Main Pathway And Key Gene Contribute To The Increasing AsA Concentration In Cucumber Leaves Under Nitrogen Deficiency

Cucumber(Cucumis sativus L.) is an important crop worldwide, its growth and yield often depends on the nitrogen supply. In cucumber cultivation, large quantities of nitrogen fertilizer are applied to achieve high yields. However, excess nitrogen fertilizers can not only pollute water but also be harmful to plant development and quality. It is important to study the genes in response to nitrogen deficiency in cucumber seedlings that can contribute to reducing the amount of nitrogen fertilizer. Cucumber seedlings(Cucumis sativus L. 9930) cultivated in nutrient solution in greenhouse was used in this study. When the fifth leaves appeared, equal numbers of seedlings were transplanted into new boxes containing reformative Hoagland solution with(12 m M) or without(0 m M) nitrogen, named as CK and ND(nitrogen deficiency), respectively. The treatments lasted 48 hours. In the present study, the effects of nitrogen deficiency on the L-ascorbic acid(As A) pathway in cucumber leaves were analyzed at the transcriptional, enzymatic, and metabolic levels. The main results are as follows:1. The anti-oxidase system of cucumber seedlings was induced under nitrogen deficiency. During the experiment, the anthocyanin concentration increased gradually in ND leaves, the net photosynthetic rate in ND leaves declined after 24 HND(hours of nitrogen deficiency), but the chlorophyll concentration did not differ significantly between ND and control leaves. MDA concentration in ND leaves began to increase at 12 HND to a higher level compare with control. Exposure of cucumber seedlings to nitrogen deficiency caused a significant induction of O2?- in the leaf tissue and the H2O2 concentration increased significantly by 12 HND. The SOD, CAT and POD activities in ND leaves were markedly higher than that in the control, and APX showed higher activity in ND leaves than in control leaves at 12 and 24 HND.2. The level of ascorbic acid increased in N-limited cucumber seedlings. The concentration of As A and T-As A(Total As A) were significantly higher in ND leaves than in control leaves.3. The activities of enzymes and transcript levels of genes involved in ascorbate recycling pathway decreased or remained unchanged under nitrogen deficiency. GR and DHAR showed lower activities in ND leaves than in control leaves; AO and MDHAR showed higher activities in ND leaves than in control leaves only at 48 HND. The activities of these enzymes decreased which was consistent with the changes in their transcript levels.Generally, the transcript levels of GR2, AO2, MDHARs and DHAR2 were lower in ND leaves than in control leaves.4. Myo-insitol pathway was activated under nitrogen deficiency, while the other three pathways were nearly unchanged. Therefore, MIOX1 might be the key gene that contributed to the accumulation of As A in cucumber seedlings under nitrogen deficiency.In our study, the transcript level of GMP1 was up-regulated under nitrogen deficiency, while the enzyme activity of GMP did not change significantly. These findings suggested that the L-galactose and L-gulose pathways might not contribute to the accumulation of As A in leaves of cucumber under nitrogen deficiency.The changes in GLDH activity as well as in Gal UR indicated that nitrogen deficiency did not change the D-galacturonic acid pathway in cucumber, while MIOX activity and its transcript level increased notably under nitrogen deficiency.The activities of enzymes and transcript levels of genes involved in the myo-inositol pathway increased dramatically, as did the ascorbate concentration in nitrogen-deficient cucumber seedlings. As the precursor of As A, myo-inositol concentration peaked at 6 HND and then gradually decreased. The transcript level of MIOX1 genes was approximately 30 times higher than control at 6 HND, followed by a significant increase in enzyme activity at 12 and 24 HND.5. As A concentration in the tobacco seedlings infected with agrobacterium carring MIOX1 increased remarkablely, which indicated that MIOX1 probably regulated the synthesis of As A in response to nitrogen deficiency.Overall, we speculated that nitrogen deficiency motivated the antioxidant defense system and the accumulation of As A. Changes in both transcriptional and enzymatic levels of As A biosynthesis pathways indicated that the accumulation of As A in nitrogen deficiency cucumber seedlings might be attributed to AO, GR and the myo-inositol pathway. MIOX as an important enzyme in myo-inositol pathway might involve in this metabolic process.