A Sucrose-Induced MYB (SIMYB) Transcription Factor Promoting

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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A Sucrose-Induced MYB (SIMYB) Transcription Factor Promoting Proanthocyanidin Accumulation in the Tea Plant (Camellia sinensis) Peiqiang Wang,†,§ Guoliang Ma,† Lingjie Zhang,‡ Yan Li,‡ Zhouping Fu,† Xinyi Kan,† Yahui Han,†,§ Haiyan Wang,‡ Xiaolan Jiang,† Yajun Liu,‡ Liping Gao,*,‡ and Tao Xia*,† †

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui 230036, China School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, China § College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China Downloaded via UNIV OF WINNIPEG on January 29, 2019 at 12:47:45 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: Proanthocyanidins (PAs, also called condensed tannins), are an important class of secondary metabolites and exist widely in plants. Tea (Camellia sinensis) is rich in PAs and their precursors, (−)-epicatechin (EC) and (+)-catechin (C). The biosynthesis of PAs is constantly regulated by many different MBW complexes, consisting of MYB transcription factors (TFs), basic-helix−loop−helix (bHLH) TFs, and WD-repeat (WDR) proteins. These regulatory factors can be environmentally affected, such as by biotic and abiotic stresses. In this study, we revalidated the effect of sucrose treatment on tea branches, and a sucrose-induced MYB (SIMYB) TF was screened and studied. Phylogenetic analysis indicted that this SIMYB TF belonged to MYB subgroup 5, named CsMYB5b. Heterologous expression of CsMYB5b in tobacco strongly induced PA accumulation, through up-regulating the key target genes LAR or ANRs. In addition, CsMYB5b restored PA production in the seed coat of A. thaliana tt2 mutant and rescued its phenotype. Yeast two-hybrid assay demonstrated CsMYB5b can interact directly with CsTT8 (an AtTT8 ortholog) and CsWD40 protein. Linking to the expression profiling of CsMYB5b and the PA accumulation pattern in tea plants suggest that the CsMYB5b acts as an important switch for the synthesis of monomeric catechins and PAs. Therefore, these data provide insight into the regulatory mechanisms controlling the biosynthesis of PAs. KEYWORDS: sucrose-induced MYB, proanthocyanidins, monomeric catechins, Camellia sinensis

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INTRODUCTION PAs, including oligomers and polymers, which are produced from the condensation of flavan-3-ol units, are a large class of plant secondary metabolites ubiquitously present in many plants.1−3 The function of PAs in plants is mainly associated with the ability to protect plants against attack by microbes and pathogens and bind metal ions, thereby playing an important role in promoting plants to adapt to harsh environments.4,5 PAs also have been reported to be beneficial for human and animal health due to its antioxidant activity.6,7 And, flavan-3ols and PAs greatly contribute to the astringency of wine, tea, and fruits, such as persimmon (Diospyros kaki).8,9 PAs are the end products of the flavonoid biosynthetic pathway, in which the precursors of flavonols and anthocyanins are also produced (Figure 1).10,11 The important structural genes dihydrof lavonol 4-reductase (DFR), anthocyanidin reductase (ANR), and leucoanthocyanidin reductase (LAR) involved in PA biosynthesis have been reported in many species.12−14 In A. thaliana (Arabidopsis thaliana), PAs contains only epicatechin (EC)-initiating units and no detectable catechin (C)-initiating unit; BANYULS (ANRs) had recently been shown to catalyze the conversion of anthocyanidin to EC, while a readily recognizable LAR orthologus gene was not present.15 With respect to the research of LARs, Tanner first reported the function of LAR purified from a kind of PA-rich plant Desmodium uncinatum, of which the recombinant DuLAR could catalyze the leucoanthocyanidins to produce afzelechin (AFZ), C, and gallocatechin (GC) in vitro.16 But, Pang and © XXXX American Chemical Society

Figure 1. A simplified flavonoid metabolic pathway in plants.

Received: November 10, 2018 Revised: January 15, 2019 Accepted: January 15, 2019

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DOI: 10.1021/acs.jafc.8b06207 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

