Hui-Cong Wang

Differential Expression of Anthocyanin Biosynthetic Genes in Relation to Anthocyanin Accumulation in the Pericarp of Litchi Chinensis Sonn

Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m−2 among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red (‘Kuixingqingpitian’, ‘Xingqiumili’, ‘Yamulong’and ‘Yongxing No. 2′), unevenly red (‘Feizixiao’ and ‘Sanyuehong’) and fully red (‘Meiguili’, ‘Baila’, Baitangying’ ’Guiwei’, ‘Nuomici’ and ‘Guinuo’). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the pericarp anthocyanin concentration (r = 0.84). These results suggest that UFGT played a predominant role in the anthocyanin accumulation in litchi as well as pericarp coloration of a given cultivar.

De novo assembly and characterization of fruit transcriptome in Litchi chinensis Sonn and analysis of differentially regulated genes in fruit in response to shading

BackgroundLitchi (Litchi chinensis Sonn.) is one of the most important fruit trees cultivated in tropical and subtropical areas. However, a lack of transcriptomic and genomic information hinders our understanding of the molecular mechanisms underlying fruit set and fruit development in litchi. Shading during early fruit development decreases fruit growth and induces fruit abscission. Here, high-throughput RNA sequencing (RNA-Seq) was employed for the de novo assembly and characterization of the fruit transcriptome in litchi, and differentially regulated genes, which are responsive to shading, were also investigated using digital transcript abundance(DTA)profiling.ResultsMore than 53 million paired-end reads were generated and assembled into 57,050 unigenes with an average length of 601 bp. These unigenes were annotated by querying against various public databases, with 34,029 unigenes found to be homologous to genes in the NCBI GenBank database and 22,945 unigenes annotated based on known proteins in the Swiss-Prot database. In further orthologous analyses, 5,885 unigenes were assigned with one or more Gene Ontology terms, 10,234 hits were aligned to the 24 Clusters of Orthologous Groups classifications and 15,330 unigenes were classified into 266 Kyoto Encyclopedia of Genes and Genomes pathways. Based on the newly assembled transcriptome, the DTA profiling approach was applied to investigate the differentially expressed genes related to shading stress. A total of 3.6 million and 3.5 million high-quality tags were generated from shaded and non-shaded libraries, respectively. As many as 1,039 unigenes were shown to be significantly differentially regulated. Eleven of the 14 differentially regulated unigenes, which were randomly selected for more detailed expression comparison during the course of shading treatment, were identified as being likely to be involved in the process of fruitlet abscission in litchi.ConclusionsThe assembled transcriptome of litchi fruit provides a global description of expressed genes in litchi fruit development, and could serve as an ideal repository for future functional characterization of specific genes. The DTA analysis revealed that more than 1000 differentially regulated unigenes respond to the shading signal, some of which might be involved in the fruitlet abscission process in litchi, shedding new light on the molecular mechanisms underlying organ abscission.

LcMYB1 Is a Key Determinant of Differential Anthocyanin Accumulation among Genotypes, Tissues, Developmental Phases and ABA and Light Stimuli in Litchi chinensis

The red coloration of litchi fruit depends on the accumulation of anthocyanins. The anthocyanins level in litchi fruit varies widely among cultivars, developmental stages and environmental stimuli. Previous studies on various plant species demonstrate that anthocyanin biosynthesis is controlled at the transcriptional level. Here, we describe a litchi R2R3-MYB transcription factor gene, LcMYB1, which demonstrates a similar sequence as other known anthocyanin regulators. The transcription levels of the LcMYB1 and anthocyanin biosynthetic genes were investigated in samples with different anthocyanin levels. The expression of LcMYB1 was strongly associated with tissue anthocyanin content. LcMYB1 transcripts were only detected in anthocyanin-accumulating tissues and were positively correlated with anthocyanin accumulation in the pericarps of 12 genotypes. ABA and sunlight exposure promoted, whereas CPPU and bagging inhibited the expression of LcMYB1 and anthocyanin accumulation in the pericarp. Cis-elements associated with light responsiveness and abscisic acid responsiveness were identified in the promoter region of LcMYB1. Among the 6 structural genes tested, only LcUFGT was highly correlated with LcMYB1. These results suggest that LcMYB1 controls anthocyanin biosynthesis in litchi and LcUFGT might be the structural gene that is targeted and regulated by LcMYB1. Furthermore, the overexpression of LcMYB1 induced anthocyanin accumulation in all tissues in tobacco, confirming the function of LcMYB1 in the regulation of anthocyanin biosynthesis. The upregulation of NtAn1b in response to LcMYB1 overexpression seems to be essential for anthocyanin accumulation in the leaf and pedicel. In the reproductive tissues of transgenic tobacco, however, increased anthocyanin accumulation is independent of tobaccos endogenous MYB and bHLH transcriptional factors, but associated with the upregulation of specific structural genes.

