Rougui Wuyi Rock tea (WRT) with special flavor can be affected by multiple factors that are closely related to the culturing regions of tea plants. The present research adopted non-targeted metabolomics based on liquid chromatography–mass spectrometry (LC-MS) and gas chromatography–mass spectrometry (GC-MS), aroma activity value method (OAV), and chemometrics to analyze the characteristic metabolites of three Rougui WRTs from different culturing regions. The results of sensory evaluation showed that the three Rougui Wuyi Rock teas had significantly different flavor qualities, especially in taste and aroma. Rougui (RG) had a heavy and mellow taste, while cinnamon-like odor Rougui (GPRG) and floral and fruity odor Rougui (HGRG) had a thick, sweet, and fresh taste. The cinnamon-like odor was more obvious and persistent in GPRG than in RG and HGRG. HGRG had floral and fruity characteristics such as clean and lasting, gentle, and heavy, which was more obvious than in RG and GPRG. The results of principal component analysis (PCA) showed that there were significant metabolic differences among the three Rougui WRTs. According to the projection value of variable importance (VIP) of the partial least squares discriminant analysis (PLS–DA), 24 differential non-volatile metabolites were identified. The PLSR analysis results showed that rutin, silibinin, arginine, lysine, dihydrocapsaicin, etc. may be the characteristic non-volatiles that form the different taste outlines of Rougui WRT. A total of 90 volatiles, including aldehydes, alcohols, esters, and hydrocarbons, were identified from the three flavors of Rougui WRT by using GC-MS. Based on OAV values and PLS-DA analysis, a total of 16 characteristic volatiles were identified. The PLSR analysis results showed that 1-penten-3-ol, α-pinene, 2-carene, β-Pinene, dehydrolinalool, adipaldehyde, D-limonene, saffron aldehyde, and 6-methyl-5-hepten-2-one may be the characteristic volatiles that form the different aroma profile of Rougui WRT. These results provide the theoretical basis for understanding the characteristic metabolites that contribute to the distinctive flavors of Rougui WRT.

The epitranscriptomic mark N 6-methyladenosine (m6A), which is the predominant internal modification in RNA, is important for plant responses to diverse stresses. Multiple environmental stresses caused by the tea-withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, the effects of the m6A-mediated regulatory mechanism on flavor-related metabolic pathways in tea leaves remain relatively uncharacterized. We performed an integrated RNA methylome and transcriptome analysis to explore the m6A-mediated regulatory mechanism and its effects on flavonoid and terpenoid metabolism in tea (Camellia sinensis) leaves under solar-withering conditions. Dynamic changes in global m6A level in tea leaves were mainly controlled by two m6A erasers (CsALKBH4A and CsALKBH4B) during solar-withering treatments. Differentially methylated peak-associated genes following solar-withering treatments with different shading rates were assigned to terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylation can directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related transcripts and also indirectly influence the flavonoid, catechin, and theaflavin contents by triggering alternative splicing-mediated regulation. Our findings revealed a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a link between the m6A-mediated regulatory mechanism and the formation of tea flavor under solar-withering conditions.

The microRNA156 (miR156) family, one of the first miRNA families discovered in plants, plays various important roles in plant growth and resistance to various abiotic stresses. Previously, miR156s were shown to respond to drought stress, but miR156s in tea plants (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. Herein, we identify 47 mature sequences and 28 precursor sequences in tea plants. Our evolutionary analysis and multiple sequence alignment revealed that csn-miR156s were highly conserved during evolution and that the rates of the csn-miR156 members’ evolution were different. The precursor sequences formed typical and stable stem–loop structures. The prediction of cis-acting elements in the CsMIR156s promoter region showed that the CsMIR156s had diverse cis-acting elements; of these, 12 CsMIR156s were found to be drought-responsive elements. The results of reverse transcription quantitative PCR (RT-qPCR) testing showed that csn-miR156 family members respond to drought and demonstrate different expression patterns under the conditions of drought stress. This suggests that csn-miR156 family members may be significantly involved in the response of tea plants to drought stress. Csn-miR156f-2-5p knockdown significantly reduced the Fv/Fm value and chlorophyll content and led to the accumulation of more-reactive oxygen species and proline compared with the control. The results of target gene prediction showed that csn-miR156f-2-5p targeted SQUAMOSA promoter binding protein-like (SPL) genes. Further analyses showed that CsSPL14 was targeted by csn-miR156f-2-5p, as confirmed through RT-qPCR, 5′ RLM-RACE, and antisense oligonucleotide validation. Our results demonstrate that csn-miR156f-2-5p and CsSPL14 are involved in drought response and represent a new strategy for increasing drought tolerance via the breeding of tea plants.