photoperiods and a constant light intensity (150−200 μmol m−2 s−1). The tobacco (Nicotiana tabacum cv. “G28”) was used for genetic assays, and N. benthamiana was used for subcellular protein localization experiments. The plants were grown in an environmental chamber with a constant temperature of 25 ± 3 °C and 12/12 h (light/dark) photoperiods and a constant light intensity (150−200 μmol m−2 s−1). The tea plant cultivar Camellia sinensis cv. “Shuchazao” was cultivated in the Experimental Tea Garden of Anhui Agricultural University (latitude 31.86° N, longitude 117.27° E). With respect to the abiotic stress, the tender tea branches (approximately 10 cm long) were sampled and cultured immediately in water for 1 d and then subjected to the sucrose treatment (90 mM sucrose for 9 h). Two or three tea branches were inserted into the tissue culture bottles which were filled with 90 mM sucrose solution (at least three bottles per treatment). At the same time, the control samples were cultivated in deionized water. All the tea branches were grown in an environmental chamber with a constant temperature of 25 ± 3 °C and a constant light intensity (150−200 μmol m−2 s−1). After 9 h, two leaves on the tea branches were picked and immediately frozen in liquid nitrogen for RNA extraction. The de novo transcriptome sequencing and data analysis of fresh tea leaves were performed by BGI Gene Tech Co., Ltd. (Shenzhen, China) using an Illumina 2000 platform. Cloning of CsMYB5b, CsTT8, and CsWD40. Total RNA was isolated from the tissues of tea plants using an RNAiso mate and RNAiso Plus (Takara, Dalian, China) according to the supplier’s instructions. The quality and quantity of RNA were verified by agarose gel electrophoresis and NanoVue plus (GE Healthcare, Waukesha, WI, USA). Total RNA was used to generate cDNA by reverse transcription using a PrimeScript RT Reagent Kit (Takara, Dalian, China). According to the cDNA sequences of CsMYB5b (KY827397), CsTT8 (MH618663), and CsWD40 (MH618664), the ORF primers (Table S1) were designed by Primer5 software to synthesize the CDS of them by end-to-end PCR using Phusion High-Fidelity DNA polymerase (Thermo Scientific, Vilnius, Lithuania). Samples were subjected to the following procedure: 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s, 60 °C for 30 s, 72 °C for 45 s, and a final extension at 72 °C for 10 min. The resulting products were purified by electrophoresis on 1.2% agarose gel, ligated into a pEASY-Blunt Simple T vector (TransGen Biotech, Beijing, China) and subsequently introduced into Escherichia coli DH5α for sequencing. Agrobacterium-Mediated Transformation of A. thaliana and Tobacco. The ORFs of the CsMYB5b were ligated into the entry vector pDONR207 using the Gateway BP Enzyme mix (Invitrogen, Carlsbad, CA, USA). The primers used for vector construction are listed in Table S1. After sequence confirmation, the entry vectors were introduced into the destination vector pCB2004 using the Gateway LR Clonase enzyme (Invitrogen, Carlsbad, CA, United States). The pCB2004 vectors carrying CsMYB5b were transformed into Agrobacterium tumefaciens (A. tumefaciens) strain C58C1 and EHA105 by electroporation. The Agrobacteria were inoculated on solidified LB medium with 50 mg/L kanamycin and 50 mg/L spectinomycin at 28 °C for approximately 48 h. A positive EHA105 colony harboring the vector pCB2004-CsMYB5b was confirmed by PCR. Then, the A. tumefaciens was cultured in liquid LB medium until the OD600 of the cell suspension reached 0.4−0.8. The A. tumefaciens cells were centrifuged at 5000g at 4 °C for 10 min and used for genetic transformation of Arabidopsis by following the foral-dip method as previously described.37 In the same way, A. tumefaciens EHA105 was transformed into tobacco using a previously reported leaf disc protocol.38 Extraction and Detection of Anthocyanins and PAs. The polyphenols were extracted from transgenic tobacco flowers using the following protocol. The fresh petals (0.1 g) from approximately 10 independent transgenic tobacco plants were ground into fine powder in liquid nitrogen. Powdered samples were completely suspended in 1 mL of 80% methanol plus 0.2% hydrochloric acid (HCl) in a 2.0 mL polypropylene tube, vortexed for 30 s, and then sonicated for 15 min

Wang found abundant EC was accumulated in the tobacco overexpressing CsLARs, with a small amount of C detected.12,17 Recently, a new research study indicated that silencing LAR could promote the accumulation of insoluble PAs in Medicago truncatula.18 In terms of DFR, research studies always pay attention to its role in participating in the biosynthesis of anthocyanins.19,20 But, in early decades, it was reported that DFR purified from the cells of Douglas fir could convert DHQ to leucocyanidin, which can release carbocation and participate in the polymerization of PAs.21 The PA biosynthetic genes are activated or inhibited by a transcriptional complex, consisting of R2R3-MYB, basic-helix− loop−helix (bHLH), and WD-repeat (WDR) proteins, called MBW complex.22−24 And, these regulatory factors are environmentally affected, such as by biotic and abiotic stresses, thereby promoting or inhibiting the accumulation of PAs. In Poplar, a pathogen-, wound-, and ultraviolet B-responsive R2R3MYB transcription factor (TF) MYB134 regulating PA biosynthesis in resistance to the stresses was identified.25 Additionally, salicylic acid (SA) promoted the biosynthesis of flavan-3-ol and PAs in Poplar by activating the MYB-bHLHWD40 complex against rust proliferation and infection.26 Jiang reported that low temperature treatment facilitated the accumulation of PAs and anthocyanin in CsMYB5e transgenic tobacco.27 Sucrose, acting not only as a carbon source for energy storage but also a signal involved in metabolic processes, plays an important role in plant growth.28,29 In A. thaliana, sucrose induced anthocyanin biosynthesis through up-regulating the positive TFs and down-regulating the negative TF (MYBL2) involved in the flavonoid pathway.30,31 Sucrose was also reported to act as a signal molecule, activating PAP1 (MYB75) and promoting anthocyanin accumulation.30 However, the sucrose-induced TFs regulating the biosynthesis of flavan-3-ol and PAs have rarely been reported in plants. Liu et al. had reported that MdSnRK1.1 interacts with MdJAZ18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation by MBW complex in apple.32 The tea plant is rich in polyphenolic compounds, including PAs and esterified catechins, both of which are derived from monomeric catechins.33 The structural genes related to the biosynthesis of PAs and esterified catechins have been studied clearly in the tea plant.8,34 In our previous research, the transcriptomic and metabolic analysis showed the sucrose treatment on tea plants (tea seedlings grown on medium) in vitro promoted the accumulation of polyphenols, including anthocyanins, catechins, and PAs.35 And, the sucrose-induced anthocyanin regulator had been verified,36 while the PA regulators which can be controlled by sucrose have not yet been identified. In this study, to further search for the target MYB TFs, we revalidated the effect of short-time sucrose treatment on tea branches, and a sucrose-induced MYB (SIMYB) transcription factor was screened and studied. Heterologous expression of SIMYB in tobacco and A. thaliana strongly induced PA accumulation, through up-regulating the key target genes LAR or ANRs. Here, we provided sufficient genetic evidence to demonstrate this SIMYB played an important role in regulating the PA biosynthesis.