Patterns of enzyme activities and gene expressions in sucrose metabolism in relation to sugar accumulation and composition in the aril of Litchi chinensis Sonn.

Sucrose metabolism enzymes, including invertase (EC 3.2.1.26), sucrose synthase (SS, EC 2.4.1.13), and sucrose phosphate synthase (SPS, EC 2.4.1.14), are key factors that determine fruit sugar accumulation and composition. Sugar concentration and sugar composition in the arils of 42 litchi cultivars were determined at maturity. The cultivars were grouped into three types according to their hexose/sucrose ratio. Five cultivars of each type were selected to monitor the activities and gene expressions of enzymes related to sucrose metabolism. Pattern changes in the arils of four cultivars with different sugar concentrations and compositions were traced from around 40 d after anthesis to full maturity. Highly significant positive correlations were observed between hexose/sucrose ratios and the activities and expression levels of soluble acid invertase (SAI) and SS among the 15 cultivars tested. The increase in hexose/sucrose ratio was accompanied by enhanced acid invertase (AI) and SS activities and the expression of their genes in Feizixiao (FZX) and Heiye (HY). By contrast, no significant correlation was observed between hexose/sucrose ratio and SPS. These results indicate that the sugar composition in litchi aril depends mainly on the sucrose cleavage enzymes AI and SS and not on the sucrose synthetic enzyme SPS. The cultivar Nuomici, which had the highest sugar content among the cultivars studied, displayed significantly lower activities of cell wall acid invertase, SAI, neutral invertase, and SS and lower expression levels of SAI and SS compared with HY, the cultivar with the lowest sugar content. The inconsistent patterns of sugar accumulation and activities and expressions of sucrose metabolism enzymes suggest that these sucrose metabolism enzymes are not necessarily related to sugar accumulation.

Sugar and acid compositions in the arils of Litchi chinensis Sonn.: cultivar differences and evidence for the absence of succinic acid

Summary Quantitative and qualitative changes in sugars and organic acids were investigated in two litchi (Litchi chinensis Sonn.) cultivars, ‘Feizixiao’ and ‘Nuomici’, during aril development, and their levels measured in another six cultivars (‘Guiwei’, ‘Sanyuehong’, ‘Jizuili’, ‘Xuehuaizi’, ‘Dahongli’ and ‘Yuhebao’) at maturity. Glucose, fructose and sucrose were the predominant sugars. Litchi cultivars could be classified into three types based on their sugar composition: 1) monosaccharide-prevalent types including cvs. ‘Feizixiao’, ‘Xuehuaizi’ and ‘Yuhebao’; 2) disaccharide-prevalent types including cvs. ‘Guiwei’, ‘Jizuili’ and ‘Nuomici’; and 3) intermediate types including cvs. ‘Sanyuehong’ and ‘Dahongli’. Differences in sugar compositions between cultivars were associated with differences in the activities of certain key enzymes. Succinic acid, which was previously reported to be one of the major organic acids in litchi arils, was found to be absent in all cultivars tested in this study. The major organic acid in the litchi aril was malic acid; others included tartaric, citric and ascorbic acids. Malic acid levels increased during the early stages of aril development, followed by a dramatic decrease as the fruit approached maturity. The ratio of malic acid to tartaric acid varied from 2.6–5.7 between cultivars. The concentration of ascorbic acid decreased with fruit development until 2 weeks before harvest, when it started to increase slightly as the fruit approached full maturity. Ascorbic acid levels varied considerably among the eight cultivars tested. Monosaccharide-prevalent cultivars contained more ascorbic acid than disaccharide-prevalent cultivars.

