Molecular Cloning and Characterization of Galactinol Synthases in

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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

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Journal of Agricultural and Food Chemistry

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Molecular cloning and characterization of galactinol synthases in Camellia

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sinensis with different responses to biotic and abiotic stressors

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Yu Zhou†, Yan Liu†, Shuangshuang Wang, Cong Shi, Ran Zhang, Jia Rao, Xu Wang, Xungang

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Gu, Yunsheng Wang, Daxiang Li, Chaoling Wei*

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State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130

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Changjiang Road West, Hefei, Anhui 230036, China

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9

†

10

*

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E-mail: [email protected]

These authors contributed equally to this work.

Corresponding author: Chaoling Wei

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1

ACS Paragon Plus Environment


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ABSTRACT

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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

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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

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that the CsGolS1 isoform was sensitive to water deficit, low temperature, and abscisic acid, while

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CsGolS2 and CsGolS3 were sensitive to pest attack and phytohormones. The gene regulation and

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RFOs determination results indicated that CsGolS1 was primarily related to abiotic stress and

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CsGolS2 and CsGolS3 were related to biotic stress. GolS-mediated biotic stress adaptations have

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not been studied in depth, so further analysis of this new biological function is required.

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KEYWORDS: Camellia sinensis, Galactinol synthase, Biofunctional investigation, Gene

26

regulation, abiotic stress, biotic stress

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INTRODUCTION

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Leaf wilting, root necrosis, and nutrient deficiency are common symptoms in plants subjected to

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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

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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

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vitreous state that protects macromolecules6-7; RFOs are also essential for phloem transport

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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.

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Galactinol synthase (GolS: EC 2.4.1.123) belongs to glycosyltransferase family 8 and catalyzes

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the

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(UDP-D-galactose) to myo-inositol12. GolS was first detected in a crude extract of maturing pea

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seeds13 and was partially purified from mature cucumber14; the GolSs from legume seeds and

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cucurbit leaves were the first to biochemical characterized15. Recently, GolS genes have been

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isolated and characterized from various plant varieties including hybrid poplar 11, Brassica napus

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L.16, Coffea arabica L.1, Salvia miltiorrhiza17, grape18, Medicago falcate19, chestnut20, Cicer

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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

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abiotic stress1, 11, 16-20. While few studies have focused on the relationship between GolS gene

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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



53

pathogen infection) responses; the mechanisms underlying GolS-mediated biotic stress responses

54

require further investigation11, 22.

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Tea plant (Camellia sinensis) is one of the most important commercial crops in China and other

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Asian countries. As an evergreen woody plant, tea plants are susceptible to various biotic and

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abiotic stressors. Drought, freezing injury, plant diseases, and pests attack are the important biotic

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and abiotic stressors frequently affecting tea production23. Although GolSs have been isolated and

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characterized in many plant varieties, GolS-mediated biotic and abiotic stress adaptation in C.

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sinensis has not been previously studied, despite this process is significant to tea plant cultivation

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and crop yields. This study, therefore, aimed to identify and characterize GolS genes in tea plant

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that were up-regulated under biotic and abiotic stress conditions. This information would be useful

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for further characterization of abiotic and biotic stress responses in tea plants, and to breed new

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lines with improved resistance capabilities.

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MATERIALS AND METHODS

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Preparation of plant materials and treatments. Two-year-old tea plant clone cuttings (Camellia

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sinensis ‘Shuchazao’) were obtained from the Dechang tea plantation (Shucheng, China), which

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grown under controlled environment (12 h for day/night rhythm with 3000 lx light intensity at

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25°C) The low-temperature groups were treated at 0°C and 4°C, and the control group was kept at

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25 °C. A total of 15 branches for each temperature group were inserted into floral foam and

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incubated at the specified temperatures for 3, 6, 9, 12, and 24 h. The drought group was watered

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for 5 days and the control every 1–2 days; samples were collected and analyzed on day 5, 8, 11, 14,

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and 17. In the pest-attacked group, tea geometrids (Ectropis oblique) at the third larval stage were

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placed on tea plants (10 geometrids per individual tea plant). After one-third of each leaf was

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consumed, the pests were removed and the surviving leaves were collected within 24 h. Tea plant

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leaves from the non-attacked group were used as the control. The phytohormone groups were

4


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evenly sprayed with 1.0 mM salicylic acid (SA), 100 µM abscisic acid (ABA), or 1 mM methyl

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jasmonate (MeJA, containing 0.05% Tween-20) until the leaves were wet; cuttings sprayed with

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0.05% Tween-20 or distilled water were used as the control. Leaves of the same age and position

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were collected 3, 6, 12, 24, and 48 h after chemical treatments.

