Subscriber access provided by University of Newcastle, Australia
Article
Molecular cloning and characterization of galactinol synthases in Camellia sinensis with different responses to biotic and abiotic stressors Yu Zhou, Yan Liu, Shuangshuang Wang, Cong Shi, Ran Zhang, Jia Rao, Xu Wang, Xungang Gu, Yunsheng Wang, Daxiang Li, and Chaoling Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00377 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 11, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
Journal of Agricultural and Food Chemistry
1
Molecular cloning and characterization of galactinol synthases in Camellia
2
sinensis with different responses to biotic and abiotic stressors
3
4
Yu Zhou†, Yan Liu†, Shuangshuang Wang, Cong Shi, Ran Zhang, Jia Rao, Xu Wang, Xungang
5
Gu, Yunsheng Wang, Daxiang Li, Chaoling Wei*
6
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130
7
Changjiang Road West, Hefei, Anhui 230036, China
8
9
†
10
*
11
E-mail:
[email protected] These authors contributed equally to this work.
Corresponding author: Chaoling Wei
12
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
13
ABSTRACT
14
Galactinol synthase (GolS) is a key biocatalyst for the synthesis of raffinose family
15
oligosaccharides (RFOs). RFOs accumulation plays a critical role in abiotic stress adaptation, but
16
the relationship between expression of GolS genes and biotic stress adaptation remains unclear. In
17
this study, two GolS genes were found to be highly up-regulated in a transcriptome library of
18
Ectropic oblique-attacked Camellia sinensis. Three complete GolS genes were then cloned and
19
characterized. Gene transcriptional analyses under biotic and abiotic stress conditions indicated
20
that the CsGolS1 isoform was sensitive to water deficit, low temperature, and abscisic acid, while
21
CsGolS2 and CsGolS3 were sensitive to pest attack and phytohormones. The gene regulation and
22
RFOs determination results indicated that CsGolS1 was primarily related to abiotic stress and
23
CsGolS2 and CsGolS3 were related to biotic stress. GolS-mediated biotic stress adaptations have
24
not been studied in depth, so further analysis of this new biological function is required.
25
KEYWORDS: Camellia sinensis, Galactinol synthase, Biofunctional investigation, Gene
26
regulation, abiotic stress, biotic stress
27
2
ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
Journal of Agricultural and Food Chemistry
28
INTRODUCTION
29
Leaf wilting, root necrosis, and nutrient deficiency are common symptoms in plants subjected to
30
biotic and abiotic stress 1. Many studies have focused on plant defense mechanisms (i.e., direct and
31
indirect defenses) with the aim of enhancing sustainable agricultural production. For example, the
32
lipoxygenase pathway, which includes two biosynthetic routes mediated by allene oxide synthase
33
or hydroperoxide lyase, was found to be an important indirect defense pathway for stress
34
adaptation in various plants2-4. The direct defense pathway includes raffinose family
35
oligosaccharides (RFOs) such as raffinose, stachyose, and verbascose that are derived from
36
sucrose and activate galactose moieties (donated by galactinol); these RFOs are extensively
37
distributed in various plants5. RFOs as plant storage carbohydrates (galactosyl-oligosaccharides)
38
were found to have important roles in abiotic stress adaptation by assisting in the formation of a
39
vitreous state that protects macromolecules6-7; RFOs are also essential for phloem transport
40
functions8. When plants are subjected to abiotic stress (e.g., cold, drought, heat, or mechanical
41
injury), RFOs accumulation occurs and osmotic pressure is regulated to maintain stable cells 1, 9-11.
42
Galactinol synthase (GolS: EC 2.4.1.123) belongs to glycosyltransferase family 8 and catalyzes
43
the
44
(UDP-D-galactose) to myo-inositol12. GolS was first detected in a crude extract of maturing pea
45
seeds13 and was partially purified from mature cucumber14; the GolSs from legume seeds and
46
cucurbit leaves were the first to biochemical characterized15. Recently, GolS genes have been
47
isolated and characterized from various plant varieties including hybrid poplar 11, Brassica napus
48
L.16, Coffea arabica L.1, Salvia miltiorrhiza17, grape18, Medicago falcate19, chestnut20, Cicer
49
arietinum L.21, and many others. Most studies showed that GolS expression was regulated by
50
various abiotic stressors (cold, drought, or heat) and that GolS activity enhanced plant tolerance to
51
abiotic stress1, 11, 16-20. While few studies have focused on the relationship between GolS gene
52
expression and biotic stress adaptations, GolSs might also regulate biotic stress (pest attack or
first step
of
RFOs
biosynthesis
by
converting
uridine diphosphate-D-galactose
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
53
pathogen infection) responses; the mechanisms underlying GolS-mediated biotic stress responses
54
require further investigation11, 22.
55
Tea plant (Camellia sinensis) is one of the most important commercial crops in China and other
56
Asian countries. As an evergreen woody plant, tea plants are susceptible to various biotic and
57
abiotic stressors. Drought, freezing injury, plant diseases, and pests attack are the important biotic
58
and abiotic stressors frequently affecting tea production23. Although GolSs have been isolated and
59
characterized in many plant varieties, GolS-mediated biotic and abiotic stress adaptation in C.
60
sinensis has not been previously studied, despite this process is significant to tea plant cultivation
61
and crop yields. This study, therefore, aimed to identify and characterize GolS genes in tea plant
62
that were up-regulated under biotic and abiotic stress conditions. This information would be useful
63
for further characterization of abiotic and biotic stress responses in tea plants, and to breed new
64
lines with improved resistance capabilities.
