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Involvement of Three CsRHM Genes from Camellia sinensis in UDP-rhamnose Biosynthesis Xinlong Dai, Guifu Zhao, Tianming Jiao, Yingling Wu, Xinmin Li, Kan Zhou, Liping Gao, and Tao Xia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01870 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018
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Involvement of Three CsRHM Genes from Camellia sinensis in UDP-rhamnose
2
Biosynthesis
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Xinlong Dai 1, 2, Guifu Zhao 1, Tianming Jiao1, Yingling Wu1, Xinmin Li2
5
Kang Zhou2 Liping Gao2*and Tao Xia1*
6 7
1
8
230036, China
9
2
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui
School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, China
10 11 12
Corresponding author:
13
Tao Xia, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei,
14
Anhui 230036, China
15
Tel: 86-551-65786003, Fax: 86-551-65785833, E-mail:
[email protected]; Liping Gao, School of Life
16
Science, Anhui Agricultural University, 130 West Changjiang Rd, Hefei, Anhui 230036 China
17
Tel: 86-551-65786232, Fax: 86-551-65785729,
[email protected]
18
ORCID
19
Tao Xia: 0000-0003-0814-2567
20
Liping Gao: 0000-0002-0348-4608
21 22 23 24 25 26 27
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Abstract UDP-rhamnose synthase (RHM), the branch-point enzyme controlling the
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nucleotide
sugar
interconversion
pathway,
converts
31
UDP-rhamnose. As a rhamnose residue donor, UDP-L-rhamnose is essential for the
32
biosynthesis of pectic polysaccharides and secondary metabolites in plants. In this
33
study, three CsRHM genes from tea plants (Camellia sinensis) were cloned and
34
characterized. Enzyme assays showed that three recombinant proteins displayed RHM
35
activity and were involved in the biosynthesis of UDP-rhamnose in vitro. The
36
transcript profiles, metabolite profiles, and mucilage location suggest that the three
37
CsRHM genes likely contribute to UDP-rhamnose biosynthesis and may be involved
38
in primary wall formation in C. sinensis. These analyses of CsRHM genes and
39
metabolite profiles provide a comprehensive understanding of secondary metabolite
40
biosynthesis and regulation in tea plants. Moreover, our results can be applied for the
41
synthesis of the secondary metabolite rhamnoside in future studies.
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Keywords: UDP-rhamnose synthase, Camellia sinensis, recombinant protein, enzyme assays,
43
expression analysis
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UDP-D-glucose
into
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1. Introduction
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Tea is one of the three most popular nonalcoholic beverages worldwide, in which
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the high content of phenolic compounds provides the health benefits.1 The flavonoids
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in tea are the main flavor components and functional ingredients and include acylated
60
glycosylated flavonols, O-glycosylated flavonols, and C-glycosylated flavones.2
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Flavonol glycosides, which are often converted from aglycones in a reaction catalyzed
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by UDP-glycosyltransferases involving various nucleotide sugar donors (including
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UDP-glucose, UDP-galactose, UDP-rhamnose, UDP-xylose, UDP-mannose, and
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UDP-glucuronic acid),3-5 play a role in inducing a tea infusion’s silky, mouth-drying,
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and mouth-coating sensation at very low threshold concentrations.6, 7 However, in tea
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plants, the genes involved in the biosynthesis of nucleotide sugars such as
67
UDP-L-rhamnose (UDP-Rha) remain unknown. In addition, the new shoots of tea
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plants are used as the raw material for making tea. The tenderness of the new shoots is
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crucial for tea quality and is determined by the degree of lignification of the cell wall
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and the content of pectin.
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Pectins include three main classes of polysaccharides: homogalacturonan (HGA),
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rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II).8 HGA plays a
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critical role in pectin cross-linking, which is a component of A. thaliana mucilage
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consisting of individual (1,4)-α-linked galacturonic acid (GalA) residues.9-11 RG-I is
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the primary component
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(1,2)-α-L-rhamnosyl and (1,4)-α-GalA.8,12 RG-II, a dimer covalently cross-linked by a
of seed
mucilage,
consisting
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the
alternating
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borate diester bond, is a component of a structurally complex polysaccharide that
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exists in primary cell walls.13,14
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As a rhamnose residue donor, UDP-Rha is required for the biosynthesis of pectic
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polysaccharides of plant cell walls8 and various secondary metabolites (flavonoids,
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terpenoids, and saponins) in plants.15 In tea shoots, numerous phenolic compounds
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accumulate, including various flavonol glucosides and flavonol rhamnosides.16,17 Their
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content is directly related to the bitter taste of tea. Nguema-Ona reported that
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L-rhamnose participates in the biosynthesis of some O-linked glycoproteins involved
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in growth, morphogenesis, and responses to various stresses.18
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The biosynthesis of UDP-Rha occurs in bacteria, fungi, and plants. However, the
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biosynthetic pathway of UDP-Rha in the new shoots of tea plants remains unclear. In
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bacteria,
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thymidylyltransferase; EC 2.7.7.24), RmlB (coding dTDPD-glucose 4,6-dehydratase;
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EC 4.2.1.46), RmlC (coding dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase; EC
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5.1.3.13), and RmlD (coding dTDP-6-deoxy-L-lyxo-4-hexulose reductase; EC
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1.1.1.133) genes has been identified to be responsible for the biosynthesis of
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dTDP-rhamnose (dTDP-Rha)19-22 (Fig. 1A). In fungi, the main nucleotide diphosphate
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rhamnose form of UDP-Rha is synthesized in a two-step reaction catalyzed by two
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enzymes:
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UDP-4-keto-6-deoxyglucose-3,5-epimerase-4-reductase (U4k6dG-ER)23 (Fig. 1B).
