The MADS-box transcription factor CsAGL9 plays essential roles in seed setting in Camellia sinensis.

The number of seed setting (NSS) is an important biological trait that affects tea propagation and yield. In this study, the NSS of an F1 tea population (n = 324) generated via a cross between 'Longjing 43' and 'Baihaozao' was investigated at two locations in two consecutive years. Quantitative trait locus (QTL) mapping of the NSS was performed, and 10 major QTLs were identified. In total, 318 genes were found in these 10 QTLs intervals, and 11 key candidate genes were preliminarily identified. Among them, the MADS-box transcription factor AGAMOUS LIKE 9 (CsAGL9, CSS0037962) located in the most stable QTL (qNSS2) was identified as a key gene affecting the NSS. CsAGL9 overexpression in Arabidopsis promoted early flowering and significantly decreased the length and number of pods and number of seeds per pod. Transcriptome analysis demonstrated that the auxin pathway, a key hormone pathway regulating plant reproduction, was highly affected in the transgenic lines. The auxin pathway was likewise the most prominent in the gene co-expression network study of CsAGL9 in tea plants. In summary, we identified CsAGL9 is essential for seed setting using QTL mapping integrated with RNA-seq, which shed a new light on the mechanism NSS of seed setting in tea plants.

Effects of Curcumin on Oxidative Stress and Ferroptosis in Acute Ammonia Stress-Induced Liver Injury in Gibel Carp (Carassius gibelio).

This study investigated the potential role of curcumin (CUR) in preventing oxidative stress and ferroptosis induced by ammonia exposure in gibel carp. Experimental fish (initial weight: 11.22 ± 0.10 g, n = 150) were fed diets supplemented with or without 0.5% CUR for 56 days, followed by a 24 h ammonia (32.5 mg/L) exposure. Liver damages (aspartate aminotransferase (AST), alanine aminotransferase (ALT), adenosine deaminase (ADA), and alkaline phosphatase (ALP)) and oxidative stress enzyme activities (reactive oxygen species (ROS), malondialdehyde (MDA); and the content of antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) were induced by ammonia stress. The antioxidant capacity was decreased, as indicated by inhibited gene expression of nuclear factor erythroid 2-related factor 2 (nrf2), heme oxygenase-1 (ho-1), catalase (cat), and sod. Ferroptosis was induced by ammonia stress, as suggested by upregulated mRNA levels of nuclear receptor coactivator 4 (ncoa4), transferrin receptor 1 (tfr1), and iron-responsive element-binding protein 2 (ireb2), and downregulated expression of glutathione peroxidase 4 (gpx4), ferroportin (fpn), and ferritin heavy chain 1 (fth1). In addition, both mRNA and protein levels of ferroptosis markers acyl-CoA synthetase long-chain family member 4 (ACSL4) and prostaglandin-endoperoxide synthase 2 (PTGS2) were upregulated, while cystine/glutamate antiporter (SLC7A11) was downregulated. However, liver injury and ferroptosis in fish induced by ammonia could be attenuated by CUR. Collectively, these findings demonstrate that CUR ameliorates oxidative stress and attenuates ammonia stress-induced ferroptosis. This study provides a new perspective on potential preventive strategies against ammonia stress in gibel carp by dietary CUR.

Taurine Alleviates Cadmium-Induced Toxicity via Genetically Specific Strategies in Two Strains of Gibel Carp (Carassius gibelio).