65
MATERIALS AND METHODS
66
Preparation of plant materials and treatments. Two-year-old tea plant clone cuttings (Camellia
67
sinensis ‘Shuchazao’) were obtained from the Dechang tea plantation (Shucheng, China), which
68
grown under controlled environment (12 h for day/night rhythm with 3000 lx light intensity at
69
25°C) The low-temperature groups were treated at 0°C and 4°C, and the control group was kept at
70
25 °C. A total of 15 branches for each temperature group were inserted into floral foam and
71
incubated at the specified temperatures for 3, 6, 9, 12, and 24 h. The drought group was watered
72
for 5 days and the control every 1–2 days; samples were collected and analyzed on day 5, 8, 11, 14,
73
and 17. In the pest-attacked group, tea geometrids (Ectropis oblique) at the third larval stage were
74
placed on tea plants (10 geometrids per individual tea plant). After one-third of each leaf was
75
consumed, the pests were removed and the surviving leaves were collected within 24 h. Tea plant
76
leaves from the non-attacked group were used as the control. The phytohormone groups were
4
ACS Paragon Plus Environment
Page 4 of 28
Page 5 of 28
Journal of Agricultural and Food Chemistry
77
evenly sprayed with 1.0 mM salicylic acid (SA), 100 µM abscisic acid (ABA), or 1 mM methyl
78
jasmonate (MeJA, containing 0.05% Tween-20) until the leaves were wet; cuttings sprayed with
79
0.05% Tween-20 or distilled water were used as the control. Leaves of the same age and position
80
were collected 3, 6, 12, 24, and 48 h after chemical treatments.
81
To determine the constitutive expression levels of GolS genes in different organs, apical buds,
82
developing leaves, mature leaves, young stems, roots, petals, flower buds, and fruits were
83
collected and evaluated. Unless specified otherwise, all treatments in this study were performed in
84
triplicate, and the collected samples were immediately frozen in liquid nitrogen and stored at
85
-80 °C until use.
86
Nucleotide and amino acid sequence analyses. Phylogenetic analysis of the CsGolS gene
87
sequences was performed using BLAST searches in the National Center for Biotechnology
88
Information database (http://www.ncbi.nih.gov:/BLAST/). The possible open reading frame (ORF),
89
signal peptide, theoretical isoelectric point (pI), and the molecular mass (Mw) predictions were
90
performed using online programs (ExPASy and SignalP 4.1)24-25. The putative domains were
91
identified using the InterPro database (http://www.ebi.ac.uk/interproscan/). The transmembrane
92
topology predictions of the CsGolS genes were carried out with TMHMM 2.0 software. Disulfide
93
bonds
94
(http://scratch.proteomics.ics.uci.edu/). Multiple sequence alignment of selected amino acids was
95
carried out using CLUSTAL_X (version 2.0), and the phylogenetic tree was constructed using
96
MEGA v6.0 with bootstrap values determined using 1000 replications26.
97
RNA extraction and cDNA synthesis. Total RNA was extracted from plant leaves (200 mg) using
98
an RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions.
99
The total RNA quality was examined by agarose electrophoresis and spectrophotometric
100
measurement at an absorbance ratio of A260/A280. First-strand cDNA was synthesized from total
101
RNA using a PrimeScript RT Reagent Kit (Takara) following the manufacturer's instructions.
were
predicted
in
the
Scratch
Protein
5
ACS Paragon Plus Environment
Predictor
Server
Journal of Agricultural and Food Chemistry
102
Molecular cloning of the full-length cDNAs of CsGolS genes. Full-length cDNAs of CsGolS
103
genes were cloned using a SMARTTM RACE Kit (Clontech, Dalian China) according to the
104
manufacturer's instructions. Primers used in 3′-RACE and 5′-RACE were designed to each partial
105
cDNA sequence for the three CsGolS genes initially obtained from tea plant transcription library.
106
The primer sequences (including three validating primer pairs) used in 3′-RACE and 5′-RACE are
107
P1 to P13 (Supplementary Table 1); the full-length cDNA sequences were tested using the S1000
108
PCR detection system (primers P14 to P19). The PCR conditions were 95 °C for 5 min followed
109
by 30 cycles of 94 °C for 30 s, 56 °C for 45 s, and 72 °C for 60 s. The PCR products were purified
110
by agarose electrophoresis and a DNA purification kit (Qiagen, Valencia, CA). The purified DNA
111
products were cloned into the pMD19-T Vector and transformed into Escherichia coli DH5α. The
112
recombinant plasmids were enriched and sequenced using the ABI PRISM 3730XL Sequencing
113
System (Applied Biosystems, Foster City, CA).
114
Protein expression and purification. The ORFs of CsGolS1, CsGolS2 and CsGolS3 were
115
amplified by PCR with pfu polymerase (Takara) using the cDNA generated from total RNA of
116
Camellia sinenesis leaves, and the gene-specific primers P30 to P35 were used (NotI and XhoI
117
sites underlined, Supplementary Table 1). After PCR amplification, base A was added to the end of
118
the PCR product by rTaq (Takara). The modified DNA fragments inserted into the pMD-18T
119
vector and transformed into E. coli DH5α resulting in pMD-CsGolS1, pMD-CsGolS2, and
120
pMD-CsGolS3. The cloned plasmids and pET-32a (+) were digested with the respective restriction
121
enzymes (NotI and XhoI), and the CsGolS ORFs were cloned into pET-32a (+) by ligation to
122
construct pET-CsGolS1, pET-CsGolS2, and pET-CsGolS3. After confirmation, the recombinant
123
plasmids were transformed into E. coli BL21 (DE3) pLysS cells (Novagen, Shanghai China). The
124
transformed cells were incubated in LB broth containing ampicillin (50 µg/mL) at 16, 25, and
125
37 °C overnight for optimal temperature evaluation. Until the cell density reached 0.6 (OD600
126
absorbance), the broth was induced by adding a range of Isopropyl β-D-1-thiogalactopyranoside 6
ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
Journal of Agricultural and Food Chemistry
127
concentrations (IPTG, 0.25-2.0 mM) for different periods of time (1–5 h). The optimal
128
overexpression conditions were evaluated by examining the recombinant protein yield and quality
129
using SDS-PAGE. His-tagged recombinant proteins in disrupted cells were purified by affinity
130
chromatography with nickel-nitrilotriacetic acid agarose resin (Ni-NTA, Qiagen, CA) and the
131
purified recombinant proteins were dissolved in phosphate buffer (pH 7.0).