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MATERIAL AND METHODS

Plant Materials. A. thaliana wild-type (Ecotype Columbia 0) and tt2 mutant (SALK_005260) used for genetic transformation were grown in a chamber at 21 ± 3 °C with 16/8 h (light/dark) B


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Figure 2. Phylogenetic analysis and qRT-PCR analysis of the selected MYB transcription factors from subgroups 5, 6, and 7. (a) A phylogenetic tree of subgroups 5, 6, and 7 MYB transcription factors (TFs) from C. sinensis and A. thaliana. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates by MEGA version 5.0. The numbers indicate the confidence percentages. (b) The relative expression level of selected MYB TFs in tea branches treated by sucrose compared to the control. The ratio values for each gene in the control were set as 1.0 after they were divided by its own value. Values less or higher than 1.0 mean their transcription abundances are decreased or increased in tea branches treated by sucrose. All the values are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. The asterisks indicate the significance level (n = 3, **P < 0.01) based on a Tukey’s honestly significant difference test. at a low temperature (10 °C). After centrifugation at 16000g for 10 min, the supernatant was transferred to a new tube. The residues were repeated once as mentioned above to obtain a final volume of 2.0 mL extracts. All extracts were centrifuged at 16000g for 10 min and then filtered with a 0.22 μm membrane (Millipore, Billerica, MA, USA). The absorption measured at 530 nm of the extracts was used to evaluate the anthocyanins in the transgenic tobacco flowers. In addition, the extracts mentioned above were also analyzed by dimethylaminocinnamaldehyde (DMACA) reaction assay and ultrahigh-performance liquid chromatography−mass spectrometry (UPLC-MS) (Agilent, Ca, USA), respectively.39 Briefly, 0.3 mL of 80% methanol extracts were mixed with 1 mL of DMACA (0.2% DMACA, w/v) for 1 min, and then the mixture was measured at 640 nm. The resulting absorbance values were used to quantify the total soluble PAs in the samples. The insoluble PAs (polymeric PAs) in the residues were quantified using the butanol−HCl hydrolysis method described previously.34 Extraction and detection of soluble and insoluble PAs from transgenic A. thaliana plants (more than 5 independent biological replicates) and different tea organs (three biological replicates per organs) were performed following the above protocol, except 0.02 g of mature seeds and 0.1 g of tea samples were replaced. UPLC coupled with triple-quadrupole mass spectrometry (QQQ-MS) (Agilent, CA, USA) was used to quantify the PA-dimers (procyanidin B1, B2, B3, and B4) and PA-trimers in tea. Subcellular Localization. The full-length coding sequence of CsMYB5b lacking its termination codon was cloned into the entry vector pDONR207 using the Gateway BP Enzyme mix (Invitrogen), with specific primers. The newly extracted plasmids after sequencing were then introduced into the destination vector, named pGWB5 (fused with GFP at the C-terminal) using the LR Clonase enzyme (Invitrogen). After sequencing, the plasmid pGWB5-CsMYB5b was transformed into A. tumefaciens strain EHA105 as described above. Selection of a positive colony for cultivation and infiltration of N. benthamiana was performed following a reported protocol.40 After infection for 48 h, leaves were examined and observed using an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). Yeast Two Hybrid (Y2H) Assay. To construct the vectors used in the Y2H system, the primers with restriction sites were designed (Table S1). In order to avoid the self-activation caused by the Cterminal activation domain of MYBs, the full-length sequence of CsMYB5b was amplified with the specific primers and inserted into the Gal4 activation domain of the vector pGADT7 (Clotech). The ORFs of CsTT8 and CsWD40 were cloned into the Gal4 binding

