Functional Research Of Nitric Oxide In The Regulation Of Rice Flowering And Response To Abiotic Stresses

Nitric oxide (NO) has been shown to play an important role in a range of plant growth and development processes. NO also participates in plant response to various biotic and abiotic stresses, such as cold, drought, salt, heat and heavy metal stresses, and pathogen infection. Our knowledge about the roles of NO in plants has been mainly achieved by exogenous application of NO donors or scavengers. However, it is still not clear whether the application of the pharmacological compounds reflect the true physiological effects of NO. Additionally, NO functions always depend on its location and concentration, plant species, as well as plant developmental stages. Thus, in different plant species, NO may play different roles in the same physiological processes. Therefore, it is suggested that in order to assess the involvement of NO in development and stress signaling, plant materials with altered NO content should be used. Rice, as the most important food crop in Asia, is still relatively less for the functional research of NO, due to the lack of rice materials with altered endogenous NO levels.In this study, we overexpressed rat nNOS in japonica rice Zhonghuall and obtained transgenic rice plants with higher NOS activity and endogenous NO levels. Additionally, we explored the tolerance of transgenic plants to drought, high salinity and cadmium stresses and analysed the physiological changes in response to these stresses. Further, we also compared and analyzed developmental phenotype, including shoot length and flowering time, between the wild type and the transgenic plants. The main results are listed below:1. Mannitol and NaCl stresses increased both NOS activity and NO content in rice. The treatment with L-NAME repressed both mannitol and NaCl-mediated increase in NOS activity, resulting in a lower NO concentration, suggesting that both drought and salt stresses can modulate NO content, at least partially through NOS activity.2. We overexpressed the nNOS gene in japonica rice Zhonghuall (ZH11) by inserting the coding region of the rat nNOS into the pUbiO plant expression vector, which was then introduced into ZH11 via Agrobacterium-mediated transformation. Further analyses showed nNOS overexpression increases NOS activity, leading to higher endogenous NO levels in the transgenic rice lines.3. nNOS-overexpressing plants with higher NO content showed improved tolerance to both drought and salt stresses compared to the wild type.4. Further analyses indicated that nNOS-overexpressing plants had lower water loss rate and stomatal conductance, and showed higher relative water content (RWC) proline accumulation, less lipid peroxidation and reduced electrolyte leakage under drought and salt conditions than the wild type. Moreover, nNOS-overexpressing plants accumulated less H2O2, due to the observed up-regulation of OsCATA, OsCATB and OsPOXl. In agreement, the activities of CAT and POX were higher in transgenic rice than the wild type under stress conditions. Additionally, the expression of six tested stress-responsive genes, including OsDREB2A, OsDREB2B, OsSNACI, OsSNAC2, OsLEA3 and OsRD29A, in nNOS-overexpressing plants was higher than that in the wild type under drought and high salinity conditions. Taken together, our results suggest that nNOS overexpression suppresses the stress-enhanced electrolyte leakage, lipid peroxidation and H2O2 accumulation, and promotes proline accumulation and the expression of stress-responsive genes under stress conditions for promoting higher tolerance to drought and salt stresses.5. nNOS-overexpressing plants showed improved tolerance to cadimium stress. Further analyses indicated that nNOS-overexpressing plants exhibited higher proline accumulation, less lipid peroxidation and reduced H2O2 content under cadimium conditions than wild type. qRT-PCR results showed that the expression of three cadmium transporter genes, including HMA2, HMA3 and HMA9, in nNOS-overexpressing plants were similar to the wild type under cadmium conditions. These results suggest that nNOS overexpression suppresses cadimium-induced lipid peroxidation and H2O2 accumulation, and promotes proline accumulation under stress condition for higher higher tolerance to cadmium stress.6. Two independent nNOS-overexpressing transgenic lines, OE-1 and OE-26, showed a dwarf and late flowering phenotype. These two lines had about 2 times NO content compared to the wild type, which were also higher than other transgenic lines. Our qRT-PCR data showed that the expression of three flowering genes, including Ehdl, Hdl and Hd3a, in nNOS-overexpressing plants was lower than that in the wild type under short-day condition.