The Mechanism Study Of Spinach Hemoglobin (SoHb) Under Nitrate Stress

Soil secondary salinization has become one of the main obstacles in facilities vegetables grow in our country, which seriously restricting the sustainable development of modern agriculture. Researches showed that the main anion in greenhouse soil is NO3-, which is about56%-76%of the total anion. Hemoglobin is widely exists in plants, animals and microbes, which plays important roles under abiotic stress. Non-symbiotic hemoglobin1improves plants’ abiotic stress resistance, such as salt stress, water stress and cold stress. In this study, we studied the relationship of SoHb expression, NO concentration, and nitrate reductase (NR) activity under nitrate stress in two spinach cultivars with different resistance. Besides, exogenous NO donor SNP, NO scavenger cPTIO, NR and NOS inhibitors were used to explore the potential function and mechanism of SoHb under NO3stress. At last, by using transgenic Arabidopsis thaliana, we further study the function and mechanism of SoHb under nitrate stress. The main results are as followes:(1) SoHb expression, NO content, and NR activity in root and leaf of salt-tolerant spinach cultivar (’Chaoji’) and salt-sensitive spinach cultivar (’Daye’) under NO3-stress were analyzed. The results showed that the SoHb expression increased in root and leaf of’Chaoji’after160mM NO3-stress treatment for24h. The SoHb expression was weak in leaf of’Daye’ and the expression in root decreased after160mM NO3-stress treatment for24h. The NO concentration in roots and leaves of spinach increased after110-210mM NO3-stress treatment for24h. In the’Chaoji’ root, NR activity increased with the increase of the N03-concentration, while the NR activity increased first and then decreased after nitrate treatment in ’Daye’.(2) The SoHb expression, NO content, and NR activity in ’chaoji’ and ’Daye’ were studied by adding exogenous SNP and cPTIO. The results showed that the fluorescence of NO increased by adding SNP under nitrate treatment in’Chaoji’root, while in ’Daye’ the fluorescence decreased. The expression of SoHb decreased significantly after adding SNP and cPTIO in two cultivars. The NR activity increased strongly with SNP under nitrate stress.(3) The SoHb expression, NO content, and NR activity in ’Chaoji’ and ’Daye’ were studied by adding Na2WO4and L-NAME. The results showed that the NO fluorescence increased under nitrate treatment. When Na2WO4were added, NO content decreased. However, the fluorensence changed a litter after L-NAME was added. The NR activity decreased significantly after Na2WO4was added, while the NR activity increased when added L-NAME, which means that the NO production might through NR. The SoHb expression increased in root of’Chaoji’and decreased in ’Daye’ after adding Na2WO4and L-NAME.(4) The open reading frame (ORF) sequence of SoHb cDNA were obtained by RT-PCR. The ORF cDNA sequence of SoHb was504bp, encoding168amino acids. Sequence alignment showed that SoHb had high identity with the other plants, and with the highest identity of84.2%with Beta vulgaris. Phylogenetic analysis indicated that SoHb belongs to non symbiotic hemoglobin1(nsHb1) family.(5)The over-expression vector pK2GW7-35s-SoHb and RNAi-vector pK2GW7-35-Soh-I-SoHb were constructed and transformed into Arabidopsis. The homozygous transgenic Arabidopsis thaliana plants were obtained after kanamycin screening, genomic DNA PCR and RT-PCR detection. The phenotypic analysis showd that the height of overexpression SoHb transgenic plants were higher than wild type (WT) plants and RNAi plants. The flowering time of SoHb overexpression transgenic plant was earlier than WT plants and the RNAi plants have delayed flowering time. Seed germination rate of SoHb overexpression transgenic plants were higher than the WT plants, suggesting that SoHb overexpression improved nitrate stress tolerance. When the overexpression transgenic seedling were treated with nitrate solution, the NO content decreased with the increasing treatment time, while in WT plants, the NO increased. The results suggested that the function of overexpression of SoHb might be related with the NO content.