Responses of Secondary Metabolites and Transcriptomes in Tea Cultivar ‘Zhong Ming 6’ (Camellia Sinensis) under Blue Light and Red Light Journal Name: Plant Growth Regulation

Light is one of the most prestigious environmental sign, which stimulates plant metabolite consequences. Zhong Ming 6 (ZM6) is a green tea (Camellia sinensis) cultivar with highly accumulating TGGP. Here, three kinds of supplemental light wavelengths including blue light (BL,200 µmol m −2 s −1 ), red light(RL,200 µmol m −2 s −1 ), and White Light (WL/CK, 200 µmol m −2 s −1 ) were applied to explore their effects on the transcriptomes and metabolomics of young shoots (one bud and two leaves) in tea plants. Interestingly, arti�cial BL and RL signi�cantly affect the secondary metabolites and transcriptome factors of tea plants. Here, BL extensively dominated the multiple physiological actions with secondary metabolism. In addition, RL could induce plant growth, development, and photosynthesis. Thoroughly, the identi�cation of eight structural genes and 34 transcription factors (TFs) signi (cid:0) cantly correlated with total catechin (TC) and anthocyanin. Due to the upregulations of the CsGSTF1 gene in the �avonoid biosynthesis pathway, anthocyanin production was maximum under BL. Then, CsMYB75, bHLH-MYC, and other R2R3-MYBs were highly upregulated in BL to increase the accumulation of TC and anthocyanins in tea plants. Again, the CsMYB4 gene was highly signi�cant and positively correlated with TC and anthocyanin accumulation under RL. We indicated that BL is more feasible due to the number of functional metabolites (gallic acid, caffeine, TC, TGGP (1, 2, 6-tri-O-galloyl-β-D-glucopyranose), and anthocyanin) being supreme. For taste, quality, and dynamic indigenous mechanism of the tea plant, RL is also suitable that increase Chlorophyll content and tea yield.


Introduction
Tea (Camellia sinensis) is the globally most effective non-alcoholic beverage for its' bene cial effects in human health (Bai et al., 2021) as well as adding GDP in many countries (Fu et al., 2015). The extracts of green pertained from Camellia sinensis species under Theaceae family bearing several kinds of secondary metabolites, for example, catechins, anthocyanin (Liao et al., 2021), caffeine (Parvez et al., 2021), Gallic acids (Jovanovic et al., 2021), etc. Flavan 3-ols, namely catechins, is also known as the principal element of polyphenols in the young shoot of tea plants (Guo et al., 2017) with high anti-oxidant and reduced the high level of blood glucose, various kinds of renal function, hepatic and lipid parameters in animals (Nazir et al., 2021). By adding extra anti-oxidant substances, the human body uses them safely and has extracted them from food and plants (Bae et al., 2020). The phenylpropanoid and avonoid biosynthetic pathways are the essential pathways for catechins and anthocyanin production ( Zhang et al., 2018).
During growing period, light affects the tea plant as an environmental factor which, effortlessly stimulates the secondary metabolites (catechins, anthocyanin, etc.) accumulation to enhance the tea quality (Liao et al., 2021). Particularly, BL and RL increased these secondary metabolites' activity . Plant received certain types of photoreceptor under different kinds of light wavelength to regulated secondary metabolites Zheng et al., 2019). It is also a crucial signaling factor to the production of yield Ye et al., 2021). In addition, photosynthetic ability is very essential, as it directly contributes to plant growth and productivity (Sperdouli et al., 2021). However, photosynthetic pathways have been detrimentally affected in light stress than other abiotic oppressions (Nouri et al., 2015).
Firstly, the bene ts of BL and RL having 470 nm and 660 nm respective single-wavelengths investigated by (Fu et al., 2015). Again, the metabolic content and growth rates of greenhouse or indoor vertical farms' plants mainly perform in LEDs (Sng et al., 2021). It can be replaced both broad-spectrum WL and narrow wavelengths of LED lights that were particularly signi cant to increase plant production  Anthocyanin, a pink-purple color group of secondary metabolites (Gould et al., 2000), is another principal compound of avonoid biosynthesis pathways in plants that works as an anti-oxidant in the plant and reduced several diseases in human body . It also used as stress-reducing, color creator, (Liao et al., 2021) and delay senescence of plants under various biotic and abiotic conditions (Agati et al., 2012). Light also stimulates the anthocyanin synthesis in plant metabolic pathways .
Besides, RL (630 nm) could accumulates more anthocyanin to increase the aroma and tea quality to compare with natural light (260mn) (Lin et al., 2021). Nevertheless, the signal network and mechanism in light for regulating the anthocyanin synthesis still keep unclear . However, we observed, there is no exact investigation of the relationship between BL and RL yet on the secondary metabolites (Catechin and anthocyanin) of ZM6. In current time, LEDs have been used rapidly as substitute light sources (Bantis et al., 2018). It has high quality, quantity, adjustability, durability, and is used as energysaving, and has successfully applied to such crops as cucumber , pepper (Brown et al., 1995), banana (Nhut et al., 2002), tomato (Li et al., 2017), and grape (Poudel et al., 2008) We observed that the regulating factors of different polyphenols, molecular mechanisms, and phenotypes changes in both BL and RL are still unknown. In this study, to determine the RNA-Seq analysis and secondary metabolites in different LED wavelengths, the young shoots of tea (ZM6) were selected. So, the genes involved in secondary metabolites were identi ed under BL and RL. Furthermore, how different wavelengths of LED light affect the biosynthetic pathways to determine the chlorophyll content, secondary metabolites, and plant growth in tea plants.

