Plant Growth Regul DOI 10.1007/s10725-017-0321-0
ORIGINAL PAPER
Identification and characterization of cationic amino acid transporters (CATs) in tea plant (Camellia sinensis) Lin Feng1 · Tianyuan Yang1 · Zhaoliang Zhang1 · Fangdong Li1 · Qi Chen1 · Jun Sun1 · Chengying Shi1 · Weiwei Deng1 · Mingmin Tao1 · Yuling Tai1 · Hua Yang1 · Qiong Cao1 · Xiaochun Wan1
Received: 8 June 2017 / Accepted: 7 September 2017 © Springer Science+Business Media B.V. 2017
Abstract Amino acids are constituents of proteins, precursors of many secondary metabolites and nitrogen carriers in plants. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. However, the amino acid transport in tea plant is rarely reported. In this study, six cationic amino acid transporter (CAT) family genes were cloned. Phylogenetic analysis categorized these CsCATs into four subgroups. These CsCATs all contain the 12–14 transmembrane domains and the conserved CAT motifs. Their expression was tissue-specific, with higher expression levels in root and stem and correlated to the abundances of key free amino acids such as Theanine. Some CsCATs expression responded to some abiotic stress conditions and to the exogenous application of theanine (Thea), glutamine or ethylamine hydrochloride, an ethylamine precursor for Thea biosynthesis. Our results indicated that the CsCATs expression is regulated by amino acid contents and is sensitive to abiotic stresses. These findings shed light on the mechanism of amino acid transport in tea plants. Keywords Tea plant (Camellia sinensis L.) · Cationic amino acid transporter · Theanine · Abiotic stress · Amino acid homeostasis
Electronic supplementary material The online version of this article (doi:10.1007/s10725-017-0321-0) contains supplementary material, which is available to authorized users. * Xiaochun Wan
[email protected] 1
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, China
Introduction Camellia sinensis, also commonly known as tea plants, is grown for the production of beverage tea, which is popular worldwide. Tea consumption is associated with many physiological benefits to human health (Cabrera et al. 2006). Tea is made from shoot tips, and tender leaves of Camellia sinensis and its characteristic umami flavor is primarily dependent on the abundance of free amino acid in the beverage (Sorrequieta et al. 2010). Amino acids are essential for plant growth and development, not only as constituents of proteins, but also as precursors of many secondary metabolites and nitrogen carriers in plants. Transport across intracellular membranes and translocation of amino acids within the plant mediated by membrane amino acid transporters is critical to leaf development (Boggio et al. 2000) and thus significantly contributes to tea flavor. Tea plants preferably uptake ammonium nitrogen and assimilate into amino acids in roots (Ruan et al. 2016; Oh et al. 2008). Then, amino acids such as glutamine (Gln), theanine (Thea), and glutamate (Glu) are translocated from roots to the shoots via the xylem transpiration stream (Oh et al. 2008). However, the molecular mechanism controlling amino acid transport in tea plant is largely unknown. Amino acid transporters (AATs) are membrane-localized proteins that mediate the intracellular and intercellular movement of amino acids, generally from biosynthetic organs (source tissues) to utilization organs (sink tissues) via vascular tissues (xylem and phloem) (Schmidt et al. 2007; Hirner et al. 2006; Svennerstam et al. 2008, 2011). The tempo-spatial expression and function of AATs in plants coordinate the movement of amino acids and plant metabolism throughout plants (Okumoto et al. 2002). Cationic amino acid transporters (CATs) are a part of the AATs family in plants, and they play an important role in