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To determine the constitutive expression levels of GolS genes in different organs, apical buds,

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developing leaves, mature leaves, young stems, roots, petals, flower buds, and fruits were

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collected and evaluated. Unless specified otherwise, all treatments in this study were performed in

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triplicate, and the collected samples were immediately frozen in liquid nitrogen and stored at

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-80 °C until use.

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Nucleotide and amino acid sequence analyses. Phylogenetic analysis of the CsGolS gene

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sequences was performed using BLAST searches in the National Center for Biotechnology

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Information database (http://www.ncbi.nih.gov:/BLAST/). The possible open reading frame (ORF),

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signal peptide, theoretical isoelectric point (pI), and the molecular mass (Mw) predictions were

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performed using online programs (ExPASy and SignalP 4.1)24-25. The putative domains were

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identified using the InterPro database (http://www.ebi.ac.uk/interproscan/). The transmembrane

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topology predictions of the CsGolS genes were carried out with TMHMM 2.0 software. Disulfide

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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

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MEGA v6.0 with bootstrap values determined using 1000 replications26.

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RNA extraction and cDNA synthesis. Total RNA was extracted from plant leaves (200 mg) using

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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


Predictor

Server


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Molecular cloning of the full-length cDNAs of CsGolS genes. Full-length cDNAs of CsGolS

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genes were cloned using a SMARTTM RACE Kit (Clontech, Dalian China) according to the

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manufacturer's instructions. Primers used in 3′-RACE and 5′-RACE were designed to each partial

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cDNA sequence for the three CsGolS genes initially obtained from tea plant transcription library.

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The primer sequences (including three validating primer pairs) used in 3′-RACE and 5′-RACE are

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P1 to P13 (Supplementary Table 1); the full-length cDNA sequences were tested using the S1000

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PCR detection system (primers P14 to P19). The PCR conditions were 95 °C for 5 min followed

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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

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by agarose electrophoresis and a DNA purification kit (Qiagen, Valencia, CA). The purified DNA

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products were cloned into the pMD19-T Vector and transformed into Escherichia coli DH5α. The

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recombinant plasmids were enriched and sequenced using the ABI PRISM 3730XL Sequencing

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System (Applied Biosystems, Foster City, CA).

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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

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Camellia sinenesis leaves, and the gene-specific primers P30 to P35 were used (NotI and XhoI

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sites underlined, Supplementary Table 1). After PCR amplification, base A was added to the end of

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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

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pMD-CsGolS3. The cloned plasmids and pET-32a (+) were digested with the respective restriction

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enzymes (NotI and XhoI), and the CsGolS ORFs were cloned into pET-32a (+) by ligation to

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construct pET-CsGolS1, pET-CsGolS2, and pET-CsGolS3. After confirmation, the recombinant

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plasmids were transformed into E. coli BL21 (DE3) pLysS cells (Novagen, Shanghai China). The

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transformed cells were incubated in LB broth containing ampicillin (50 µg/mL) at 16, 25, and

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37 °C overnight for optimal temperature evaluation. Until the cell density reached 0.6 (OD600

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absorbance), the broth was induced by adding a range of Isopropyl β-D-1-thiogalactopyranoside 6


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concentrations (IPTG, 0.25-2.0 mM) for different periods of time (1–5 h). The optimal

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overexpression conditions were evaluated by examining the recombinant protein yield and quality

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using SDS-PAGE. His-tagged recombinant proteins in disrupted cells were purified by affinity

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chromatography with nickel-nitrilotriacetic acid agarose resin (Ni-NTA, Qiagen, CA) and the

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purified recombinant proteins were dissolved in phosphate buffer (pH 7.0).

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Galactinol synthase activity assay. The purified recombinant proteins (CsGolSs) were used for

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the galactinol synthase activity assay. A total reaction volume of 50 µL comprising 20.0 mM

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myoinositol, 4.0 mM UDP-Gal, 50 mM HEPES (PH7.0), 2.0 mM dithiothreitol, 4.0 mM MnCl2,

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4.0 µg BSA, and 10.0 µL recombinant protein (0.1 mg/mL) was obtained; the supernatant of the

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pET-32a (+) transformant and phosphate buffer were used as negative controls 27; The enzymatic

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reaction was performed at 30 °C for 5 h, and was stopped by the addition of 50 µL of 100%

