Effects of grafting on citrulline content and expression of key enzyme genes in watermelon fruits

Abstract: 【Objective】In recent years, with the progress of agricultural production conditions and scientific research level as well as the adjustment of agricultural economic policies, watermelon production in China has developed rapidly. Continuous cropping has impeded the agricultural production, resulting in severe soil-borne diseases and thus restricting the sustainable development of cucurbit crops. In practical production, grafting is widely used to solve the above-mentioned problems. Considerable practical experience and researches indicated that watermelon grafting could overcome some continuous cropping obstacles, and grafting onto a suitable rootstock could make watermelon plants grow vigorously, improve the water and fertilizer absorption, enhance resistance to diseases, raise the seedling tolerance to high temperature and salt stress, and increase the yield of watermelon. Previous studies have shown that watermelon fruit is rich in citrulline. On exposure to drought, intensive light and salt stress, citrulline was the primary component (49%) of the amino acids pool, and the citrulline content could accumulate osmotically. It was important to help plants to maintain the osmotic pressure and enhance the ability of stress tolerance. It was suggested that citrulline content could act as a potent scavenger in watermelons by protecting the green tissues from the oxidative stress during drought conditions. Citrulline was identified as the most potent scavenger of hydroxyl radicals than mannitol, proline, and glycine betaine, so some scholars suggested that citrulline can be used as a biomarker to study stress resistance of plants.So far, some studies have shown that grafting can change citrulline contents in watermelon fruits, but the current research is just limited to the determination of citrulline content. There is limited information available about the citrulline synthesis pathway and the mechanism of citrulline accumulation. This study aims to investigate the impact of grafting on citrulline accumulation in developing watermelon fruits and transcriptional regulation of key genes of the citrulline biosynthetic pathway to elucidate the possible mechanism influencing citrulline accumulation in grafted watermelon and make it clear the reason for citrulline accumulation in watermelon fruits by grafting.【Methods】'SBD black'characterized by low citrulline content was selected as the scion and grafted to the pumpkin rootstock. Self-rooted'SBD black'served as the control. The flesh samples were taken 10 days after pollination. In order to ensure the consistency of fruit development, three fruits with the same pollination date, shape, size and node position were sampled from each variety. Then the samples were taken every 7 days until the fruit was over ripe (45 days after pollination) . The watermelon fruit was cut into two halves and central flesh was used to measure the citrulline contents, and the tissues were immediately frozen in liquid nitrogen and then quickly stored at-80 ℃ untill use. Citrulline was extracted by Methanol-HCl in 55 ℃ waterbath30 min after watermelon tissues were homogenized, followed by decolorization with active carbon, under the action of a mixture of phosphoric acid and sulfuric acid, boiled 30 min. in the dark for colorreaction by the diacetyl monoxime. The UV-visible spectrophotometric method was used to determine the citrulline content in watermelon fruits indirectly, and all data were repeated three times. The other half of the central flesh of watermelons was extruded with water, wrapped in tin foil and immediately put into liquid nitrogen for quick freezing. Later it was stored in a refrigerator at-80 ℃ for RNA extraction. Then the RNA was eversely transcribed into cDNA. The key genes of citrulline biosynthesis pathway in watermelon fruits were quantified by Roche LightCycler 480 Real Time PCR System.【Results】Compared to'SBD black'and'Xijiaqiangsheng'grafted onto pumpkin rootstock could increase citrulline contents in'SBD black'during the development of watermelon fruits, and the citrulline content in'SBD black'grafted watermelon fruit was higher than that of non-grafted'SBD black'. The citrulline content of both'SBD black'and grafted'SBD black'reached the maximum at 38 days, and the citrulline content of'SBD black'grafted on pumpkin increased by 63.0% than non-grafted watermelon; Compared the difference in the gene expression related to citrulline biosynthesis between grafted and non-grafted watermelons during fruit development, it was suggested that the expression of NAGK and GAT genes in grafted and non-grafted watermelon fruits differed greatly in the late stage of fruit development, and the expression of both genes in'SBD black'-grafted watermelon was lower than that from non-grafted'SBD black', while the expression of'SBD black'Arginase gene in grafted watermelon fruits was higher than that in non-grafted watermelon fruits.【Conclusion】The citrulline content of'SBD black'-grafted watermelon was higher than that with non-grafted'SBD black'one during fruit development.'Xijiaqiangsheng'grafted onto the pumpkin rootstockcan increase the citrulline content in watermelon. The differences in the citrulline content between grafted'SBD black'and non-grafted watermelons was attributed to the high expression of the upstream genes NAGK and GAT as well as the low expression of the downstream genes Arginase in grafted watermelon. The'Xijiaqiangsheng'grafting can regulate the expression of genes involved in the citrulline biosynthesis and ultimately regulate citrulline contents.