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amino acid transport and nitrogen homeostasis. All CAT proteins in animal cells mediate N a+-independent transport of cationic L-amino acids (Hatzoglou et al. 2004). In Arabidopsis, nine CAT transporters (AtCAT1-9) have been identified so far. These CATs contain 14 conserved transmembrane (TM) domains. They are expressed in root, stem, leaf, flower, and siliques (Frommer et al. 1995, Su et al. 2004; Hammes et al. 2006; Yang et al. 2010, 2013a, 2014a, 2014b, 2015). All these transporters exhibit distinctive subcellular locations and tempo-spatial expression patterns (Yang et al. 2014a, 2015; Hammes et al. 2006, Su et al. 2004). In tomatoes, SICAT9 is shown to be a tonoplast Glu/Asp/GABA exchanger that strongly affects the accumulation of these amino acids during fruit ripening (Snowden et al. 2015). Thea is a unique non-protein amino acid in tea plants. The main synthesis of Thea in roots is carried out by theanine synthetase using Glu and ethylamine as substrates (Ashihara 2015) and then transported to other parts of the plant. Thea has a similar chemical structure with Gln and Glu, which also undergo long-distance transport in tea plants (Oh et al. 2008). Given the structural similarities of these three amino acids, it is possible that they may undergo the same transport system in tea plants. In Arabidopsis, CAT members AtCAT1, AtCAT8, and AtCAT9 are the major transporters for Gln and Glu. Moreover, AtCAT1 and AtCAT3 expression is mainly observed in vascular tissues of roots, leaves and developing siliques, and AtCAT8 also has a strong expression in vasculature-enriched petiole and vein (Frommer et al. 1995, Su et al. 2004; Yang et al. 2010, 2015; Snowden et al. 2015). Furthermore, stress induced responses in tea plants can lead to changes in the abundance of free amino acids (Wang et al. 2016). Numerous studies have found that the expression of amino acid transporter genes, such as AtCAT1 in Arabidopsis and SlCAT2 in tomato, are regulated by developmental and environmental signals, light, osmotic stress and pathogenic infection (Liu and Bush 2006; Yang et al. 2010, 2013). However, the expression of CsCATs under different stress conditions is not well known in tea plants. In the present study, the potential role of CAT transporters in the long-distance transport of amino acids in tea plants is studied. Six CsCATs genes in tea plants were identified, and their phylogenetic relationship with CATs of other plant species was analyzed. The transmembrane (TM) domains and conserved CAT motifs of were also investigated. Tempo-spatial expression patterns of CsCATs in tea plants were also examined under exogenous amino acids and abiotic stresses. Our data indicated that the CsCATs expression is regulated by amino acid contents and sensitive to abiotic stresses. These findings shed light on the mechanism of amino acid transport in tea plants.
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Materials and methods Plant materials and growth conditions Two-year-old tea plants propagated using cuttings (Camellia sinensis L. cv. “Shuchazao”) were obtained from Dechang tea plantation (Shucheng, China) and used for the experiments in this study. These plants were grown in pots containing soil and placed in a growth chamber with 23 ± 0.5 °C, a photoperiod of 12 h and relative humidity 70%. In shade treatment, tea plants were covered by black shading nets (20 ± 5% light transmittance) for 3 days. In cold treatment, tea plants were put at 4 °C for 3 days. For salt, Abscisic acid (ABA), and polyethylene glycol (PEG) treatments, tea seedlings were grown in light and watered four times every day. They were respectively treated with NaCl (300 mM), 100 µM ABA and 10% PEG-6000, as following previously published protocol (Cao et al. 2015). The second and third mature leaves below the top bud were collected after 3 days treatment, washed with distilled water, and immediately frozen in liquid nitrogen and stored at −80 °C. For the amino acid and ethylamine hydrochloride (EtHCl) treatments, tea shoot cuttings were hydroponically cultured in nutrient solution. Nutrient solution was prepared as previously described (Oh et al. 2008) and was changed every 3 days until treatment started. Amino acid Thea, Gln, Glu or EtHCl was added into the nutrient solution to a final concentration of 5 mM. Fibrous roots and the second and third mature leaves below the top bud were collected from ten plants at 4 and 21 h post treatment. Samples collected at start time point of treatment (0 h) were used as controls (CK). All samples were frozen in liquid nitrogen and stored at −80 °C. For all treatments, three biological replicates were performed and analyzed. CAT gene search and phylogenetic analysis (1) Database screening and sequence collection Cationic amino acid transporter’ was used as an gene annotation term to search the databases for Arabidopsis thaliana (http://www.arabidopsis.org/), Vitis vinifera (http:// www.Genoscope.cns.fr/spip/Vitis-vinifera-whole-genome. html), Theobroma cacao (http://www.Cacaogenomedb. org/), Actinidia spp. actinidia kiwifruit (http://bioinfo.bti. cornell.edu/cgi-bin/kiwi/home.cgi), Populus tremula (http:// genome.jgi-psf.org/Poptr1-1/ Poptr1-1.home.html), and Coffea canephora (http://coffee-genome.org/). All selected sequences were blasted against NCBI database (https://blast. ncbi.nlm.nih.gov/Blast.cgi) and each candidate sequence with CAT domain was confirmed using the Pfam (http:// pfam.xfam.org/). To find putative CAT members in the tea genome (unpublished), ‘cationic amino acid transporter’ was