Our previous studies in gibel carp (Carassius gibelio) have shown that cadmium (Cd) exposure elicits deleterious effects depending on the genetic background, and thus we hypothesized that mitigation via nutritional intervention may vary between strains. Therefore, two gibel carp strains (the A and F strains) were fed diets supplemented with 0% or 1% taurine for 8 weeks prior to 96 h Cd exposure, and the responses of antioxidant pathways, endoplasmic reticulum (ER) stress, autophagy, and apoptosis were investigated. The results showed that taurine supplementation had no effect on the growth performance of gibel carp. After Cd exposure, histological damage to mitochondria and ER, induction of oxidative stress and antioxidant responses, occurrence of ER stress, and apoptotic signals were observed in the livers. Upon the diet effects, taurine supplementation alleviated the ER-stress-induced autophagy and apoptosis after Cd exposure and stimulated antioxidant pathways. Regarding the difference between strains, taurine played a protective role in alleviating Cd toxicity through the antioxidant response, ER stress, and autophagy in the F strain, whereas such effects were achieved by the attenuation of apoptosis in the A strain. Taken together, our results demonstrate the potential use of taurine in the mitigation of heavy metal toxicity in aquatic organisms.

Integrated transcriptome and hormonal analysis of naphthalene acetic acid-induced adventitious root formation of tea cuttings (Camellia sinensis).

BACKGROUND: Tea plant breeding or cultivation mainly involves propagation via cuttings, which not only ensures the inheritance of the excellent characteristics of the mother plant but also facilitates mechanized management. The formation of adventitious root (AR) determines the success of cutting-based propagation, and auxin is an essential factor involved in this process. To understand the molecular mechanism underlying AR formation in nodal tea cuttings, transcriptome and endogenous hormone analysis was performed on the stem bases of red (mature)- and green (immature)-stem cuttings of 'Echa 1 hao' tea plant as affected by a pulse treatment with naphthalene acetic acid (NAA). RESULTS: In this study, NAA significantly promoted AR formation in both red- and green-stem cuttings but slightly reduced callus formation. External application of NAA reduced the levels of endogenous indole-3-acetic acid (IAA) and cytokinin (TZR, trans-zeatin riboside). The number of DEGs (NAA vs. CK) identified in the green-stem cuttings was significantly higher than that in the red-stem cuttings, which corresponded to a higher rooting rate of green-stem cuttings under the NAA treatment. A total of 82 common DEGs were identified as being hormone-related and involved in the auxin, cytokinin, abscisic acid, ethylene, salicylic acid, brassinosteroid, and jasmonic acid pathways. The negative regulation of NAA-induced IAA and GH3 genes may explain the decrease of endogenous IAA. NAA reduced endogenous cytokinin levels and further downregulated the expression of cytokinin signalling-related genes. By the use of weighted gene co-expression network analysis (WGCNA), several hub genes, including three [cellulose synthase (CSLD2), SHAVEN3-like 1 (SVL1), SMALL AUXIN UP RNA (SAUR21)] that are highly related to root development in other crops, were identified that might play important roles in AR formation in tea cuttings. CONCLUSIONS: NAA promotes the formation of AR of tea cuttings in coordination with endogenous hormones. The most important endogenous AR inductor, IAA, was reduced in response to NAA. DEGs potentially involved in NAA-mediated AR formation of tea plant stem cuttings were identified via comparative transcriptome analysis. Several hub genes, such as CSLD2, SVL1 and SAUR21, were identified that might play important roles in AR formation in tea cuttings.

Systematic Investigation and Expression Profiles of the Nitrate Transporter 1/Peptide Transporter Family (NPF) in Tea Plant (Camellia sinensis).

NRT1/PTR FAMILY (NPF) genes are characterized as nitrate and peptide transporters that played important roles in various substrates transport in plants. However, little is known about the NPF gene in tea plants. Here, a total of 109 CsNPF members were identified from the tea plant genome, and divided into 8 groups according to their sequence characteristics and phylogenetic relationship. Gene structure and conserved motif analysis supported the evolutionary conservation of CsNPFs. Many hormone and stress response cis-acting elements and transcription factor binding sites were found in CsNPF promoters. Syntenic analysis suggested that multiple duplication types contributed to the expansion of NPF gene family in tea plants. Selection pressure analysis showed that CsNPF genes experienced strong purifying selective during the evolution process. The distribution of NPF family genes revealed that 8 NPF subfamilies were formed before the divergence of eudicots and monocots. Transcriptome analysis showed that CsNPFs were expressed differently in different tissues of the tea plant. The expression of 20 CsNPF genes at different nitrate concentrations was analyzed, and most of those genes responded to nitrate resupply. Subcellular localization showed that both CsNPF2.3 and CsNPF6.1 were localized in the plasma membrane, which was consistent with the characteristics of transmembrane proteins involved in NO3- transport. This study provides a theoretical basis for further investigating the evolution and function of NPF genes.

