Characterization of pectin methylesterase gene family and its possible role in juice sac granulation in navel orange (Citrus sinensis Osbeck).

BACKGROUND: Citrus is one of the most important fresh fruit crops worldwide. Juice sac granulation is a physiological disorder, which leads to a reduction in soluble solid concentration, total sugar, and titratable acidity of citrus fruits. Pectin methylesterase (PME) catalyzes the de-methylesterification of homogalacturonans and plays crucial roles in cell wall modification during plant development and fruit ripening. Although PME family has been well investigated in various model plants, little is known regarding the evolutionary property and biological function of PME family genes in citrus. RESULTS: In this study, 53 non-redundant PME genes were identified from Citrus sinensis genome, and these PME genes were divided into four clades based on the phylogenetic relationship. Subsequently, bioinformatics analyses of gene structure, conserved domain, chromosome localization, gene duplication, and collinearity were performed on CsPME genes, providing important clues for further research on the functions of CsPME genes. The expression profiles of CsPME genes in response to juice sac granulation and low-temperature stress revealed that CsPME genes were involved in the low temperature-induced juice sac granulation in navel orange fruits. Subcellular localization analysis suggested that CsPME genes were localized on the apoplast, endoplasmic reticulum, plasma membrane, and vacuole membrane. Moreover, yeast one-hybrid screening and dual luciferase activity assay revealed that the transcription factor CsRVE1 directly bound to the promoter of CsPME3 and activated its activity. CONCLUSION: In summary, this study conducts a comprehensive analysis of the PME gene family in citrus, and provides a novel insight into the biological functions and regulation patterns of CsPME genes during juice sac granulation of citrus.

MdSCL8 as a Negative Regulator Participates in ALA-Induced FLS1 to Promote Flavonol Accumulation in Apples.

Apples (Malus domestica) are rich in flavonols, and 5-aminolevulinic acid (ALA) plays an important role in the regulation of plant flavonoid metabolism. To date, the underlying mechanism of ALA promoting flavonol accumulation is unclear. Flavonol synthase (FLS) is a key enzyme in flavonol biosynthesis. In this study, we found that ALA could enhance the promoter activity of MdFLS1 in the 'Fuji' apple and improve its expression. With MdFLS1 as bait, we screened a novel transcription factor MdSCL8 by the Yeast One-Hybrid (Y1H) system from the apple cDNA library which we previously constructed. Using luciferase reporter assay and transient GUS activity assay, we verified that MdSCL8 inhibits the activity of MdFLS1 promoter and hinders MdFLS1 expression, thus reducing flavonol accumulation in apple. ALA significantly inhibited MdSCL8 expression. Therefore, ALA promoted the expression of MdFLS1 and the consequent flavonol accumulation probably by down-regulating MdSCL8. We also found that ALA significantly enhanced the gene expression of MdMYB22 and MdHY5, two positive regulators of MdFLS. We further demonstrated that MdMYB22 interacts with MdHY5, but neither of them interacts with MdSCL8. Taken together, our data suggest MdSCL8 as a novel regulator of MdFLS1 and provide important insights into mechanisms of ALA-induced flavonol accumulation in apples.

High-spatiotemporal-resolution transcriptomes provide insights into fruit development and ripening in Citrus sinensis.

Citrus fruit has a unique structure with soft leathery peel and pulp containing vascular bundles and several segments with many juice sacs. The function and morphology of each fruit tissue are different. Therefore, analysis at the organ-wide or mixed-tissue level inevitably obscures many tissue-specific phenomena. High-throughput RNA sequencing was used to profile Citrus sinensis fruit development based on four fruit tissue types and six development stages from young fruits to ripe fruits. Using a coexpression network analysis, modules of coexpressed genes and hub genes of tissue-specific networks were identified. Of particular, importance is the discovery of the regulatory network of phytohormones during citrus fruit development and ripening. A model was proposed to illustrate how ABF2 mediates the ABA signalling involved in sucrose transport, chlorophyll degradation, auxin homoeostasis, carotenoid and ABA biosynthesis, and cell wall metabolism during citrus fruit development. Moreover, we depicted the detailed spatiotemporal expression patterns of the genes involved in sucrose and citric acid metabolism in citrus fruit and identified several key genes that may play crucial roles in sucrose and citric acid accumulation in the juice sac, such as SWEET15 and CsPH8. The high spatial and temporal resolution of our data provides important insights into the molecular networks underlying citrus fruit development and ripening.

Genomic and transcriptomic analyses of Citrus sinensis varieties provide insights into Valencia orange fruit mastication trait formation.

