CYTOCHROME P450s genes are a large gene family in the plant kingdom. Our earlier transcriptome data revealed that a CYTOCHROME P450 gene of Citrus sinensis (CsCYT75B1) was associated with flavonoid metabolism and was highly induced after drought stress. Here, we characterized the function of CsCYT75B1 in drought tolerance by overexpressing it in Arabidopsis thaliana. Our results demonstrated that the overexpression of the CsCYT75B1 gene significantly enhanced the total flavonoid contents with increased antioxidant activity in transgenic Arabidopsis. The gene expression results showed that several genes that are responsible for the biosynthesis of antioxidant flavonoids were induced by 2-12 fold in transgenic Arabidopsis lines. After 14 days of drought stress, all transgenic lines displayed an enhanced tolerance to drought stress along with accumulating antioxidant flavonoids with lower superoxide radicals and reactive oxygen species (ROS) than wild type plants. In addition, drought-stressed transgenic lines possessed higher antioxidant enzymatic activities than wild type transgenic lines. Moreover, the stressed transgenic lines had significantly lower levels of electrolytic leakage than wild type transgenic lines. These results demonstrate that the CsCYT75B1 gene of sweet orange functions in the metabolism of antioxidant flavonoid and contributes to drought tolerance by elevating ROS scavenging activities.

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Grafting is an ancient technique used for plant propagation and improvement in horticultural crops for at least 1,500 years. Citrus plants, with a seed-to-seed cycle of 5–15 years, are among the fruit crops that were probably domesticated by grafting. Poncirus trifoliata, a widely used citrus rootstock, can promote early flowering, strengthen stress tolerance, and improve fruit quality via scion–rootstock interactions. Here, we report its genome assembly using PacBio sequencing. We obtained a final genome of 303 Mb with a contig N50 size of 1.17 Mb and annotated 25,680 protein-coding genes. DNA methylome and transcriptome analyses indicated that the strong adaptability of P. trifoliata is likely attributable to its special epigenetic modification and expression pattern of resistance-related genes. Heterografting by using sweet orange as scion and P. trifoliata as rootstock and autografting using sweet orange as both scion and rootstock were performed to investigate the genetic effects of the rootstock. Single-base methylome analysis indicated that P. trifoliata as a rootstock caused DNA demethylation and a reduction in 24-nt small RNAs (sRNAs) in scions compared to the level observed with autografting, implying the involvement of sRNA-mediated graft-transmissible epigenetic modifications in citrus grafting. Taken together, the assembled genome for the citrus rootstock and the analysis of graft-induced epigenetic modifications provide global insights into the genetic effects of rootstock–scion interactions and grafting biology.

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BackgroundCitrus fruits are consumed freshly or as juice to directly provide various dietary flavonoids to humans. Diverse metabolites are present among Citrus genera, and many flavonoids biosynthetic genes were induced after abiotic stresses. To better understand the underlying mechanism, we designed experiments to overexpress a UDP-GLUCOSYL TRANSFERASE gene from sweet orange (Citrus sinensis) to evaluate its possible function in metabolism and response to stress.ResultsOur results demonstrated that overexpression of Cs-UGT78D3 resulted in high accumulation of proanthocyanidins in the seed coat and a dark brown color to transgenic Arabidopsis seeds. In addition, the total contents of flavonoid and anthocyanin were significantly enhanced in the leaves of overexpressed lines. Gene expression analyses indicated that many flavonoid (flavonol) and anthocyanin genes were up-regulated by 4–15 folds in transgenic Arabidopsis. Moreover, after 14 days of high light stress, the transgenic Arabidopsis lines showed strong antioxidant activity and higher total contents of anthocyanins and flavonoids in leaves compared with the wild type.ConclusionOur study concluded that the citrus Cs-UGT78D3 gene contributes to proanthocyanidins accumulation in seed coats and confers tolerance to high light stress by accumulating the total anthocyanin and flavonoid contents with better antioxidant potential (due to photoprotective activity of anthocyanin) in the transgenic Arabidopsis.

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