Huini Xu

Overexpression of CsNMAPK in tobacco enhanced seed germination under salt and osmotic stresses

In this research, biological function of CsNMAPK, encoding a mitogen-activated protein kinase of cucumber, was investigated under salt and osmotic stresses. Northern blot analysis showed that the expression of CsNMAPK was induced by salt and osmotic stresses in the cucumber root. In order to determine whether CsNMAPK was involved in plant tolerance to salt and osmotic stresses, transgenic tobacco plants constitutively overexpressing CsNMAPK were generated. Northern and Western blot analysis showed that strong signals were detected in the RNA and protein samples extracted from transgenic lines, whereas no signal was detected in the wild type tobacco, indicating that CsNMAPK was successfully transferred into tobacco genome and overexpressed. The results of seed germination showed that germination rates of transgenic lines were significantly higher than wild type under high salt and osmotic stresses. In addition, seed growth of transgenic lines was much better than wild type under salt and osmotic stresses. These results indicated that overexpression of CsNMAPK positively regulated plant tolerance to salt and osmotic stresses.

Overexpression of spinach non-symbiotic hemoglobin in Arabidopsis resulted in decreased NO content and lowered nitrate and other abiotic stresses tolerance.

A class 1 non-symbiotic hemoglobin family gene, SoHb, was isolated from spinach. qRT-PCR showed that SoHb was induced by excess nitrate, polyethylene glycol, NaCl, H2O2, and salicylic acid. Besides, SoHb was strongly induced by application of nitric oxide (NO) donor, while was suppressed by NO scavenger, nitrate reductase inhibitor, and nitric oxide synthase inhibitor. Overexpression of SoHb in Arabidopsis resulted in decreased NO level and sensitivity to nitrate stress, as shown by reduced root length, fresh weight, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm), and higher malondialdehyde contents. The activities and gene transcription of superoxide dioxidase, and catalase decreased under nitrate stress. Expression levels of RD22, RD29A, DREB2A, and P5CS1 decreased after nitrate treatment in SoHb-overexpressing plants, while increased in the WT plants. Moreover, SoHb-overexpressing plants showed decreased tolerance to NaCl and osmotic stress. In addition, the SoHb-overexpression lines showed earlier flower by regulating the expression of SOC, GI and FLC genes. Our results indicated that the decreasing NO content in Arabidopsis by overexpressing SoHb might be responsible for lowered tolerance to nitrate and other abiotic stresses.

Spinach 14-3-3 protein interacts with the plasma membrane H(+)-ATPase and nitrate reductase in response to excess nitrate stress.

To investigate the function of 14-3-3 protein in response to excess nitrate stress, a 14-3-3 protein, designated as So14-3-3, was isolated from spinach. Phylogenetic analysis demonstrated that So14-3-3 belongs to non-ε group of 14-3-3 superfamily. Real time-quantitative RT-PCR and western blot analysis showed that So14-3-3 was induced by excess nitrate stress in spinach roots and leaves. After nitrate treatment, the phosphorylated H(+)-ATPase and nitrate reductase (NR) increased and decreased respectively. Co-Immunoprecipitation (Co-IP) suggested that the interaction of So14-3-3 with the phosphorylated H(+)-ATPase enhanced, but reduced with phosphorylated NR in spinach roots after nitrate treatment. Besides, 5 proteins interacted with So14-3-3 were found by Co-IP and LC-MS/MS analysis. So14-3-3 overexpressing transgenic tobacco plants showed enhanced tolerance to nitrate treatment at the germination and young seedlings stage. The transgenic plants showed longer root length, lower malondialdehyde (MDA), H2O2, protein carbonyl contents, relatively higher soluble sugar and protein contents, than the WT plants after nitrate treatment. The phosphorylation levels of H(+)-ATPase in transgenic plants were higher than the WT plants after nitrate treatment, whereas NR were lower. Additionally, in transgenic plants, the interaction of So14-3-3 with phosphorylated H(+)-ATPase and NR increased and decreased more than the WT plants under nitrate stress, leading to higher H(+)-ATPase and NR activities in transgenic plants. These data suggested that So14-3-3 might be involved in nitrate stress response by interacting with H(+)-ATPase and NR.

PHYSIOLOGICAL RESPONSES OF TWO CUCUMBER CULTIVARS TO NITRATE STRESS

Effect of nitrate (NO3 −) on lipid peroxidation, activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), soluble sugar, and proline accumulation were investigated in cucumber [Cucumis sativus L. cv. ‘Xintaimici’ (salt-tolerant) and ‘Shennongchunwu’ (salt-sensitive)] seedlings treated with 14 (control) and 140 mM NO3 − for seven days. The results showed that excess NO3 − increased malondialdehyde (MDA) content and electrolyte leakage in cucumber roots of both cultivars, and the degree of increase was higher in ‘Shennongchunwu’. The activities of SOD, CAT, POD, and APX increased in the roots of ‘Xintaimici’, but decreased in roots of ‘Shennongchunwu’, with respect to the control plants. Compared to the control, the soluble sugar and proline content increased in the cucumber roots of both cultivars, especially in ‘Xintaimici’. These results suggested that ‘Xintaimici’ tolerance to NO3 − stress may partly be due to higher activities of antioxidant enzymes and accumulation of osmotic regulation substances.

