Effects of salt and drought stresses and light quality on vitamin C content and expression of synthetic genes in kiwifruit leaves

Abstract: 【Objective】Ascorbic acid (Vitamin C) is an important agronomic trait of fruits and vegeta-bles. Kiwifruit is rich in ascorbic acid. However, the effects of abiotic stresses on vitamin C synthesis re-main largely unclear. Here, we investigated the changes in vitamin C content in the leaves of kiwifruit (Actinidia eriantha) under different abiotic stress environments and analyzed the relative expression lev-els of key synthetic genes in the pathway of vitamin C synthesis. The results of this research will clarify the regulatory mechanism of vitamin C accumulation under abiotic stresses in kiwifruit.【Methods】Actinidia eriantha‘Huate’was selected as the experimental cultivar because of its high vitamin C content in fruit (>600 mg·100 g^-1 fruit weight). Annual branches (about 1-2 cm thick and 50-100 cm long) with fresh leaves and tissue culture plantlets were incubated in a greenhouse for salt, drought and light treat-ments. Firstly, four salt treatments, including three salt concentrations (NaCl solutions at 3, 6 and 9 g·L^-1)and clean water treatment (CK), were set to evaluate the effect of salt concentration on vitamin C syn-thesis in kiwifruit. Each treatment had 5 replicates. The leaf materials were sampled at 0 d, 2 d, 4 d and 6 d for Vc content determination and gene expression analysis. The leaf samples were freeze-dried and pretreated in liquid nitrogen, the RNA was extracted and reverse transcribed into cDNA for gene expres-sion analysis. Secondly, the A. eriantha‘Huate’samples were treated with different osmotic condi- tions, namely, the control (treated with clean water, CK), mild (with 5% PEG6000 solution), moderate (10% PEG6000 solution) and severe (20% PEG6000 solution) osmotic stress. The sampling time and strategy were consistent with those of the salt treatments. Thirdly, four different light treatments, including white light, red light, blue light and red & blue light (red light∶blue light =1∶1), were designed in an artificial climate chamber. The wavelength of white, red and blue light were 450-465 nm, 650-700 nm and 465-480 nm, respectively. The light intensity was（200±5）μmol·m^-2·s^-1; the ambient temperature was (24±1) ℃. The materials were treated in darkness for 48 h in advance, and then treated in different light for 12 h. The leaves were sampled in darkness, 1 h, 2 h, 6 h and 12 h under light treatment. The content of vitamin C in A. eriantha‘Huate’was determined with high performance liquid chromatogra-phy (Shimadzu liquid chromatograph). The expression levels of key metabolic genes of vitamin C path-way under different treatments were analyzed by real-time fluorescence quantitative PCR. Excel 2016 software was used for statistical analysis of the data, SPSS 21.0 software was employed for one-way analysis of variance, LSD, Tukey and Duncan’s multiple range tests, and Photoshop was used for plot-ting. Different lowercase letters indicate significant differences (p＜0.05).【Results】Salt treatment experiments confirmed that low salt concentration (3 g·L^-1 NaCl solution) significantly promoted vitamin C synthesis in A. eriantha‘Huate’leaves, while moderate and high salt concentrations caused damage on the experimental materials, resulting in no significant change in vitamin C content in their leaves. The vitamin C was significantly increased under osmotic stress treatments with the increase in stress time. In the light experiment, the concentration of vitamin C in A. eriantha leaves decreased under dark condition, whereas vitamin C increased sharply by more than five times within two hours of exposure to light. Of that, the mixed blue and red light treatment had the highest vitamin C accumulation, while the monochromatic blue light or red light had the least effect on vitamin C accumulation, even less than that in white light. Real-time fluorescence quantitative PCR results showed that the relative expression level of AceGGP in salt stress treatment was higher than that in the control group, indicating that the in-creased accumulation of vitamin C content under salt stresses might be related to the up-regulated expression of AceGGP gene. The expression levels of AceGME, AceGGP and AceGPP were increased by osmotic stress, which might be related to the increase in vitamin C accumulated in kiwifruit leaves. Compared with white light, the expression levels of AceGME, AceGMP and AceGGP genes were signifi-cantly increased under blue light (p＜0.05), but there was no significant difference in AceGPP ex-pression, which inferred that blue light may be an important factor affecting vitamin C synthesis.【Conclusion】The abiotic stress conditions, conducive to elevation of vitamin C content in kiwifruit were identified, and the key genes of vitamin C synthesis affected by different abiotic stresses were ex-plored, providing data support for the subsequent study on the mechanism of vitamin C biosynthesis in response to different environmental conditions in kiwifruit. In addition, our results suggest that main-taining appropriate environmental conditions in commercial production will facilitate the production of kiwifruit with high vitamin C content.