Valencia orange (Citrus sinensis Osbeck) (VO) is a type of late-ripening sweet orange whose ripening occurs 4 to 5 months later than that of the mid-ripening common sweet orange (CO). Notably, the mastication trait of VO fruit is inferior to that of CO fruit. To date, how inferior pulp mastication trait forms in VO has not been determined. In this study, 13 VO varieties and 12 CO varieties were subjected to whole-genome resequencing. A total of 2.98 million SNPs were identified from 25 varieties, and a SNP molecular marker was developed to distinguish VO and CO. Moreover, 144 and 141 genes identified by selective sweep analysis were selected during VO and CO evolution, respectively. Based on gene functional enrichment analysis, most of the selected VO genes were related to the stress response and lignin biosynthesis. Simultaneously, we comparatively analyzed the transcriptome profiles of peel and pulp tissues among three VO varieties and three CO varieties, and the results demonstrated differences in lignin biosynthesis between VO and CO fruits. Furthermore, coexpression network analysis was performed to identify hub genes of lignin-related and variety-specific networks, which included CsERF74, CsNAC25, CsHSFB3, CsSPL4/13, etc. Overall, this study provides important insights into the mastication trait formation of Valencia orange fruit.

Global tissue-specific transcriptome analysis of Citrus sinensis fruit across six developmental stages.

Citrus sinensis fruit is a type of nonclimacteric fruit that mainly consists of four tissues: the epicarp, albedo, segment membrane and juice sac. The fruit quality is determined by the characteristics of these four tissues. However, our knowledge of the molecular processes that occur in these four tissues during citrus fruit development and ripening is limited. Tissue-specific transcriptomes provide a comprehensive and detailed molecular regulatory network of citrus fruit development and ripening. In our study, we collected four types of tissue from C. sinensis fruits at six developmental stages. A total of 72 libraries were constructed from 24 samples (each sample had three replicates), and the transcriptomes were sequenced by an Illumina HiSeq 4000. The comprehensive analyses of the transcriptomes from the four tissues and six developmental stages presented here provide a valuable resource for the discovery of the molecular networks underlying citrus fruit development and ripening.

Genome-wide comprehensive analysis of transcriptomes and small RNAs offers insights into the molecular mechanism of alkaline stress tolerance in a citrus rootstock.

Alkaline stress has serious-negative effects on citrus production. Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka) (Cj) is a rootstock that is tolerant to alkaline stress and iron deficiency. Trifoliate orange (Poncirus trifoliata (L.) Raf.) (Pt), the most widely used rootstock in China, is sensitive to alkaline stress. To investigate the molecular mechanism underlying the tolerance of Cj to alkaline stress, next-generation sequencing was employed to profile the root transcriptomes and small RNAs of Cj and Pt seedlings that were cultured in nutrient solutions along a three pH gradient. This two-level regulation data set provides a system-level view of molecular events with a precise resolution. The data suggest that the auxin pathway may play a central role in the inhibitory effect of alkaline stress on root growth and that the regulation of auxin homeostasis under alkaline stress is important for the adaptation of citrus to alkaline stress. Moreover, the jasmonate (JA) pathway exhibits the opposite response to alkaline stress in Cj and Pt and may contribute to the differences in the alkaline stress tolerance and iron acquisition between Cj and Pt. The dataset provides a wealth of genomic resources and new clues to further study the mechanisms underlying alkaline stress resistance in Cj.

Comparative Analysis of miRNAs and Their Target Transcripts between a Spontaneous Late-Ripening Sweet Orange Mutant and Its Wild-Type Using Small RNA and Degradome Sequencing.

Fruit ripening in citrus is not well-understood at the molecular level. Knowledge of the regulatory mechanism of citrus fruit ripening at the post-transcriptional level in particular is lacking. Here, we comparatively analyzed the miRNAs and their target genes in a spontaneous late-ripening mutant, "Fengwan" sweet orange (MT) (Citrus sinensis L. Osbeck), and its wild-type counterpart ("Fengjie 72-1," WT). Using high-throughput sequencing of small RNAs and RNA degradome tags, we identified 107 known and 21 novel miRNAs, as well as 225 target genes. A total of 24 miRNAs (16 known miRNAs and 8 novel miRNAs) were shown to be differentially expressed between MT and WT. The expression pattern of several key miRNAs and their target genes during citrus fruit development and ripening stages was examined. Csi-miR156k, csi-miR159, and csi-miR166d suppressed specific transcription factors (GAMYBs, SPLs, and ATHBs) that are supposed to be important regulators involved in citrus fruit development and ripening. In the present study, miRNA-mediated silencing of target genes was found under complicated and sensitive regulation in citrus fruit. The identification of miRNAs and their target genes provide new clues for future investigation of mechanisms that regulate citrus fruit ripening.