132
Galactinol synthase activity assay. The purified recombinant proteins (CsGolSs) were used for
133
the galactinol synthase activity assay. A total reaction volume of 50 µL comprising 20.0 mM
134
myoinositol, 4.0 mM UDP-Gal, 50 mM HEPES (PH7.0), 2.0 mM dithiothreitol, 4.0 mM MnCl2,
135
4.0 µg BSA, and 10.0 µL recombinant protein (0.1 mg/mL) was obtained; the supernatant of the
136
pET-32a (+) transformant and phosphate buffer were used as negative controls 27; The enzymatic
137
reaction was performed at 30 °C for 5 h, and was stopped by the addition of 50 µL of 100%
138
ethanol. After 25.0 µg phenyl α-D-glucoside was added as internal standard, the reaction mixture
139
was incubated at 80 °C for 30 min, passed through a 10000 MW cutoff filter (NANOSEPTM
140
Microconcentrators, Pall Filtron), and evaporated to dryness under a nitrogen stream. Trace water
141
in the resulting residue was removed by phosphorus pentoxide in a desiccator overnight. The
142
thoroughly dried residue was derivatized with 200 µL trimethylsilyl imidazole: pyridine (1:1, v/v)
143
at 80 °C for 45 min, and analyzed for phenyl α-D-glucoside and galactinol using a gas
144
chromatography-mass spectrometer (GC-MS, Shimadzu, Japan) as previously described28. The
145
Zebron ZB-MultiResidue-2 capillary column (30 m × 0.25 mm i.d, 0.25 µm film thickness,
146
Guangzhou FLM Scientific Instrument Co., Ltd., Guangzhou, China) was used for analyte
147
separation.
148
Quantitative real-time PCR analysis (qRT-PCR). SYBR Green qRT-PCR amplification was
149
performed on a CFX96 Touch real-time PCR detection system (BIO-RAD, CA) with a total
150
volume of 25.0 µL containing 12.5 µL of 2× SYBR Premix Ex Taq (TaKaRa), 2.0 µL of cDNA
151
template (100 ng/µL), 0.5 µL (10 µmol/L) of forward primer, 0.5 µL (10 µmol/L) of reverse 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 28
152
primer, and 9.5 µL of Milli-Q water. Three technical replicates were run for each sample using the
153
following cycling parameters: 95 °C for 3 minutes followed by 40 cycles of 94 °C for 45 s,
154
60/55 °C for 30 s, and 72 °C for 20 s. The gene β-Actin was selected as an internal control for
155
qRT-PCR amplification and the specific primers P20 to P29 listed in Supplementary Table 1 were
156
used for qRT-PCR amplification29. The amplification efficiencies of all genes tested in this study
157
ranged from 90% to 110%. Data were analyzed according to the threshold cycle (Ct). The relative
158
changes in gene expression were quantified using the 2-∆∆Ct method30. Significant differences were
159
determined
160
(www.chinadps.net).
161
Northern blot analysis. To validate the qRT-PCR results, northern blot analyses using RNA
162
obtained from different tissues (apical buds, developing leaves, mature leaves, young stems, roots,
163
petals, flower buds, and fruits) of C. sinensis ‘Shuchazao’ without biotic or abiotic stress and RNA
164
obtained from leaves of drought-stressed plants were conducted. A total of 3 g of frozen sample
165
was ground in liquid nitrogen and RNA was isolated using an RNAprep Pure Plant Kit (QIAGEN)
166
according to the manufacturer’s instructions. Total RNA quantification was determined using a
167
QubitTM (Invitrogen, Shanghai China) and Northern blotting was performed as previously
168
described1.
169
RFOs extraction and determination. Samples treated at 4 °C for 3, 6, 12, and 24 h were used for
170
the abiotic stress group analyses; samples collected from 3, 6, 12, and 24 h after Ectropis oblique
171
attack were used for the biotic stress group analyses. For each sample, 0.3 g of fresh leaves were
172
used for the extraction of RFOs (i.e., raffinose, stachyose, and verbascose). The extraction method
173
and chromatography analyses were conducted as previously described 31.
174
Statistical analysis. Three biological replicates were analyzed for each treatment and GraphPad
175
Prism software was used for data analysis. Data are presented as means ± SD. Significant
176
differences were determined using the Student’s t-test with p values < 0.05 considered to be
using
Duncan’s
multiple
range
tests,
calculated
8
ACS Paragon Plus Environment
using
DPS
software
Page 9 of 28
Journal of Agricultural and Food Chemistry
177
statistically significant.
178
RESULTS
179
Isolation and sequence analysis of CsGolS genes. In our preliminarily studies, three EST
180
sequences were selected from an Ectropic oblique-attacked tea plant transcription library; these
181
genes were found to be similar to GolS genes using BLAST searching against the GenBank
182
database. The full-length sequences of the three CsGolS genes, designated CsGolS1, CsGolS2, and
183
CsGolS3 (1386 bp, 1574 bp, and 1350 bp, respectively) were obtained by 3' and 5' RACE-PCR.