Plant Materials and Light Treatments
The experiment was carried out in the growth chamber of Tea Research Institute, CAAS, Hangzhou, to investigate the different phenotypic traits and molecular mechanisms of ZM6. It was derived from a Camellia sinensis (green tea cultivar) under arti cial LEDs (light-emitting diodes) light. One and half-yearold tea plants (30cm in length) were cultivated for seven days in grown culture under white LED light. Tea plants were from the germplasm collection garden to use as sample selection materials. They were grown in Plastic Pots (30×30×20cm) containing a soil matrix in an environment adjusting growth chamber. The growth conditions of this experiment were at 25 ± 2°C with a 14 L/10 D photoperiod, and the RH (relative humidity) was at 70 ± 5%. It subjected the LED lamps horizontally above the plant canopy inside the chamber. The average PPF (photosynthetic photon ux) at the plant canopy level of each treatment was at 200 µmolm−2·s-1 by adjusting the number of LED lamps and the distance between the LED lamps and tea plants. Then it placed in a normal condition for one day due to acclimatizing before light quality treatments. After that, they were transferred to three controlled plastic boxes (each light) for ten days under WL (Control/CK), BL, and RL LED wavelength of 200-µmol m-2 s-1 during the 14-h photoperiod (Additional le S6). After ten days, we collected the young shoots (one bud and two leaves) of each treatment for transcriptome and metabolome analyses and executed under three biological replicates.

HPLC analysis
The estimation of catechins contents of HPLC was narrated by (Wei et al., 2015). In this study, we expelled the tea samples (0.2g, fresh weight) with 5ml 70% (v/v) methanol in a water bath.
Then placed it for 10 min at 70°C. Then shaken after a 5 min interval. After that centrifuged this liquid at 12000r/min for 10 min and then taken into a 10-ml volumetric ask. Then repeated these steps with a nal volume of 10ml. The extracts were then ltered through 0.45µm Millipore lters before the injection.

Identi cation of DEGs (differential gene expressions)
The

Quantitative Real-Time PCR (qRT-PCR) Validation for DEGs
Here, we selected eighteen genes to con rm the RNA-seq results for qRT-PCR (real-time PCR) analysis. Then analyzed the data by using three biological replications with means ± SD (n =3). The performance of qRT-PCR and RNA extraction precisely followed by   Zhong Ming 6 (ZM6) was derived from a green tea cultivar of Camellia sinensis in China. We mainly focused on young shoots (a bud with two leaves) for phenotypical supervision under three LED wavelengths. After ten days of light quality treatments, we found unique phenotypes among the CK, BL, and RL in the second leaf of the tea plant. It observed that a crimpy phenotype with comparatively reducing leaf area was in the new leaf under BL (Fig. 1a). In contrast, the newly grown shoots of tea plants provided the spacious phenotype with maintaining growth under supplemental RL (Fig. 1a).

Correlation Analysis of Gene Expression and Catechins Accumulation
Although the seeding of ZM6 produced enough leaf growth in CK, they displayed a lower photosynthetic capacity. Oppositely, chlorophyll content was higher under RL than BL (Fig. 1b)

Quantitative Real-Time PCR for Validation of Transcriptome Data
FPKM values were used to measure the level of gene expression in the tea plant. By con rming the results of RNA-Seq, the selection of eighteen genes derived from avonoid biosynthesis, regulation, and transformation for validation of qRT-PCR analysis. This study showed the qRT-PCR and RNA-Seq results in Fig. 4. It was clear that the tea genes' expression patterns were estimated by qRT-PCR and highly compatible with the RNA-seq (Additional le S4). The results of RNA-seq and qRT-PCR data con rmed that the methods of RNA-seq analysis provided feasible data. The correlation analysis was positively and signi cantly correlated (r = 0.78**) in this study. The data identi ed the principal genes involved in avonoids biosynthesis in tea plants.