a
gene
cluster
including
RmlA
(coding
UDP-glucose-4,6-dehydratase
glucose-1-phosphate
(UG4,6-Dh)
and
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In plants, the biosynthesis of UDP-Rha is catalyzed by a trifunctional enzyme in
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a three-step reaction, with UDP-D-glucose (UDP-Glc) as a substrate and NAD+ and
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NADPH as cofactors14,24 (Fig. 1C). An excellent study in A. thaliana demonstrated
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that three proteins of UDP-rhamnose synthase (RHM) are trifunctional enzymes that
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convert UDP-Glc to UDP-Rha.25,26 Although biochemical evidence supporting the role
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of RHM in UDP-Rha biosynthesis has been provided in A. thaliana,25 comprehensive
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research on the function of RHM in vitro and in vivo is still lacking. Conversely, many
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researchers are focusing on herbs such as A. thaliana but not on woody plants.
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In this study, we cloned and characterized three homologous CsRHM genes from
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tea plants (Camellia sinensis). Biochemical evidence showed that the proteins are
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encoded by three CsRHM genes and exhibit RHM activity in vitro. Transcript profiles,
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metabolite profiles, and mucilage location suggested that the CsRHM genes are likely
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to be involved in the biosynthesis of UDP-Rha compounds and might participate in the
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formation of the primary cell wall in tea plants.
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2. Materials and Methods
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2.1 Plant materials and chemicals
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Tea plants (C. sinensis (L.) O. Kuntze cv. “Shuchazao”), including buds, first
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leaves, second leaves, third leaves, fourth leaves, mature leaves, old leaves, young
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stems, tender roots, and flowers, were collected from the experimental tea garden of
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Anhui Agricultural University (Anhui, China) during early spring for use in this study.
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Samples were divided into three parts: one part was used for analyzing the total RNA
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extracted and gene expression, a second for determining the total nucleotide sugar
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extracted, and a third for histochemical staining assays in tea plants.
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The nucleotide sugar donor UDP-Glc and ruthenium red were obtained from
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Sigma-Aldrich (www.sigmaaldrich.com/israel.html). NAD+ and NADPH were
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obtained from Solarbio (Shanghai, China). E.coli DH5α and BL21 (DE3) (TransGen
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Biotech, Beijing, China) were used as the host strain and expression strain for
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prokaryotic expression, respectively.
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2.2 Analysis of CsRHM gene sequences
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In this study, to screen the genes involved in the biosynthesis of UDP-Rha in tea
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plants, three CsRHM sequences were identified from tea plant transcriptome data
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using sequence homology search. In addition, using the same method, protein
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sequences from other species (including bacteria and fungi) participating in the
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biosynthesis
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(https://www.ncbi.nlm.nih.gov/).
of
UDP-Rha
were
obtained
from
the
NCBI
website
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The protein sequences of the three CsRHM genes were aligned with those of
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corresponding genes from other species using DNAMAN 7.0 (Lynnon, Canada). An
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unrooted phylogenetic tree was constructed using MEGA 5.0 based on the ClustalW
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multiple alignment through the neighbor-joining method with 1000 bootstrap
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replications.
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2.3 cDNA cloning
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Total RNA was extracted from tea plants using RNAiso-mate for Plant Tissue
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(Takara, Dalian, China; Code: D325A) and RNAiso Plus (Takara, Dalian, China;
140
Code: D9108B), according to the manufacturer’s instructions. Subsequently, cDNA
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was reverse transcribed from total RNA using PrimeScript RT Master Mix (Takara,
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Dalian, China; Cat: RR036A). The 5′-RACE and 3′-RACE based on ESTs from C.
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sinensis (Bioproject ID PRJNA343349) were performed using a SMARTer™ RACE
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cDNA Amplification Kit (Clontech, USA; Cat. Nos 634923 and 634924). The full
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lengths of the three candidate genes were obtained by assembling them based on the 5′
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and 3′ sequences. Subsequently, the ORF sequence of the three candidate genes was
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amplified using Phusion® High-Fidelity DNA Polymerase (New England Biolabs,
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MA, USA) with gene-specific primers from C. sinensis. To determine the exact
149
sequences, the polymerase chain reaction (PCR)-amplified product was cloned into the
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PESY-T1 vector (TransGen, Beijing, China). All primer sequences used in this study
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are listed in Table S1.