Vitamin C Attenuates Oxidative Stress, Inflammation, and Apoptosis Induced by Acute Hypoxia through the Nrf2/Keap1 Signaling Pathway in Gibel Carp (Carassius gibelio).

Previous studies have found that vitamin C (VC) has protective effects in fish. However, the efficacy of VC on hypoxia-induced liver injury in fish remains unknown. Therefore, to investigate the protective mechanism of VC on liver injury after acute hypoxic stimulation in fish, gibel carp were fed a diet containing VC for eight weeks, then were subjected to acute hypoxia stimulation. The specific growth rate of fish was increased by the supplementation of VC. Plasma stress markers (glucose, lactic acid, and cortisol) were decreased by the VC supplementation. Moreover, the levels of the inflammatory cytokines (tnf-α, il-2, il-6, and il-12) were increased by enhancing the Nrf2/Keap1 signaling pathway. Upregulation of the antioxidant enzymes activity (CAT, SOD, and GPx); T-AOC; and anti-inflammatory factors (il-4 and tgf-ß) highlighted the antioxidant and anti-inflammatory activities of VC. The results showed that VC reduced the apoptotic index of the fish hypothalamus. The expression of GRP78 protein in the liver and endoplasmic reticulum stress and apoptosis induced by hypoxia were inhibited by VC. Taken together, the results indicate that VC can attenuate oxidative damage, inflammation, and acute hypoxia induced apoptosis in gibel carp via the Nrf2/Keap1 signaling pathway. The results identify a new defense strategy of gibel carp in response to hypoxic conditions.

Chemical characterization of Tianshan green tea polysaccharides and its protective effects on cell oxidative injury.

The purpose of this study was to analyze the chemical characterization of Tianshan green tea polysaccharides (TSPS), and evaluate its antioxidant activity by chemical-based and cellular-based antioxidant models in vitro. The results showed that the TSPS were composed of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and fucose with a molar ratio of 14.5:33.5:10.5:6.5:111.5:22.3:59.5:51: 1.0, and an average molecular weight of 19.49 kDa. TSPS exhibited excellent antioxidant ability to DPPH radical, hydroxyl radical, and ABTS radical, and enhanced the ferric-reducing power (FRAP). The antioxidation model of LO2 and HepG2 cells was established, and found that TSPS had no significant toxicity to either of the two cells at the range of 0.1-5 mg/mL, but clearly protected cells from H2 O2 -induced apoptosis and significantly reduced intracellular ROS level. In addition, the activities of antioxidant-associated enzymes were detected in LO2 cells, which suggested that TSPS could significantly improve the activities of SOD and CAT enzyme when the concentration was higher than 0.5 mg/mL. Furthermore, TSPS activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway by promoting Nrf2 nuclear translocation and inhibited the expression of Kelch-like ECH-associated protein 1 (Keap-1) and enhanced the expression of heme oxygenase-1 (HO-1). PRACTICAL APPLICATIONS: Tianshan green tea, a local variety in Fujian Province, belongs to unfermented tea. Polysaccharide is considered as the most promising component in Tianshan green tea. This study showed that TSPS had excellent antioxidant activity and had no significant toxicity to cells, which provides a scientific foundation and new idea for its further development and application in functional foods.

Responses of tea plants (Camellia sinensis) with different low-nitrogen tolerances during recovery from nitrogen deficiency.