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also used as a search term, then CAT domain (TIGR00906 and pfam13520) obtained from the Pfam website (http:// pfam.xfam.org/) was employed as a query to identify all possible CAT genes in tea. After removing the redundant sequences, the remaining protein sequences were select as the candidate sequences after BLASTA confirmation against NCBI database. (2) Phylogenetic, conserved domain and motif analysis Phylogenetic analysis was performed with the MEGA4 program (Kumar et al. 2004) using the neighbour-joining method (Saitou and Nei 1987) and bootstrapping with 1000 replicates. The open reading frame (ORF) and deduced amino acids of CsCAT genes were predicted using the Editseq software then confirmed by BLAST in NCBI database (http://www. ncbi.nlm.gov/BLAST). Length of protein sequence, molecular weight (MW) and isoelectric point (pI) were predicted by Compute/Mw tool (http://web.expasy.org/). TM regions were predicted using TMHMM Server v.2.0 (Krogh et al. 2001). The software Multiple Em for Motif Elicitation (MEME) (http://meme-suite.org/tools/meme) was used for conserved domains search with the following parameters: the width of a motif was between 5 aa and 50 aa and the number of motif was 20. Conserved amino acid sequences in CsCAT family were analyzed using DNAMAN. Cloning of CsCATs genes Based on above-described analyses, a total of 15 contigs were identified as CATs genes in tea. Subsequently, 6 contigs were used for designing specific primers for cDNA cloning. Then 5′ RACE and 3′ RACE PCR reactions were performed using SMART RACE cDNA amplification kit (Clonetech, CA, USA) following the manufacturer’s instruction to get the full length of cDNAs. The amplified and purified PCR products were cloned into the pMD18-T vector (Takara, China) and then sequenced. Total RNA extraction and real‑time quantitative RT‑PCR analysis For gene expression analysis, total RNA was extracted from the roots or leaves of tea plants subjected to different treatments using the standard TRIzol (Invitrogen) protocol. First-strand cDNAs were synthesized from 0.5 µg total RNA from each sample using Promega Reverse Transcription System kit. Real-time quantitative RT-PCR reaction was performed using 1 µl cDNA(100 ng/µl) product and 0.4 µM of each primer in a 20 µl reaction volume with SYBR Premix Ex TaqTM II (Perfect Real Time; Promega). GAPDH gene (Song et al. 2016) was used as an internal
control. Gene specific primers (P-1 to P-9, Table S1) were designed using Primer Premier 5 software. Three replicates of each PCR reaction were conducted in a Bio-Rad C1000 Thermal Cycler (Bio-Rad, USA) using the following program: 94 °C for 3 min, followed by an optimum of 29 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. Data were analyzed according to the threshold cycle (Ct), and relative changes in gene expression were quantified using the 2 −∆∆Ct method (Livak and Schmittgen 2001) and 2−∆Ct (Schmittgen and Zakrajsek 2000). Statistical and multivariate analysis Data were expressed as means ± SE from three independent biological replicates for all chemical assays. Statistical analysis was performed by one-way analysis of variance (ANOVA) using the software SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The HPLC data matrix was subjected to multivariate analysis with SIMCA-P 11.5 software (Umetrics AB, Umea, Sweden). Supervised partial least-squares discriminant analysis (PLS-DA), a more advanced multivariate method for understanding the inter-class variation, was performed to separate different variations among samples in a clearer and more straightforward way (Trygg et al. 2007). A range of metabolites was selected to represent the variables of importance in the projection (VIP) based on the PLS-DA analysis (Ku et al. 2010).