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2.4 Heterologous expression and recombinant protein purification
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Full-length coding sequences were subcloned into the expression vector
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pMAL-c2X (New England Biolabs) at the BamHI and PstI restriction sites under the
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control of a tac promoter. The identity of the cloned gene was confirmed through
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sequence
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5′-TGCGTACTGCGGTGATCAAC-3′
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5′-CTGCAAGGCGATTAAGTTGG-3′ (http://www.lifetechnologies.com/). The three
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recombinant
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pMAL-c2X-CsRHMc were transformed into E. coli NovaBlue (DE3) competent cells
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(Novagen, Schwalbach, Germany). The expression strain harboring the recombinant
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plasmids was grown at 37 °C in 200 mL Luria–Bertani medium containing 100
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µg·mL−1 ampicillin and 2 g·L−1 glucose. Subsequently, protein expression was
analysis
with
plasmids
the
of
following
sequencing and
pMAL-c2X-CsRHMa,
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primers:
pMAL-C2X-F pMAL-C2X-R
pMAL-c2X-CsRHMb,
and
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induced
by
adding
isopropyl-β-D-thiogalactopyranoside
(IPTG)
at
a
final
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concentration of 0.5 mM when the OD600 reached approximately 0.4–0.6. This culture
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was incubated at 28 °C for 24 h. The cells were harvested by centrifugation and stored
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at −20 °C overnight. Fusion proteins were purified through affinity chromatography
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using maltose-binding protein (MBP) resin (New England Biolabs), according to the
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manufacturer’s protocol. The identity of the obtained protein fraction was confirmed
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by electrophoresis on a 12% SDS polyacrylamide gel stained. The purified
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recombinant protein was used for the activity assay in vitro.
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2.5 UDP-Rha synthesis assay in vitro
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During the initial screening, each reaction mixture (total volume of 100 µL)
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contained 100 mM Tris–HCl (pH 7.5), 3 mM UDP-Glc or 3 mM UDP-Gal, 3 mM
175
NAD+, 3 mM NADPH, and 15 µg of purified fusion proteins. Each reaction mixture
176
was incubated at 35 °C for 60 min. To determine the optimum pH for the catalytic
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activity of the three purified fusion proteins with 3 mM UDP-Glc as a substrate, assays
178
were performed at pH 4.0–11.0 with 0.5 pH increments using different buffer
179
solutions, including 100 mM acid-sodium citrate buffer (pH 5.0–8.0), 100 mM
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Tris–HCl buffer (pH 7.0–10.0), 100 mM phosphate buffer (pH 7.0–9.5), and 100 mM
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Na2CO3/NaHCO3 buffer (pH 9.0–11.0). The effect of temperature on catalytic activity
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was determined at a series of temperatures (20, 25, 30, 35, 40, and 45 °C) in phosphate
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buffer of pH 9.5 for 30 min.
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To analyze the kinetic parameters (Km and Vmax) of UDP-Glc for three
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recombinant proteins, namely CsRHMa, CsRHMb, and CsRHMc, purified enzymes
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(20 µg) were incubated in reaction mixtures containing 3 mM NAD and 3 mM
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NADPH (as cofactors) and 100 mM phosphate buffer (pH 7.5) in a final volume of
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100 µL. The concentration of the tested substrate UDP-Glc ranged from 0 to 4 mM.
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Each assay mixture was incubated at 35 °C for 15 min.
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All of the reaction mixtures were terminated through heat treatment (100 °C) for
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10 min after incubation and were repeated in triplicate. The enzyme reaction samples
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were centrifuged at 14,720 x g for 10 min and analyzed using high-performance liquid
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chromatography (HPLC), as previously described.25 The kinetic parameters Km and
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Kcat of UDP-Glc for CsRHMa, CsRHMb, and CsRHMc proteins were calculated using
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Hyper 32 (http:// hyper32.software.informer.com/).
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2.6 HPLC and nuclear magnetic resonance analysis
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The HPLC system from Agilent Technologies (RHMo Alto, CA, USA) was used
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in this study and comprised a Venusil XBP C18 reverse phase column (4.6 × 251 mm,
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Agela Technologies), quaternary pump with a vacuum degasser, thermostated column
200
compartment, and autosampler; the protocol of the HPLC system was described
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previously.25 The mobile phase consisted of 20 mM of triethylamine acetate buffer
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(pH 7.5) with a flow rate of 0.5 mL·min−1. The HPLC-purified enzyme products from
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UDP-Glc of CsRHMb were lyophilized and dissolved in 500 µL of 99.97% D2O. The
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proton nuclear magnetic resonance (1H-NMR) and carbon-13 nuclear magnetic
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resonance (13C-NMR) spectra of the products of UDP-Rha were acquired on a Bruker
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AVANCE AV 600-MHz nuclear magnetic resonance (NMR) spectrometer from
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Agilent Technologies (RHMo Alto, CA, USA) at 22 °C. For each sample,
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high-resolution
one-dimensional
water-suppressed
and
two-dimensional
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water-suppressed correlation spectroscopy, total correlation spectroscopy (TOCSY),
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and nuclear overhauser effect spectroscopy (NOESY) experiments were performed.
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The data were processed and analyzed using MestReNova v. 5.2.5.
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2.7 Gene expression analysis
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Gene-specific primers for semiquantitative reverse transcription PCR (RT-PCR)
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analysis were verified through the efficiency and specificity of amplicons using
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melting curve analysis and are listed in Table S1. The conditions for the quantitative
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RT-PCR (RT-qPCR) used in the study were described in a previous report.27 The
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semiquantitative RT-PCR analysis of three CsRHM genes expressions in buds, first
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leaves, second leaves, third leaves, fourth leaves, mature leaves, old leaves, young
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stems, tender roots, and flowers was performed. The housekeeping gene,
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a calibrator in all
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RT-qPCR measurements. The resultant data were expressed as the mean of three
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replicates.