184
The full-length sequences of CsGolS1, CsGolS2, and CsGolS3 were submitted to GenBank
185
database with the accession numbers JX624168, KP757767, and KP757768, respectively. The
186
CsGolS1 putative protein was 339 aa long with a calculated MW of 38.97 kDa and a theoretical pI
187
of 5.20. The CsGolS2 putative protein was 338 aa long with a calculated MW of 38.69 kDa and a
188
theoretical pI of 4.96. The CsGolS3 putative protein was 326 aa long with a calculated MW of
189
37.80 kDa and a theoretical pI of 4.80. No signal peptide was observed in the three CsGolSs by
190
analyzing the protein sequence with SignalP 4.1 and no internal transmembrane segment was
191
found by the transmembrane topology predictions. Scratch protein predictor analysis showed that
192
no disulfide bonds were detected for these proteins, and the ratio of helix, strand, and loop in the
193
secondary structure was 23.89:10.91:65.19 for CsGolS1, 23.67:10.06:66.27 for CsGolS2, and
194
29.14:11.96:58.90 for CsGolS3.
195
Multiple sequence alignment and phylogenetic analysis. The BLASTp analysis showed that the
196
aa sequence of CsGolS1 and CsGolS2 displayed the highest homology (85.0% and 84.7% identity)
197
with a GolS protein from Coffea arabica (GenBank no. ADM92588), while CsGolS3 displayed
198
the highest homology (77.9% identity) with a GolS protein from Manihot esculenta (GenBank no.
199
AGC51778). Moreover, 73.1% identity was observed between CsGolS1 and CsGolS3, and 70.5%
200
identity was observed between CsGolS2 and CsGolS3. The protein sequences of CsGolS1 and
9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
201
CsGolS2 showed the highest similarity (92%) to each other. Multiple sequence alignment of the
202
three CsGolSs with highly homologous GolS proteins from Coffea arabica and Manihot esculenta
203
indicated that the three functional domains of the CsGolSs were conserved in GolS proteins
204
(Figure 1). The active residues in the three functional domains consisted of a manganese-ligation
205
motif (DXD), a conserved serine (S) phosphorylation site, and a typical hydrophobicity
206
pentapeptide (APSAA) at the c-terminal.
207
The phylogenetic analysis showed that GolSs from different plants examined in this study were
208
evenly distributed in four major classes (Figure 2). CsGolS3 clustered in Class Ι and was closely
209
related to CaGolS2, while CsGolS1 and CsGolS2 clustered in Class III and were closely related to
210
CaGolS1. We, therefore, hypothesized that CsGolS3 was likely to possess different functions from
211
CsGolS1 and CsGolS2. The result of this phylogenetic analysis was consistent with the multiple
212
sequence alignment of the CsGolS aa sequences that showed that, for the manganese-ligation
213
motif, CsGolS3 had the aa residues DGD while CsGolS1 and CsGolS2 had DAD. CaGolS1 (Class
214
III) has previously been shown to be constitutively expressed under natural field conditions and
215
was sensitive to various environmental stresses, while expression of CaGolS2 (Class Ι) was not
216
detected unless the plants were cultivated under extreme drought conditions or in a high salt
217
environment1.
218
Production of CsGolS recombinant protein and its hydrolytic activities. Three CsGolS genes
219
were efficiently expressed as a soluble protein fraction in E. coli BL21 (DE3) pLysS cells. The
220
recombinant protein yield and quality results indicated that the optimum expression condition was
221
25 °C with 0.25 mg/mL IPTG induction for 3 h. The apparent MW of recombinant CsGolS proteins
222
was about 50 kDa, and the MW of fusion proteins (including Trx A, His-Tag, and S-Tag
223
recombinant proteins) determined by SDS-PAGE was slightly bigger than the theoretical MW
224
described above (Figure 3).
225
Enzymatic activity assays showed that when the substrates of myoinositol and UDP-Gal existed in 10
ACS Paragon Plus Environment
Page 10 of 28
Page 11 of 28
Journal of Agricultural and Food Chemistry
226
the reaction systems, galactinol was synthesized by fusion proteins of CsGolS1, CsGolS2, and
227
CsGolS3 (Figure 4), indicating that the three CsGolS fusion proteins showed GolS activity.
228
Further quantitative determination showed that the specific activity of the CsGolS1, CsGolS2, and
229
CsGolS3 fusion proteins was 0.19 µmol/min·mg, 0.08 µmol/min·mg, and 0.30 µmol/min·mg,
230
respectively.
231
CsGolSs constitutive expression levels in different tea plant organs. The CsGolSs constitutive
232
expression results showed that three CsGolSs genes had quite different transcription patterns
233
(Supplemental Figure 1). The highest level of CsGolS1 transcription was detected in mature leaves
234
and the next highest in roots; the highest transcription level of CsGolS2 was detected in flower
235
buds followed by mature leaves; and the highest transcription level of CsGolS3 was detected in
236
fruits followed by mature leaves. For all three genes, the transcription levels in other organs were
237
very low. As the three CsGolS genes were constitutively expressed in the mature leaves, this tissue
238
was selected for expression analyses in the subsequent abiotic and biotic stress studies.
239
Northern blot validation for CsGolS1 transcription. For the validation of CsGolS expression
240
patterns determined using qRT-PCR, CsGolS1 transcription levels in different organs under natural
241
field conditions and CsGolS1 transcription levels in mature leaves under drought stress were
242
further examined by Northern blotting. These results showed that CsGolS1 expression in mature
243
leaves was much higher than in the other organs, with the next highest expression observed in
244
roots; the level of CsGolS1 expression in other organs was low. These results were completely
245
consistent with those obtained using qRT-PCR (Supplemental Figure 1, Supplemental Figure 2A).