BACKGROUND: Tea plants have high nitrogen (N) consumptions, whereas molecular and physiological responses of tea plants to N recovery are still unclear. RESULTS: By using non-invasive micro-test technology (NMT), 15 N tracer technique, ultra-performance liquid chromatography (UPLC), and transcriptome sequencing technology, we investigated the N recovery-induced changes in N absorptions, N tissue distributions, contents of free amino acids (FAAs), and global transcription of the low-N tolerant and intolerant tea genotypes [i.e. Wuniuzao (W) and Longjing43 (L)]. The results showed that the phenotype of Wuniuzao was better than that of Longjing43 under low-N condition. The N absorption and utilization of Wuniuzao were superior to Longjing43 under N recovery. The γ-aminobutyric acid (GABA) ratio (N recovery/N deficiency) in the root of Wuniuzao was significantly higher than that of Longjing43, while the glutamic acid ratio in the root of Wuniuzao was significantly lower than that of Longjing43. This findings suggested that Wuniuzao tended to enhance the GABA synthesis, while Longjing43 tended to inhibit the GABA synthesis under N recovery. The key genes in response to N recovery in Wuniuzao included N transport (AMT and NRT), N transformation (NR, NirA, and GAD), and amino acid transport (GAT) genes. In addition, some ribosome and flavonoid biosynthesis genes might help to maintain proteome homeostasis. CONCLUSION: The N absorption and transport, and the conversion abilities of key amino acids (Glu and GABA) might improve the adaptability of tea plants to N recovery, which provided a basis for the breeding of N efficient tea varieties. © 2021 Society of Chemical Industry.

Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis).

BACKGROUND: Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. RESULTS: The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0-8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5'-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). CONCLUSIONS: Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

Identification of Regulatory Networks and Hub Genes Controlling Nitrogen Uptake in Tea Plants [Camellia sinensis (L.) O. Kuntze].

Nitrogen (N) uptake, as the first step of N metabolism, is a key limiting factor for plant growth. To understand the gene expression networks that control N absorption and metabolism in tea plants, we analyzed transcriptomes in the young roots of two groups of tea plants with significantly different growth rates under different N treatments (0, 0.2, and 2 mM). Using pairwise comparisons and weighted gene co-expression network analyses (WGCNA), we successfully constructed 16 co-expression modules. Among them, a specific module (turquoise) that substantially responded to the low N treatment was identified. Based on KEGG analysis, the relative genes that enriched in the "N metabolism" pathways were used to construct gene co-expression networks of N metabolism. Finally, a high-affinity ammonium (NH4+) transporter designated CsAMT1.2 was identified as a hub gene in the N metabolism network in tea plant roots and the gene expression could be highly induced by N resupply. The gene functional analysis revealed that CsAMT1.2 could make functional complementation of MEP1, MEP2, and MEP3 genes in 31019b yeast cells and improve NH4+ uptake rate in 31019b at low NH4+ level. Thus, CsAMT1.2 was a key gene controlling N uptake in tea plants and might play a vital role in promoting NH4+ uptake from the environment in tea roots. This study provided a useful foundation for improving the NUE in tea plantations.

Dietary Scenedesmus ovalternus improves disease resistance of overwintering gibel carp (Carassius gibelio) by alleviating toll-like receptor signaling activation.