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2.8 Extraction and analysis of nucleotide sugars
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Total nucleotide sugars in tea plants (content in 2 g of buds, first leaves, second
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leaves, third leaves, fourth leaves, mature leaves, old leaves, young stems, tender roots,
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and flowers) were extracted as follows. Step 1: the samples with 5:1 (m/m)
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polyvinylpolypyrrolidone were ground in liquid nitrogen using a pestle and mortar.
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Step 2: the fine powders were washed with 2 mL of water and pelleted by
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centrifugation at 14,720 x g for 15 min. Step 3: after centrifugation, the residues were
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re-extracted twice with 1 mL of water. Step 4: the pooled supernatant was extracted
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three times with an equal volume of ethyl acetate and then centrifuged at 14,720 x g
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for 10 min. Step 5: after centrifugation, the supernatants (ethyl acetate phase) were
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removed, and the water phase was washed with 70% aqueous ethanol. Step 6: the
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mixed sample was gently swirled for 10 min at room temperature and then centrifuged
235
at 14,720 x g for 10 min. Step 7: after centrifugation, the supernatants were collected
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and freeze-dried. Finally, the samples were dissolved in 0.2 mL of water and stored at
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–20 °C before HPLC analysis.
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2.9 Tissue slicing and staining
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Tea tissues slices were obtained from fresh organs (including second leaves, old
240
leaves, young stems, tender roots, and upper and lower epidermis of second leaves) as
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supporter samples. A fresh plant organ was cut into 0.5 cm × 0.5 cm sections. The
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supporter sample containing the tea organ or epidermis was sliced by freehand
243
sectioning and observed under a light microscope (XQT-2, COIC).28,29 Images of the
244
section were recorded before and after staining. The section was stained with 0.01%
245
(w/v) ruthenium red.30,31 Sufficient reagent was added to one side of the section and
246
absorbed into it with tissue paper on the opposite side for about 5 min. After staining,
247
the excess reagent on the surface of the section was completely removed with tissue
248
paper. The stained tissues were observed under a light microscope, and the mucilage
249
appeared pink.
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2.10 Accession numbers
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The GenBank accession numbers and sequence data used in this study are listed
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in Supplementary sequence information.
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3. Results
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3.1 Cloning and sequence analysis of CsRHM genes
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In this study, three CsRHM sequence fragments were screened from the tea
256
transcriptome sequencing database (Bioproject ID PRJNA343349) through homology
257
search using AtRHM as the homologous sequence. Subsequently, the sequences of
258
full-length cDNA were amplified using PCR with gene-specific primers (Table S1),
259
and the proteins of the three candidate genes were named CsRHMa, CsRHMb, and
260
CsRHMc. The predicted protein molecular weights (MWs) and isoelectric point values
261
are listed in Table S2.
262
To investigate the evolutionary relationships of CsRHMs with other plant RHMs,
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a phylogenetic tree, including 267 plant RHM homologous candidates, was
264
constructed using the neighbor-joining method. Phylogenetic analysis revealed that,
265
except for an unresolved polytomy root (including alga, pteridophyta, and bryophyta),
266
the angiosperm RHMs were divided into two groups (cluster I and cluster II) (Fig. 2
267
and Fig. S1). CsRHMa was grouped into cluster I, which consisted of monocot RHMs
268
(HvRHM from Hordeum vulgare subsp, BdRHM from Brachypodium distachyon,
269
OsRHM from Oryza sativa, and SbRHM from Sorghum bicolor) and dicot RHMs
270
(CcRHM from Coffea canephora, NnRHM from Nelumbo nucifera, JrRHM from
271
Juglans regia, and GhRHM from Gossypium hirsutum Linn). CsRHMb and CsRHMc
272
were grouped into cluster II, which consisted of dicot RHMs from Vitis vinifera,
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Glycine max, Gossypium hirsutum, Ricinus communis, and A. thaliana (Fig. 2 and Fig.
274
S1).
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Multiple sequence alignment revealed that the angiosperm RHMs were highly
276
homologous at the protein level. Although CsRHMa, CsRHMb, and CsRHMc were
277
distributed in different branches of the phylogenetic tree, the three CsRHM proteins
278
showed 84.2%–89.8% sequence identity. Moreover, the CsRHM proteins and
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AtRHMs displayed 80.7%–87.4% similarity (Table S3). Plant RHM proteins not only
280
shared high identity on the amino acid level, but also contained two functional units
281
(the N-terminal and the C-terminal) (Fig. 3 and Fig. S2). In vitro enzyme analysis
282
revealed that the N-terminal region of AtRHM2 displayed UDP-Glc 4,6-dehydratase
283
activity, and that the C-terminal region displayed both UDP-4K6DG 3,5-epimerase
284
and UDP-4KR 4-keto-reductase activity.25 Similar to other plant RHMs, the three
285
CsRHMs also contained two functional units (Fig. 3 and Fig. S2), and each unit
286
harbored a putative NAD(P)(H)-binding motif (GxxGxxG/A) and a conserved
287
catalytic triad (YxxxK) motif (Fig. 3 and Fig. S2). The above mentioned analysis
288
suggests that the three CsRHMs are trifunctional enzymes that convert UDP-Glc to
289
UDP-Rha.