246
Under drought stress, CsGolS1 transcription levels determined by Northern blotting were also
247
consistent with those determined using qRT-PCR, with expression decreasing for the first 5 days,
248
highly up-regulated on the day 14, and then recovered to the same level as the control day 17
249
(Supplemental Figure 2B, Figure 5A). The Northern blot results in different organs and
250
environmental conditions indicated that the tea plant CsGolSs transcription levels determined by 11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
251
qRT-PCR in this study were reliable.
252
Abiotic and biotic stress regulations of CsGolS expression in tea plant. Under drought stress,
253
the expression of CsGolS1 was down-regulated for the first 5 days (0.3-fold), returned to the level
254
of the control on d 11, was rapidly up-regulated 2.6-fold (compared with the control) on d 14, and
255
returned to control levels on d 17 (Figure 5A). Under low-temperature stress, the expression of
256
CsGolS1 was up-regulated 26.6-fold (compared with the control) after 6 h at 0 °C, and 30-fold
257
after 3 h at 4 °C; these time points had the highest CsGolS1 expression for each respective
258
temperature treatment. After the maximum expression points, CsGolS1 expression decreased
259
rapidly at both temperatures and reached the control level at 9 h (Figure 5B). These results
260
indicated that low-temperature stress of 4 °C resulted in a more rapid change in CsGolS1
261
expression than 0 °C. The expression levels of CsGolS2 and CsGolS3 did not change significantly
262
with low temperatures or drought stress (data not shown).
263
In the pest-attacked group, CsGolS1 showed only limited up-regulation in the first 3 h, and then
264
rapidly returned to control levels. Comparatively, CsGolS2 expression was very sensitive to pest
265
attack, with its expression upregulated 5.8-fold on 6 h after pest attack; CsGolS2 expression
266
quickly returned to control levels between 6 and 12 h. Similar to CsGolS2, the expression of
267
CsGolS3 was up-regulated 4.2-fold after 6 h and then rapidly returned to the control level after 9 h
268
(Figure 5C). These results suggested that the CsGolS2 and CsGolS3 isoforms were much more
269
sensitive to pest attack than the CsGolS1 isoform.
270
The expression patterns of CsGolS1 and CsGolS2 in response to ABA were similar, with both
271
slightly down-regulated in the first 3 h before being steadily up-regulated until 24 h, while the
272
expression of CsGolS3 was consistently up-regulated from 0 to 9 h before rapidly returning to
273
control levels (Figure 5E). Of the three genes, CsGolS2 expression was most sensitive to SA
274
(Figure 5D), suggesting that it may play a role in the disease response. Compared to SA and ABA,
275
the phytohormone MeJA had limited effects on CsGolS1 and CsGolS2 expression, while CsGolS3 12
ACS Paragon Plus Environment
Page 12 of 28
Page 13 of 28
Journal of Agricultural and Food Chemistry
276
expression was sensitive to MeJA and increased 6.3-fold after 3 h before rapidly returning to
277
control levels (Figure 5F). The phytohormone treatments showed that CsGolS1 and CsGolS2 had
278
similar responses to ABA and MeJA while CsGolS2 and CsGolS3 had similar responses to SA.
279
Tea plant RFOs change under abiotic and biotic stresses. Under low-temperature stress, the
280
raffinose concentration began to increase at 6 h, reached the maximum concentration of 0.367
281
mg/g fresh leaves at 12 h (p < 0.05), and then returned to the level of the control by 24 h (Figure
282
6A). Conversely, verbascose was not detected in control or low temperature-treated samples for
283
the first 6 h; at 12 h the maximum verbascose concentration of 0.051 mg/g fresh leaves was
284
observed before levels reduced slightly at 24 h (Figure 6C). With low-temperature treatment
285
raffinose levels increased significantly only at 12 h, while verbascose levels were significantly
286
higher only at 12 and 24 h. Under pest attack stress, the raffinose concentration began to increase
287
after 3 h hour and quickly increased to the maximum concentration of 0.922 mg/g fresh leaves at 6
288
hour. After 6 h, the raffinose concentration reduced to approximately the same level as at 3 h and
289
was still significantly higher than in the control (Figure 6B). Verbascose was significantly higher
290
on 6 h after pest attack and increased continuously to 24 h (0.078 mg/g, Figure 6D). Stachyose was
291
not detected in any samples examined (Figure 6). Comparing the RFO results of the
292
low-temperature experiment to the pest attack experiment indicated that RFO synthesis was
293
regulated by both abiotic and biotic stress, but that biotic stress had a greater effect than abiotic
294
stress.
295
DISCUSSION
296
Many studies have shown that the abiotic stressors can regulate the expression of GolS genes, and
297
therefore, result in RFOs accumulation. RFOs are important carbohydrate storage and assist plants
298
to overcome adverse environments1, 11, 16-20. GolS genes expression was regulated by pest attack in
299
hybrid poplar, suggesting that RFOs may also be involved in combatting biotic stress
13
ACS Paragon Plus Environment
11, 22.
Journal of Agricultural and Food Chemistry
300
However, only a few studies have focused on the relationships between GolS expression and plant
301
biotic stress adaptations, and the mechanism of GolS-mediated biotic stress adaptation has not
302
been characterized in depth. In this study, two significantly up-regulated GolS genes (CsGolS2 and
303
CsGolS3) were identified in the transcriptome library of Ectropic oblique-attacked tea plant.