domain of pGBKT7 vector. After sequencing, the newly extracted plasmids were transformed into the yeast strain AH109 following the manufacturer’s instructions. Yeast cells containing both vectors were first tested on synthetically defined (SD) media lacking Leu (Leucine) and Trp (Tryptophan) to confirm the feasibility of experiment. Then, the yeast cells were tested on (SD) media (with X-α-gal, 50 μg/mL) lacking Leu, Trp, Ade (Adenine), and His (Histidine) to confirm the protein interaction. Quantitative Real Time (qRT) PCR and Statistical Analysis. The newly synthesized cDNA of tea and tobacco was diluted to 25% (v/v) with deionized water before being used as the template. The qRT-PCR was performed in a reaction mixture volume of 20 μL containing 1.1 μL of cDNA template, 0.8 μL of forward and reverse primer (10 μM), and 10 μL of SYBR Green PCR Master Mix (Takara). The PCR cycling parameters and relative expression level were set and calculated by a previously published method, respectively.41 The significance analysis was analyzed by one-way ANOVA followed by Tukey’s test using GraphPad Prism 5 statistical software. The accession numbers for tobacco genes used in qRT-PCR are as follows: NtDFR (KF927020.1), NtANS (AB289447), NtANR1 (XM_016656914.1), NtANR2 (XM_009786976.1), and NtLAR (XM_016641079.1).

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RESULTS Identification of a Sucrose-Induced MYB (SIMYB) Transcription Factor in the Tea Plant. The sucrose treatment on tea plants promoted the accumulation of polyphenols, especially anthocyanins and PAs in previous research.35 To further search for the PA-related MYB TFs regulated by sucrose, we revalidated the effect of short-time sucrose treatment (9 h) on tea branches. It has been reported that MYB TFs classified in subgroup 5, 6, and 7 are involved in the regulation of the biosynthesis of polyphenols.36,42 In this study, RNA-seq analysis (PRJNA494888) showed that some PA regulators (MYB subgroup 5), anthocyanin regulators (MYB subgroup 6), and flavonol regulators (MYB subgroup 7) were up-regulated with different degrees (Figure 2a and Table S2). Among them, one MYB TF (TEA031375.1) in subgroup 5 was up-regulated by more than 15-fold [log2 ratio (9 hsucrose/9 h-control) > 4]. In addition, quantitative real-time PCR (qRT-PCR) was performed to check the reliability of RNA-seq results. The results also confirmed that the selected C


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Figure 3. Phylogenetic relationships and sequence comparison of two types PA-related MYB transcription factors from different species. (a) The phylogenetic tree of PA-related MYB transcription factors (TFs) from the tea plant and other species. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates by MEGA version 5.0. The numbers indicate the confidence percentage. The protein sequence GenBank accession numbers are LcMYB14 (ARK19308.1), AtTT2 (Q9FJA2), LjTT2a (BAG12893.1), LjTT2b (BAG12894.1), LjTT2c (BAG12895.1), TaMYB14 (AFJ53054.1), MtMYB14 (AFJ53058.1), PtMYB134 (ACR83705.1), MdMYB9 (ABB84757), VvMYBPA2 (ACK56131), GhMYB38 (AAK19618), CsMYB5a (KY827396), CsMYB5b (KY827397), GhMYB10 (AAK19615.1), GhMYB36 (AAK19617.1), PtrMYB115 (XM_002302608.2), AtMYB5 (NP_187963.1), DkMYB4 (BAI49721.1), MtMYB5 (XP_003601609.1), PH4 (AAY51377.1), BNLGHi233 (AAK19611.1), VvMYBPA1 (CAJ90831), CsMYB 5d (KY827399), CsMYB5e (KY827400), VvMYB5a (AAS68190), VvMYB5b (AAX51291), and TcMYBPA (ADD51352.1). (b) Sequence alignment of two types of CsMYB TFs from tea plant and other selected species. The R2 and R3 domains are marked with a black line. The bHLH motifs are marked by a red box. The TT2 motif is marked by a blue box. Sequence alignment was performed using the DNAMAN program (Lynnon Corporation, San Ramon, CA, USA). The identical amino acids are marked by white letters on a black background; the conservative amino acids (similarity > 75%) are marked by a dark gray background. Similar amino acids (similarity > 50%) are marked by black letters on a light gray background; other amino acids are marked by black letters on a white background (similarity < 50%).

relationship with CsMYB5a, and both of them belonged to TT2-type PA regulators. CsMYB 5d and CsMYB5e belonged to MYB5-type PA regulators, which mainly regulated mucilage accumulation in seed coats and trichome branching in leaves, and played a minor role in regulation of PA biosynthesis.49,50 Sequence alignment showed both TT2-type and MYB5-type regulators were R2R3MYB family TFs, and all of them had the bHLH binding domain, which is indispensable to form a ternary MBW complex (Figure 3b). But, only the clade I family regulators have the TT2 specific motif, which plays a major role in regulation of PA biosynthesis. Subcellular Localization of CsMYB5b. In order to identify the subcellular localization of CsMYB5b in plants, GFP fusion is an efficient approach used for structure genes and transcription factors.51,52 The ORF of CsMYB5b fused with GFP at the C-terminus, driven by the 35S promoter was introduced into N. benthamiana (Figure 4a). In addition, a negative empty vector control (GFP) and a positive nuclear localization marker (AtMYB75-GFP) were also introduced into N. benthamiana. After cocultivation for 48 h, the confocal microscopy observation showed that CsMYB5b-GFP was localized in the nucleus, which was consistent with positive nuclear localization marker (Figure 4b). It indicates CsMYB5b is a nuclear localization transcription factor. CsMYB5b Promotes PA Accumulation in A. thaliana. Following the phylogenetic analysis and sequence alignment, CsMYB5b was predicted as a TT2-type PA regulator. To test it, a genetic complementation experiment was performed by introduction of a constitutively expressed CsMYB5b into A.