Results Identification of six CsCATs in tea plants Bioinformatic analyses were performed to look for CAT genes in tea plants, and 15 putative CsCAT genes were identified. To further evaluate these putative CAT genes, the presence of cationic amino acid transport permease 2A0303 domain (TIGR00906) and amino acid permease AA_permease_2 domain (pfam13520) was examined. Nine candidate genes did not contain complete domain sequences and were not further pursued in this study. The remaining six candidate genes were found to contain the complete sequences of the two domains. RACE PCR method was then used to obtain the full-length ORF of the six CsCATs. Basic information, including the ORF length, the number of amino acids, pI, MW and number of TM, were predicted and listed in Table 1. Based on the amino acid similarities between CsCATs and AtCATs, these genes were named as CsCAT1, CsCAT2, CsCAT5, CsCAT6, CsCAT8 and CsCAT9 (Table 2).
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Table 1 Six tea CsCATs examined in this study
Plant Growth Regul Gene name Accession number ORF (bp) AA
Conserved domain
PI
MW (KD) TMs
CsCAT1 CsCAT2 CsCAT5 CsCAT6 CsCAT8 CsCAT9
2A0303, AA_permease_2 2A0303, AA_permease_2 2A0303, AA_permease_2 2A0303, AA_permease_2 2A0303, AA_permease_2 2A0303, AA_permease_2
8.17 6.5 8.13 8.34 9.37 7.66
65.6 68.2 64.1 63.1 65.3 61.3
KY709681 KY709679 KY709680 KY709682 KY709684 KY709683
1824 1932 1767 1758 1779 1719
607 643 588 485 592 569
14 12 14 14 13 12
ORF open reading frame, AA the numbers of amino acid residues, pI theoretical isoelectric point, MW molecule weight, TMs transmembrane domains
Table 2 The similarity of amino acid sequences of CsCATs with Arabidopsis homologues
Identity (%)
CsCAT1
CsCAT2
CsCAT5
CsCAT6
CsCAT8
CsCAT9
CsCAT1 CsCAT2 CsCAT5 CsCAT6 CsCAT8 CsCAT9 AtCAT1 AtCAT2 AtCAT3 AtCAT4 AtCAT5 AtCAT6 AtCAT8 AtCAT9
1 20.8 50.2 30.4 47.9 24 63.5 23.9 23 23.2 58.4 36.1 48.6 22.7
1 19.4 19.7 20.3 24.64 18.8 48.7 46.9 44.3 20 21 19.1 23.8
1 31.8 52.7 23.6 51.7 23.5 24.2 21.8 70 32.7 55.7 23.5
1 35.2 26.4 36.1 23.9 23 23.2 58.4 63.5 48.6 22.7
1 27 24 26.7 28.3 25.7 26 25.5 71.4 26.4
1 48.6 19.7 22 22 61.2 35.4 24 72
Phylogenetic analysis of CsCATs To explore the phylogenetic relationship between CsCATs and CATs in Arabidopsis, V. vinifera, C. canephora, T. cacao, P. tremula and Actinidia spp. actinidia kiwifruit, a phylogenetic tree was built based on their amino acid sequences. The CATs were classified into four subgroups (Fig. 1). Subgroup 1 contains the members of CAT1, CAT5, and CAT8, whereas subgroup 2 includes CAT6 and CAT7. Moreover,, subgroup 3 includes CAT2, CAT3, and CAT4, whereas subgroup 4 includes only CAT9. The subgroups of CsCATs were consistent with CATs in other species. These results indicated that CsCATs are evolutionarily conserved. These results also showed that the number of CAT genes varies across species. There are 9, 7, 5, 7, 6, 6, and 6 members in Arabidopsis, V. vinifera, C. canephora, T. cacao, P. tremula, Actinidia spp.actinidia kiwifruit and tea plant, respectively. CAT5, CAT6, CAT8 and CAT9 orthologs were found in all species. In contrast, CAT7 orthologs were only found in Arabidopsis. Orthologs of CAT3 were found only in Arabidopsis and P. tremula. Meanwhile orthologs of CAT4 were found in Arabidopsis,
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T. cacao and P. tremula. The absence of CAT3, 4 and 7 in tea plants may suggest that specific environmental adaptation could have resulted in the loss of these CsCATs. Conserved CAT motifs and TM domains in CsCATs Twenty conserved motifs of CATs from the seven species were extracted by the MEME program based on protein sequences (Fig. 2). We found that the conserved motifs 1, 2, 3, 4, 6 and 7 were widely distributed among all CsCAT members (Fig. 3). In contrast, other motifs were more specific. For example, motif 20 was only found in CsCAT6. Motifs 5, 11 and 13 were specific to CsCAT1, CsCAT5, CsCAT8, while motif 16 and 18 were specific to CsCAT2 and CsCAT9 (Fig. S1). The TM domains of CsCATs were also predicted using the TMHMM server. We found that the number of TM domains in CsCATs varied from 12 to 14 (Table 1). The length and amino acid composition of these domains were conserved in these CsCATs (Fig. 3). These findings further demonstrated that CsCATs are conserved in protein sequence and structure.
