cotyledon margin and central part around midrib vascular bundle was detected on 22 days (Stage S1), 26 days, respectively. The PCD order in different regions of the cotton cotyledon was consistent with morphological changes. However, the timing of PCD was earlier than morphological changes.(3)The net photosynthetic rate of cotyledon increased progressively to a plateau at 14 days (Stage G), and stayed relatively constant from 14 to 18 days, then decreased gradually. At the time of PCD occurrence, photosynthetic rate decreased rapidly, which suggest that the onset of PCD and decline of cotyledon function was asynchronously. The onset of decrease in the soluble protein content of cotyledon were detected at 18 days, earlier than the occurrence of PCD, which indicate the onset of decrease in capacity of synthesize substance earlier than the occurrence of PCD. The dry weight of cotyledon decreased at 22 days, while the water content remained nearly constant throughout the course of the study (90 %). Both parameters, therefore, indicate trends in the rate of accumulation and translocation of cotyledon metabolites during PCD.(4)A gradual decrease in SOD and CAT activity was observed after 14 DAE, which was earlier than the onset of senescence and cell death. The MDA content in cotyledon increased during the developmental phases. This increase was noted between 14 and 18 days. The results of our experiments also proved, the occurrence of PCD in cotyledon cells was result from the decline in capability of disassembles reactive oxygen species.(5)In terms of bioinformatics and by means of BLASTX program and Arabidopsis thaliana MIPS (http://www.mips.biochem.mpg.de/) on line analytical utility, 216 of expressed sequence tags (ESTs) obtained in our lab were analyzed and grouped. Of these 216 cDNAs, 151 clones (69.9%) show significant homologies to previously known genes, while the remaining 65 do not match any known sequences. 151 unique ESTs are assigned to twelve different categories based on their putative functions generated by BLAST analysis. These SAG-encoded proteins are likely to participate in macromolecule degradation, nutrient recycling, detoxification of oxidative metabolites, and signaling and regulatory events. 65 ESTs maybe new SAGs induced in cotton.(6)The expression pattern of a selection of genes was confirmed using northern hybridization. Northern hybridization confirmed that MAP kinase-like protein (F7A1) is expressed at early stages, but thereafter its transcript levels drop. This may suggest it participation in the initiation phase of PCD. Cysteine protease, whose product is involved in the terminal phase of leaf senescence, accumulated to high levels only in the yellowing cotyledons. This may suggest it participation in the decomposition and recycle of protein.