Changes of carotenoid content and expression analysis of associated genes in loquat fruits at different developmental stages under shading conditions

【Objective】Loquat (Eriobotrya japonica Lindl.) belongs to the Rosaceae family and is an evergreen subtropical fruit crop native to China. Loquat fruit is rich in carotenoids. Differences in carotenoid content and components make loquat fruit show yellow and white colors. Light is an important environmental factor affecting carotenoid accumulation in fruit. Some studies have shown that shading is beneficial to improving fruit appearance and intrinsic quality, but some studies have shown that it may affect fruit carotenoid accumulation. For loquat, it has been reported that shading has varying effects on the quality and color of loquat fruits, whereas the underlying molecular regulation mechanism remains unexplored. Further, although important genes on the carotenoid synthesis pathway of loquat have been identified, the expression pattern of these genes under shading conditions needs to be explored.【Methods】A yellow-fleshed loquat Zaojia 90 was treated with non-shade and shade treatments on fruits at the green fruit stage (S1). The treatment group was shaded using opaque fruit bags and the control groupwas non-shaded using mesh bags. Fruit treatments were preceded by fruit thinning, and 3-4 small fruits of uniform length were retained for subsequent experiments. The effects of shading on pigment concentration and expression of genes related to carotenoid synthesis in fruits at different developmental periods were investigated. After recording the appearance of peel and flesh at different developmental stages under shading and non-shading conditions, the chlorophyll a, chlorophyll b and carotenoid contents were also determined at each stage. In addition, changes in the expression of 14 relevant genes related to carotenoid biosynthesis were analyzed by quantitative real-time PCR (qRT-PCR) to explore the carotenoid accumulation pattern in the peel and flesh of the fruit under non-shade versus shade conditions. EjHY5 gene was cloned and analyzed to examine its expression characteristics in different tissue parts during different developmental periods under shading and non-shading conditions. The transient transformed tobacco was used for subcellular localization. To investigate the potential relationship between EjHY5- 1 and carotenoid synthesis process, virus- induced silencing (VIGS) was used to silence the EjHY5 gene in fruits and its expression was analyzed by qRT-PCR. The expression and promoter analysis of carotenoid synthesis- related genes PSY1 and PDS were also performed, which initially explored the potential relationship between EjHY5, and PSY1 and PDS.【Results】Fruits at each developmental stage were separated from the peel and flesh and assayed for pigment content and expression of related genes, respectively. Fruit color changed from green to yellow, while chlorophyll a, chlorophyll b, and carotenoid contents showed a decrease at the color change stage (S2), suggesting that chlorophyll degradation was the main cause of fruit color change. Shading caused an early greening of the peel of Zaojia 90, and resulted in a decrease in carotenoid content of both peel and flesh at the maturity stage (S4). Carotenoid content of the flesh was significantly reduced compared to the peel, indicating that shading had a greater effect on carotenoid accumulation in the flesh than in the peel. Shading reduced the nutritional value of the loquat fruit. Besides, shading had greater effect on carotenoid accumulation in the flesh than in the peel. qRT-PCR results showed that the expression trend of 14 genes related to carotenoid synthesis changed after shading. At the S2 stage, the expression of LCYb was up-regulated in the peel of shadingtreated fruits, while the expression of ZEP was significantly down-regulated, which may promote the accumulation of β-carotene. However, the detection of pigment content revealed a significant decrease in carotenoid content. qRT-PCR results showed that DXS, DXR, PSY1, and PDS, which are related to lycopene synthesis, showed a trend of down- regulation of expression in the peel of shading-treated fruits, which was hypothesized to indirectly affect the synthesis of β-carotene. Among them, DXR and PDS are potentially important genes in response to shading that significantly reduces carotenoid content of peel at the S2 stage. At the S4 stage, the carotenoid content in flesh under shading conditions significantly decreased, and PSY2A was significantly down- regulated in expression, suggesting that PSY2A may be a key gene affecting the final accumulation of carotenoids in fruit under shading conditions. Based on the loquat genome, EjHY5-1 and EjHY5-2 were cloned and their sequences and expression properties were studied. Both EjHY5-1 and EjHY5-2 were localized in the nucleus. EjHY5-1 was 594 bp in length and encoded 197 amino acids, while EjHY5-2 was 495 bp in length and encoded 164 amino acids. qRT-PCR results showed that EjHY5-1 was mainly expressed in the peel and EjHY5-2 was mainly expressed in the flesh. Light is an important environmental factor affecting the expression of EjHY5. After shading, the expression of EjHY5-1 in the peel appeared to be significantly or highly significantly down-regulated at late developmental stages, while the expression of EjHY5-2 gene appeared to highly significantly increase, suggesting that there may be different light-responsive processes between the two in the peel. Virus-induced gene silencing results showed that the expressions of EjPSY1 and EjPDS in the fruit signifi-cantly decreased after EjHY5-1 silencing. The promoters of EjPSY1 and EjPDS contained G- box elements that HY5 recognized. EjHY5-1 may play a regulatory role in them.【Conclusion】Shading had an important effect on the pigment content and expression of genes related to carotenoid synthesis in loquat fruits. It had greater effect on carotenoid accumulation in the peel. DXR and PDS were potential candidates responsible for the significant decrease of carotenoids in the peel of shading fruits at the color change stage. The down- regulation of PSY2A at the maturity stage under shading conditions may have affected carotenoid accumulation in the flesh. In addition, the expression of EjHY5-1 and EjHY5-2 was tissue-specific and closely related to light. The expressions of PSY1 and PDS genes significantly decreased in loquat fruit after the silencing of EjHY5-1 gene. Since the PSY1 and PDS promoter regions contained G- box elements, EjHY5- 1 may have transcriptional regulatory effects on PSY1 and PDS genes. Further studies are necessary to verify the potential function of EjHY5.