Cloning And Characterization Of Senescence And Nitrogen Resorption Associated Genes During Cotton Leaf Senescence

In order to unravel the biochemical pathways and decipher the molecular mechanisms involved in leaf senescence and nitrogen resorption during leaf senescence. Suppression subtractive hybridization (SSH) was first used to generate a cDNA libraries enriched for transcripts differentially expressed in developmental senescence cotyledons of a upland cotton cultivar Shannong fengkang 6, 416 produced readable sequences were successfully generated. EST cluster analysis indicated that the sequences represented 216 unique senescence-associated genes (SAGs).The putative functions of these 216 ESTs were identified by comparing them to previously reported databases using the BLASTX program (BLAST Against NR). Homologies that showed e-value less than 1×e-10 with more than 100 nucleotides were considered significant. According to this criterion, 151 ESTs which represent known genes were found. Functional classification of the ESTs was carried out according to the functional categories of Arabidopsis proteins. By Sequin (Version 6.25) program, all these unique ESTs have been submitted to GenBank dbEST with accession numbers from DV437859 to DV438009. Of the other 65 ESTs, 34 unique ESTs which have no homologies identified were defined as unknown genes, 31 unique ESTs have some homology to sequences in GenBank but do not meet the cutoff criteria, were defined as unknown genes either. It's possible all these unknown ones are new genes during cotton leaf senescence.151 known genes were classified into different categories according to the functional classification for Arabidopsis thaliana MIPS. The results are as follows: 12 functional categories exsist in senescing cotton leaf : metabolism; energy; transcription, protein fate (folding, modification, destination); cellular transport, transport facilitation and transport routes; cellular communication/signal transduction mechanism; cell rescue, deference and virulence; interaction with the environment; cell fate; protein synthesis; biogenesis of cellular components and unknown protein. The largest group of genes with known function is metabolism-related genes and cell rescue, deference and virulence-related genes. Senescence-associated genes isolated in our study, which participate in cellular protein degradation processes, three protein degradation pathways were proposed during cotton leaf senescence: the ubiquitin/proteosome pathway, the vacuolar and autophagic (APG) pathway and the chloroplast degradation pathway. Meanwhile, two unique biochemical cycles exist for N recycling were proposed: GS1/GOGAT cycle,GDH/GOGAT cycle and AspAT/AS cycle. In order to further decipher the mechanism involved in N-resorption, Northern hybridization was performed to detect the expression of several pivotal genes involved in protein degradation and N-resorption. The results indicate that the ubiquitin/proteosome pathway occurs during the whole process of leaf senescence. However, the pathway is weak at the early stage, and increases with the development of cotyledon, then decreases again to the terminal phase. Contrary to the ubiquitin/proteosome pathway, the vacuolar and autophagic (APG) pathway doesn't take place at the early stage, however, it increases rapidly to the terminal phase.The increased expression of NADH-GOGAT and GS1 during leaf senescence, indicating that GS1/GOGAT cycle is the key pathway during cotton cotyledon senescence. GDH activity is weaker, and increases little during cotyledon senescence, implying that GDH/GOGAT cycle may be the accessorial pathway. Likely, AspAT and AS are the key enzymes involved in N-resorption because of their high expression during cotyledon senscence. Hence, AspAT/AS cycle was proposed to be the main pathway of N-resorption during cotton cotyledon senescence.