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ethanol. After 25.0 µg phenyl α-D-glucoside was added as internal standard, the reaction mixture

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was incubated at 80 °C for 30 min, passed through a 10000 MW cutoff filter (NANOSEPTM

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Microconcentrators, Pall Filtron), and evaporated to dryness under a nitrogen stream. Trace water

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in the resulting residue was removed by phosphorus pentoxide in a desiccator overnight. The

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thoroughly dried residue was derivatized with 200 µL trimethylsilyl imidazole: pyridine (1:1, v/v)

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at 80 °C for 45 min, and analyzed for phenyl α-D-glucoside and galactinol using a gas

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chromatography-mass spectrometer (GC-MS, Shimadzu, Japan) as previously described28. The

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Zebron ZB-MultiResidue-2 capillary column (30 m × 0.25 mm i.d, 0.25 µm film thickness,

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Guangzhou FLM Scientific Instrument Co., Ltd., Guangzhou, China) was used for analyte

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separation.

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Quantitative real-time PCR analysis (qRT-PCR). SYBR Green qRT-PCR amplification was

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performed on a CFX96 Touch real-time PCR detection system (BIO-RAD, CA) with a total

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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



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primer, and 9.5 µL of Milli-Q water. Three technical replicates were run for each sample using the

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following cycling parameters: 95 °C for 3 minutes followed by 40 cycles of 94 °C for 45 s,

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60/55 °C for 30 s, and 72 °C for 20 s. The gene β-Actin was selected as an internal control for

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qRT-PCR amplification and the specific primers P20 to P29 listed in Supplementary Table 1 were

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used for qRT-PCR amplification29. The amplification efficiencies of all genes tested in this study

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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

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obtained from different tissues (apical buds, developing leaves, mature leaves, young stems, roots,

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petals, flower buds, and fruits) of C. sinensis ‘Shuchazao’ without biotic or abiotic stress and RNA

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obtained from leaves of drought-stressed plants were conducted. A total of 3 g of frozen sample

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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.

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RFOs extraction and determination. Samples treated at 4 °C for 3, 6, 12, and 24 h were used for

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the abiotic stress group analyses; samples collected from 3, 6, 12, and 24 h after Ectropis oblique

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attack were used for the biotic stress group analyses. For each sample, 0.3 g of fresh leaves were

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used for the extraction of RFOs (i.e., raffinose, stachyose, and verbascose). The extraction method

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and chromatography analyses were conducted as previously described 31.

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Statistical analysis. Three biological replicates were analyzed for each treatment and GraphPad

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Prism software was used for data analysis. Data are presented as means ± SD. Significant

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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


using

DPS

software

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statistically significant.

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RESULTS

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Isolation and sequence analysis of CsGolS genes. In our preliminarily studies, three EST

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sequences were selected from an Ectropic oblique-attacked tea plant transcription library; these

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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

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CsGolS3 (1386 bp, 1574 bp, and 1350 bp, respectively) were obtained by 3' and 5' RACE-PCR.

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The full-length sequences of CsGolS1, CsGolS2, and CsGolS3 were submitted to GenBank

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database with the accession numbers JX624168, KP757767, and KP757768, respectively. The

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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

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37.80 kDa and a theoretical pI of 4.80. No signal peptide was observed in the three CsGolSs by

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analyzing the protein sequence with SignalP 4.1 and no internal transmembrane segment was

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found by the transmembrane topology predictions. Scratch protein predictor analysis showed that

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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.

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Multiple sequence alignment and phylogenetic analysis. The BLASTp analysis showed that the

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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

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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



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

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indicated that the three functional domains of the CsGolSs were conserved in GolS proteins

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(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

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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

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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


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the reaction systems, galactinol was synthesized by fusion proteins of CsGolS1, CsGolS2, and

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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

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CsGolS3 fusion proteins was 0.19 µmol/min·mg, 0.08 µmol/min·mg, and 0.30 µmol/min·mg,

230

respectively.

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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



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


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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


11, 22.


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


Page 14 of 28

Page 15 of 28


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



349

stress.

350

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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


IPTG

(supernatant);

4,

Page 21 of 28


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



Page 22 of 28

Figure 1

22


Page 23 of 28


Figure 2

23



Page 24 of 28

Figure 3

24


Page 25 of 28


Figure 4

25



Page 26 of 28

Figure 5

26


Page 27 of 28


Figure 6

27



TOC graphic 94x45mm (300 x 300 DPI)


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Molecular Cloning and Characterization of Galactinol Synthases in

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