| Tea plant(Camellia sinensis),which originated in southwest China,is one of most economically important crop plant around the world.Oolong tea is a semi-fermented tea with high flavor,which is produced mainly in Fujian,Guangdong,and Taiwan provinces in China.Additionally,it is very popular among consumers because of its unique floral and fruity aroma and mellow taste.Withering is the first step for improving flavors during the postharvest processing of oolong tea,which is closely related to the subsequent tea processing steps and is important for the development of the unique flavor of oolong tea.Traditional withering treatments include solar-withering and indoor-withering.In these two treatments,fresh leaves were affected by various environmental stresses to varying degrees.Environmental stress can induce plants to produce a variety of secondary metabolites,which can help plants resist external stress.Secondary metabolites endow tea with unique flavor and quality.Flavonoids are the most important secondary metabolites in tea,which are closely related to the taste formation and the main reason for the bitterness and astringency in oolong tea.At present,studies on the regulatory mechanism of secondary metabolites induced by stress mainly focus on transcriptional and metaboilic levels,especially the transcriptional changes of key biosynthetic genes induced by environmental stress directly affect the fluctuation of flavor-related metabolites.Decades of studies have demonstrated that multiple environmental stresses caused by withering treatments result in a series of multidimensional responses extending from genes to metabolites,and further affects the formation of oolong tea flavor.Although there is evidence that nc RNA and epigenetic modification play indispensable roles in various biological processes such as environmental stress response and metabolic regulation,the research on nc RNA and epigenetic regulation in tea leaves lagged far behind model plants.However,our knowledge regarding the upstream regulation mechanism of the flavor-related genes and relevant metabolites is still limited.In this study,tea plants(Camellia sinensis cv.tieguanyin)were used as materials.From the perspective of multi-transcriptomics,this study systematically explored the potential causes of the metabolic regulation of flavonoids and terpenoids and the differences in flavor quality between solar-withering and indoor-withering.A cascade regulatory network of lnc RNA-mi RNA-m RNA and circ RNA-mi RNA-m RNA centered on mi RNA was constructed to participate in the flavonoid and terpenoid metabolic pathways during tea-withering stage.It reveals the indispensability of solar-withering during the manufacturing process of oolong tea.In addition,from the perspective of RNA methylation modification,the molecular regulatory mechanism of RNA methylation as an upstream regulatory factor involved in the flavonoid and terpenoid metabolism under solar-withering with different light intensities was investigated,and the molecular mechanism of moderate shading during solar-withering promoting the flavor formation of oolong tea was clarified.The main results were as follows:1 Transcriptional regulation of flavonoid and terpenoid metabolism during withering stage of oolong teaTranscriptome sequencing was used to analyze the difference among the fresh leaves(FL),indoor-withered leaves(IW)and solar-withered leaves(SW)of oolong tea.A total of 10793 differentially expressed genes(DEGs)were identified from the three samples.KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in flavonoid biosynthesis,terpenoid biosynthesis,plant hormone signal transduction and spliceosome pathways.Further analysis revealed that the transcriptional inhibition of flavonoid biosynthesis-related genes,the transcriptional enhancement of terpenoid biosynthesis-related genes,as well as the jasmonic acid signal transduction and the alternative splicing mechanism jointly contributed to the flavor formation of high floral and fruity aroma and low bitterness in solar-withered leaves.These findings reveal the importance of solar-withering treatment in the formation of high-quality flavor of oolong tea and the detailed mechanism of transcriptional regulation and alternative splicing in the flavor-related metabolic pathways.2 lnc RNA-mediated regulation of flavonoid and terpenoid metabolism during withering stage of oolong teaOn the basis of lnc RNA sequencing,we systematically analyzed and identified lnc RNAs in fresh leaves,indoor-withered leaves and solar-withered leaves,and analyzed the lnc RNA-mediated regulation of flavonoid and terpenoid metabolism during withering stage of oolong tea.First,the changes of flavor metabolites and phytohormones before and after withering treatments were detected.The results showed that the total flavonoid and total catechin contents as well as the abundance of five individual catechins,especially the galloylated catechins(CG,GCG,ECG,and EGCG),were significantly lower in the SW than in the FL and IW,whereas the opposite pattern was observed for the lignin content.Moreover,the(Z)-3-hexenal,(E)-2-hexenal,and terpenoid volatiles,which represent a large proportion of the volatiles in oolong tea,may be the most affected by the withering process,especially solar-withering,which can trigger a significant increase in the content of these terpenoid volatiles and a decrease in the abundance of hexenal volatiles.The contents of JA and Me JA,which can be used as signal molecules to promote the biosynthesis and accumulation of related volatile metabolites,were significantly higher