This study was conducted to investigate the effect of dietary Scenedesmus ovalternus on the growth and disease resistance of gibel carp (Carassius gibelio) during overwintering. Gibel carp (initial body weight: 90.39 ± 0.33 g) were fed with diets containing 0% or 4% Scenedesmus ovalternus (DS0 and DS4) for 4 weeks during the early overwintering period, and then all fish were left unfed during the late overwintering period. A bacterial challenge test using Aeromonas hydrophila was subsequently conducted. The 4% Scenedesmus ovalternus diet had no effect on the growth of gibel carp (P > 0.05), but did improve the survival rate after the challenge (P ≤ 0.05). In the DS0 group, the bacterial challenge decreased the contents of complement 3 (C3), immunoglobulin M (IgM), interleukin 2 (IL2) and tumor necrosis factor α (TNFα) in fish (P < 0.05); in the DS4 group, the challenge increased total antioxidant capacity (T-AOC) and myeloperoxidase (MPO) activity but decreased IL2 and TNFα contents (P < 0.05). The activities of MPO and contents of C3, IgM and TNFα were higher in the DS4 group than that fed the DS0 diet after bacterial challenge (P < 0.05). Compared to pre challenge, the expression levels of toll like receptor 2 (TLR2), toll like receptor 3 (TLR3), toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), TIR-domain-containing adapter-inducing interferon ß (TRIF), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα), transforming growth factor ß (TGFß), interleukin 1ß (IL1ß), tumor necrosis factor α1 (TNFα1) and interleukin 10 (IL10) in the head kidney of gibel carp were induced after challenge (P < 0.05). Gibel carp fed the DS4 diet showed lower expression of TGFß in head kidney before the challenge and lower expression of TLR2, TLR3, TLR4, TIRAP, TRIF, IκBα, TNFα1, IL10 and TGFß after the challenge than that fed the DS0 diet (P < 0.05). Overall, Scenedesmus ovalternus supplement enhanced the resistances of gibel carp against A. hydrophila after overwintering via the TLR signaling pathway.

Identification of a Novel Gene Encoding the Specialized Alanine Decarboxylase in Tea (Camellia sinensis) Plants.

Theanine, a unique amino acid in Camellia sinensis, accounts for more than 50% of total free amino acids in tea and has a significant contribution to the quality of green tea. Previous research indicated that theanine is synthesized from glutamic acid (Glu) and ethylamine mainly in roots, and that theanine accumulation depends on the availability of ethylamine which is derived from alanine (Ala) decarboxylation catalyzed by alanine decarboxylase (AlaDC). However, the specific gene encoding AlaDC protein remains to be discovered in tea plants or in other species. To explore the gene of AlaDC in tea plants, the differences in theanine contents and gene expressions between pretreatment and posttreatment of long-time nitrogen starvation were analyzed in young roots of two tea cultivars. A novel gene annotated as serine decarboxylase (SDC) was noted for its expression levels, which showed high consistency with theanine content, and the expression was remarkably high in young roots under sufficient nitrogen condition. To verify its function, full-length complementary DNA (cDNA) of this candidate gene was cloned from young roots of tea seedlings, and the target protein was expressed and purified from Escherichia coli (E. coli). The enzymatic activity of the protein for Ala and Ser was measured in vitro using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). The results illustrated that the target protein could catalyze the decarboxylation of Ala despite of its high similarity with SDC from other species. Therefore, this novel gene was identified as AlaDC and named CsAlaDC. Furthermore, the gene expression levels of CsAlaDC in different tissues of tea plants were also quantified with quantitative real-time PCR (qRT-PCR). The results suggest that transcription levels of CsAlaDC in root tissues are significantly higher than those in leaf tissues. That may explain why theanine biosynthesis preferentially occurs in the roots of tea plants. The expression of the gene was upregulated when nitrogen was present, suggesting that theanine biosynthesis is regulated by nitrogen supply and closely related to nitrogen metabolism for C. sinensis. The results of this study are significant supplements to the theanine biosynthetic pathway and provide evidence for the differential accumulation of theanine between C. sinensis and other species.

Characteristics of Free Amino Acids (the Quality Chemical Components of Tea) under Spatial Heterogeneity of Different Nitrogen Forms in Tea (Camellia sinensis) Plants.

