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Alternative splicing (AS) was an important post-transcriptional mechanism that involved in plant resistance to adversity stress. WRKY transcription factors function as transcriptional activators or repressors to modulate plant growth, development and stress response. However, the role of alternate splicing of WRKY in cold tolerance is poorly understood in tea plants. In this study, we found that the CsWRKY21 transcription factor, a member of the WRKY IId subfamily, was induced by low temperature. Subcellular localization and transcriptional activity assays showed that CsWRKY21 localized to the nucleus and had no transcriptional activation activity. Y1H and dual-luciferase reporter assays showed that CsWRKY21 suppressed expression of CsABA8H and CsUGT by binding with their promoters. Transient overexpression of CsABA8H and CsUGT reduced abscisic acid (ABA) content in tobacco leaves. Furthermore, we discovered that CsWRKY21 undergoes AS in the 5'UTR region. The AS transcript CsWRKY21-b was induced at low temperature, up to 6 folds compared to the control, while the full-length CsWRKY21-a transcript did not significantly change. Western blot analysis showed that the retention of introns in the 5'UTR region of CsWRKY21-b led to higher CsWRKY21 protein content. These results revealed that alternative splicing of CsWRKY21 involved in cold tolerance of tea plant by regulating the protein expression level and then regulating the content of ABA, and provide insights into molecular mechanisms of low temperature defense mediated by AS in tea plant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Alternative splicing (AS) increases the diversity of transcripts and proteins through the selection of different splice sites and plays an important role in the growth, development and stress tolerance of plants. With the release of the reference genome of the tea plant (Camellia sinensis) and the development of transcriptome sequencing, researchers have reported the existence of AS in tea plants. However, there is a lack of a platform, centered on different RNA-seq datasets, that provides comprehensive information on AS.To facilitate access to information on AS and reveal the molecular function of AS in tea plants, we established the first comprehensive AS database for tea plants (TeaAS, http://www.teaas.cn/index.php ). In this study, 3.96 Tb reads from 66 different RNA-seq datasets were collected to identify AS events. TeaAS supports four methods of retrieval of AS information based on gene ID, gene name, annotation (non-redundant/Kyoto encyclopedia of genes and genomes/gene ontology annotation or chromosomal location) and RNA-seq data. It integrates data pertaining to genome annotation, type of AS event, transcript sequence, and isoforms expression levels from 66 RNA-seq datasets. The AS events resulting from different environmental conditions and that occurring in varied tissue types, and the expression levels of specific transcripts can be clearly identified through this online database. Moreover, it also provides two useful tools, Basic Local Alignment Search Tool and Generic Genome Browser, for sequence alignment and visualization of gene structure.The features of the TeaAS database make it a comprehensive AS bioinformatics platform for researchers, as well as a reference for studying AS events in woody crops. It could also be helpful for revealing the novel biological functions of AS in gene regulation in tea plants.

Background: Alternative splicing (AS) may generate multiple mRNA splicing isoforms from a single mRNA precursor using different splicing sites, leading to enhanced diversity of transcripts and proteins. AS has been implicated in cold acclimation by affecting gene expression in various ways, yet little information is known about how AS influences cold responses in tea plant (Camellia sinensis)., Results: In this study, the AS transcriptional landscape was characterized in the tea plant genome using high-throughput RNA-seq during cold acclimation. We found that more than 41% (14,103) of genes underwent AS events. We summarize the possible existence of 11 types of AS events, including the four common types of intron retention (IR), exon skipping (ES), alternative 5' splice site (A5SS), and alternative 3' splice site (A3SS); of these, IR was the major type in all samples. The number of AS events increased rapidly during cold treatment, but decreased significantly following de-acclimation (DA). It is notable that the number of differential AS genes gradually increased during cold acclimation, and these genes were enriched in pathways relating to oxidoreductase activity and sugar metabolism during acclimation and de-acclimation. Remarkably, the AS isoforms of bHLH transcription factors showed higher expression levels than their full-length ones during cold acclimation. Interestingly, the expression pattern of some AS transcripts of raffinose and sucrose synthase genes were significantly correlated with sugar contents., Conclusion: Our findings demonstrated that changes in AS numbers and transcript expression may contribute to rapid changes in gene expression and metabolite profile during cold acclimation, suggesting that AS events play an important regulatory role in response to cold acclimation in tea plant.

Volatile fatty acid derivatives (VFADs) produced in tea plants ( Camellia sinensis ) not only have been shown to function as defense compounds but also impart a "fresh green" odor to green tea products; however, little is known about alternative splicing (AS) of genes in regulating the production of VFADs in plants. In this study, the contents of VFADs and corresponding transcriptome profiles were obtained in five different months (April, June, August, September, and October). Correlation analysis identified seven unique transcripts of enzyme-coding genes ( CsLOX2 , CsLOX4 , CsADH4 , CsADH8 , and CsADH10 ), which are responsible for regulating VFAD biosynthesis; four AS transcripts of these genes ( CsLOX2 , CsLOX4 , CsADH4 , and CsADH8 ) were validated by RT-PCR. By employing the gene-specific antisense oligodeoxynucleotide-mediated reduction method, we found the expression levels of alternatively spliced transcripts of CsLOX4-iso1 , CsLOX4-iso2 , and CsADH4-iso3 were lower, and the contents of cis -3-hexenol were correspondingly reduced in the leaves of tea plant; this result suggested that the AS play important roles in regulating biosynthesis of VFADs in C. sinensis . Our results provide new insights into the important contribution of AS events in regulating the VFAD biosynthesis in tea plant.

