The Physiological Mechanism Of Leaf Senescence Regulated By 6-BA And Nitrogen And Identification Of Senescence-Related Genes In Wheat And Cotton

Pre-senescence of plants or leaves is one of limiting factor for crop photosynthetic potential, yield and quality that occurred during crop production. Therefore, it is invaluable in theory and practice to explore the physiological and molecular mechanisms related to the pre-senescence, by which to further elevate the crop productivity. Based on the related previous studies, the physiological mechanism of senescence characterizations regulated by senescence-delaying substance 6-Benzyladenine (6-BA) and nitrogen were studied. In the meantime, the differential expressed genes during leaf natural senescence, regulated by 6-BA and nitrogen were identified by modern molecular approach. Using the techniques of gene cloning, expression and genetic transformation, two novel superoxide dismutase (SOD) genes from wheat (Triticum aestivum L.) were isolated and functional analyzed in this paper. The main results were as follows:1. Compared with the control (CK), exogenous 6-BA could sustain higher chlorophyll contents, increase the catalase (CAT) activity, and decrease the over-oxidation of the cellular cytoplasm membrane during the late-senescence of the leaves in wheat. This is one of the important physiological reasons that 6-BA delays the senescence of plants or the leaves. In crop production, the concentrations and amounts of applied 6-BA should depend on the maturity properties and leaf senescence characterizations of the cultivars.2. Compared to normal-N, low-N decreased the plant height, leaf area per plant, fresh weight and dry weight. The chlorophyll a, b contents, photosynthetic rate, soluble sugar content, and soluble protein content were all also decreased by low-N. It is found that the regulation of cellular senescence degree by nitrogen supply levels were mainly due to the dual control of SOD activity and peroxidase (POD) activity, little effect was resulted from CAT activity. Therefore, it is one of important factors that the speeds of the metabolism in carbon and nitrogen were lowered in resulting in the pre-senescence in leaves.3. Based on cDNA-AFLP approach, 9, 20, and 16 differential up-regulated genes at 15 d, 30 d, and 45 d during the leaf natural senescence were identified, corresponding to 13, 8 and 4 differential down-regulated genes identified at the same time points. Bioinformation analysis indicated that the up-regulated genes were classified into 4 subgroups, including cellular metabolism, protein synthesis, signal transduction, and cellular protection; whereas the down-regulated genes were classified into 3 subgroups, including cellular metabolism, signal transduction, and cellular protection. After 15 d and 30 d of 6-BA treatment, there were 13 and 8 up-regulated genes, and 2 and 4 down-regulated genes sub-grouped into cellular metabolism, protein synthesis, signal transduction and cellular protection were identified. After 15 d of low-N treatment, there were 13 and 4 up- and down-regulated genes, respectively, sub-grouped into cellular metabolism, transcription regulation, protein synthesis, signal transduction, and cellular protection identified. It is suggested that there existed complicate gene-regulating networks and metabolic pathways that control the leaf senescence in leaf natural senescence, and regulated by 6-BA and low-N.4. Compared to the control (CK), exogenous 6-BA increased the plant height, leaf numbers per plant, leaf area per plant and dry weight per plant in cotton. It also enhanced the chlorophyll content, soluble protein content, and the photosynthetic rate. But the positive effects of 6-BA on above parameters were obvious in 33B, the senescing-easy cultivar, compared to in Fengkang6, the senescing-delaying cultivar. 6-BA increased the SOD activity and the POD activity, decreased the MDA content. Therefore, the capability that 6-BA could delay the leaf senescence were mainly due to the improvement of the balancing status between the production and scavenging of the active oxygen. It could have much better effects to apply 6-BA in the cotton cultivars with senescing-easy properties.5. Using the cultivar 33B and Fengkang6 as the experimental materials, the effects of nitrogen levels on leaf photoset rate and senescence were studied. The results indicated that the photosynthetic rate (P_n) in Fengkang6 were higher than in 33B.With the prolonging of the treatments, the chlorophyll a, b, carotene contents and soluble protein content were all gradually decreased. Compared to 33B, Fengkang6 had higher values in above parameters at the late-treatment duration. Fengkang6 also had higher SOD activity at this time phase. Compared to 33B, Fengkang6 had lower MDA contents at two tested nitrogen levels. Therefore, the lower over-oxidation in the cellular cytoplasm membrane could possibly be the physiological basis for the senescing-delaying cotton cultivar Fengkang6.6. Based on cDNA-AFLP approach, the differential expressed genes at 15 d and 30 d after 6-BA treatment were identified. Among them, there were 13 up-regulated genes and 22 down-regulated genes, sub-grouped into cellular metabolism, transcription regulation, protein synthesis, protein fate, signal transduction, and cellular protection. It is found that the Ca²⁺ signal and its signal cascade were perhaps involved in the regulation of the leaf natural senescence in cotton. After the treatment of 6-BA, 12 and 6 in totaled up-regulated genes, and 5 and 14 down-regulated genes were identified at 15 d and 30 d, respectively. It is suggested that 6-BA regulated the leaf senescence by the auxin signal pathway at some extent. In this study, after low-N treatment of 15 d and 30 d, 16 and 10 in totaled up-regulated genes and 4 and 20 down-regulated genes were identified, respectively.7. Based on the nucleic sequence of SOD1.1, the wheat CuZnSOD gene released in GenBank, two novel wheat CuZnSOD genes bamed as TaSOD1.1 and TaSOD1.2 were cloned and characterized. It was found that the cDNA full length of TaSOD1.1 and TaSOD1.2 were 980 bp and 1121 bp, respectively, all encoding 201 amino acids. A 45-aa length of transit peptide at the N-terminal and a 79-aa conserved CuZn-SOD domain were respectively located in TaSOD1.1 and TaSOD1.2. Phylogenetic analysis indicated that the query SODs, most of CuZnSODs, could be classified into four subgroups. Compared with the control (CK), the abundance of TaSOD1.1 transcripts did not change under drought, salt, low and high temperature conditions, but the TaSOD1.2 transcripts were strongly induced by the above abiotic stresses, which was in accordance with the elevated SOD activities in leaves in the above stress treatments to some extent, suggesting its involvement in the plant's acclimation and tolerance to the above abiotic stresses by possibly reducing the amount of the harmful ROS from enhancement of the SOD activity.8. Using the transgenic tobacco plants with overexpression of TaSOD1.1 and TaSOD1.2 genes to be the experimental materials, the functions of TaSOD1.1 and TaSOD1.2 in senescence-delaying and abiotic stress tolerance capabilities were assayed. There were not obvious effects on the leaf senescence-delaying in the leaves of transgenic plants compared to the control (CK). But, under the salt and low temperature stress, the transgenic tobacco plants could increase the capabilities for above stresses by the regulation at the transcription and translation levels. Compared with the CK, the chlorophyll a, b and carotenoid contents, and soluble sugar content, soluble protein content, were all increased in the leaves of transgenic plants, companying the lower MDA content. Therefore, TaSOD1.1 and TaSOD1.2 perhaps have important application potential in generation of crop genotypes (cultivars) based on the genetic engineering method in the future.