304
Therefore, the CsGolS regulation in the tea plant was considered to be important biological agent
305
for adaptation to biotic stress. On the basis of this assumption, three GolS isoforms were cloned
306
and characterized from tea plant and their expression in response to abiotic and biotic stressors
307
was investigated.
308
The transcript regulation analyses under abiotic and biotic stress showed that the expression level
309
of CsGolS1 was significantly affected by abiotic stresses (water deficiency, low temperature, and
310
ABA treatment); conversely, other than moderate up-regulation following SA treatment, CsGolS1
311
expression did not change significantly with the biotic stressors of pest attack and MeJA. This
312
suggests that the CsGolS1 isoform is primary related to abiotic stress responses, as well as being
313
somewhat responsive to specific biotic stress (i.e., tea plant disease). The result of CsGolS1
314
regulation was consistent with the majority of GolS studies in other plants11, 17-18, 20. In contrast to
315
CsGolS1 isoform, the expression of CsGolS2 and CsGolS3 showed only minor changes with the
316
abiotic stressors of water deficiency and low temperature. The expression of CsGolS2 and
317
CsGolS3 was significantly regulated by biotic stressors including Ectropic oblique attack, SA
318
treatment (related to plant disease), and MeJA treatment (related to pest attack). This suggests that
319
the CsGolS2 and CsGolS3 isoforms should primarily relate to biotic stress adaptation, which is
320
consistent with the new biological role hypothesized in hybrid poplar22. RFOs concentrations in
321
samples subjected to abiotic and biotic stresses also supported the CsGolSs regulation results, the
322
RFO synthesis was significantly up-regulated by pest attack and that the influence of biotic stress
323
was more important than low temperature as abiotic stress. Taken together, these results suggested
324
that the CsGolS1 isoform was primarily related to abiotic stress and CsGolS2 and CsGolS3
14
ACS Paragon Plus Environment
Page 14 of 28
Page 15 of 28
Journal of Agricultural and Food Chemistry
325
isoforms were closely related to biotic stress. Interestingly, ABA treatment displayed significantly
326
upregulated CsGolS2 and CsGolS3 expression, suggesting that these two isoforms might also be
327
related to some unknown abiotic stress, although the two isoforms did not respond to the abiotic
328
stressors of water deficiency and low temperature. The results of gene regulation and RFOs
329
determination were not consistent with the phylogenetic analysis, which suggested that CsGolS3
330
may have a different biological role from CsGolS1 and CsGolS2, but the actual reasons are need
331
to be further investigated. The role of the three isoforms in biotic stress responses in C. sinensis
332
represents a new biofunctional role for the GolS family in tea plant.
333
Acknowledgements
334
We would like to thank the native English speaking scientists of Elixigen Company (Huntington
335
Beach, California) for editing our manuscript.
336
Funding
337
This work was supported by the National Natural Science Foundation of China (NSFC no.
338
31171608, 31671949, 31301248), the Changjiang Scholars and Innovative Research Team in
339
University (grant number IRT1101), the Anhui Natural Science Foundation (1608085J08,
340
1608085QC57), and the Project of Universities Leading Talent Team of Nutrition and Safety of
341
Agricultural Products in Anhui Province.
342
Supporting Information Available
343
Supplemental Table 1. Primers used in this study.
344
Supplemental Figure 1. Expression of CsGolS genes in different tea plant organs grown under
345
field conditions determined using qRT-PCR. Data represent the means ± SD (n = 3) of three
346
biological replicates. Different letters above bars represent significant differences at p < 0.05.
347
Supplemental Figure 2. CsGolS1 transcription level validation by Northern blotting. A, CsGolS1
348
transcripts in different tea plant tissues; B, CsGolS1 transcripts in mature leaves following drought 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
349
stress.
350
References
351
[1] dos Santos, T.B.; Budzinski, I.G.; Marur, C.J.; Petkowicz, C.L.; Pereira, L.F.; Vieira, L.G.
352
Expression of three galactinol synthase isoforms in Coffea arabica L. and accumulation of
353
raffinose and stachyose in response to abiotic stresses. Plant Physiol. Biochem. 2011, 49, 441-448.
354
[2] Halitschke, R.; Ziegler, J.; Keinanen, M.; Baldwin, I.T. Silencing of hydroperoxide lyase and
355
allene oxide synthase reveals substrate and defense signaling crosstalk in Nicotiana attenuate.
356
Plant J. 2004, 40, 35-46.
357
[3] De Moraes, C.M.; Mescher, M.C.; Tumlinson, J.H. Caterpillar-induced nocturnal plant
358
volatiles repel conspecific females. Nature 2001, 410, 577-580.
359
[4] Kessler, A.; Baldwin, I.T. Defensive function of herbivore-induced plant volatile emissions in
360
nature. Science 2001, 291, 2141-2144.
361
[5] Lehle, L.; Tanner, W. Synthesis of raffinose-type sugars, In V Ginsberg, ed, Methods in
362
Enzymology, Complex Carbohydrates Part B, Academic Press, New York, 1972, 28, 522-529.
363
[6] Leopold, A.C.; Sun, W.Q., Bernal-Lugo, I. The glassy state in seeds: analysis and function.
364
Seed Sci. Res. 1994, 4, 267-274.
365
[7] Hincha, D.K.; Zuther, E.; Heyer, A.G. The preservation of liposomes by raffinose family
366
oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions.
367
Biochim. Biophys. Acta 2003, 1612, 172-177.
368
[8] Ayre, B.G.; Keller, F.; Turgeon, R. Symplastic continuity between companion cells and the
369
translocation stream: long-distance transport is controlled by retention and retrieval mechanisms in
370
the phloem. Plant Physiol. 2003, 131, 1518-1528.