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Fig. 1 Phylogenetic relationship between CsCATs and CATs in Arabidopsis (At), V. vinifera (Vv), C. canephora (Cc), T. cacao (Tc), P. tremula (Pt) and Actinidia spp.actinidia kiwifruit (Ac). The tree was generated using MEGA4.0 with the NJ method. Bootstrap values are indicated by numbers at branches. Red diamonds indicate tea genes
Tempo‑spatial expression patterns of CsCATs and the abundances of key amino acids in tea plants Tissue-specific gene expression can provide important information of gene function. Therefore, CsCATs expression was examined in various tissues, including root, stem, leaf and flower (Fig. 4a). Gene-specific primers used in the expression analysis were showed in Table S1. CsCAT9 highly expressed in all the tested tissues and the rest of CsCATs was more specifically expressed in root and stem (Fig. 4b). Moreover, higher levels of Gln, Glu and Thea were found in roots and stems than in leaves and flowers (Fig. 4c). These results indicate the positive correlation between the abundance of amino acids and expression of CsCATs. Distinct expression patterns of CsCATs in responding to exogenous addition of Thea, Gln and EtHCl Correlation of CsCATs expression with the abundances of amino acids exogenously applied or endogenously present in tea plants was further examined. Leaf abundances of Gln and Glu were significantly reduced in the plants exogenously
applied with Thea, but not significantly affected by Gln and EtHCl applications (Fig. 5a, b). However, Thea content in leaves were increased due to the application of Gln, EtHCl, or Thea (Fig. 5c). Gln application resulted in the reduction of Glu content in roots at 21 h after (Fig. 5d) and reduction of Gln in roots was also noted due to the application of Thea, Glu and EtHCl (Fig. 5e). In contrast, Thea content in roots was not affected by exogenous application of any of the three compounds (Fig. 5f). Exogenous addition of the compounds exhibited more significant effects on the changes in the abundances of Thea, Gln and Glu in leaves than in roots (Fig. 5). Expression levels of different CsCATs in leaves and roots determined by qRT-PCR varied after the exogenous addition of Thea, Gln or EtHCl. All the exogenous addition of the tested compound resulted in reduction of the expression level of all CsCAT genes in different extents shortly (4 h) after the treatment, except for CsCAT8, which was enhanced (Fig. 6). However, as treatment proceeded for 21 h, the expression of CsCAT1, -2, -6, and -9 was increased differentially, dependent on specific compound addition. In leaves CsCAT1, -6, and -9 reached their highest levels when Gln was supplied. While CsCAT2 and -8 peaked when Thea and EtHCl were applied, respectively. In roots the expression of CsCAT1, -2, -5 and -9 was similar to their expression patterns in leaves. But the expression of CsCAT6 in roots was down-regulated through the experimental observation period no matter what compound was applied (Fig. 6k). Moreover, expression of CsCAT8 in roots was enhanced slightly, not as significantly as in leaves (Fig. 6f, m). CsCATs expression was responsive to abiotic stresses It has also been reported that the expression of amino acid transporters is regulated by abiotic stresses (Tegeder 2014). To explore the roles of the CsCATs under different abiotic stresses, we treated tea plants with cold, drought and salt stresses and abiotic stress-related phytohormone ABA for 3 days. Partial least-squares discriminant analysis (PLS) was first performed on the metabolite profiles to examine amino acid profile changes under different treatments. The PLS analysis score plot showed a clear separation between the non-treated control (CK), shade, cold PEG and salt treatment groups (Fig. 7a), demonstrating that treated plants had metabolic profiles distinct from untreated tea plants. A list of top metabolites (VIP) was generated by PLS analysis, illustrating that Thea (VIP = 1.44 > 1) and Glu (VIP = 1.27 > 1) exhibited large contribution to the cluster formation within the amino acid metabolite profiles (Table S2). The abundance of Thea, Glu and Gln were determined by HPLC, which revealed that Glu and Gln contents were increased in tea leaves by all these stresses treatments (Fig. 7b). Thea
