Yonghua Qin

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.

Isolation and differential expression analysis of self-compatibility-related genes from mature pistils of 'Shatangju' mandarin (Citrus reticulata Blanco)

Summary The objective of this research was to identify self-compatibility (SC)-related genes that are up-regulated in ‘Shatangju’ mandarin (Citrus reticulata Blanco). A suppression-subtractive hybridisation (SSH) cDNA library was constructed using self-compatible ‘Shatangju’ cDNA as the tester, and self-incompatible ‘Wuzishatangju’ mandarin cDNA as the driver. Among the 949 positive clones obtained in the size range of 200 – 700 bp, 158 showed up-regulated expression in ‘Shatangju’ mandarin. Several SC-related genes, such as those for the S1 self-incompatibility locus-linked pollen 3.15 protein (S1-protein), the ubiquitin-activating enzyme E1 (ubiquitin-E1), a zinc finger (C2H2 type) family protein (zinc-finger protein), the nuclear inhibitor of protein phosphatase-1 (nnpp-1), ubiquitin, molecular chaperones, and senescence-related proteins were identified. Semi-quantitative reverse transcription-PCR (sqRT-PCR) and quantitative real-time PCR (qPCR) were used in this study to elucidate the differential expression patterns of five of these SC-related genes. The results showed that expression levels of the S1-protein and of ubiquitin-E1 in ‘Shatangju’ ovaries and anthers were up-regulated by 60-fold and seven-fold, respectively, compared to the same tissues in ‘Wuzishatangju’ mandarin. The expression levels of the zinc-finger protein and the ubiquitin protein in ‘Shatangju’ leaves and buds were higher than in leaves and buds of ‘Wuzishatangju’. Expression levels of the nnpp-1 gene in the filaments and anthers of ‘Shatangju’ were three-fold and four-fold higher, respectively, than in the same tissues of ‘Wuzishatangju’. These results suggest that the S1-protein and ubiquitin-E1 might play an important role in the self-compatible reaction of ‘Shatangju’ mandarin. This information could provide new knowledge to understand SC, not only in citrus, but also in other fruit.

Comparative transcript profiling of gene expression between self-incompatible and self-compatible mandarins by suppression subtractive hybridization and cDNA microarray

AbstractSelf-incompatibility (SI) is an important trait of Citrus plants that is exploited by farmers to produce seedless fruit. However, the molecular mechanism of SI in Citrus is not well understood. Wuzishatangju (Citrus reticulata Blanco) (SI) is an excellent seedless cultivar selected from a seedy Shatangju cultivar (self-compatible, SC) through spontaneous bud mutation. The two cultivars are therefore excellent materials for studying the mechanisms of SI and/or SC in Citrus. In this study, an integrative strategy combining eight suppression subtractive hybridization libraries with cDNA microarray was used to study the molecular mechanisms that differ between Wuzishatangju and Shatangju (control) mandarins. A custom microarray screen resulted in a total of 1,830 up- or down-regulated clones (false discovery rate <0.05 and a fold change

Transcriptome changes between compatible and incompatible graft combination of Litchi chinensis by digital gene expression profile.

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Identifying differentially expressed genes in pollen from self-incompatible "Wuzishatangju" and self-compatible "Shatangju" mandarins.

≧2) obtained from 9,810 positive clones. The expression of genes involved in embryonic development, ubiquitination pathway, Ca2+-signaling pathway, gibberellins, and auxin was significantly up-regulated in SI Wuzishatangju compared with SC Shatangju mandarin. The microarray analysis suggested that the ubiquitin-mediated proteolysis pathway might be involved in the SI reaction of Wuzishatangju. Additionally, our research highlighted some main genes (mitogen-activated protein kinase, SI S1 family protein, ubiquitin-conjugating factor E4-like, auxin transporter protein 1, and gibberellin receptor) that participate in the SI reaction of Wuzishatangju and could be beneficial for understanding the evolution of SI systems and for breeding seedless citrus fruits in the future.