DEGs (Differentially Expressed Genes) in C. sinensis under Various Light Qualities
The application of each LQ treatment with CK was estimated the DEGs by using the DESeq2 R package.
Here, various LQ were used to explore the molecular comparison in tea plants. There were some DEGs under BL (2938), while the DEGs in RL (1942) was comparatively lower (Fig. 4). Interestingly, samples clustering were derived from the different LQ that was similar to our results of catechin distribution (Fig. 2). It suggested that samples' difference in the light quality played a dominant role. Moreover, leaf samples under BLvsWL were far from the RLvsWL groups, revealing a signi cant difference between them. Thus, the nine groups were divided into three groups, namely group CK (CK-1, CK-2, and CK-3), BL (BL-1, BL-2, and BL-3), and RL (RL-1, RL-2, and RL3). The various expression of genes were analyzed between-group BL vs WL, group RL vs WL. Total 2938 DEGs were identi ed between-group BL and WL, 1920 with genes up regulated and 1018 down regulated. Similarly, 1942 DEGs were identi ed in the group of RL vs WL (Fig. 4). The enrichment analysis of the KEGG pathway was estimated the putative DEGs function. The comparative group of BL vs WL and RL vs WL were obtained 762 and 526 DEGs to reference responsible pathways (Fig. 3). Both phenylpropanoid biosynthesis (vvi00940) and avonoid biosynthesis (vvi00941) were placed among these delegate pathways in two pairs.
Because of the enrichment of DEGs between BL vs CK and RL vs CK, we further analyzed the global metabolic pathways of DEGs. Additional les (S1 and S3), most DEGs were annotated to Photosynthesis, Ribosome, carbon metabolism, Phenylpropanoid biosynthesis, avonoid metabolism, Plant hormone signal transduction, and amino acid metabolism. Among these DEGs, photosynthesis, amino acid, and Ribosome in energy metabolism were signi cantly high that representing the energy metabolism of tea plant shoots under BL and RL. In the enrichment analysis of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, 336 DEGs annotated in the 10 most signi cant pathways (FDR < 0.05). It included the avone and avonol biosynthesis, Phenylpropanoid biosynthesis, avonoid metabolism, Plant hormone signal transduction, amino acid metabolism, photosynthesis, Ribosome, Oxidative phosphorylation; Protein processing in the endoplasmic reticulum, Ubiquitin mediated proteolysis, Glycolysis or Gluconeogenesis under BL (Additional le S1 and S3).
On the other hand, the enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, 296 DEGs were annotated in the ten most signi cant pathways (FDR < 0.05). It included Photosynthesis, Phenylpropanoid biosynthesis, avonoid metabolism, Plant hormone signal transduction, amino acid metabolism, and Photosynthesis -antenna proteins, Carbon xation in photosynthetic organisms, Ribosome, MAPK signaling pathway -plant, Carbon metabolism under RL (Additional le S1 and S3).
Overall, many DEGs showed that the distinct expression patterns between CK and BL were related to photosynthesis and avonoid metabolism. Interestingly, the 32 DEGs annotated to Catechin biosynthesis were upregulated signi cantly in BL (Fig. 7). Oppositely, PSI-H genes from Photosystem further suggested that the high-energy RL regulated energy metabolism in tea plants (Fig. 1b). Twenty-three functional genes associated with catechins biosynthesis and 34TFs were identi ed in the DEGs with the expression patterns of BL > RL.