Nitrogen (N) forms are closely related to tea quality, however, little is known about the characteristics of quality chemical components in tea under the spatial heterogeneity of different N forms. In this study, a split-root system, high performance liquid chromatography (HPLC), and root analysis system (WinRHIZO) were used to investigate free amino acids (FAAs) and root length of tea plants under the spatial heterogeneity of different N forms. Uniform. (U.) ammonium (NH4⁺) (both compartments had NH4⁺), U. nitrate (NO3-) (both compartments had NO3-), Split. (Sp.) NH4⁺ (one of the compartments had NH4⁺), and Sp. NO3- (the other compartment had NO3-) were performed. The ranking of total FAAs in leaves were as follows: U. NH4⁺ > Sp. NH4⁺/Sp. NO3- > U. NO3-. The FAA characteristics of Sp. NH4⁺/Sp. NO3- were more similar to those of U. NO3-. The contents of the important FAAs (aspartic acid, glutamic acid, and theanine) that determine the quality of tea, increased significantly in U. NH4⁺. The total root length in U. NH4⁺ was higher than that in the other treatments. More serious root browning was found in U. NO3-. In conclusion, NH4⁺ improved the accumulations of FAAs in tea leaves, which might be attributed to the root development.

A coupled role for CsMYB75 and CsGSTF1 in anthocyanin hyperaccumulation in purple tea.

Cultivars of purple tea (Camellia sinensis) that accumulate anthocyanins in place of catechins are currently attracting global interest in their use as functional health beverages. RNA-seq of normal (LJ43) and purple Zijuan (ZJ) cultivars identified the transcription factor CsMYB75 and phi (F) class glutathione transferase CsGSTF1 as being associated with anthocyanin hyperaccumulation. Both genes mapped as a quantitative trait locus (QTL) to the purple bud leaf color (BLC) trait in F1 populations, with CsMYB75 promoting the expression of CsGSTF1 in transgenic tobacco (Nicotiana tabacum). Although CsMYB75 elevates the biosynthesis of both catechins and anthocyanins, only anthocyanins accumulate in purple tea, indicating selective downstream regulation. As glutathione transferases in other plants are known to act as transporters (ligandins) of flavonoids, directing them for vacuolar deposition, the role of CsGSTF1 in selective anthocyanin accumulation was investigated. In tea, anthocyanins accumulate in multiple vesicles, with the expression of CsGSTF1 correlated with BLC, but not with catechin content, in diverse germplasm. Complementation of the Arabidopsis tt19-8 mutant, which is unable to express the orthologous ligandin AtGSTF12, restored anthocyanin accumulation, but did not rescue the transparent testa phenotype, confirming that CsGSTF1 did not function in catechin accumulation. Consistent with a ligandin function, transient expression of CsGSTF1 in Nicotiana occurred in the nucleus, cytoplasm and membrane. Furthermore, RNA-Seq of the complemented mutants exposed to 2% sucrose as a stress treatment showed unexpected roles for anthocyanin accumulation in affecting the expression of genes involved in redox responses, phosphate homeostasis and the biogenesis of photosynthetic components, as compared with non-complemented plants.

Characteristics of NH4+ and NO3- fluxes in tea (Camellia sinensis) roots measured by scanning ion-selective electrode technique.

As a vital beverage crop, tea has been extensively planted in tropical and subtropical regions. Nitrogen (N) levels and forms are closely related to tea quality. Based on different N levels and forms, we studied changes in NO3- and NH4+ fluxes in tea roots utilizing scanning ion-selective electrode technique. Our results showed that under both single and mixed N forms, influx rates of NO3- were much lower than those of NH4+, suggesting a preference for NH4+ in tea. With the increase in N concentration, the influx rate of NO3- increased more than that of NH4+. The NH4+ influx rates in a solution without NO3- were much higher than those in a solution with NO3-, while the NO3- influx rates in a solution without NH4+ were much lower than those in a solution with NH4+. We concluded that (1) tea roots showed a preference for NH4+, (2) presence of NO3- had a negative effect on NH4+ influx, and (3) NH4+ had a positive effect on NO3- influx. Our findings not only may help advance hydroponic tea experiments but also may be used to develop efficient fertilization protocols for soil-grown tea in the future.