Aluminium-inhibited NO3- uptake is related to Al-increased H2O2 content and Al-decreased plasma membrane ATPase activity in the root tips of Al-sensitive black soybean

In this study, Al-sensitive black soybean (Glycine max (L.) Merr.) specimens were treated in Hoagland solutions containing 50-400µM Al for 1-4 days. The measurement for NO3- uptake showed that the NO3- uptake decreased gradually as the Al concentration and treatment time increased, suggesting that Al stress significantly reduced the NO3- uptake by soybean. Under 100-µM Al stress for 4 days, the plasma membrane (PM) ATPase activity (inorganic phosphate (Pi) release), H+ pump activity, phosphorylation of PM ATPase and its interaction with 14-3-3 protein in soybean root tips were all smaller than those in the root tips of control plants. The addition of 150µM Mg2+ in Al treatment solutions significantly alleviated the Al inhibition of NO3- uptake in soybean. The presence of Mg2+ in a 100-µM Al solution pronouncedly enhanced PM ATPase activity, H+ pump activity, phosphorylation of PM ATPase and its interaction with 14-3-3 protein in soybean root tips. The application of 2mM ascorbic acid (AsA, an H2O2 scavenger) in Al treatment solutions significantly decreased Al-inhibited NO3- uptake in soybean. The cotreatment of soybeans with 2mM AsA and 100µM Al significantly reduced H2O2 accumulation and increased the PM ATPase activity, H+ pump activity, phosphorylation of PM H+-ATPase and its interaction with 14-3-3 protein in soybean root tips. The evidence suggested that Al-inhibited NO3- uptake is related to Al-increased H2O2 content and Al-decreased phosphorylation of PM ATPase and its interaction with 14-3-3 protein as well as PM ATPase activity in the root tips of soybean.

Overexpression of a peroxidase gene ( AtPrx64 ) of Arabidopsis thaliana in tobacco improves plant’s tolerance to aluminum stress

Key messageAtPrx64 is one of the peroxidases gene up-regulated in Al stress and has some functions in the formation of plant second cell wall. Its overexpression may improve plant tolerance to Al by some ways. Studies on its function under Al stress may help us to understand the mechanism of plant tolerance to Al stress.AbstractIn Arabidopsis thaliana, the expressions of some genes (AtPrxs) encoding class III plant peroxidases have been found to be either up-regulated or down-regulated under aluminum (Al) stress. Among 73 genes that encode AtPrxs in Arabidopsis, AtPrx64 is always up-regulated by Al stress, suggesting this gene plays protective roles in response to such stress. In this study, transgenic tobacco plants were generated to examine the effects of overexpressing of AtPrx64 gene on the tolerance to Al stress. The results showed that overexpression of AtPrx64 gene increased the root growth and reduced the accumulation of Al and ROS in the roots. Compared with wild type controls, transgenic tobaccos had much less soluble proteins and malondialdehyde in roots and much more root citrate exudation. The activity of plasma membrane (PM) H+-ATPase, the phosphorylation of PM H+-ATPase and its interaction with 14-3-3 proteins increased in transgenic tobaccos; moreover, the content of lignin in root tips also increased. Taken together, these results showed that overexpression of AtPrx64 gene might enhance the tolerance of tobacco to Al stress.

Physiological response and transcription profiling analysis reveals the role of H 2 S in alleviating excess nitrate stress tolerance in tomato roots

Soil secondary salinization caused by excess nitrate addition is one of the major obstacles in greenhouse vegetable production. Excess nitrate inhibited the growth of tomato plants, while application of 100 μM H2S donor NaHS efficiently increased the plant height, fresh and dry weight of shoot and root, root length, endogenous H2S contents and L-cysteine desulfhydrases activities. NaHS altered the oxidative status of nitrate-stressed plants as inferred by changes in reactive oxygen species (ROS) accumulation and lipid peroxidation accompanied by regulation of the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX). Besides, NaHS increased the nitric oxide (NO) and total S-nitrosothiols (SNOs) contents, nitrate reductase (NR) activities and decreased the S-nitrosoglutathione reductase (GSNOR) activities under nitrate stress. Furthermore, microarray analysis using the Affymetrix Tomato GeneChip showed that 5349 transcripts were up-regulated and 5536 transcripts were down-regulated under NaHS and excess nitrate stress treatment, compared to the excess nitrate stress alone. The differentially expressed genes (log2 fold change >2 or <  -2) of up-regulated (213) and down-regulated (271) genes identified were functionally annotated and subsequently classified into 9 functional categories. These categories included metabolism, signal transduction, defence response, transcription factor, protein synthesis and protein fate, transporter, cell wall related, hormone response, cell death, energy and unknown proteins. Our study suggested exogenous NaHS might enhance excess nitrate stress tolerance of tomato plants by modulating ROS and reactive nitrogen species (RNS) signaling and downstream transcriptional adjustment, such as defence response, signal transduction and transcription factors.