Background: Alternative 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., Results: In 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., Conclusion: Our 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.

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., (� The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

SUMMARY: Flavan‐3‐ols are abundant in the tea plant (Camellia sinensis) and confer tea with flavor and health benefits. We recently found that alternative splicing of genes is likely involved in the regulation of flavan‐3‐ol biosynthesis; however, the underlying regulatory mechanisms remain unknown. Here, we integrated metabolomics and transcriptomics to construct metabolite–gene networks in tea leaves, collected over five different months and from five spatial positions, and found positive correlations between endogenous jasmonic acid (JA), flavan‐3‐ols, and numerous transcripts. Transcriptome mining further identified CsJAZ1, which is negatively associated with flavan‐3‐ols formation and has three CsJAZ1 transcripts, one full‐length (CsJAZ1‐1), and two splice variants (CsJAZ1‐2 and ‐3) that lacked 3′ coding sequences, with CsJAZ1‐3 also lacking the coding region for the Jas domain. Confocal microscopy showed that CsJAZ1‐1 was localized to the nucleus, while CsJAZ1‐2 and CsJAZ1‐3 were present in both the nucleus and the cytosol. In the absence of JA, CsJAZ1‐1 was bound to CsMYC2, a positive regulator of flavan‐3‐ol biosynthesis; CsJAZ1‐2 functioned as an alternative enhancer of CsJAZ1‐1 and an antagonist of CsJAZ1‐1 in binding to CsMYC2; and CsJAZ1‐3 did not interact with CsMYC2. In the presence of JA, CsJAZ1‐3 interacted with CsJAZ1‐1 and CsJAZ1‐2 to form heterodimers that stabilized the CsJAZ1‐1–CsMYC2 and CsJAZ1‐2–CsMYC2 complexes, thereby repressing the transcription of four genes that act late in the flavan‐3‐ol biosynthetic pathway. These data indicate that the alternative splicing variants of CsJAZ1 coordinately regulate flavan‐3‐ol biosynthesis in the tea plant and improve our understanding of JA‐mediated flavan‐3‐ol biosynthesis. Significance Statement: Alternative splicing is an important post‐transcriptional mechanism that plays vital roles in plant development and metabolism. This study identified CsJAZ1 and its alternative splicing variants (CsJAZ1‐2 and CsJAZ1‐3) that formed homo‐ and heterodimers, bound to CsMYC2, and coordinately repressed the jasmonic acid‐mediated flavan‐3‐ol biosynthesis. These findings provide evidence that the coordination of JAZ splice variants play important roles in regulating plant secondary metabolism. [ABSTRACT FROM AUTHOR]

• The AS events of the genes involved in the withering process of fresh tea leaves were systematically identified. • 25.1 % of the flavor-related genes and 40.1 % of the transcription factors underwent AS. • The expression of AS transcripts of most genes was more significantly correlated with the changes in the corresponding aroma content than the normal transcripts. The flavor quality of tea, particularly its aroma, can be substantially enhanced by moderate withering of fresh leaves; however, this enhancement process cannot be attributed alone to the transcriptional regulation. Alternative splicing (AS) plays a key role as a post-transcriptional regulatory mechanism in regulating plant growth and development, stress response, and metabolism; however, it remains unclear whether AS is involved in tea aroma quality formation during the withering of fresh leaves. In this study, AS events of the expressed genes were systematically characterized based on the transcriptomic data from fresh leaves withered for different time intervals (0, 2, 4, 6, 8 and 10 h). A total of 88,976 AS events from 13,995 genes were identified. The highest number of differential alternative splicing (DAS) was found in samples after 4 h of withering and then gradually decreased; however, the number of DAS remained higher after 10 h of withering than after 2 h of withering. Furthermore, 25.1 % of the flavor-related genes and 40.1 % of the transcription factors underwent AS, and most of them were enriched in the hormone signaling pathway and the aroma volatile synthesis pathways. A total of 89 volatiles were identified from the withering samples. The relative contents of the volatiles increased with the increase in withering time and were significantly higher than those in the control sample after 4 h of withering. Compared to the expression of the normal transcripts, the expression of AS transcripts of most genes was more significantly correlated with the changes in the corresponding aroma content. These results suggest that AS was involved and played a key role in tea aroma quality formation during withering. Our results further revealed the complexity of tea flavor quality formation and provided new insights regarding its regulatory mechanisms. [ABSTRACT FROM AUTHOR]