Gene expression analysis of bud and leaf color in tea.

Purple shoot tea attributing to the high anthocyanin accumulation is of great interest for its wide health benefits. To better understand potential mechanisms involved in purple buds and leaves formation in tea plants, we performed transcriptome analysis of six green or purple shoot tea individuals from a F1 population using the Illumina sequencing method. Totally 292 million RNA-Seq reads were obtained and assembled into 112,233 unigenes, with an average length of 759 bp and an N50 of 1081 bp. Moreover, totally 2193 unigenes showed significant differences in expression levels between green and purple tea samples, with 1143 up- and 1050 down-regulated in the purple teas. Further real time PCR analysis confirmed RNA-Seq results. Our study identified 28 differentially expressed transcriptional factors and A CsMYB gene was found to be highly similar to AtPAP1 in Arabidopsis. Further analysis of differentially expressed genes involved in anthocyanin biosynthesis and transportation showed that the late biosynthetic genes and genes involved in anthocyanin transportation were largely affected but the early biosynthetic genes were less or none affected. Overall, the identification of a large number of differentially expressed genes offers a global view of the potential mechanisms associated with purple buds and leaves formation, which will facilitate molecular breeding in tea plants.

Transcriptome analysis reveals self-incompatibility in the tea plant (Camellia sinensis) might be under gametophytic control.

BACKGROUND: Self-incompatibility (SI) is under genetic control and prevents inbreeding depression in angiosperms. SI mechanisms are quite complicated and still poorly understood in many plants. Tea (Camellia sinensis L.) belonging to the family of Theaceae, exhibits high levels of SI and high heterozygosity. Uncovering the molecular basis of SI of the tea plant may enhance breeding and simplify genomics research for the whole family. RESULTS: The growth of pollen tubes following selfing and crossing was observed using fluorescence microscopy. Self-pollen tubes grew slower than cross treatments from 24 h to 72 h after pollination. RNA-seq was employed to explore the molecular mechanisms of SI and to identify SI-related genes in C. sinensis. Self and cross-pollinated styles were collected at 24 h, 48 h and 72 h after pollination. Six RNA-seq libraries (SP24, SP48, SP72, CP24 CP48 and CP72; SP = self-pollinated, CP = cross-pollinated) were constructed and separately sequenced. In total, 299.327 million raw reads were generated. Following assembly, 63,762 unigenes were identified, and 27,264 (42.76 %) unigenes were annotated in five public databases: NR, KOG, KEGG, Swiss-Port and GO. To identify SI-related genes, the fragments per kb per million mapped reads (FPKM) values of each unigene were evaluated. Comparisons of CP24 vs. SP24, CP48 vs. SP48 and CP72 vs. SP72 revealed differential expression of 3,182, 3,575 and 3,709 genes, respectively. Consequently, several ubiquitin-mediated proteolysis, Ca(2+) signaling, apoptosis and defense-associated genes were obtained. The temporal expression pattern of genes following CP and SP was analyzed; 6 peroxidase, 1 polyphenol oxidase and 7 salicylic acid biosynthetic process-related genes were identified. The RNA-seq data were validated by qRT-PCR of 15 unigenes. Finally, a unigene (CL25983Contig1) with strong homology to the S-RNase was analyzed. It was mainly expressed in styles, with dramatically higher expression in self-pollinated versus cross-pollinated tissues at 24 h post-pollination. CONCLUSIONS: The present study reports the transcriptome of styles after cross- and self-pollination in tea and offers novel insights into the molecular mechanism behind SI in C. sinensis. We believe that this RNA-seq dataset will be useful for improvement in C. sinensis as well as other plants in the Theaceae family.

Transcriptome Analysis Reveals Key Flavonoid 3'-Hydroxylase and Flavonoid 3',5'-Hydroxylase Genes in Affecting the Ratio of Dihydroxylated to Trihydroxylated Catechins in Camellia sinensis.