371
[9] Maruyama, K.; Takeda, M.; Kidokoro, S.; Yamada K., Sakuma Y., Urano K., Fujita M.,
16
ACS Paragon Plus Environment
Page 16 of 28
Page 17 of 28
Journal of Agricultural and Food Chemistry
372
Yoshiwara K., Matsukura S., Morishita Y., Sasaki R., Suzuki H., Saito K., Shibata D., Shinozaki
373
K., Yamaguchi-Shinozaki K. Metabolic pathways involved in cold acclimation identified by
374
integrated analysis of metabolites and transcripts regulated by DREB1A and DREB2A. Plant
375
Physiol. 2009, 150, 1972-1980.
376
[10] Nishizawa, A.; Yabuta, Y.; Shigeoka, S. Galactinol and raffinose constitute a novel function to
377
protect plants from oxidative damage. Plant Physiol. 2008, 147, 1251-1263.
378
[11] Unda, F.; Canam, T.; Preston, L.; Mansfield, S.D. Isolation and characterization of galactinol
379
synthases from hybrid poplar, J. Exp. Bot. 2012, 63, 2059-2069.
380
[12] Peterbauer, T.; Richter, A. Biochemistry and physiology of raffinose family oligosaccharides
381
and galactosyl cyclitols in seeds. Seed Sci. Res. 2001, 11, 185-197.
382
[13] Frydman, R.B.; Neufeld, E.F. Synthesis of galactosylinositol by extracts from peas. Biochem.
383
Biophys. Res. Commun. 1963, 12, 121-125.
384
[14] Pharr, D.M.; Sox, H.N.; Locy, R.D., Huber S.C. Partial characterization of the galactinol
385
forming enzyme from leaves of Cucumis sativus L., Plant Sci. Lett. 1981, 23, 25-33.
386
[15] Keller, F.; Pharr, D.M. Metabolism of carbohydrates in sinks and sources: galactosyl-sucrose
387
oligosaccharides , in Photoassimilate Distribution in Plants and Crops: Source-Sink Relationships ,
388
eds Zamski, E.; Schaffer, A. A. New York, 1996, 157-184.
389
[16] Li, X.; Zhuo, J.; Jing, Y.; Liu, X.; Wang, X. Expression of a GALACTINOL SYNTHASE
390
gene is positively associated with desiccation tolerance of Brassica napus seeds during
391
development. J. Plant Physiol. 2011, 168, 1761-1770.
392
[17] Wang, D.; Yao, W.; Song, Y.; Liu, W.; Wang, Z. Molecular characterization and expression of
393
three galactinol synthase genes that confer stress tolerance in Salvia miltiorrhiza, J. Plant Physiol.
394
2012, 169, 1838-1848.
17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
395
[18] Pillet, J.; Egert, A.; Pieri, P.; Lecourieux, F.; Kappel, C.; Charon, J.; Gomes, E.; Keller, F.;
396
Delrot, S.; Lecourieux, D. VvGOLS1 and VvHsfA2 are involved in the heat stress responses in
397
grapevine berries, Plant Cell Physiol. 2012, 53, 1776-1792.
398
[19] Zhuo, C.; Wang, T.; Lu, S.; Zhao, Y.; Li, X.; Guo, Z. A cold responsive galactinol synthase
399
gene from Medicago falcate (MfGolS1) is induced by myo-inositol and confers multiple tolerances
400
to abiotic stresses, Physiol Plant, 149 (2012) 67-68.
401
[20] Ibáñez, C.; Collada, C.; Casado, R.; González-Melendi, P.; Aragoncillo, C.; Allona, I. Winter
402
induction of the galactinol synthase gene is associated with endodormancy in chestnut trees. Trees
403
2013, 27, 1309 - 1316.
404
[21] Gangola, M.P.; Jaiswal, S.; Kannan, U.; Gaur, P.M.; Baga, M.; Chibbar, R.N. Galactinol
405
synthase enzyme activity influences raffinose family oligosaccharides (RFO) accumulation in
406
developing chickpea (Cicer arietinum L.) seeds. Phytochemistry 2016, 125, 88-98.
407
[22] Philippe, R.N.; Ralph, S.G.; Mansfield, S.D.; Bohlmann, J. Transcriptome profiles of hybrid
408
poplar (Populus trichocarpa x deltoides) reveal rapid changes in undamaged, systemic sink leaves
409
after simulated feeding by forest tent caterpillar (Malacosoma disstria). New Phytol. 2010, 188,
410
787-802.
411
[23] Deng, W.W.; Wu, Y.L.; Li, Y.Y.; Tan, Z.; Wei, C.L. Molecular Cloning and Characterization of
412
Hydroperoxide Lyase Gene in the Leaves of Tea Plant (Camellia sinensis), J. Agric. Food. Chem.
413
2016, 64, 1770-1776.
414
[24] Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R.D.; Bairoch, A. ExPASy: The
415
proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 2003, 31,
416
3784-3788.
417
[25] Bendtsen, J.D.; Nielsen, H.; von Heijne, G.; Brunak, S. Improved prediction of signal peptides:
418
SignalP 3.0. J. Mol. Biol. 2004, 340, 783-795. 18
ACS Paragon Plus Environment
Page 18 of 28
Page 19 of 28
Journal of Agricultural and Food Chemistry
419
[26] Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. (). MEGA6: Molecular
420
Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725-2729.
421
[27] Ueda, T.; Coseo, M.P.; Harrell, T.J.; Obendorf, R.L. A multifunctional galactinol synthase
422
catalyzes the synthesis of fagopyritol A1 and fagopyritol B1 in buckwheat seed. Plant Sci. 2005,
423
168, 681–690.