genes were susceptible to sucrose treatment (Figure 2b). And, the same candidate PA regulator (TEA031375.1) was the most sensitive to sucrose. Linking the RNA-seq and qRT-PCR results, the candidate gene was known as a sucrose-induced MYB (SIMYB) TF. To further study the function of this gene, we cloned and named it CsMYB5b (KY827397). Identification of Two Types of PA-Related Regulators in the Tea Plant. MYB family TFs regulating the biosynthesis of PAs have been reported in several plants, mainly divided into two categories of TT2-type (a major PA regulator) and MYB5-type (with a minor role in PA biosynthesis), such as AtMYB5 and AtTT2 from A. thaliana, MtMYB14 and MtMYB5 from Medicago truncatula, VvMYBPA1 and VvMYBPA2 from grapevine (Vitis vinifera), and DkMYB2 and DkMYB4 from persimmon (Diospyros kaki).43−47 All of them positively regulate the biosynthesis of PAs through forming the MBW complex with bHLH and WD40. To obtain more specific PA-related regulators in tea plant, homologous sequences were searched in the transcriptome and genomic data of tea by the BLAST method.41,48 Four candidate genes with high sequence similarity were screened and cloned, named as CsMYB5a (KY827396), CsMYB5b, CsMYB 5d (KY827399), and CsMYB5e (KY827400), encoding R2R3MYB TFs. In order to further classify the candidate MYB TFs from tea plant, a phylogenetic tree was constructed with other known MYBs involved in the regulation of PA biosynthesis (Figure 3a). The phylogenetic tree showed that all PA-related MYB TFs could be grouped into two main clades, namely, clade I (TT2-type) and clade II (MYB5-type). CsMYB5b had a closer D


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CsMYB5b Promotes PA Accumulation in Tobacco. Another model plant tobacco, in which a slight accumulation of PAs was detected, was used for studying the biosynthesis and regulation of PAs. In order to functionally characterize CsMYB5b which can promote the biosynthesis of PAs, we also overexpressed CsMYB5b in tobacco, generating several positive transgenic tobacco with different transcription levels. The control tobacco was transformed with an empty vector PCB2004 (Figure 7). Different lines of CsMYB5b-transformed tobacco produced pale-pink flowers, losing the pink flower pigmentation characteristic of empty vector control tobacco (Figure 7a, b). After the petals of tobacco carrying CsMYB5b with 80% methanol/0.2% HCl were extracted and stained with DMACA reagent, the generated blue coloration indicated that PA-like compounds were accumulated in CsMYB5b transgenic tobacco (Figure 7c). The spectrophotometric determination at 640 and 530 nm confirmed an increase of DMACA-reactive compounds and a reduction in anthocyanins in tobacco overexpressing CsMYB5b, respectively, as the transcription levels of the CsMYB5b increased (Figure 7d). UPLC-MS/MS analysis showed not only catechin monomer (m/z 289.0, EC and C) was accumulated but also PA-dimer (m/z 577.0, procyanidin B1 and B2) and PA-trimer (m/z 865.0, EC-EC-EC) were detected in the extracts of petals of CsMYB5b transgenic tobacco (Figure 7e). MS/MS analysis further confirmed the fragment features of PA-dimer and PA-trimer. No apparently elevated product was detected in the extracts of tobacco harboring the empty vector. It is intriguing that the content of EC was much more than that of C, and the content of procyanidin B2 (EC-EC) was much more than that of procyanidin B1 (EC-C) (Figure S1). No other PA-trimer was detected except for EC-trimer. In addition, butanol/HCl hydrolysis showed the insoluble PA levels significantly increased in the residues of transgenic CsMYB5b tobacco lines (data not shown). To investigate the gene expression difference in transgenic CsMYB5b tobacco compared with the control, qRT-RCR was performed. Following the qRT-RCR result, some important structural genes involved in the flavonoid pathway, such as NtDFR, NtANS, NtANRs, and NtLAR, were all up-regulated, especially NtANRs and NtLAR (Figure S2). That is why large amounts of soluble and insoluble PAs were accumulated in the tobacco overexpressing CsMYB5b. In other words, these structural genes may be the target genes of CsMYB5b in tobacco. The primers of these structural genes used for qRTPCR are listed in Table S3. Gene Expression Profile of the Sucrose-Induced MYB (SIMYB) TF and PA Accumulation Pattern in Tea. Though CsMYB5b could be induced by sucrose, its expression profile in different tea organs was unclear. To investigate the expression profile of it, qRT-PCR was performed (Figure 8a). CsMYB5b exhibits high transcription levels in tender leaves, especially in the buds, medium expression levels in stem and roots, and low expression levels in the mature leaves (Figure 8b). Tea is rich in polyphenolic compounds. Soluble PAs contain monomeric catechins and oligomeric PAs. Some polymeric PAs with a high degree of polymerization are always insoluble. The PA accumulation pattern (including the content and distribution) in different tea organs was detected. Quantification analysis showed the soluble PAs mainly accumulated in tender leaves, and only a small amount was detected in roots