290
3.2 Identification of CsRHMs catalyzing UDP-L-rhamnose biosynthesis
291
The ORF sequences of the three CsRHM genes were first expressed with a MBP
292
tag in E. coli. Subsequently, the three recombinant proteins were purified through
293
affinity chromatography using amylose resin. The MWs of the three recombinant
294
CsRHM proteins were all approximately 114 kDa, and degradation fragments were
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not detectable in the SDS-PAGE gel (Fig. S3). This was consistent with the predicted
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MWs of CsRHMa (~75.75 kDa), CsRHMb (~75.91 kDa), and CsRHMc (~75.65 kDa)
297
plus the MBP tag (~42.5 kDa).
298
To evaluate the biochemical activities of the three recombinant proteins, a
299
standard enzyme activity assay was performed at 35 °C for 1 h with a mixture
300
containing 3 mM NAD+, 3 mM NADPH, 3 mM UDP-Glc or 3 mM UDP-Gal, and 20
301
µg of the recombinant proteins. Subsequently, the enzyme products were analyzed
302
using reversed-phase HPLC. The results of HPLC analysis indicated that compared
303
with the control, a product peak (defined as product A) was detected through UV260
304
absorbance at the retention time of 13.2 min in the catalyzed reactions (Fig. 4A).
305
To confirm the identity of the product, approximately 10 mg of product A was
306
collected through prep-HPLC from a scaled-up reaction assay of the rCsRHMb protein.
307
Subsequently, the product was lyophilized and dissolved again in 500 µL of 99.95%
308
D2O; the chemical structure was analyzed using 1H-NMR and 13C-NMR spectroscopy.
309
The results of NMR spectroscopic analysis of product A are summarized in Table 1
310
and Fig. S4. Representative spectral signals δH 5.980 (1H,d,8.4) and δH 7.961 (1H,d,8.4)
311
corresponded to the H5 and H6 of uracil. δH 6.000 (1H,d,3.6), δH 4.387 (1H,dd, 3.0,1.6),
312
δH 4.240 (1H,dd,3.0,1.8), δH 4.291 (1H,dd, 2.4,2.4), and δH 4.208 (1H,d,2.4)
313
corresponded to the H1–5 of ribose, respectively. Similarly, δH 5.210 (1H,d,1.6), δH
314
4.102 (1H,dd, 3.0,1.6), δH 3.643 (1H,dd,3.0,2.4), δH 3.385 (1H,dd, 9.6,7.8), δH 3.453
315
(1H,dd, 6.0,3.0), and δH 1.326 (3H,d,6.0) corresponded to the H1–6 of rhamnose,
316
respectively. Moreover, the representative spectral signals δC 165.99, δC 151.47, δC
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141.50, and δC 102.48 corresponded to the C2, C4, C5, and C6 of uracil residues,
318
respectively; δC 95.33, δC 72.68, δC 83.12, δC 42.07, and δC 10.44 corresponded to the
319
C1, C2, C3, C4, and C5 of ribose residues, respectively; and δC 102.16, δC 72.08, δC
320
72.65, δC 73.66, δC 64.74, and δC 16.68 corresponded to the C1, C2, C3, C4, C5, and C6
321
of rhamnose residues, respectively. In addition, two-dimensional TOCSY and NOESY
322
(Fig. 4B) spectra showed that the representative spectral signals H1, H2, H3, H4, H5,
323
and H6 corresponded to the chemical structure of rhamnose.25 These chemical shift
324
values and coupling constants for product A were consistent with the chemical
325
structure of UDP-Rha based on a previous study.25 Overall, these results showed that
326
all three CsRHM proteins exhibited the capacity for converting UDP-Glc to UDP-Rha
327
in vitro.
328
For the biochemical characterization of the three rCsRHM proteins, the optimum
329
pH, optimum temperature, and kinetic parameters were obtained by measuring
330
enzyme activities with UDP-Glc as a substrate. These results showed that rCsRHMa
331
and rCsRHMb displayed maximum activity from pH 8.0 to 9.5 (Fig. 5A,B) and
332
temperature 35 to 50 °C (Fig. 5E,F). However, rCsRHMc showed lower pH optima
333
(7.5–8.5) and temperature optima (25–35 °C) than rCsRHMa and rCsRHMb (Fig.
334
5C,G).
335
Kinetic parameters were calculated using an appropriate non-linear regression
336
software Hyper 32.32 The Km values of the three rCsRHM proteins were 448.5 ± 62,
337
201.0 ± 33, and 969.2 ± 137 µM, with Vmax values of 83.33 ± 15, 112.11 ± 17, and
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55.56 ± 11 µM·min−1, respectively (Table 2). Kinetic analysis showed that rCsRHMa
339
and rCsRHMb displayed higher catalytic efficiency than rCsRHMc.
340
3.3 Expression patterns of CsRHM genes in tea plants
341
In this study, to characterize the functions of CsRHMa, CsRHMb, and CsRHMc,
342
their relative expression levels in different tea organs (including buds, first leaves,
343
second leaves, third leaves, fourth leaves, mature leaves, old leaves, young stems,
344
tender roots, and flowers) were analyzed through semiquantitative RT-PCR analysis.