in SW than FL and IW.Subsequently,FL,IW,and SW underwent a transcriptome sequencing analysis after r RNA removal.An average of 12.66 Gb of clean data were obtained per sample,and 32036 lnc RNAs were identified.We then identified the enriched KEGG pathways among the target genes of the DE-lnc RNAs.Target genes of the DE-lnc RNAs are mainly enriched in flavonoid metabolism,terpenoid metabolism and JA and Me JA biosynthesis-related linoleic acid/linolenic acid metabolism pathways.The regulatory relationship and expression levels of DE-lnc RNAs and their target genes in the above-mentioned three pathways were analyzed.high FLS,CCR,CAD,and HCT transcript levels and seven up-regulated lnc RNAs as well as low4 CL,CHI,F3 H,and F3’H transcript levels and three down-regulated lnc RNAs in the SW are closely associated with the low abundance of flavonoids and catechins in the SW.Because of the regulation of related lnc RNAs,the expression of genes involved in the flavonoid biosynthetic pathway is inhibited,whereas the expression of genes related to the lignin biosynthetic pathway is up-regulated,thereby decreasing the amounts of total flavonoids and catechins,especially the galloylated catechins,in the SW.In the terpenoid metabolism,SW had nine up-regulated genes(DXS,CMK,HDS,HDR,AACT,MVK,PMK,GGPPS,and TPS),three up-regulated lnc RNAs,and six down-regulated lnc RNAs related to terpenoid metabolism,which are key regulatory factors causing high accumulation of terpenoids in SW.High JA and Me JA contents may have a cumulative effect on terpenoid levels.In addition,two lnc RNAs(LTCONS＿00026271 and LTCONS＿00020084)identified from JA/Me JA biosynthesis and signal transduction pathway can synergically promote terpenoids through the regulatory mechanism of lnc RNA-mi RNA-m RNA mediated by e TM,together with high JA/Me JA content synergistically promoted the accumulation of terpenoids in SW.In conclusion,compared with IW,lnc RNAs in SW is involved in the metabolic regulation of flavonoids and terpenoids through direct regulation of target genes and e TM-mediated lnc RNA-mi RNA-m RNA modules,promoting the low flavonoid and catechin contents as well as high terpenoid content in SW,thus effectively removing bitter taste,improving tea palatability,and further promoting the formation of floral and fruity aroma in oolong tea.The molecular mechanism of improving tea flavor by solar-withering treatment was preliminarily elucidated.3 mi RNA-mediated regulation of flavonoid and terpenoid metabolism during withering stage of oolong teaSmall RNA sequencing was used to systematically explore and identify mi RNAs from fresh leaves,indoor-withering leaves and solar-withering leaves.A total of 168 known mi RNAs belonging to 44 mi RNA families and 62 novel mi RNAs were identified.We then identified the enriched KEGG pathways among the target genes of the DE-mi RNAs.Target genes of the DE-mi RNAs are mainly enriched in Plant hormone signal transduction and ABC transporters.The expression of DE-mi RNAs and their target genes related to above-mentioned pathways are related to the accumulation of flavonoids and terpenoids.In addition,DE-mi RNAs also regulated the transport of flavor metabolites and plant hormones through mi RNA-m RNA modules,thus affecting the accumulation of flavor metabolites and improving the flavor quality of oolong tea.IAA and GA contents are important for the accumulation of flavonoid and terpenoid in SW,and are controlled via the IAA-mi R167-ARF-GH3 and GA-mi R171b-DELLA-MYC2 modules.Circ RNA also affects the transport capacity of flavor metabolites and plant hormones through the circ RNA-mi RNA-m RNA cascade transcriptional regulation mediated by circ RNA,resulting in the decrease of flavonoid and catechin contents and the increase of terpenoid content in SW.The aforementioned regulation mechanism promoted the dissipation of bitter taste and the formation of mellow taste and strong fragrance of oolong tea after solar-withering treatment.These results revealed the molecular regulatory mechanism of circ RNA-mi RNA-m RNA cascaded regulation mechanism centered on mi RNA to promote the formation of high-quality oolong tea after solar-withering treatment.To improve the overall understanding of the chain regulation of "nc RNA-m RNA-protein-function",to comprehensively and systematically improve the multifaceted regulatory network mediated by nc RNA during withering stage of oolong tea,and to emphasized that solar-withering played a more crucial role in improving the flavor quality of oolong tea than indoor-withering.4 Mechanism of RNA methylation regulatory genes during withering stage of oolong teaOn the basis of chromosome-scale genome of tea plants,RNA methylation regulatory genes were comprehensively mined.After thoroughly screened the whole genome of tea plant,a total of nine m6 A writer,sixteen m6 A eraser,and nine m6 A reader genes were ultimately ascertained.And 30 m6 A regulatory genes were randomly distributed on13 chromosomes,and the remaining four genes were located on the unanchored contigs.Then,the evolutionary relationships and gene structure of RNA methylation regulatory genes were analyzed on the genome-wide scale.Instead of variation in conserved domain and motif,the gain or loss of exons and introns in gene sequence may contribute to the functional diversification of m6 A regulatory genes.WGD are not only the major force acting upon the expansion of m6 A regulatory genes,but also lay the foundation for triggering the functional innovation of these genes.The combined variations in gene sequence and expression abundance of m6 A regulatory