Proteomics Analysis Of Cotton Somatic Embryogenesis And Functional Characterization Of GhTZF1

Reactive oxygen species(ROS) was considered as byproducts of plant metabolism, which may cause oxidative damage to plant. Also, it may act as molecular signal to modulate pant growth and developmental processes as well as stress responses. The dual role of ROS was depended on its cellular content. Plant cells contain an array of protective and repair systems to maintain cellular ROS homeostasis. Under normal developmental processes, the steady state of ROS was well controlled, while, it could be perturbed by varieties of environmental factors, leading to oxidative stress.The maintainance of ROS homeostasis was important for plant metabolism processes. 1. ROS homeostasis regulates somatic embryogenesis via the regulation of auxin signaling in cottonSomatic embryogenesis(SE) is a process by which somatic cells tranform into embryogenic cells, go through a development process similarly to zygotic embryo development, and eventually develop to a new plant. SE is a versatile model for understanding the mechanisms of plant embryogenesis and a useful tool for plant propagation. To decipher the intricate molecular program and potentially to control the parameters affecting the frequency of SE, a proteomics approach based on two-dimensional gel electrophoresis(2-DE) combined with MALDI-TOF/TOF was used. A total of 149 unique differentially expressed proteins(DEPs) were identified at different stages of cotton SE compared with the initial control(0 h explants). Based on GO analysis, most of the DEPs were involved in cellular metabolism, stimuli response, development and reproductive process. The expression profile and functional annotation of these DEPs revealed that SE activated stress-related proteins, including several reactive oxygen species(ROS)-scavenging enzymes, such as APX, GST, SOD and thioredoxin. The expression patterns of most of the ROS-scavenging enzymes were consistant between trancriprit level and protein expression level. Further experiments were performed to confirm the role of ROS-scavenging enzymes, suggesting the involvement of ROS homeostasis during SE in cotton. Suppressing the expression of specifically identified Gh APX proteins resulted in the inhibition of dedifferentiation. Accelerated redifferentiation was observed in the suppression lines of Gh APXs or Gh GSTL3, in parallel with the accumulation of endogenous H2O2 content. These results indicated that these ROS scavenging-related genes may through regulating ROS homeostasis to regulate cotton SE development. Consistently, disrupting endogenous redox homeostasis through the application of high concentration of DPI, H2O2, BSO or GSH inhibited the dedifferentiation of cotton explants. Mild oxidation induced through BSO treatment facilitated the transition from embryogenic calluses(ECs) to somatic embryos. The content of free IAA was attenuated by DPI or H2O2 treatment. Also, the metabolism of auxin and the expressions of auxin-related genes were altered by DPI or H2O2 treatment as compared with normal control. The metabolism of auxin and the expressions of auxin-related genes during SE were also altered in Gh APXs suppression lines as compared with wild type. Indicating that auxin homeostasis was altered through the perturbation of ROS homeostasis by chemical treatments or suppression of ROS-scavenging proteins. These results show that stress responses are activated during SE, and may regulate the ROS homeostasis by interacting with auxin signaling. 2. Gh TZF1 regulates drought stress responses and delays leaf senescence by inhibiting reactive oxygen species accumulation in transgenic ArabidopsisRedox homeostasis is important for plants to be able to maintain cellular metabolism, and disrupting cellular redox homeostasis will cause oxidative damage to cells and adversely affect plant growth. In this study, a cotton CCCH-type tandem zinc finger gene, which had a plant unique TZF domain, was characterized and defined as Gh TZF1. Gh TZF1 was isolated from a cotton cell wall regeneration SSH library in our previous research. It was predominantly expressed during early cell wall regeneration, and it was expressed in various vegetative and reproductive tissues, especially was induced by leaf senescence. The expression of Gh TZF1 was substantially up-regulated by a variety of abiotic stresses, such as PEG and salt. Gh TZF1 also responds to Me JA and H2O2 treatment. Overexpression of Gh TZF1 enhanced drought tolerance and delayed drought-induced leaf senescence in transgenic Arabidopsis. Subsequent experiments indicated that dark- and Me JA-induced leaf senescence was also attenuated in transgenic plants. The amount of H2O2 in transgenic plants was attenuated under both drought conditions and with Me JA-treatment. The activity of SOD and POD was higher in transgenic plants than in wild type plants under drought condition. q RT-PCR analysis revealed that overexpression of Gh TZF1 reduced the expression of oxidative-related senescence-associated genes(SAGs) under drought conditions. Overexpression of Gh TZF1 also enhanced oxidative stress tolerance, which was determined by measuring the expression of a set of antioxidant genes and SAGs that were altered in transgenic plants during H2O2 treatment. Hence, we conclude that Gh TZF1 may serve as a regulator in mediating drought stress tolerance and subsequent leaf senescence by modulating the reactive oxygen species homeostasis.