MicroRNA166 (miR166) is a highly conserved plant miRNA that plays a crucial role in plant growth and the resistance to various abiotic stresses. However, the miR166s in tea (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. This study identified 30 mature miR166s and twelve pre-miR166s in tea plants. An evolutionary analysis revealed that csn-miR166s originating from the 3′ arm of their precursors were more conserved than the csn-miR166s derived from the 5′ arm of their precursors. The twelve pre-miR166s in tea were divided into two groups, with csn-MIR166 Scaffold364-2 separated from the other precursors. The Mfold-based predictions indicated that the twelve csn-MIR166s formed typical and stable structures comprising a stem-loop hairpin, with minimum free energy ranging from −110.90 to −71.80 kcal/mol. An analysis of the CsMIR166 promoters detected diverse cis-acting elements, including those related to light responses, biosynthesis and metabolism, abiotic stress defenses, and hormone responses. There was no one-to-one relationship between the csn-miR166s and their targets, but most csn-miR166s targeted HD-Zip III genes. Physiological characterization of tea plants under drought stress showed that leaf water content proportionally decreased with the aggravation of drought stress. In contrast, tea leaves’ malondialdehyde (MDA) content proportionally increased. Moreover, the cleavage site of the ATHB-15-like transcript was identified according to a modified 5′ RNA ligase-mediated rapid amplification of cDNA ends. The RT-qPCR data indicated that the transcription of nine csn-miR166s was negatively correlated with their target gene.

Fujian province, an important tea-producing area in China, has abundant tea cultivars. To investigate the genetic relationships of tea plant cultivars in Fujian province and the characteristics of the tea plant varieties, a total of 70 tea cultivars from Fujian and other 12 provinces in China were subjected to restriction site-associated DNA sequencing (RAD-seq). A total of 60,258,975 single nucleotide polymorphism (SNP) sites were obtained. These 70 tea plant cultivars were divided into three groups based on analyzing the phylogenetic tree, principal component, and population structure. Selection pressure analysis indicated that nucleotide diversity was high in Southern China and genetically distinct from cultivars of Fujian tea plant cultivars, according to selection pressure analysis. The selected genes have significant enrichment in pathways associated with metabolism, photosynthesis, and respiration. There were ten characteristic volatiles screened by gas chromatography–mass spectrometry (GC–MS) coupled with multivariate statistical methods, among which the differences in the contents of methyl salicylate, 3-carene, cis-3-hexen-1-ol, (E)-4-hexen-1-ol, and 3-methylbutyraldehyde can be used as reference indicators of the geographical distribution of tea plants. Furthermore, a metabolome genome-wide association study (mGWAS) revealed that 438 candidate genes were related to the aroma metabolic pathway. Further analysis showed that 31 genes of all the selected genes were screened and revealed the reasons for the genetic differences in aroma among tea plant cultivars in Fujian and Southern China. These results reveal the genetic diversity in the Fujian tea plants as well as a theoretical basis for the conservation, development, and utilization of the Fujian highly aromatic tea plant cultivars.

Wuyi Rock Tea (WRT) is cherished for its exceptional “rock flavor” and its quality shows obvious regional differences. However, the flavor characteristics of Primary Wuyi Rock Teas (PWRTs) from different production areas remain unclear. Here, the Camellia sinensis var. sinensis cv. ‘Rougui’ and ‘Shuixian’, two quintessential cultivars for making WRT, planted in Zhengyan, Banyan, at high elevations, and Waishan production areas were used to make PWRTs. We conducted a comprehensive comparison of the sensory attributes, volatile organic compounds (VOCs), and macro-compositions of PWRTs of ‘Rougui’ and ‘Shuixian’ cultivars from different producing areas. Sensory evaluation indicated that both ‘Rougui’ and ‘Shuixian’ PWRTs from Zhengyan exhibited the best flavor qualities, followed by those from Banyan, at high altitudes, and Waishan production areas. The results of the determination and analysis of VOCs showed 680 VOCs in ‘Rougui’ and ‘Shuixian’ PWRTs, and that the different production areas mainly influenced the quantitative pattern of VOCs and rarely the qualitative composition. Integrated multivariate statistical analysis methods revealed that benzyl alcohol, hotrienol, butanoic acid, 2-methyl-, hexyl ester, benzene, (2-nitroethyl)-, and geranyl isobutyrate may be the key VOCs affecting the aroma differences in PWRTs from different production areas. In addition, water-extractable substances, tea polyphenols, caffeine, and free amino acids may be the important macro-compositions that distinguish PWRTs from different production areas. The metabolite basis for differences in the flavor qualities of PWRTs across production areas was elucidated, which may be helpful for the production of high-quality WRT.