Correlation Analysis within Candidate Genes and Catechins Groups
HPLC analysis provided the determination of catechins shown in Fig. 2A and Fig. 2B. This analysis showed the six categories of catechins, namely GC, EGC, C, EC, EGCG, and ECG. We found, TC content signi cantly varied among the different wavelengths of light with the sequence of light > BL >RL. The TC contents in RL were remarkably lower than BL. For individual catechins, EGCG accounted (224mg/g or about 58%) of the TC contents in BL of leaves, followed by EGC. Both catechins come from trihydroxylated catechins. It indicated that the synthesis of trihydroxylated catechins added a remarkable contribution under BL and RL in the tea plant. While EGCG, EGC, and ECG (trihydroxylated catechins) were the dominant catechin components in BL (totally about 92%) and red light (90%). The results provided a signi cant change for the trihydroxylated catechins biosynthesis under BL that signi cantly more than RL. Again, this HPLC analysis also determined the accumulation of GA (gallic acid), CAF (caffeine), Gallocatechin gallate (GCG), and TGGP (1, 2, 6-tri-O-galloyl-β-D-glucopyranose) that signi cantly more in BL than RL ( Fig. 2A and S5).

Anthocyanin Contents in Tea Young Shoot of Selected Light Qualities
Total 1F3′5′H, 2DFR, 1GSTF12, 1(3GT), and 3UFGT genes were upregulated under BL but down regulated under RL in avonoid biosynthesis pathway for synthesis and degraded the anthocyanin production in RL. Moreover, the young shoots of the tea plant inhibited the anthocyanin synthesis due to the downregulated expression of DEGs by the effects of RL whereas increased in BL (Fig. 2C).

Analysis of DETFs (Differentially Expressed Transcription Factors)
TFs are essential regulatory factors that regulated the plant growth and yield. Again, for the accumulation of plant avonoids, the bHLH and MYB TFs genes were played a signi cant part in this study. According to our RNA-seq data, the selected 34 DETFs (differentially expressed transcription factors) under twelve families from the BL vs WL and RL vs WL group (Fig. 6b and Fig. 7). Among these DETFs, the most exorbitant TF families were MYB (9, 28.13%) and bHLH (8, 25%) followed by (6, 18.75%), WRKY (3, 9.38%), UNE (3, 9.38%), and TCP (3, 9.38%). Interestingly, maximum genes of MYB and bHLH were signi cantly upregulated in BL and balanced with related structural DEGs expression involving avonoid biosynthesis (Fig. 7). The regulatory genes were expressed by the TFs. Here, TFs played signi cant roles for catechins regulation. Among them, CsMYB75 (Cha01g017900) showed to be highly similar to CsMYB75 (114319222) (Wei et al., 2019) a key regulator of anthocyanin production Moreover, in the RL wavelength, a CsMYB4 (Cha08g011300) may be a homologous gene of MYB4 in Arabidopsis thaliana . For BL wavelength, bHLH-MYC and R2R3-MYB (Cha04g003640) were also played an important role for catechin and anthocyanin accumulation that signi cantly similar to R2R3-MYBs gene (Zheng et al., 2019) also identi ed in these DETFs. Further research of these TFs would condense our intelligence of regulatory system for catechin and anthocyanin biosynthesis (Fig. 7).  (Tian et al., 2019). Our study was schematic to explore the effective of a selected application of BL and RL to form plant growth and development with different kind of secondary metabolism. Even though, a comparative investigation of which DEGs respond to BL and RL in catechins and anthocyanin metabolic pathways of tea plants. For this region, we focused on the transcriptional and metabolic changes and key processes elicited in tea plant shoots in response to BL and RL wavelengths. We also expressed that the supplemental light qualities aggravated the phenotypical differences (Fig. 1a) and various metabolic factors (Fig. 2) in tealeaves under BL and RL. It also assisted due to elucidating which intensity of LED light was more reacted between these two light wavelengths (Fig. 5).