genes may further enrich the functional diversification of the these duplicated gene members in tea plants.These factors synergistically promoted the clear division of labor among different members of m6 A regulatory genes,which contributes to the formation of the precise regulatory mechanism of m6 A modification in tea plant.We further investigated the expression patterns of RNA methylation regulatory genes in fresh leaves,indoor-withered leaves and solar-withered leaves,and explored the specific roles of three types of RNA methylation regulatory factors in m6 A modification.It was found that m6 A reader genes mediated AS mechanism may be the main reason for inhibiting flavonoid biosynthesis.Meanwhile,the down-regulation of m6 A writer gene transcription abundance and up-regulation of m6 A eraser gene transcription abundance further synergistically reduced the accumulation of flavonoids.Subsequently,the relationship between RNA methylation regulatory genes and methylation level as well as the potential biological function of RNA methylation during tea-withering process were verified by si RNA-mediated transient transcription inhibition system.High expression levels of DNA demethylase genes can stimulate the transcription of RNA demethylase genes through removing the DNA methylation marks,thereby indirectly inhibiting the flavonoid biosynthesis and improving the palatability of oolong tea.RNA methylation and DNA methylation interact with methylation regulatory genes to form negative feedback to control flavonoid accumulation in tea leaves within a reasonable range.This study provided a new perspective for the preliminary study of the potential mechanism of m6 A regulatory gene in precise regulation of tea quality,and opened up new perspectives for understanding m6A-mediated regulatory mechanism on the formation of tea palatability during withering stage of postharvest processing.5 Metabolic regulation of flavor metabolites in oolong tea by RNA methylation-mediated light intensity of witheringThrough integrated RNA methylome and transcriptome analyses,the effects of m6 A modification on flavor quality of oolong tea under solar-withering with different light intensities were investigated.It was found that m6 A modifications were widely distributed on the m RNAs of tea plant,and m6 A modification levels changed dynamically under solar-withering with different light intensities.Solar-withering triggered a significant decrease in m6 A levels,concomitant with a negative correlation between light intensity and globally m6 A abundance within a certain range.Analysis of the expression levels of m6 A regulatory genes revealed that the dynamic of total m6 A levels was mainly regulated by m6 A eraser genes,and Cs ALKBH4-mediated RNA demethylation may play a crucial role under solar-withering with different light intensities.Next,KEGG pathway analysis showed that the identified DMGs were mainly enriched in terpenoid biosynthesis pathway,and m6 A peaks in DMGs were all distributed within 3′UTR and around the stop codon.The m6 A abundance of terpenoid biosynthesis-related DMGs dropped sharply from FL to SW3,but exhibited a distinct rebound from SW3 to SW4.Similar to the above result,the expression levels of DMGs and terpenoid content showed similar trends under solar-withering with different light intensities.These findings revealed that Cs ALKBH4-mediated RNA demethylation may directly activate terpenoid biosynthesis by removing m6 A marks and enhancing the stability of corresponding m RNAs,thereby enormously improving the aroma quality of oolong tea under solar-withering.From FL to SW3,there were positive correlations between light intensity and the expression levels of terpenoid biosynthesis-related genes as well as terpenoid content.Within a certain range,high light intensity helped promote terpenoid biosynthesis and the formation of a high-quality aroma in oolong tea.Excessively high light intensity in SW4 arrested Cs ALKBH4-mediated RNA demethylation,thereby destabilizing the terpenoid biosynthesis-related genes,and ultimately leading to a significant decrease in the terpenoid abundance.Under solar-withering with different light intensities,RNA demethylation might be involved in the regulation of AS events by modulating the m6 A abundance and expression levels of spliceosome-related genes.The down-regulation of Cs4 CL,Cs4CL-a,and Cs F3’H-a as well as up-regulation of Cs GPX3,Cs APX1,and Cs APX1-a in solar-withered leaves synergistically dampened the flavonoid biuosynthesis and expedited the flavonoid catabolism,jointly promoting the conversion of bitter and astringent substances(flavonoid and catechin)into mellow-tasting theaflavin.Compared with other light intensities of solar-withering,SW3 had the lowest content of catechins and high content of theaflavins,indicating that moderate sunlight intensity,rather than lack or excessive light intensity,could,effectively reduce bitterness and astringency and enhance the mellow taste of tea infusions.Together,these findings highlighted that RNA demethylation indirectly affect the accumulation of flavonoids,catechins and theaflavins by triggering AS-mediated regulatory mechanism,thereby improving the palatability of oolong tea.In conclusion,the above findings revealed the molecular regulation mechanism of RNA methylation in the flavor formation of oolong tea during withering stage of postharvest processing,and the molecular mechanism of moderate shading during solar-withering promoting the flavor formation of oolong tea was elucidated. |
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