Anthocyanins generate the red color in the pericarp of Litchi chinensis. UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT, EC. 2.4.1.91) stabilizes anthocyanidin by attaching sugar moieties to the anthocyanin aglycone. In this study, the function of an UFGT gene involved in the biosynthesis of anthocyanin was verified through heterologous expression and virus-induced gene silencing assays. A strong positive correlation between UFGT activity and anthocyanin accumulation capacity was observed in the pericarp of 15 cultivars. Four putative flavonoid 3-O-glycosyltransferase-like genes, designated as LcUFGT1 to LcUFGT4, were identified in the pericarp of litchi. Among the four UFGT gene members, only LcUFGT1 can use cyanidin as its substrate. The expression of LcUFGT1 was parallel with developmental anthocyanin accumulation, and the heterologously expressed protein of LcUFGT1 displayed catalytic activities in the formation of anthocyanin. The LcUFGT1 over-expression tobacco had darker petals and pigmented filaments and calyxes resulting from higher anthocyanin accumulations compared with non-transformed tobacco. In the pericarp with LcUFGT1 suppressed by virus-induced gene silencing, pigmentation was retarded, which was well correlated with the reduced-LcUFGT1 transcriptional activity. These results suggested that the glycosylation-related gene LcUFGT1 plays a critical role in red color formation in the pericarp of litchi.

A fluoride export gene (CsFEX) was newly found and isolated from Camellia sinensis, and its functions in detoxifying F were investigated in transgenic Escherichia coli and Arabidopsis thaliana. CsFEX contains two crcB domains, which is the typical structure in plants. The expression of CsFEX in C. sinensis is tissue-specific and related to maturity of leaves, and its expression is significantly induced by F treatments in different tissues of C. sinensis, particularly in leaves. Additionally, the growth of C. sinensis, E. coli, and A. thaliana can all be inhibited by F treatment. However, the growth of CsFEX-overexpression E. coli was increased with lower F content under F treatment compared to the control. Similarly, the germination and growth of CsFEX-overexpression A. thaliana were enhanced with lower F content under F treatment compared to the wild type. CsFEX relieves F toxicity in the transgenic E. coli and A. thaliana by alleviating F accumulation.

The post-phloem unloading pathway and the mechanism of sugar accumulation remain unclear in litchi fruit. A combination of electron microscopy, transport of phloem-mobile symplasmic tracer (carboxyfluorescein, CF) and biochemical and molecular assays was used to explore the post-phloem transport pathway and the mechanism of aril sugar accumulation in litchi. In the funicle, where the aril originates, abundant plasmodesmata were observed, and CF introduced from the peduncle diffused to the parenchyma cells. In addition, abundant starch and pentasaccharide were detected and the sugar concentration was positively correlated with activities of sucrose hydrolysis enzymes. These results clearly showed that the phloem unloading and post-phloem transport in the funicle were symplastic. On the other hand, imaging of CF showed that it remained confined to the parenchyma cells in funicle tissues connecting the aril. Infiltration of both an ATPase inhibitor [eosin B (EB)] and a sucrose transporter inhibitor [p-chloromercuribenzene sulfonate (PCMBS)] inhibited sugar accumulation in the aril. These results indicated an apoplasmic post-phloem sugar transport from the funicle to the aril. Although facilitated diffusion might help sucrose uptake from the cytosol to the vacuole in cultivars with high soluble invertase, membrane ATPases in the aril, especially tonoplast ATPase, are crucial for aril sugar accumulation. The expression of a putative aril vacuolar membrane sucrose transporter gene (LcSUT4) was highly correlated with the sugar accumulation in the aril of litchi. These data suggest that apoplasmic transport is critical for sugar accumulation in litchi aril and that LcSUT4 is involved in this step.

Cell wall invertase (CWIN) are known to play important roles in seed development. However, most reports to date have focused on a single gene family member, and have mainly investigated CWIN functions during the filling stage of seed development. In this study, we found significant lower levels of CWIN protein and activity associated with seed abortion in the Litchi chinensis cultivar “Nuomici.” We identified five litchi CWIN genes and observed that the expression of LcCWIN5 was limited to the flower tissues and decreased sharply with fruit development. Silencing of LcCWIN5 expression before 28 DAA (cell division stage) resulted in perturbed liquid endosperm development, smaller seeds, and higher seed abortion rate, while silencing after 28 DAA (filling stage) had no effect on seed development. In contrast, LcCWIN2 was mostly expressed in the funicle and seed coat, and increased with fruit development. Decreased LcCWIN2 expression and CWIN activity during early seed filling coincided with smaller seeds in the cultivar “Feizixiao.” Silencing of LcCWIN2 caused a reduction in the seed size without inducing seed abortion. We propose that CWIN activity in seed maternal tissues during cell division stage is likely due to LcCWIN5 expression, which regulates early seed development. On the other hand, CWIN activity during the filling stage is due to the expression of LcCWIN2, which may promote carbon import by creating a sucrose gradient. Comparable LcCWIN5 expression, but much lower CWIN activity, detected in the funicle of “Nuomici” is consistent with post-translational regulation.

The available components in the flesh of litchi seem insufficient to interpret its wide and significant physiological effects. Some unusual compounds, including myo-inositol, inositol methyl derivatives and γ-aminobutyric acid (GABA) were identified as main constituents in the flesh of litchi. Their concentrations varied among cultivars but remain relatively constant during development. Litchi flesh was shown to contain moderate myo-inositol (0.28-0.78 mg g(-1) FW), ascorbic acid (0.08-0.39 mg g(-1) FW) and phenolics (0.47-1.60 mg g(-1) FW), but abundant l-quebrachitol (1.6-6.4 mg g(-1) FW) and GABA (1.7-3.5 mg g(-1) FW). The concentration of GABA in the flesh of litchi was about 100 times higher than in other fruits. And l-quebrachitol is not a common component in fruits. The biological and physiological activities of inositols, inositol derivatives and GABA have been extensively documented. These compounds are probably important compositional characteristic contributing to the widely shown health benefits of litchi.