Sequence differences in LcFGRT4 alleles are responsible for the diverse anthocyanin composition in the pericarp of Litchi chinensis

Plant S-phase kinase-associated protein 1 (SKP1) genes play crucial roles in plant development and differentiation. However, the role of SKP1 in citrus is unclear. Herein, we described a novel SKP1-like gene, designated as CrWSKP1, from “Wuzishatangju” (Citrus reticulata Blanco). The cDNA sequence of CrWSKP1 is 779 base pairs (bp) and contains an open reading frame (ORF) of 477 bp. The genomic sequence of the CrWSKP1 gene is 1296 bp with two exons and one intron. CrWSKP1 has high identity with SKP1-like genes from other plant species within two conserved regions. Approximately 85% of pollen tubes of self-pollinated CrWSKP1 transgenic tobaccos became twisted at four days after self-pollination. Pollen tube numbers of self-pollinated CrWSKP1 transformants entering into ovules were significantly fewer than that of the control. Seed number of self-pollinated CrWSKP1 transformants was significantly reduced. These results suggested that the CrWSKP1 is involved in the self-incompatibility (SI) reaction of “Wuzishatangju”.

Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

Plant grafting has been practiced widely in horticulture and proved as a useful tool in science. However, the mechanisms of graft healing or graft incompatibility remain poorly understood. In this study, Litchi chinensis cv. ‘Jingganghongnuo’ homograft (‘J/J’) and ‘Jingganghongnuo’/‘zhuangyuanhong’ heterograft (‘J/Z’) as compatible and incompatible combination, respectively, was used to study transcriptional changes between incompatible and compatible graft during graft union formation. Anatomical observation indicated that three stages (2 h, 14 d and 21 d after grafting) were critical for graft union formation and selected for high-throughput sequencing. Results indicated 6060 DEGs were differentially expressed in the compatible combination and 5267 DEGs exhibiting in the incompatible one. KEGG pathway enrichment analysis revealed that DEGs were involved in metabolism, wound response, phenylpropanoid biosynthesis and plant hormone signal transduction. The expression of 9 DEGs annotated in auxin pathway was up-regulated in compatible combination than that in incompatible combination. The IAA concentration confirmed that the IAA might promote the graft compatibility. In addition, 13 DEGs related to lignin biosynthesis were differentially expressed during graft healing process. Overall, our results provide abundant sequence resources for studying mechanisms underlying graft compatibility and establish a platform for further studies of litchi and other evergreen fruit trees.

Comparative transcriptome analyses of a late-maturing mandarin mutant and its original cultivar reveals gene expression profiling associated with citrus fruit maturation

Red pitaya (Hylocereus polyrhizus) fruit is a high-value, functional food, containing a high level of betalains. Several genes potentially related to betalain biosynthesis, such as cytochrome P450-like (CytP450-like), have been identified in pitaya fruit, while their transcriptional regulation remains unclear. In this work, the potential involvement of a WRKY transcription factor, HpWRKY44, in regulating CytP450-like1 expression in pitaya fruit was examined. HpWRKY44, a member of the Group 1 WRKY family, contains two conserved WRKY motifs and is localized in the nucleus. HpWRKY44 also exhibits trans-activation ability. Gene expression analysis showed that the expression of HpCytP450-like1 and HpWRKY44 increased steadily during pitaya fruit coloration, which corresponded with the production of elevated betalain levels in the fruit. HpWRKY44 was also demonstrated to directly bind to and activate the HpCytP450-like1 promoter via the recognition of the W-box element present in the promoter. Collectively, our findings indicate that HpWRKY44 transcriptionally activates HpCytP450-like1, which perhaps, at least in part, contributes to betalain biosynthesis in pitaya fruit. The information provided in the current study provides novel insights into the regulatory network associated with betalain biosynthesis during pitaya fruit coloration.