345
Fig. 6B shows the differential expression levels of the three CsRHM genes in
346
different tissues. The three genes (i.e., CsRHMa, CsRHMb, and CsRHMc) displayed
347
similar expression profiles and were highly expressed in tender tissues (including first,
348
second, and third leaves; young stems; and tender roots; Fig. 6B). Although the
349
expression pattern of CsRHMc was similar to the patterns of CsRHMa and CsRHMb,
350
the transcript levels of CsRHMa (30 cycles) and CsRHMb (30 cycles) were
351
predominantly higher than that of CsRHMc (36 cycles) in all tea organs (Fig. 6B).
352
Intriguingly, CsRHMb was not only detected in young leaves, stems, and roots but
353
also specifically expressed in flowers (Fig. 6B).
354
3.4 Metabolite profiles of nucleotide sugars and mucilage
355
The major nucleotide sugar compounds UDP-Glc, UDP-Gal, and UDP-Rha in
356
different tea organs were measured through HPLC. The results indicated that
357
accumulations of UDP-Glc, UDP-Gal, and UDP-Rha were highest in buds and young
358
leaves (first, second, and third leaves), followed by young stems and tender roots;
359
however, the lowest accumulation was found in mature leaves, old leaves, and flowers
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(Fig. 6C), which was relatively consistent with the transcript profile of the three
361
CsRHM genes.
362
Mucilage is a complex mixture composed of pectic polysaccharides; it can be
363
stained with ruthenium red and conveniently visualized under a light microscope. In
364
this study, to investigate the accumulation profiles and histochemical localization of
365
mucilage in tea plant samples, sections of different tea organs (including second leaves,
366
old leaves, young stems, tender roots, and the upper and lower epidermis of second
367
leaves) obtained from freehand sectioning were stained with ruthenium red (Fig. 6D).
368
After staining, the red-stained mucilage was visualized using a light microscope (Fig.
369
6D). Ruthenium red staining showed that mucilage could be detected easily in almost
370
all tissues. Compared with mucilage accumulation in old leaves, mucilage
371
accumulation was higher in tender organs, such as second leaves, young stems, and
372
tender roots. Moreover, the red-stained mucilage was specifically accumulated in the
373
intercellular space and primary cell wall (Fig. 6D). Overall, the transcript profiles of
374
the three CsRHM genes were consistent with the metabolite patterns of nucleotide
375
sugar compounds and the accumulation profiles of mucilage (Fig. 6B,C). These results
376
suggest that the three CsRHM genes may contribute to the biosynthesis of UDP-Rha
377
and may be involved in the formation of primary walls in C. sinensis.
378
4. Discussion
379
4.1 Protein motif analysis of the plant RHM family
380
In bacteria, a gene cluster consisting of RmlA, RmlB, RmlC, and RmlD genes is
381
responsible for the biosynthesis of dTDP-Rha from dTDP-Glc19-22 (Fig. 1A). In fungi,
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two genes UG4,6-Dh and U4k6dG-ER are responsible for the biosynthesis of
383
UDP-Rha from UDP-Glc23 (Fig. 1B). In plants, RHM1, RHM2/MUM4, and RHM3
384
genes from A. thaliana encode a trifunctional enzyme.25 Recombinant AtRHM
385
proteins display UDP-Glc 4,6-dehydratase; UDP-4K6DG 3,5-epimerase; and
386
UDP-4KR 4-keto-reductase activities and convert UDP-Glc to UDP-Rha in vitro.25 In
387
this study, we expressed and purified three CsRHM proteins from E. coli and found
388
that the three proteins showed trifunctional activity and converted UDP-Glc to
389
UDP-L-rhamnose in vitro.
390
Takuji Oka, Nemoto, and Jigami proved that the N-terminal region of RHM2
391
(1–370 amino acids) shows UDP-Glc 4,6-dehydratase activity, and that the C-terminal
392
region of RHM2 (371–667 amino acids) shows both UDP-4K6DG 3,5-epimerase and
393
UDP-4KR 4-keto-reductase activities.25 Amino acid sequence alignment (Fig. S2)
394
revealed that RmlB and UG4,6-Dh were similar to the N-terminal region of RHM and
395
CsRHMs, and all of them contained one NADPH-binding motif (GxxGxxG) and one
396
NAD+ binding site (YxxxK). The aforementioned similar structures and conserved
397
sites maybe the reason why they have similar function with dehydratase. Similarly,
398
U4k6dG-ER of fungi displayed higher identity with the C-terminal region of RHM
399
and CsRHMs and contained one NADPH-binding motif (GxxGxxG) and one NAD+
400
binding site (YxxxK) (Fig. S2). Such high sequence similarity explains the similarity
401
in epimerase and reductase functions.
