| Soil secondary salinazation is one of the major obstacles to the vegetable production in greenhouse, which seriously inhibited the growth and quality of vegetables. The main anion in soil of greenhouse is NO3. Tomato (Solatium lycopersicum L.) is widely planted in field and facilities, but soil secondary salinization has restricted tomato production. Hydrogen sulfide (H2S) is an important signal molecule, which could improve the resistance of plants to a certain degree. In this study, effects of exogenous H2S on germination of tomato seed and growth and physiological characteristics of tomato seedlings under nitrate stress were studied, besides, we analyzed the molecular mechanisms of H2S function by microarray. The main results were presented as follows:1.In this study, the effects of exogenous H2S on germination of tomato seed under nitrate stress were investigated. The results showed that the germination of tomato seed was inhibited by high concentrations of NO3- and the germination rate reduced 45% under 100 mmol·L-1 NO3-. Exogenous H2S donor NaHS of 100,200,500,800 μmol·L-1 alleviated the inhibition effects of 100 mmol·L-1 NO3-, especially 100 μmol·L-1 NaHS treatment. NaHS treatment reduced accumulations of TBARS content. Furthermore,100 μmol·L-1 NaHS treatment reduced ROS content, increased the activities of amylase, SOD, POD, CAT and APX significantly, alleviating the oxidative damage of nitrate stress to germinating seeds. Besides, the NO content was increased by NaHS treatment under nitrate stress. Our results suggested that exogenous H2S alleviated nitrate stress damage possibly through regulating antioxidant enzyme activities and enhancing NO content.2.The effects of nitrate stress with 0,50,100,150,200 mmol·L-1 concentrations were studied on growth and physiological and biochemical indexes of tomato using the hydroponic culture test. The results showed that the plant height, fresh weight, dry weight and relative water content decreased with the increasing of nitrate concentration after treated for 6 days, while the root to shoot ratio increased first and then decreased. The contents of TBARS, ROS, H2O2 and PC of tomato seedlings increased with the increasing of nitrate concentration. The activities and gene expression of SOD, POD, CAT and APX of tomato seedlings decreased with the increasing of nitrate concentration. The contents of proline, soluble sugar and soluble protein of tomato seedlings increased with the increasing of nitrate concentration. Besides,50 mmol·L-1 NO3 had little effect on the plants, while 100 mmol·L01 NO3 inhibited the growth of tomato seedlings, and caused damage to the membrane lipid and decreased the activity of antioxidant enzymes (SOD, POD, CAT and APX). However, the increased osmotic adjustment substances could adjust themselves to avoid nitrate stress.3.The effects of exogenous NaHS on growth and physiological and biochemical indexes of tomato under nitrate stress were studied. The results showed that exogenous H2S donor NaHS of 50,100,300 μmol·L-1 alleviated the inhibition effects of 100 mmol·L-1 NO3-, especially 100 μmol·L-1 NaHS treatment. In comparison with the 100 mmol·L-1 NO3-treatment, the plant height, fresh weight, dry weight and relative water content of tomato seedlings increased by adding 100 μmol·L-1 NaHS treatment for 10 days, while the root to shoot ratio decreased. And 100 μmol·L-1 NaHS treatment reduced the contents of TBARS, ROS, H2O2 and PC of tomato seedlings. Furthermore,100 μmol·L-1 NaHS treatment increased the activities of SOD, POD, CAT and APX significantly and the contents of osmotic, alleviating the oxidative damage of nitrate stress to growth of tomato seedlings. Besides, the H2S content was increased by NaHS treatment under nitrate stress. Our results suggested that exogenous NaHS alleviated nitrate stress damage possibly through regulating antioxidant enzyme activities and osmotic substances, and enhancing H2S content.4.To real the molecular mechanism of the H2S function, microarrary analysises were performed using Affymetrix tomato genechip arrays of the tomato seedling in response to nitrate(100 mmol·L-1), NaHS(100μmol·L-1), and nitrate and NaHS. The results showed that the expression of 10773,9122, and 10885 genes changed, respectively. The genes changed more than two folds were 503 (up-regulated 250, and down-regulated 253),430 (up-regulated 190, down-regulated 240),484 (up-regulated 213, down-regulated 271), as compared with that in control seedling, respectively. Bioinformatics analysis revealed that function of the differentially expressed genes involved in metabolism, signal transduction, stress tolerance, transcription factor, transporter, protein synthesis and degradation, hormone responsive, cell wall. RT-PCR was used to verify the expression patterns of 24 positive clones. The results indicated that these microarray data were reliable.5. By using pharmacological and biochemical approaches, we reported the effects of H2S and NO on tomato seedlings exposed NO3-stress. The results showed that in comparison with the 100 mmol·L-1 NO3- treatment alone, both 100 μmol·L-1 NaHS and 100 μmol·L-1 SNP decreased the lipid peroxidation of tomato seedling. Total activities of antioxidant enzymes, including SOD, POD and APX were modulated differentially, thus leading to the alleviation of oxidative damage. Effects of H2S above were reversed by HT and PAG, the specific scavenger and inhibitor of NO, and cPTIO, the specific scavenger of NO. By using fluorescence microscope method, further results showed that NO production increased significantly after the NaHS treatment regardless of whether NO3- stress was applied or not, all of which were obviously inhibited by HT, PAG and cPTIO. Therefore, the above protective roles of H2S might be related to the induction of endogenous NO as well as H2S-triggered NO production was confirmed. |
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