Figure 4. Subcellular localization of CsMYB5b. (a) Plasmid construct used for subcellular localization of CsMYB5b in Nicotiana benthamiana. The GFP fused with CsMYB5b is driven by the 35S promoter. (b) Images of CsMYB5b-GFP protein in mesophyll cells of Nicotiana benthamiana obtained using confocal microscopy. The empty vector and nuclear marker (AtMYB75-GFP) were used as negative and positive controls, respectively. Bar = 30 μm.

thaliana tt2 mutant. More than 10 herbicide-resistant T1 transgenic lines were generated. As shown in Figure 5, overexpression of CsMYB5b in A. thaliana tt2 mutant complemented the PA-deficient phenotype, leading to PA accumulation in the seed coats (Figure 5a), which suggested that CsMYB5b is a functional ortholog of AtTT2. The PA contents in the seed coats, including soluble and insoluble PAs, were determined. Compared with tt2, the contents of PAs in CsMYB5b-tt2 transgenic lines were greatly increased, which almost had the equal contents of PAs in wildtype A. thaliana (Figure 5b, c). UPLC-MS/MS results showed that the EC (m/z 289.0/245.0) and PA-dimer (procyanidin B2, m/z 577.0/289.0) were reaccumulated in CsMYB5b-tt2 transgenic lines (Figure 5d). The above data indicated that CsMYB5b plays an important role in regulating the biosynthesis of PAs. The biosynthesis of PA and anthocyanin is always controlled by a ternary MBW complex, consisting of MYB, bHLH TFs, and WD40 protein. To test whether CsMYB5b also can interact directly with the cofactors bHLH and WD40, we screened orthologs of AtTT8 and AtWD40 in tea plant transcriptome and cloned them; we named them CsTT8 and CsWD40, respectively. Then, we determined the protein interaction using the yeast two hybrid (Y2H) system (Figure 6). It is evident that all the transformants of fusion constructs (pGADT7−CsMYB5b + empty vector pGBKT7, pGADT7− CsMYB5b + pGBKT7−CsTT8, and pGADT7−CsMYB5b + pGBKT7−CsWD40) can grow well on the selective SD/Leu/-Trp medium (Figure 6a), whereas only transformants of pGADT7−CsMYB5b + pGBKT7−CsTT8 and pGADT7− CsMYB5b + CsWD40 can grow well on selective SD/-Leu/Trp/-His/-Ade/X-α-gal medium and show X-α-galactosidase activity (Figure 6b). The Y2H assay demonstrated that both CsTT8 and CsWD40 can interact with CsMYB5b. E


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Figure 5. Genetic complementation of A. thaliana PA-deficient tt2 mutant by constitutively expressing CsMYB5b. (a) The mature seeds from A. thaliana wild-type (WT), tt2 mutant, and two independent transgenic lines of tt2-CsMYB5b. (b) The content of soluble PAs in mature seeds of the plants mentioned above. All data are the means of three biological replicates. The asterisks indicate the significance level (n = 3, ***P < 0.001) based on a Tukey’s honestly significant difference test. (c) The content of insoluble PAs in mature seeds of the plants mentioned above. (d) The MRM (multiple reaction monitoring) chromatogram (in negative ion mode) of catechins (m/z 289.0/245.0) and PA-dimers (m/z 577.0/289.0) in the extracts of soluble PAs from WT, tt2 mutant, and transgenic line of tt2-CsMYB5b.

(Figure 8c). The insoluble PAs are mainly distributed in the root, and the content of insoluble PAs in root is far more than that in other tea organs (Figure 8d). With further characterization of soluble PAs using UPLC-QQQ-MS, we found PAdimers (procyanidian B1, B2, B3, and B4) and PA-trimer (ECEC-EC) were mainly accumulated in stems and roots (Figure 8e, f). It is intriguing that procyanidian B2 and B4 are the major components of PA-dimers. The accumulation pattern of PA-dimers and PA-trimer is somewhat inconsistent with that of total soluble PAs. The reasons for this phenomenon may be that a large amount of esterified catechins, including (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) account for a large proportion in the leaves of the aerial parts, which confer on tea its unique flavor and health function.33,34 Linking to the expression profile of CsMYB5b and the PA accumulation pattern in tea, the results indicated that CsMYB5b played a vital role in controlling catechin accumulation, not only regulating the biosynthesis of polymeric PAs but also the monomeric catechins.

Figure 6. The direct interaction of CsMYB5b with CsTT8 and CsWD40 in yeast two-hybrid assay. The interaction was tested in the assay using the His/Ade auxotrophs and X-α-gal (bottom panel).