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Fig. 2 Conserved motifs in the CATs. The MEME program was used to search the motifs, with the width of the motifs ranging from 6 to 50 and a site distribution of zero or one occurrence per sequence. The height of each letter represents the specific amino acid conservation in each motif
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Fig. 3 Multiple sequence alignment and TM domains of CsCATs. Identical (100%), conservative (75–99%), and blocks (50–74%) of similar amino acid residues are shaded in deep blue, cherry red, and
light blue, respectively. The red line represents the position of the TM domains. The conserved motifs 1, 2, 3, 4, 6, and 7 are marked (in order) by black lines
content was also increased by shading, cold and salt treatments (Fig. 7b), which is consistent with previous reports (Ku et al. 2010; Deng et al. 2013; Taochy et al. 2015; Wang et al. 2016). Lastly, the expression of CsCATs was quantified by qRT-PCR analysis (Fig. 7c). The expression of all CsCATs, except CsCAT8, was downregulated by shading, indicating an inducing effect of light on CsCATs expression. CsCAT2 and 5 were significantly upregulated, whereas CsCAT8 and 9 were downregulated, by cold, drought/PEG and salt treatments. Similarly, CsCAT2 was upregulated, while CsCAT8 was downregulated, by ABA treatment (Fig. 7c). These results indicated that CsCATs could respond to abiotic stresses differentially and may be involved in abiotic stress
response by mediating intercellular, intracellular and longdistance amino acid transport.
Discussion CsCATs are evolutionarily conserved CsCATs are important for the amino acid transport in tea plants. However, the molecular mechanism of CsCATs in tea plants remains unclear. In this study, a total of six putative CsCATs genes were identified in Camellia sinensis. Genetic information and transmembrane domains of these CsCATs were predicted using various web
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Fig. 4 The contents of key amino acids and CsCATs expression in different tissues of tea plants. a The tissue samples of tea plant used for tissue-specific CsCATs expression analysis, b Thea, Gln and Glu
contents determined by HPLC, c Relative expression of CsCATs determined by quantitative RT-PCR. Relative expression levels refer to fold differences in CsCATs transcripts use 2−∆ct method
Fig. 5 Thea, Gln and Glu content in the leave (a–c) and roots (d–f) of the tea plants exogenously applied with 5 mM Thea, 5 mM Gln or 5 mM EtHCl for 4 or 21 h. Data are means ± SE of three inde-
pendent replicates. Asterisks represent significant difference between treated one and control using one way ANOVA analysis, *P < 0.05, **P < 0.01
servers. These six CsCATs all contain the cationic amino acid transport permease 2A0303 domain (TIGR00906) and the amino acid permease AA_permease_2 domain
(pfam13520). Characteristic 12–14 TM domains and conserved CAT motifs were also predicted (Fig. 3). Consistent with other reports, CsCATs were classified into
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Fig. 6 Relative expression of CsCATs in tea leave (a–f) and root (h– m) treated with 5 mM Thea, 5 mM Gln and 5 mM EtHCl after 4 or 21 h. Relative expression levels referred to fold differences in CsCATs transcripts compared with the level in tea plants without any treat-
ment, using 2 −∆∆ct method. Data are means ± SE of three independent replicates. Asterisks represent significant difference between treated one and non-treated control by one way ANOVA analysis, *P < 0.05, **P < 0.01
four subgroups, similar to CATs in Arabidopsis, grape, coffee, cocoa, poplar and kiwifruit (Su et al. 2004; Wu et al. 2015; Ma et al. 2016) (Fig. 1). Subgroup 1 consists of homologs of AtCAT1, 5 and 8; Subgroup 2 consists homologs of AtCAT6 and 7; Subgroup 3 consists homologs of AtCAT2, 3 and 4; Subgroup 4 consists homolog of AtCAT9. However, the total number of CATs differs in Arabidopsis, V. vinifera, C. canephora, T. cacao, Actinidia spp.actinidia kiwifruit, P. tremula, Glycine max and Oryza sativa L. (Zhao et al. 2012; Wu et al. 2015; Ma et al. 2016; Cheng et al. 2016). This suggests that different CATs may be involved with plant’s adaptation to the different environment.