402
4.2 CsRHMs are probably responsible for the biosynthesis of UDP-Rha in tea
403
plants
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UDP-Rha is not only a rhamnose donor in the biosynthesis of various
405
L-rhamnose-containing
natural
compounds
(flavonoids,
terpenoids,
and
406
saponins)15,33,34 but also an essential compound for the biosynthesis of the primary cell
407
wall components RG-I and RG-II.8,35
408
A. thaliana epidermal cells in the seed coat provide a classical model for
409
exploring genes involved in cell wall production.36 Upon hydration, the mature seed
410
coat releases a sticky capsule of mucilage, which can be stained with ruthenium red
411
and conveniently visualized under a light microscope.37 In addition, mucilage
412
represents a readily accessible source of pectins that is not required for normal plant
413
growth and development under laboratory conditions.31 Hence, the produced mucilage,
414
which is composed of cell wall polysaccharides, provides a valuable system to
415
investigate the genes related to pectin biology.36-40
416
A previous study demonstrated the overexpression of the AtRHM1 gene, which
417
caused the rhamnose content to significantly increase in the leaf cell wall of transgenic
418
lines compared with that of wild-type lines.41 Defective seed mucilage in the MUM4-1
419
and MUM4-2 mutants and rhm2 T-DNA insertion mutants suggested that the
420
AtRHM2/MUM4 gene is responsible for the biosynthesis of UDP-Rha in vivo, and that
421
the AtRHM2 protein is essential for the biosynthesis of seed coat mucilage, which is
422
mainly composed of pectins.26,35
423
In this study, an expression analysis demonstrated that CsRHMa, CsRHMb, and
424
CsRHMc genes shared similar expression profiles and were highly expressed in the
425
tender tissues of tea plants (Fig. 6B). Additionally, metabolite profiles showed that
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UDP-Rha accumulated at low levels in mature leaves and old leaves but at a very high
427
level in shoots (Fig. 6C). The tender tissues of tea plants accumulate a large amount of
428
the flavonol rhamnoside. In addition, as a rhamnose residue donor, UDP-Rha is
429
required for the biosynthesis of rhamnoside. In this study, the expression profiles of
430
the three CsRHM genes were correlated with the accumulation patterns of main
431
nucleotide diphosphate sugars (including UDP-Glc, UDP-Gal, and UDP-Rha) (Fig.
432
6B,C). We suggest that the CsRHMs are likely to participate in the formation of these
433
secondary metabolites; however, the content of the secondary metabolites is directly
434
related to the bitter taste of tea. The results revealed that the ruthenium red-stained
435
mucilage accumulated in larger quantities in shoots and was specifically located in the
436
intercellular space and primary cell wall (Fig. 6D). Thus, enzyme activity analysis,
437
transcript profiles, metabolite profiles, and mucilage location suggest that the CsRHM
438
genes are likely to be involved in the biosynthesis of UDP-Rha and participate in the
439
formation of primary cell walls in tea plants.
440
ABBREVIATIONS USED
441
UDP, uridine diphosphate; RHM, UDP-rhamnose synthase; UDP-Glc, UDP-D-glucose;
442
UDP-Rha, UDP-rhamnose; HGA, homogalacturonan; RG-I, rhamnogalacturonan-I; RG-II,
443
rhamnogalacturonan-II; IPTG, isopropyl-β-D-thiogalactopyranoside; NMR, nuclear magnetic
444
resonance; TOCSY, total correlation spectroscopy; NOESY, nuclear overhauser effect spectroscopy;
445
GAPDH,
446
chromatography; MWs, molecular weights; MBP, Maltose-binding protein;
447
AUTHOR INFORMATION
glyceraldehyde-3-phosphate
dehydrogenase;
HPLC,
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high-performance
liquid
Journal of Agricultural and Food Chemistry
448
Corresponding Authors
449
Telephone: 86-551-65786003. Fax: 86-551-65785833. E-mail:
[email protected].
450
Telephone: 86-551-65786232. Fax: 86-551-65785729. E-mail:
[email protected]
451
Author Contributions
452
Conceived and designed the study: Liping Gao, Tao Xia, Xinlong Dai. Drafted the manuscript:
453
Liping Gao, Xinlong Dai, Yajun Liu, Yinglin Wu. Performed the experiments: Xinlong Dai, Guifu
454
Zhao, Tianming Jiao. Analyzed the data: Liping Gao, Xinlong Dai, Xiaolan Jiang. Contributed
455
reagents, materials, and analysis tools: Xinlong Dai,Yinglin Wu. All authors have read and approved
456
the final manuscript.
457
Notes
458
The authors declare no competing financial interests.
459
ACKNOWLEDGMENTS
460
This work was supported by the Natural Science Foundation of China (31470689 and
461
31570694). The Natural Science Foundation for Higher Education of Anhui Province (KJ2017A441)
462
and the Natural Science Foundation of Suzhou University (2016jb02). Anhui Major Demonstration
463
Project for the Leading Talent Team on Tea Chemistry and Health, the Chang-Jiang Scholars and the
464
Innovative Research Team in University (IRT1101). The funders had no role in the study design, data
465
collection and analysis, decision to publish, or preparation of the manuscript. We thank Prof. Jingwei
466
Hu for assistance with NMR spectroscopic analysis. This manuscript was edited by Wallace
467
Academic Editing.
468 469
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Supporting Information.
471
Figure S1. RHM family phylogenetic tree; Figure S2. Multiple sequence alignment of the RHM
472
proteins from various species at the amino acid level; Figure S3. SDS-PAGE of full-length rCsRHM
473
proteins; Figure S4. NMR spectroscopic analysis of product A; Table S1. Specific primers used in
474
this study; Table S2. Basic information of three CsRHM genes; Table S3. Sequence identities of
475
CsRHM protein.