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Figure 7. The characterization of transgenic tobacco plants overexpressing CsMYB5b. (a) Phenotypes of empty vector control (CK) and CsMYB5b transgenic tobacco flowers. (b) Semiquantitative RT-PCR analysis of the CsMYB5b and the housekeeping gene NtActin transcription levels in total RNA from the flowers. (c) DMACA staining of the extracts from CsMYB5b transgenic tobacco flowers and CK. (d) The absorbance values of the anthocyanin extracts and bluish compounds from DMACA-reactive compounds at 525 and 640 nm, respectively. All data are the means of three biological replicates. The asterisks indicate the significant level (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001) based on a Tukey’s honestly significant difference test. (e) The MRM chromatogram (in negative ion mode) of catechins (m/z 289.0), PA-dimer (m/z 577.0), and PA-trimer (m/z 865.0) in the extracts of CsMYB5b transgenic tobacco flowers. The MS/MS spectra of PA-dimer and PA-trimer ions are listed.

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DISSCUSSION Identification of the SIMYB TF Regulating PA Biosynthesis. Sugars are considered as a major regulatory molecule in addition to being essential metabolic nutrients and structural components in higher plants.28,53 A glucose sensor MdHXK1 modulates anthocyanin accumulation mainly through directly regulating the anthocyanin-related bHLH TFs in response to a glucose signal in plants.54 Sucrose was also reported to act as a signal molecule to promote anthocyanin accumulation by activating PAP1 TF.30 However, regarding the sucrose-induced TFs regulating the biosynthesis of flavan-3-ol and PAs, Liu et al. had reported that MdSnRK1.1 interacts with MdJAZ18 to regulate PA accumulation by MBW complex in response to a sucrose signal.32 In previous research, the transcriptomic and metabolic analyses showed that the sucrose treatment (2 days) on tea plants in vitro promoted the accumulation of polyphenols, including anthocyanins and catechins; several R2R3-MYBs and bHLH TFs acting as regulators of polyphenol biosynthesis were regulated.35 One sucrose-induced anthocyanin-regulator CsMYB6A had been identified and verified through heterologous expression in tobacco,36 but the sucrose-induced PA regulator in tea plants had not been identified and has attracted our attention. Among them, one EST (expressed sequence tag, CL8695.Contig1) predicted to be a PA regulator in MYB subgroup 5 was found significantly up-regulated. In this study,

RNA-seq analysis of a short-time sucrose treatment (9 h) on a tea branch was revalidated and showed a similar result. One candidate gene (TEA031375.1) that shared a same sequence with previously reported EST was up-regulated more than 15fold as compared to the controls (Table S2), and qRT-PCR also confirmed that the candidate gene (CsMYB5b) was more susceptible to sucrose treatment (Figure 2b). To date, a number of PA regulators have been reported, which are always divided into TT2-type and MYB5-type TFs. According to the phylogenetic analysis and sequence alignment, CsMYB5b was recognized as a TT2-type PA regulator, having a conserved TT2-motif (Figure 3). PA-related MYB TFs in tea plants had been reported in previous study. Overexpression of CsMYB5a (a TT2-type PA regulator) in tobacco promoted the PA accumulation, while no polymeric PA was detected in CsMYB5e transgenic tobacco unless it was treated with low temperature.27 But, neither of these two genes were induced by sucrose, no matter if it was in the transcriptome or qRT-PCR analysis. Hence, CsMYB5b had become a valuable TT2-type PA regulator for its sucroseinduced characteristic. Role of CsMYB5b in the Tea Plant. In this paper, a sucrose-induced PA regulator (SIMYB) was identified in tea. Tea is a recalcitrant woody species in regeneration from tissue culture, and no success has been reported about the genetic transformation in tea plant. Therefore, we tested the function of CsMYB5b in model plants tobacco and A. thaliana. As G


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Figure 8. Accumulation profiles of soluble PAs and insoluble PAs and relative expression level of CsMYB5b in different tissues of tea plant. (a) Images of newly grown young branch (buds, the 1st, 2nd, and 3rd leaves and mature leaves), stems, and roots. (b) The relative expression level of CsMYB5b in different organs of tea plant. (c) The accumulation profiles of soluble PAs in different organs of tea plant. (d) The accumulation profiles of insoluble PAs in different organs of tea plant. (e) The accumulation profiles of soluble oligomers (procyanidin B1, B2, B3, and B4) in different organs of tea plant. (f) The accumulation profiles of soluble oligomer PA-trimer in different organs of tea plant. We do not have the PAtrimer standard, so the values represent the peak area. All values are the means of three biological replicates, and each error bar represents the standard deviation of three replicates. The different letters (a, b, c, d, ...) indicate the significance level at P < 0.05 based on a Tukey’s honestly significant difference test.

structural genes, such as CsLARs and CsANRs, directly involved in the biosynthesis of catechins and PAs always exhibit high transcription levels in the tender organs, following previous research.34,56 The biosynthesis of polymeric PAs (such as insoluble PAs) may require an unknown catalytic mechanism, polymerization, or transportation aside from the regulation of CsMYB5b. So, the total catechin accumulation profiles are not in contradiction with the expression pattern of CsMYB5b. The Mystery of the Biosynthesis of PAs. As we all know, the biosynthesis of catechins and epicatechins is responsible by LARs and ANRs in vitro, respectively.15,16 But to date, the biosynthesis mechanism of PAs has remained unclear. Regarding the biosynthesis of PAs, a common idea is that carbocations as extension units in the polymerization process are indispensable.57−59 However, the source of carbocations is unknown.