Tissue‑specificity of CsCATs expression Amino acid transporters play many essential roles in the growth and development of plants (Liu and Bush 2006). Functional characterization of CATs is mainly performed in Arabidopsis. Tissue specific expression of AtCATs in root, stem, leaf, and flower has provided important information on their gene regulation (Su et al. 2004). AtCAT2 and AtCAT9 are highly and ubiquitously expressed in these tissues. In tea plant, the expression of CsCAT9 is similar with that of AtCAT9. Unlike AtCAT2, CsCAT2 is mainly expressed in the stem of tea plants. AtCAT1 is highly expressed in leaf and flower and expressed at lower level in root and stem (Su et al.
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Fig. 7 Partial least-squares discriminant analysis (PLS), the contents of key amino acids and CsCATs expression in leaves of tea plants treated by indicated stresses or ABA. a PLS score plot derived from the integrated HPLC data sets of untreated and shade, ABA, Cold, PEG and Salt treatment in tea plants. Data were derived from three independent biological replicates; b Thea, Gln and Glu contents in leaves determined by HPLC. Data are means ± SE of three independ-
ent replicates; c relative expression of CsCATs in leaves. Relative expression levels referred to fold differences in CsCATs transcripts compared with the level in tea plants without any treatment, using 2−∆∆ct method. Data are presented as mean ± SE with three independent replicates. Asterisks represent significant difference between treated ones and non-treated control by one way ANOVA analysis, *P < 0.05, **P < 0.01
2004). In contrast, CsCAT1 is expressed at a much higher level in root and stem than in leaf and flower (Fig. 4b). This discrepancy between the expression of CsCATs and their homologs in Arabidopsis could be explained by the fact that different amino acids are transported in both plants. In addition, the main tissue assimilating nitrogen also varies in different species. This assumption is consistent with the previous observation that nitrogen absorbed by tea plant was mainly assimilated into Gln and Thea in roots (Oh et al. 2008). CsCAT5, CsCAT8, and CsCAT1 are categorized into subgroup I, and their pattern of gene expression is similar to one another. These results suggest that subgroup I CsCATs may mainly function in amino acid transport from root to shoot. CsCAT2 may play a role in amino acid transport in shoots; CsCAT9 probably mediates intracellular transport of these amino acids, considering the CsCAT9 expression pattern and the vesicle localization of AtCAT9 (Yang et al. 2015).