476
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FIGURE CAPTION
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Figure 1. Chemical reaction and structural formula of the dTDP-Rha and UDP-Rha
601
biosynthetic pathways in bacteria, fungi, and plants. (A) In bacteria, dTDP-Rha is produced from
602
dTDP-Glc by three proteins: dTDP-Glc 4,6-dehydratase (RmlB), dTDP-4K6DG 3,5-epimerase
603
(RmlC), and dTDP-4KR 4-keto-reductase (RmlD). (B) In fungi, UDP-Rha is produced from
604
UDP-Glc by two proteins 4,6-dehydratase (UG4,6-Dh) and bifunctional 3,5-epimerase and
605
4-reductase (U4k6dG-ER). (C) In plants, UDP-Rha is produced from UDP-Glc by a trifunctional
606
RHM protein. UDP-Rha is further used in the synthesis of cell wall polysaccharides and
607
L-rhamnose-containing natural compounds.
608
Figure 2. RHM family phylogenetic tree. An unrooted phylogenetic tree was constructed using
609
MEGA 5.0 software through the neighbor-joining method. These RHM sequences were clustered
610
into five groups, including alga (blue), pteridophyta (purple), bryophyta (light blue), plant cluster I
611
(red), and plant cluster II (green). Three CsRHMs are clustered in plant cluster I and plant cluster II
612
and are indicated with triangles. Sequence information of the RHMs is shown in Table S2.
613
Figure 3. Multiple alignment of the amino acid sequences of CsRHMs with AtRHM2.
614
Conserved residues between the CsRHMs and AtRHM2 are indicated with a black column. The
615
putative highly conserved NAD(P)+ cofactor-binding (GxxGxxG/A) and active-site catalytic couple
616
(YxxxK) motifs are indicated above the sequence alignment.
617
Figure 4. Molecular identification of the enzyme reaction products of CsRHM proteins. (A)
618
HPLC analysis of the products from UDP-Glc of three purified rCsRHM proteins. The substrates and
619
corresponding products were detected through HPLC on UV260 nm absorbance. (a-1), control. (a-2),
620
(a-3), and (a-4) indicate the enzyme reaction products of the three CsRHM recombinant proteins
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621
rCsRHMa, rCsRHMb, and rCsRHMc, respectively. (a-5), UDP-Rha used as a standard. (B)
622
Structural identification of product A from UDP-Glc of the three CsRHM proteins. (b-1), TOCSY
623
spectra of product A. (b-2), NOESY spectra of product A. (b-3), the chemical structure of UDP-Rha.
624
Figure 5. Optimum condition of reactions catalyzed by three rCsRHMs. Each reaction mixture
625
(100 µL) was incubated with 3 mM UDP-Glu, 3 mM NAD, and 3 mM NADPH at different ranges of
626
pH (5.0–11.0) and temperature (20–65 °C) for 30 min. (A) Effect of reaction pH on activities of the
627
rCsRHMa. (B) Effect of reaction pH on activities of the rCsRHMb. (C) Effect of reaction pH on
628
activities of the rCsRHMc. (E) Effect of reaction temperature on activities of the three rCsRHMa. (F)
629
Effect of reaction temperature on activities of the three rCsRHMb. (G) Effect of reaction temperature
630
on activities of the three rCsRHMc. Data are presented as the average mean of three independent
631
trials ± SD. Buffer 1: 100 mM acid-sodium citrate buffer (pH 5.0–8.0); Buffer 2: 100 mM Tris–HCl
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buffer (pH 7.0–8.0); Buffer 3: 100 mM phosphate buffer (pH 7.0–10.0); and Buffer 4: 100 mM
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Na2CO3/NaHCO3 buffer (pH 9.0–11.0).
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Figure 6. Expression profiles of three CsRHM genes and accumulation profiles of nucleotide
635
sugars in different tea organs. (A) The tea samples (including buds, first leaves, second leaves,
636
third leaves, fourth leaves, mature leaves, old leaves, young stems, tender roots, and flowers) used
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for gene expression and total nucleotide sugar content analysis. (B) The transcript analysis of
638
CsRHMs in various tissues. (C) The content analysis of UDP-Gal, UDP-Glc, and UDP-Rha in
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different organs; all data points are the mean of three biological replicates, and each error bar
640
indicates the SD. (D) Histochemical localization of mucilage in different tea organs and tea callus.
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Pink represents mucilage-accumulating areas after 0.01% (w/v) ruthenium red staining; (a and g, b
642
and h, c and i, and d and j) the transverse sections of the second leaves, old leaves, young stems, and
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tender roots, respectively, before and after staining; (e and k as well as f and i) the upper and lower
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epidermis sections, respectively, before and after staining; (m–r) the high magnification image of
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different plant sections after staining. Abbreviations: is, intercellular space; pcw, primary cell wall.
646 647 Table 1. NMR spectroscopic data of UDP-Rha synthesized from UDP-Glu in a reaction 648 catalyzed by CsRHM (D2O, δ in ppm, J in Hz) 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683
Table 2. Kinetic parameters of recombinant CsRHMa, CsRHMb, and CsRHMc proteins
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684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727
Figure 1.
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Figure 3.
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Figure 5.
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