Identification of a BAHD Acyltransferase Gene Involved in Plant Growth and Secondary Metabolism in Tea Plants.

Plant acyl-CoA dominated acyltransferases (named BAHD) comprise a large appointed protein superfamily and play varied roles in plant secondary metabolism like synthesis of modified anthocyanins, flavonoids, volatile esters, etc. Tea (Camellia sinensis) is an important non-alcoholic medicinal and fragrancy plant synthesizing different secondary metabolites, including flavonoids. In the tea (C.A sinensis) cultivar Longjing 43 (LJ43), eight samples were performed into three groups for transcriptome analysis under three biological replications. Among the BAHD acyltransferase genes in tea cultivars, the expression of TEA031065 was highest in buds and young leaves following the RNA sequencing data, which was coincident with the tissue rich in catechins and other flavonoids. We then transformed this gene into wild-type Arabidopsis as an over-expression (OX) line 1 and line 2 in ½ MS media to verify its function. In the wild types (WT), the primary root length, number of secondary roots, and total root weight were significantly higher at 24%, 15%, and 53.92%, respectively, compared to the transgenic lines (OX1 and OX2). By contrast, the leaves displayed larger rosettes (21.58%), with higher total leaf weight (32.64%) in the transgenic lines than in the wild type (WT). This result is consistent with DCR mutant At5g23940 gene in Arabidopsis thaliana. Here, anthocyanin content in transgenic lines was also increased (21.65%) as compared to WT. According to the RNA sequencing data, a total of 22 growth regulatory genes and 31 structural genes with TFs (transcription factors) that are correlative with plant growth and anthocyanin accumulation were identified to be differentially expressed in the transgenic lines. It was found that some key genes involved in IAA (Auxin) and GA (Gibberellin) biosynthesis were downregulated in the transgenic lines, which might be correlated with the phenotype changes in roots. Moreover, the upregulation of plant growth regulation genes, such as UGT73C4 (zeatin), ARR15, GH3.5, ETR2, ERS2, APH4, and SAG113 might be responsible for massive leaf growth. In addition, transgenic lines shown high anthocyanin accumulation due to the upregulation of the (1) 3AT1 and (3) GSTF, particularly, GSTF12 genes in the flavonoid biosynthesis pathway. However, the TFs such as, CCoAMT, bHLH, WRKY, CYP, and other MYBs were also significantly upregulated in transgenic lines, which increased the content of anthocyanins in A. thaliana seedlings. In conclusion, a BAHD acyltransferase (TEA031065) was identified, which might play a vital role in tea growth and secondary metabolites regulation. This study increases our knowledge concerning the combined functionality of the tea BAHD acyltransferase gene (TEA031065).

Genome-Wide Identification and Expression Analysis of Isopentenyl transferase Family Genes during Development and Resistance to Abiotic Stresses in Tea Plant (Camellia sinensis).

The tea plant is an important economic crop and is widely cultivated. Isopentenyl transferase (IPT) is the first and rate-limiting enzyme of cytokinin (CK) signaling, which plays key roles in plant development and abiotic stress. However, the IPT gene family in tea plants has not been systematically investigated until now. The phylogenetic analyses, gene structures, and conserved domains were predicted here. The results showed that a total of 13 CsIPT members were identified from a tea plant genome database and phylogenetically classified into four groups. Furthermore, 10 CsIPT members belonged to plant ADP/ATP-IPT genes, and 3 CsIPTs were tRNA-IPT genes. There is a conserved putative ATP/GTP-binding site (P-loop motif) in all the CsIPT sequences. Based on publicly available transcriptome data as well as through RNA-seq and qRT-PCR analysis, the CsIPT genes which play key roles in the development of different tissues were identified, respectively. Furthermore, CsIPT6.2 may be involved in the response to different light treatments. CsIPT6.4 may play a key role during the dormancy and flush of the lateral buds. CsIPT5.1 may play important regulatory roles during the development of the lateral bud, leaf, and flower. CsIPT5.2 and CsIPT6.2 may both play key roles for increased resistance to cold-stress, whereas CsIPT3.2 may play a key role in improving resistance to high-temperature stress as well as drought-stress and rewatering. This study could provide a reference for further studies of CsIPT family's functions and could contribute to tea molecular breeding.

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.

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.

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.

Molecular cloning and expression analysis of ammonium transporters in tea plants (Camellia sinensis (L.) O. Kuntze) under different nitrogen treatments.

Ammonium is a major inorganic nitrogen source for tea plant growth and is mainly taken up and transported by ammonium transporters (AMTs). Here, we analyzed the NH4+ uptake kinetics of three tea cultivars, Longjing43 (LJ43), Zhongcha108 (ZC108) and Zhongcha302 (ZC302). The results revealed that ZC302 had a higher NH4+ uptake efficiency than the other two cultivars. The full CDS sequences of three Camellia sinensis ammonium transporter (CsAMT) genes, i.e., CsAMT1.1, CsAMT1.2 and CsAMT3.1, were cloned. Analysis of tissue-specific expression showed that CsAMT1.2 followed a root-specific expression pattern, while transcripts of CsAMT1.1 and CsAMT3.1 were mainly accumulated in leaves. The temporal course experiment on gene expression levels showed CsAMT1.1 and CsAMT3.1 followed a reciprocal expression pattern in leaves as CsAMT1.1 was up-regulated by a short time (2 h, 6 h) nitrogen (N) supply both in the leaves and buds of LJ43 and ZC108; and the expression of CsAMT3.1 in leaves was increased by a long time (72 h) N supply, particularly in ZC302. Therefore, we inferred that CsAMT1.1 and CsAMT3.1 might play important roles in photorespiratory ammonium metabolism. The expression of CsAMT1.2 was extremely high in roots and can be greatly induced by N over a short period of time, especially in ZC302; thus, we concluded CsAMT1.2 might play an important role in ammonium uptake from soils in tea plant roots.

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.