1 Introduction
Tea (Camellia sinensis ) is an important woody economic crop (Xia et al., 2020), and its leaves can be used to produce one of the world’s most important beverages (Rietveld and Wiseman, 2003). Tea plants are susceptible to attack by various pathogens and insects during their growth (Chen et al., 2020b). Tea anthracnose disease caused by fungi in the genus Colletotrichum , especially Colletotrichum gloeosporioides (Jeyaraj et al., 2019) and gray blight disease caused by Pestalotiopsis species (Chen et al., 2018), are two of the most destructive foliar diseases of tea plants and are responsible for 30–60% (Wang et al., 2021) and 10–20% of the losses of tea products on an annual basis, respectively (Chen et al., 2018, Jiang et al., 2020). Plants have evolved complex defense mechanisms to defend against pathogens (Sharifi et al., 2018). Plant hormones such as salicylic acid (SA) and jasmonic acid play key roles in defense against pathogens (Sharifi et al., 2018). SA is the primary hormone responsible for plant disease resistance, including the activation of the defense response following pathogen infection (Wang et al., 2021). Previous studies have shown that the release of volatile terpenes is one of the key mechanisms by which plants resist pathogen (Sharifi et al., 2018).
Tea possesses abundant secondary metabolites that are strongly associated with its quality and health benefits (Jiang et al., 2019, Xia et al., 2020). The release of defense-related volatiles plays an important role in mediating both local and systemic responses, as the emission of volatiles primes their defense mechanisms in response to attack by herbivores and pathogens (Bouwmeester et al., 2019; Jiang et al., 2019; Quintana-Rodriguez et al., 2015; Richter et al., 2016; Turlings and Erb, 2018). The exposure of susceptible cultivars to volatiles from resistant cultivars can significantly increase the expression of defense-related genes and confer disease resistance (Castelyn et al., 2015; Eberl et al., 2018; Quintana-Rodriguez et al., 2015; Sharifi et al., 2018). Terpenoids contribute to tea flavor via their low human odor perception thresholds (Yang et al., 2013). Monoterpenes, including linalool and geraniol, enhance the flavor and aroma of tea (Ho et al., 2015). Linalool and geraniol are two of the most abundant and odor-active monoterpenoids in tea plants, and they contribute to the pleasant floral scent of tea products (Han et al., 2016; Yang et al., 2013). Although the biosynthesis of the terpenoid pathway in tea plants has been studied, only a few terpene synthases (TPSs) and TPS genes involved in terpenoid synthesis have been identified (Zhou et al., 2017). The key gene involved in linalool formation in tea plants has been isolated and functionally verified (Liu et al., 2018). However, the key enzyme involved in geraniol biosynthesis and its biological function in tea plants remain unclear (Zhou et al., 2020).
Alternative splicing (AS) can generate different mRNA splicing isoforms from a single mRNA precursor via different splicing sites (Li et al., 2020), and this can result in diverse protein isoforms(Laloum et al., 2018). An increasing number of studies have shown that AS plays an important role in the growth, development, and abiotic and biotic stress tolerance of plants (Mi et al., 2021; Posé et al., 2013). AS is also key in the biosynthesis of secondary metabolites (Zhao et al., 2014) and the response to pathogen infection (Liu et al., 2016). AS also figures prominently in abiotic stress tolerance, especially in ABA-mediated responses (Laloum et al., 2018). More than 41% of genes undergo AS during cold acclimation, and the four main types of AS events in tea plants are intron retention, exon skipping, alternative 5′ splice site, and alternative 3′ splice site (Li et al., 2020). AS isoforms of theCsLOX2, CsLOX9 , a nd CsLOX10 genes can be induced under low-temperature treatment (Zhu et al., 2018a). AS in tea plants plays an important role in regulating the synthesis of secondary metabolites (Zhu et al., 2018b), including the synthesis of anthocyanins (Chen et al., 2020a), linalool (Liu et al., 2018), and volatile fatty acid derivatives(Xu et al., 2019). However, whether AS plays a role in the regulation of geraniol formation and biotic stress responses in tea following pathogen infection remains unclear.
Here, the first geraniol synthase (CsGES ) was identified, cloned, and functionally characterized in tea plants. The expression level of the AS isoform CsTPS1 -AS , but not the full-lengthCsTPS1 , was significantly increased following C. gloeosporioides and Neopestalotiopsis sp. infection, and the function of CsTPS1 -AS in planta was assessed. Silencing ofCsTPS1 -AS led to a decrease in the expression of defense-related and SA biosynthesis-related genes and an increase in the susceptibility of tea plants to C. gloeosporioides andNeopestalotiopsis sp. infection. The findings of this study enhance our understanding of geraniol formation in tea plants following fungal infection and provide new insights into the functions of AS isoforms during pathogen infection in plants.