Discussion
The HBL (high blue light) at 200 µmol m −2 s −1 on the growth and metabolism, such as avonoid (catechins and anthocyanin) were maximum than other low condition light . Our study revealed that the numbers of DEGs under BL in differential metabolic pathways were higher than RL in the young shoot of the tea plant ( Fig. 3 and Table 1). This result reported that TFs and metabolic pathways were extensively enhanced under BL compared to RL. BL also accumulated the secondary metabolism more than RL in the tea plants.  ., 2019). On the other hand, for plant photosynthesis, BL and RL were used as the light energy source that recognized for antibacterial activity (Lin et al., 2021). Our research has shown that the chlorophyll content and area of the second leaf (one bud and two leaves) in the tea plant were maximum under RL (Fig. 1b). Though wavebands (200-µmol m −2 s −1 ) are the same in both two lights, BL might be provided more light intensity than RL (Additional le S3). Leaf area, and plant height were comparatively reduced in BL than in RL (Liang et al., 2021). It has concluded that BL could only manage the normal plant production. This result is signi cantly and positively correlated in cucumber and tomato (Liang et al., 2021) but negatively correlated in grafted watermelon (Moosavi-Nezhad et al., 2021). In BL, leaf area was increased by 94% as compared to WL in Pakchoi (Brassica campestris) . In 'Huangjinya' (albino tea) leaves, RL might be promoted more chlorophyll content due to gene upregulation than BL (Tian et al., 2021).
The accumulation of catechin in the young shoots was maximum in BL because of gene expression in the biosynthetic pathway (Zhang et al., 2018). The HLPC analysis of our research displayed that the peak of the chromatogram of catechin composition, e.g., EGCG, EGC, ECG, EC, was signi cantly high compared to other lights ( Fig. 2A) and the total content of catechins was also maximum (Fig. 2B). FtANR and FtLAR1 genes have been cloned to the formation of ( +) C and EC were upregulated in BL but downregulated in RL in Tartary  Caffeine (1, 3, 7-trimethylxanthine)  and Gallic acid were used for the fermentation and manufacturing of tea leaves and also bene cial for human health (Trevisanato and Kim, 2000). In this study, HPLC analysis also showed that the highest accumulation of caffeine and Gallic Acid were found in BL than RL in the young shoot of ZM6 ( Fig. 2A and S5). BL also induced accumulation of caffeine content in cucumbers (Palma et al., 2021). Though caffeine accumulation was maximum under RL and WL that signi cantly reduced the avonoids in Gongmei white tea plant (Huang et al., 2021).
Again, RL also reduced the content of Gallic Acid in Protea cynaroides L as an ornamental plant.
TGGP maybe also work as a microbicide that prevents the transmission of sexual HIV in the human body (Sun et al., 2008). Our study also found that TGGP accumulation was maximum under BL by HPLC analysis (Additional le S5).
Quantitative and qualitative changes in anthocyanin can be occurred by light in the plant . Light duration, quality, and intensity regulated the anthocyanin biosynthetic process by in uencing various light receptors such as PHYs, PHOTs, CRYs, and UVR8. These photoreceptors affected the PIFs (phytochrome-interacting factors), COP1, and HY5. WDR, bHLH, and MYB used as a promoter to activate or inhibit the genes expression . BL enhanced the maximum anthocyanin accumulation in leaf vacuole (Fig. 7) of the tea plant (ZM6) by upregulating the expression of CsGSTF12 and CsMYB75 genes in this study ( Fig. 2C and Fig. 5 (Taulavuori et al., 2018). Again, the products of avonoid biosynthesis could be enhanced effectively by BL in Eruca saliva (arugula) but have no response from the Ocimunt basilican (basil) and Rumex sanguineus (bloody dock) species (Taulavuori et al., 2018). On the other hand, the activity of avonoid glycoside transferase affected by the RL, but the accumulation of avonoids was reduced in the tobacco plant. In addition, avonoids productivity could be enhanced under BL by upregulating of CHS gene in Cyclocarya paliurus leaves  Our result revealed that a total number of structural and TFs genes were 25 and 34 respectively included in avonoid productions. But maximum genes structural (Fig. 5b) and TFs (Fig. 6) were upregulated in BL vs WL and downregulated in RL vs WL that's why total catechin content (Fig. 2B) and anthocyanin accumulation (Fig. 2C) (3), and bHLH (7/8) were upregulated highly in BL (Fig. 7). Especially, CsMYB75 (Cha01g017900) may be a homologous gene of CsMYB75 (114319222) in Arabidopsis thaliana (Wei et al., 2019) was signi cantly and positively correlated with catechin and anthocyanin accumulation (Fig. 5) in both cytosol and vacuole of tea leaves (Fig. 7).
On the contrary, the structural candidate genes expression (CsPAL, Cs4CL, CsFHT, CsFLS, CsDFR, CsUFGT, and LAR) downregulated in avonoid biosynthesis pathways under RL vs WL. TC and anthocyanin content were also positively correlated under RL by upregulating of CsMYB4 (Cha08g011300) gene. It may be the homologous gene of MYB4 in Arabidopsis thaliana that inhibited avonoid accumulation , an important TFs gene in avonoid biosynthesis pathways, signi cantly upregulated in ZM6 ( Fig. 5c and Fig. 7).
In addition, we observed from the RNA seq. analysis, CsGSTF12 (Cha08g011300) may be a homologous gene of CsGSTF1 (114322623) in Arabidopsis thaliana, and Cs3GT (ChaUn26415.1) were upregulated signi cantly in BL vs WL group but down regulated in RL vs WL group (Fig. 6)