Two LcbHLH Transcription Factors Interacting with LcMYB1 in Regulating Late Structural Genes of Anthocyanin Biosynthesis in Nicotiana and Litchi chinensis During Anthocyanin Accumulation

Anthocyanin biosynthesis requires the MYB-bHLH-WD40 protein complex to activate the late biosynthetic genes. LcMYB1 was thought to act as key regulator in anthocyanin biosynthesis of litchi. However, basic helix-loop-helix proteins (bHLHs) as partners have not been identified yet. The present study describes the functional characterization of three litchi bHLH candidate anthocyanin regulators, LcbHLH1, LcbHLH2, and LcbHLH3. Although these three litchi bHLHs phylogenetically clustered with bHLH proteins involved in anthcoyanin biosynthesis in other plant, only LcbHLH1 and LcbHLH3 were found to localize in the nucleus and physically interact with LcMYB1. The transcription levels of all these bHLHs were not coordinated with anthocyanin accumulation in different tissues and during development. However, when co-infiltrated with LcMYB1, both LcbHLH1 and LcbHLH3 enhanced anthocyanin accumulation in tobacco leaves with LcbHLH3 being the best inducer. Significant accumulation of anthocyanins in leaves transformed with the combination of LcMYB1 and LcbHLH3 were noticed, and this was associated with the up-regulation of two tobacco endogenous bHLH regulators, NtAn1a and NtAn1b, and late structural genes, like NtDFR and NtANS. Significant activity of the ANS promoter was observed in transient expression assays either with LcMYB1-LcbHLH1 or LcMYB1-LcbHLH3, while only minute activity was detected after transformation with only LcMYB1. In contrast, no activity was measured after induction with the combination of LcbHLH2 and LcMYB1. Higher DFR expression was also oberseved in paralleling with higher anthocyanins in co-transformed lines. LcbHLH1 and LcbHLH3 are essential partner of LcMYB1 in regulating the anthocyanin production in tobacco and probably also in litchi. The LcMYB1-LcbHLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.

Transcriptome Profiling of Light-Regulated Anthocyanin Biosynthesis in the Pericarp of Litchi

Light is a key environmental factor that affects anthocyanin biosynthesis. To enhance our understanding of the mechanisms involved in light-regulated anthocyanin biosynthesis in the pericarp of litchi, we performed transcriptomic analyses on the basis of Illumina sequencing. Fruit clusters were bagged with double-layer Kraft paper bags at 42 days after anthesis. The bags were removed after 2 weeks. Under light conditions, anthocyanins accumulated rapidly in the pericarp. RNA sequences were de novo assembled into 75,935 unigenes with an average length of 913 bp. Approximately 74.5% of unigenes (56,601) were annotated against four public protein databases. A total of 16,622 unigenes that significantly differed in terms of abundance were identified. These unigenes are implicated in light signal perception and transduction, flavonoid biosynthesis, carotenoid biosynthesis, plant hormone signal transduction, and photosynthesis. In photoreceptors, the expression levels of UV RESISTANCE LOCUS 8 (UVR8), Phototropin 2 (PHOT2), Phytochrome B (PHYB), and Phytochrome C (PHYC) increased significantly when the fruits were exposed to light. This result indicated that they likely play important roles in anthocyanin biosynthesis regulation. After analyzed digital gene expression (DGE), we found that the light signal transduction elements of COP1 and COP10 might be responsible for anthocyanin biosynthesis regulation. After the bags were removed, nearly all structural and regulatory genes, such as UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT), MYB, basic helix-loop-helix (bHLH), and WD40, involved in the anthocyanin biosynthetic pathway were upregulated. In addition to MYB-bHLH-WD40 transcription complex, ELONGATED HYPOCOTYL (HY5), NAM/ATAF/CUC (NAC), homeodomain leucine zipper proteins (ATHBs), and FAR-RED ELONGATED HYPOCOTYL (FHY) possibly participate in light-induced responses. On the basis of DGEs and qRT-PCR validation, we observed a light-induced anthocyanin biosynthesis and regulation pattern in litchi pericarp. This study enhanced our understanding of the molecular mechanisms governing light-induced anthocyanin biosynthesis in litchi pericarp.