CsCATs may be involved in amino acid homeostasis in tea plants
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A previous study found that AtCAT9 in Arabidopsis has a (direct or indirect) effect on cellular amino acid homeostasis, which may be most relevant in the leaves after nitrogen starvation (Yang et al. 2015). Induction of AtAAP1, AtAAP2 and AtAAP6 expression by ammonium, Glu and Gln has also been observed in Arabidopsis (Guo 2004). The repression of VvAAP1 by Glu and cysteine was demonstrated in V. vinifera (Miranda et al. 2001). Our study found that CsCATs can be induced or repressed by Thea, Gln, and EtHCl treatments (Fig. 6). These treatments also affected the level of Gln, Thea, and Glu in leaves and roots (Fig. 5). Moreover, greater effects on CsCATs expression and Gln, Thea and Glu contents were observed in leaves than roots. This observation is consistent with the previous report that nitrate and ammonium had a greater effect
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on gene expression in shoots than in roots in Arabidopsis (Liu and Bush 2006). Promoter analysis has identified two cis elements that could be related to nitrogen regulation of CATs expression: GATA element for NIT-2 recognition (Fu and Marzluf 1987) and an AT-rich element (Hwang et al. 1997). Nitrogen responsive GCN4/RY- like element has also been identified in the promoter regions of AtCAT1, AtCAT5, AtLHT1, AtLHT2, AtAAP1, AtAAP3 and AtAAP4 (Bǎumlein et al. 1992). We speculate that CsCAT promoters may also contain GCN4/RY-like, GATA or AT-rich element for nitrogen induced expression. Additional cloning of CsCATs promoters is needed to examine the potential existence of these cis elements. CsCATs participate in response to abiotic stresses in tea plants Besides their roles in plant growth, CATs also play a significant role in cellular resistance to abiotic and biotic stresses (Wipf et al. 2002; Tegeder 2012; Tegeder et al. 1999; Yang et al. 2014b). Our study has found that CsCATs can be induced by cold, PEG, salt and ABA (Fig. 7c), which is consistent with the stress induction of AtProT2, AtAAP4 and AtAAP6 in Arabidopsis (Rentsch et al. 1996), HvProT in Hordeum vulgare L. (Ueda et al. 2001) and McAAT1 in Mesembryanthemum crystallinum (Popova et al. 2003). Our study has shown that the level of Thea, Gln, and Glu in tea leaves were increased under cold, PEG, salt and ABA treatments (Fig. 7b). This finding is consistent with previous reports (Ku et al. 2010; Deng et al. 2013). The production and accumulation of free amino acids, especially Thea and Glu during drought, salt, and water stress, could be interpreted as an adaptive response in tea plants. Gln carrying nitrogen molecules undergo long-distance transport and has been linked to salt (Amonkar and Karmarkar 1995), cold (Khan et al. 2004; Jia et al. 2015), drought (Vance and Zaerr 1990), and ABA stress (Khan et al. 2004; Shinozaki and Yamaguchi-Shinozaki 2000). As a unique amino acid in tea plants, Thea could be the storage form of nitrogen and also implicated in cellular response to abiotic stresses (Ku et al. 2010; Deng et al. 2013). Thus, the increased level of Thea, Gln, and Glu in leaves suggests that these amino acids contribute to abiotic stress resistance in tea plants. Alternatively, the increase of amino acid under stress conditions could be the result of reduced utilization due to lower growth rate and increased protein degradation under stress conditions. It is interesting to note that in CsCATs subgroup 1, CsCAT1 did not respond to cold, drought, salt, and ABA treatments; CsCAT5 expression was greatly induced by cold, drought and salt treatments; whereas CsCAT8 expression was downregulated by these treatments. Moreover, subgroup 3 member CsCAT2 showed similar expression pattern as
CsCAT5, and subgroup 4 member CsCAT9 showed similar expression pattern as CsCAT8 in response to these abiotic stresses. In AtCATs promoters, drought and ABA responsive elements have previously been identified (Liu and Bush 2006). These findings suggest that potential stress-responsive cis-elements may exist in the promoter regions of these genes. In this study, Six CsCATs were cloned, and CsCATs sequence and structure were analyzed. CsCATs expression was found to be tissue-specific, and inducible via amino acid levels and abiotic stresses. These results suggest that CsCATs expression is regulated by amino acid levels and abiotic stresses. Overall, this study has provided critical findings for further characterization of CsCATs functions and the mechanism of amino acid long-distance transport in tea plants. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant Number 11008389), and Graduate Innovation Fund of Anhui Agriculture University (D21) to LF, and the “Twelfth Five-Year” National Key Basic Research and Development Project (973) in China (11000206) to XW. Author Contributions XW and LF conceived this project and designed the experiments. LF performed the experiments. All authors contributed to data analysis. XW and LF wrote the manuscript with input from other authors. All authors read and approved the manuscript. Compliance with ethical standards Conflict of interest The authors declare no conflict of financial interest.
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