The ratio of dihydroxylated to trihydroxylated catechins (RDTC) is an important indicator of tea quality and biochemical marker for the study of genetic diversity. It is reported to be under genetic control but the underlying mechanism is not well understood. Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key enzymes involved in the formation of dihydroxylated and trihydroxylated catechins. The transcriptome and HPLC analysis of tea samples from Longjing43 and Zhonghuang2 under control and shading treatment were performed to assess the F3'H and F3'5'H genes that might affect RDTC. A total of 74.7 million reads of mRNA seq (2×101bp) data were generated. After de novo assembly, 109,909 unigenes were obtained, and 39,982 of them were annotated using 7 public databases. Four key F3'H and F3'5'H genes (including CsF3'5'H1, CsF3'H1, CsF3'H2 and CsF3'H3) were identified to be closely correlated with RDTC. Shading treatment had little effect on RDTC, which was attributed to the stable expression of these key F3'H and F3'5'H genes. The correlation of the coexpression of four key genes and RDTC was further confirmed among 13 tea varieties by real time PCR and HPLC analysis. The coexpression of three F3'H genes and a F3'5'H gene may play a key role in affecting RDTC in Camellia sinensis. The current results may establish valuable foundation for further research about the mechanism controlling catechin composition in tea.

Transcriptome analysis of indole-3-butyric acid-induced adventitious root formation in nodal cuttings of Camellia sinensis (L.).

Tea (Camellia sinensis L.) is a popular world beverage, and propagation of tea plants chiefly depends on the formation of adventitious roots in cuttings. To better understand potential mechanisms involved in adventitious root formation, we performed transcriptome analysis of single nodal cuttings of C. sinensis treated with or without indole-3-butyric acid (IBA) using the Illumina sequencing method. Totally 42.5 million RNA-Seq reads were obtained and these were assembled into 59,931 unigenes, with an average length of 732 bp and an N50 of 1292 bp. In addition, 1091 differentially expressed unigenes were identified in the tea cuttings treated with IBA compared to controls, including 656 up- and 435 down-regulated genes. Further real time RT-PCR analysis confirmed RNA-Seq data. Functional annotation analysis showed that many genes were involved in plant hormone signal transduction, secondary metabolism, cell wall organization and glutathione metabolism, indicating potential contributions to adventitious rooting. Our study presents a global view of transcriptome profiles of tea cuttings in response to IBA treatment and provides new insights into the fundamental mechanisms associated with auxin-induced adventitious rooting. Our data will be a valuable resource for genomic research about adventitious root formation in tea cuttings, which can be used to improve rooting for difficult-to-root varieties.

Floral transcriptome sequencing for SSR marker development and linkage map construction in the tea plant (Camellia sinensis).

Despite the worldwide consumption and high economic importance of tea, the plant (Camellia sinensis) is not well studied in molecular biology. Under the few circumstances in which the plant is studied, C. sinensis flowers, which are important for reproduction and cross-breeding, receive less emphasis than investigation of its leaves or roots. Using high-throughput Illumina RNA sequencing, we analyzed a C. sinensis floral transcriptome, and 26.9 million clean reads were assembled into 75,531 unigenes averaging 402 bp. Among them, 50,792 (67.2%) unigenes were annotated with a BLAST search against the NCBI Non-Redundant (NR) database and 10,290 (16.67%) were detected that contained one or more simple sequence repeats (SSRs). From these SSR-containing sequences, 2,439 candidate SSR markers were developed and 720 were experimentally tested, validating 431 (59.9%) novel polymorphic SSR markers for C. sinensis. Then, a consensus SSR-based linkage map was constructed that covered 1,156.9 cM with 237 SSR markers distributed in 15 linkage groups. Both transcriptome information and the genetic map of C. sinensis presented here offer a valuable foundation for molecular biology investigations such as functional gene isolation, quantitative trait loci mapping, and marker-assisted selection breeding in this important species.