shown in the Results, ectopic expression of CsMYB5b in A. thaliana and tobacco confirmed it can promote the accumulation of oligomeric and polymeric PAs in plants (Figures 5 and 7). On the basis of the above results, we speculated that CsMYB5b had the same role in regulating PA biosynthesis in tea plant. Though CsMYB5b is highly expressed in the tender vegetative organs (buds and first leaves) and stems, the polymerized catechins (PA-dimers, PA-trimer, and insoluble PAs) tend to accumulate in roots and stems (Figure 3d−f). So, it seems that the expression pattern of CsMYB5b is not directly related with the PA accumulation profiles. This is because tea is also rich in monomeric catechins (especially the esterified catechins) in the tender leaves and buds, which greatly contribute to the astringency and bitterness of tea beverages.33,55 The biosynthesis of monomeric catechins is also controlled by the TT2-type regulators. In addition, the H


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*E-mail: [email protected]. Tel.: 86-551-65786232. Fax: 86551-65785729.

Recently, a new discovery on polymerization of PAs was reported that 4β-(S-cysteinyl)-epicatechin, which served as a PA extension unit, can participate in automatic nonenzymatic polymerization with flavan-3-ols as starter units in vitro.60 In the process, 4β-(S-cysteinyl)-epicatechin released an EC carbocation. The new substrate 4β-(S-cysteinyl)-epicatechin was purified in MtMYB5-expressing Medicago hairy roots, but the structural genes directly involved in its synthesis are unknown. In the synthesis process of PAs and other secondary metabolites, the transcriptional regulation in different pathways is indispensable.61−63 The phenotypes (transparent testa) of A. thaliana tt2 and tt8 mutants are attributed to the deletion of single gene AtMYB123 or AtTT8, respectively.44,64 In the petals of wild tobacco, a slight accumulation of catechins is detected due to the extremely low transcription levels of the NtANRs and NtLARs genes. But in our experiments and the experiments of others, the sole overexpression of a PA regulator (CsMYB5b) in tobacco was proved to be sufficient to enhance the accumulation of catechins and PAs, through up-regulating the important structural genes.65 All of the results indicate that the PA regulators play a vital role in catechin and PA accumulation. As key structural genes in the biosynthesis of catechins and PAs, overexpression of CsLARs in tobacco only increased the monomeric catechin EC and C, and no PA was detected; in the CsANRs transgenic tobacco, no monomeric catechin or PA was detected, though a DMACA-reactive compound was largely accumulated.34,56,66 In our experiment, overexpression of CsMYB5b in tobacco promoted the accumulation of monomeric catechin (EC and C) and PAs (PA-dimer and PA-trimer) synchronously. qRT-PCR analysis showed that NtANRs and NtLAR were significantly up-regulated in CsMYB5b transgenic tobacco (Figure S1). Therefore, we speculate that the biosynthesis of PAs requires the coexpression of NtANRs and NtLAR. It is recognized that LARs are responsible for the formation of catechin monomers, and whether ANRs are responsible for the formation of carbocations is yet unknown.

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ORCID

Tao Xia: 0000-0003-0814-2567 Author Contributions

L.G. and T.X. conceived the experiment. P.W. performed most of experiments and wrote the draft. G.M. and L.Z. constructed the heterologous expression vectors and did the plant genetic transformation experiments (including transgenetic tobacco and A. thaliana). Yan Li, X.K. and Y.H. extracted and determined the metabolites of transgenetic tobacco and A. thaliana. Z.F. and X.J. analyzed the LC-MS data. Yajun Liu modified the language of the paper. All authors read and approved the final version of the manuscript. Funding

This work was financially supported by the Natural Science Foundation of China (31700608), the Natural Science Foundation of Anhui Province (1708085MC58), and the Chang-Jiang Scholars and the Innovative Research Team in University (IRT1101). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED PAs, proanthocyanidins; TF, transcription factor; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; DFR, dihydroflavonol 4-reductase; EC, epicatechin; C, catechin; AFZ, afzelechin; GC, gallocatechin.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b06207. Sequences of primers used for DNA cloning, construction of subcellular localization vectors, construction of heterologous expression vectors, construction of Y2H vectors, and quantitative RT-PCR of CsMYB5b in tea plants (Table S1); expression analysis of the selected MYB transcription factors (subgroup 5, 6, and 7) in the tea plant treated by sucrose (Table S2); sequences of primers used for quantitative RT-PCR in transgenic tobacco (Table S3) (PDF) The structures of B-type procyanidin B1, B2, B3, and B4 (Figure S1) and the expression levels of structural genes involved in PA biosynthesis in three independent transgenic CsMYB5b tobacco lines (Figure S2) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: 86-551-65786003. Fax: 86551-65785833. I


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A Sucrose-Induced MYB (SIMYB) Transcription Factor Promoting

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