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Oxylipins, including jasmonic acid (JA) and volatiles, are important for signaling in plants, and these are formed by the lipoxygenase (LOX) enzyme family. There is a large gap in understanding of the underlying molecular basis of their roles in tea plants. Here, we identified 11 CsLOX genes from the tea plant (Camellia sinensis), and characterized their phylogeny, gene structure and protein features into three subclasses. We then examined their enzymatic activities, LOX expression and alternative splicing of transcripts during development and in response to abiotic or biotic stresses in tea plants. In vitro expressed protein assays showed that the CsLOX2, 3 and 9 enzymatically function to produce 9/13-HPOT, 13-HPOT and 9-HPOT, respectively. CsLOX2 and CsLOX9 green fluorescent protein (GFP) fusion proteins localized to chloroplasts and the cytoplasm, respectively. RNA sequencing, quantitative reverse transcription–PCR and Northern blot analysis suggested that CsLOX5, 6 and 9 were predominantly expressed in seeds, flowers and roots, respectively. CsLOX2, 3, 4, 6 and 7 were up-regulated after attack by the insect Ectropis oblique, while CsLOX1 was induced after infection with the pathogen Glomerella cingulata. CsLOX3, 7 and 10 were up-regulated by JA but not ABA or salicylic acid. Long-term cold stress down-regulated CsLOX expression while a short duration of cold induced the expression of CsLOX1, 6 and 7. Alternatively spliced transcripts of six CsLOX genes were dynamically regulated through time and varied in relative abundances under the investigated stresses; we propose a mechanism of competing or compensating regulation between isoforms. This study improves our understanding of evolution of LOXs and regulation of their diverse functions in plants.

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Characteristic secondary metabolites, including flavonoids, theanine and caffeine, are important components of Camellia sinensis, and their biosynthesis has attracted widespread interest. Previous studies on the biosynthesis of these major secondary metabolites using next-generation sequencing technologies limited the accurately prediction of full-length (FL) splice isoforms. Herein, we applied single-molecule sequencing to pooled tea plant tissues, to provide a more complete transcriptome of C. sinensis. Moreover, we identified 94 FL transcripts and four alternative splicing events for enzyme-coding genes involved in the biosynthesis of flavonoids, theanine and caffeine. According to the comparison between long-read isoforms and assemble transcripts, we improved the quality and accuracy of genes sequenced by short-read next-generation sequencing technology. The resulting FL transcripts, together with the improved assembled transcripts and identified alternative splicing events, enhance our understanding of genes involved in the biosynthesis of characteristic secondary metabolites in C. sinensis.

BackgroundAlternative splicing (AS) regulates mRNA at the post-transcriptional level to change gene function in organisms. However, little is known about the AS and its roles in tea plant (Camellia sinensis), widely cultivated for making a popular beverage tea.ResultsIn our study, the AS landscape and dynamics were characterized in eight tissues (bud, young leaf, summer mature leaf, winter old leaf, stem, root, flower, fruit) of tea plant by Illumina RNA-Seq and confirmed by Iso-Seq. The most abundant AS (~ 20%) was intron retention and involved in RNA processes. The some alternative splicings were found to be tissue specific in stem and root etc. Thirteen co-expressed modules of AS transcripts were identified, which revealed a similar pattern between the bud and young leaves as well as a distinct pattern between seasons. AS events of structural genes including anthocyanidin reductase and MYB transcription factors were involved in biosynthesis of flavonoid, especially in vegetative tissues. The AS isoforms rather than the full-length ones were the major transcripts involved in flavonoid synthesis pathway, and is positively correlated with the catechins content conferring the tea taste. We propose that the AS is an important functional mechanism in regulating flavonoid metabolites.ConclusionOur study provides the insight into the AS events underlying tea plant’s uniquely different developmental process and highlights the important contribution and efficacy of alternative splicing regulatory function to biosynthesis of flavonoids.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1497-9) contains supplementary material, which is available to authorized users.

Tea, made from leaves of Camellia sinensis, has long been consumed worldwide for its unique taste and aroma. Terpenoids play important roles not only in tea beverage aroma formation, but also in the productivity and quality of tea plantation due to their significant contribution to light harvesting pigments and phytohormones. To date, however, the regulation of terpenoid synthase genes remains unclear. Herein, the analyses of metabolomics, sRNAs, degradome, and transcriptomics were performed and integrated for identifying key regulatory miRNA-target circuits on terpenoid biosynthesis in leaf tissues over five different months in which the amount of terpenoids in tea leaves varies greatly. Four classes of miRNA-TF pairs that might play a central role in the regulation of terpenoid biosynthesis were also uncovered. Ultimately, a hypothetical model was proposed that mature miRNAs maintained by light regulator at both the transcriptional and posttranscriptional levels negatively regulate the targets to control terpenoid biosynthesis.

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Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.