Linking cracking resistance and fruit desiccation rate to pericarp structure in litchi (Litchi chinensis Sonn.)

Summary The structure and mechanical properties (e.g. strength and extensibility) of the pericarp, the structure of the cuticle layer, and fruit desiccation rate were compared in two litchi (Litchi chinensis Sonn.) cultivars, the cracking-resistant cv. ‘Huaizhi’ and the cracking-susceptible cv. ‘Nuomici’. Pericarp strength and extensibility were higher in ‘Huaizhi’ than in ‘Nuomici’ prior to rapid aril expansion. Based on changes in pericarp structure, we propose a “Zig-zag Unfolding” model for pericarp extension driven by the expanding aril. In this model, the cristate structure of the pericarp, and the spongy tissue in the mesocarp, provide extensibility for the pericarp. We suggest that the spongy tissue contributes to cracking resistance, as it permits pericarp extension/expansion. This hypothesis is supported by the observation that ‘Huaizhi’ has thicker spongy tissue than ‘Nuomici’. The model also reveals that endocarp and mesocarp tissues beneath the ‘cracks’ are the main contributors to the tensile strength of the pericarp. However, no significant difference in thickness of these tissues was observed between the two cultivars. Post-harvest, ‘Nuomici’ lost water twice as fast as ‘Huiazhi’, suggesting that the thicker spongy tissue in the pericarp does not necessarily mean a faster desiccation rate. This study showed large differences in cuticle structure between the two cultivars, which may result in differences in desiccation rate. We suggest that the greater tendency for ‘Nuomici’ to lose water results in its higher sensitivity to drought, which in turn induces fruit cracking as well as a shorter shelf-life compared with ‘Huaizhi’.

LcGST4 is an anthocyanin-related glutathione S - transferase gene in Litchi chinensis Sonn.

Key messageA novelLcGST4was identified and characterized fromLitchi chinensis. Expression and functional analysis demonstrated that it might function in anthocyanin accumulation in litchi.AbstractGlutathione S-transferases (GSTs) have been defined as detoxification enzymes for their ability to recognize reactive electrophilic xenobiotic molecules as well as endogenous secondary metabolites. Anthocyanins are among the few endogenous substrates of GSTs for vacuolar accumulation. The gene encoding a GST protein that is involved in anthocyanin sequestration from Litchi chinensis Sonn. has not been reported. Here, LcGST4, an anthocyanin-related GST, was identified and characterized. Phylogenetic analysis showed that LcGST4 was clustered with other known anthocyanin-related GSTs in the same clade. Expression analysis revealed that the expression pattern of LcGST4 was strongly correlated with anthocyanin accumulation in litchi. ABA- and light-responsive elements were found in the LcGST4 promoter, which is in agreement with the result that the expression of LcGST4 was induced by both ABA and debagging treatment. A GST activity assay in vitro verified that the LcGST4 protein shared universal activity with the GST family. Functional complementation of an Arabidopsis mutant tt19 demonstrated that LcGST4 might function in anthocyanin accumulation in litchi. Dual luciferase assay revealed that the expression of LcGST4 was activated by LcMYB1, a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in litchi.

Functional characterization of a glucosyltransferase gene, LcUFGT1, involved in the formation of cyanidin glucoside in the pericarp of Litchi chinensis.

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.