Effects of water stress on the synthesis of volatile compounds in Marselan grape berries

Abstract: 【Objective】Water is one of the important factors that restrict agricultural production. The grape producing area in the eastern foot of Helan Mountain in Ningxia is located in the inland of northwest China, which belongs to temperate continental climate. The annual rainfall is small and the climate is dry. Relying on the Yellow River water for irrigation, however, there is excessive irrigation and unreasonable utilization, resulting in serious waste of water resources, which restricts the healthy and sustainable development of grape industry. Therefore, advocating scientific and efficient use of water resources is an important measure for agricultural sustainable development. In order to explore the effects of different water stress on the metabolism of aroma compounds synthesis during the berry development of Marselan grape, the present experiment was undertaken to obtain the technical measures of water management for improvement of the fruit quality of Marselan grape, and optimize the irrigation conditions suitable for arid and semi-arid areas as well as lay a theoretical foundation for the optimization of the cultivation and management of Marselan grape.【Methods】Taking 5-year-old Marselan grapevines as the test materials, three water treatments were set according to the water potential of leaves before dawn: no stress (control), light water stress (T1) and moderate stress (T2), and the vines were kept within the target range by controlling the irrigation amount. Extraction of free aroma: 15 g of crushed fruit samples was added into a centrifuge tube, and 1 g of cross-linked polyvinylpyrrolidone (PVPP) and 0.5 g of D-gluconolactone were added. The centrifuge tube was placed in a 4 °C refrigerator for 120 min. Then it was centrifuged at 10 000 r·min-1 for 15 min at 4 °C to obtain clear grape juice. Headspace solidphase microextraction (HS-SPME): Take 5 mL of grape juice into a 15 mL headspace bottle, add 1 g of sodium chloride, 5 μL of internal standard 2-octanol and a magnetic rotor, then quickly tighten the bottlecap, the extraction head was inserted into the sample headspace vial and placed on a magnetic stirrer for adsorption at 60 °C for 40 min. After adsorption, the extraction head was taken out and inserted into the gas chromatography injection port, and decomposed at 250 °C for 5 min. Gas chromatography separation conditions: Column: HP-INNO-Wax capillary column (length 30 m, inner diameter 0.25 mm, liquid film thickness 0.25 μm), carrier gas He (99.99%), flow rate 1.0 mL·min-1 ; Inlet temperature: 250 °C, desorption for 5 min; programmed temperature was 50 °C for 1 min, and then increased to 220 °C at a rate of 3 °C·min-1 and held for 5 min; mass spectrometry interface temperature was 280 °C, ion source temperature was 230 °C, ionization method was EI, the ionization energy was 70 eV, and the scanning range was 30-350 amu. Qualitative analysis and quantitative analysis: using the total ion current spectrum in the full ion scanning mode of mass spectrometry, the collected total ion map current was searched and data were analyzed with NIST08 and RTLPEST3 spectral libraries, combined with the retention index (Retention Index, RI) and references of identify volatile aroma components. The internal standard method was used for relative quantification, and 2-octanol was used as the internal standard to determine the relative content. The calculation formula was: relative content of aroma components (μg·L-1 ) = [peak area of each component/internal standard peak area × internal standard mass (μg)]/sample mass (kg). Grape fruit (without seeds) RNA was extracted with RNA Extraction Kit (spin column type). cDNA was synthesized by reverse transcription using TranScript kit. Fluorescence quantitative PCR was performed with Actin as the internal reference gene, and the specific sequences of VvGPPS, VvHPLA and VvCCD1 were searched in GeneBank, and primers were designed and synthesized by Shanghai Sheng Gong. Relative quantification was performed using the 2-ΔΔCt method.【Results】41, 45 and 36 volatile organic compounds were detected in the control, light stress and moderate stress, respectively, during the ripening stage of Marselan grape. The number of compound species was significantly less than that of light stress treatment. It can be seen that light water stress can significantly increase the types of volatile compounds in berries, which increased by 9.7% and 25.0% compared with the control and moderate stress, respectively. Under moderate stress, the types of volatile compounds in the berry decreased by 13.8% compared with the control. A total of 12 aldehydes and 12 alcohols were detected, and their contents increased with the development of Marselan grape berry. 120 days after flowering, the content of n-hexanol in berries treated with T1 was 54.7% and 60.9% higher than that with control and T2, respectively. Nine esters were detected, among which ethyl caprate, dibutyl phthalate, diethyl phthalate and propyl heptyl lactone were the main ester volatile substances in Marselan grape berry. Twelve kinds of volatile acids were detected, among which nonanoic acid had unpleasant pungent odor, and its content increased with berry development, indicating that water stress would aggravate the inferior aroma brought by nonanoic acid. At maturity, the expression of VvGPPS with control was higher than that of T1 and T2, which indicated that water stress would reduce the expression of this gene. The expression of VvHPLA decreased with the development of Marselan grape berry. The expression of VvCCD1 showed a downward trend, but the expression of VvCCD1 in T2-treated grape fruit was always higher than control. It was observed that the expression of VvCCD1 in T1-treated grape berry was significantly higher than control and T2 at 87th day after anthesis, which indicated that water stress treatment could effectively improve the expression of VvCCD1 in Marselan grape berry.【Conclusion】In conclusion, compared with the control, moderate water stress can significantly increase the variety and relative content of volatile organic compounds in Marcelan grape berry. Compared with the control, water stress is not conducive to the expression of VvGPPS and VvHPLA, but beneficial to the expression of VvCCD1.