424
[28] Horbowicz, M.; Obendorf R.L. Seed desiccation tolerance and storability: dependence on
425
flatulence-producing oligosaccharides and cyclitols-review and survey. Seed Sci. Res. 1994, 4,
426
385-405.
427
[29] Sun, Z.; Qi, X.; Wang, Z.; Li, P.; Wu, C.; Zhang, H.; Zhao, Y. Overexpression of TsGOLS2, a
428
galactinol synthase, in Arabidopsis thaliana enhances tolerance to high salinity and osmotic
429
stresses. Plant Physiol. Biochem., 2013, 69, 82-89.
430
[30] Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time
431
quantitative PCR and the 2-∆∆Ct Method. Methods 2001, 25, 402-408.
432
[31] Panikulangara, T.J.; Eggers-Schumacher, G.; Wunderlich, M.; Stransky, H.; Schöffl F.
433
Galactinol synthase1. A novel heat shock factor target gene responsible for heat-induced synthesis
434
of raffinose family oligosaccharides in Arabidopsis. Plant Physiol. 2004, 136, 3148-3158.
435
19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 20 of 28
436
Figure legends
437
Figure 1. Multiple sequence alignment of CsGolS1 (AGQ44777), CsGolS2 (AKS29170), and
438
CsGolS3 (AKS29171) with the galactinol synthases from other plant species. The conserved
439
catalytic residues of a manganese-ligation motif (DXD), a serine phosphorylation site (S), and a
440
typical hydrophobicity pentapeptide (APSAA) in the C-terminal are contained in the red boxes.
441
Figure 2. Neighbour-joining phylogenetic tree based on complete aa sequences of CsGolS and
442
GolS from other plant species. The multiple sequence alignment was performed using
443
CLUSTAL_X, bootstrap values are percentages of 1000 replicates, with only bootstrap values
444
above 50% shown here. GenBank accession numbers are shown in parentheses. Scale bar =
445
five substitutions per 100 aa positions.
446
Figure 3. SDS-PAGE analysis of recombinant CsGolS1, CsGolS2, and CsGolS3 proteins
447
expressed in E. coli BL21 (DE3) pLysS. A: M, protein marker; 1, pET-32a(+) without IPTG; 2,
448
pET-32a(+) with IPTG; 3, pET-32a(+)/CsGolS1 without IPTG; 4, pET-32a(+)/CsGolS1 with IPTG;
449
5, pET-32a(+) with IPTG (whole cell sonication); 6, pET-32a(+) with IPTG (precipitation of
450
sonication); 7, pET-32a (+)/CsGolS1 with IPTG (whole cell sonication); 8, pET-32a(+)/CsGolS1
451
with IPTG (precipitation of sonication); 9, pET-32a(+)/CsGolS1 with IPTG (supernatant of
452
sonication). B: M, protein marker; 1, pET-32a(+)/CsGolS2 without IPTG; 2, pET-32a(+)/CsGolS2
453
with
454
pET-32a(+)/CsGolS2 with IPTG (supernatant of sonication); 5, pET-32a(+)/CsGolS2 with IPTG
455
(precipitation of sonication); 6, pET-32a (+) without IPTG; 7, pET-32a(+) with IPTG; 8,
456
pET-32a(+) with IPTG (supernatant of sonication). C: M: Protein marker; 1, pET-32a(+)/CsGolS3
457
without IPTG; 2, pET-32a(+)/CsGolS3 with IPTG (precipitation); 3, pET-32a(+)/CsGolS3 with
458
IPTG (supernatant); 4: pET-32a(+)/CsGolS3 with IPTG (supernatant of sonication); 5:
459
pET-32a(+)/CsGolS3 with IPTG (precipitation of sonication); 6: pET-32a (+) without IPTG; 7:
460
pET-32a(+) with IPTG; 8: pET-32a(+) with IPTG (supernatant of sonication).
IPTG
(precipitation);
3,
pET-32a(+)/CsGolS2
with
20
ACS Paragon Plus Environment
IPTG
(supernatant);
4,
Page 21 of 28
Journal of Agricultural and Food Chemistry
461
Figure 4. The synthesis product of recombinant CsGolS proteins determined by gas
462
chromatography-tandem mass spectrometry. A, Standard mixture (i.e., phenyl-α-D-glucoside and
463
galactinol, 8.0 mg/L for each analyte); B. Synthesis product of PET32a(+) expressed proteins
464
(negative control); C, Synthesis product of recombinant CsGolS1; D, Synthesis product of
465
recombinant CsGolS2; E, Synthesis products of recombinant CsGolS3.
466
Figure 5. Expression of CsGolSs genes in mature leaves under abiotic and biotic stress conditions
467
determined using qRT-PCR. Gene expression in non-treated controls was set to 1.0. Data represent
468
the means ± SD (n = 3) of three biological replicates. Different letters above bars represent
469
significant differences at p < 0.05.
470
Figure 6. HPLC analysis of RFOs content in tea plants under low temperature and pest attack
471
stress. Data represent the means ± SD (n = 3) of three biological replicates. *p < 0.05, **p < 0.01
472
and ***p < 0.001, compared with the control.
21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 22 of 28
Figure 1
22
ACS Paragon Plus Environment
Page 23 of 28
Journal of Agricultural and Food Chemistry
Figure 2
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 24 of 28
Figure 3
24
ACS Paragon Plus Environment
Page 25 of 28
Journal of Agricultural and Food Chemistry
Figure 4
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 26 of 28
Figure 5
26
ACS Paragon Plus Environment
Page 27 of 28
Journal of Agricultural and Food Chemistry
Figure 6
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
TOC graphic 94x45mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 28 of 28