Coexpression of methyltransferase gene dmt50 and methylene

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Biotechnology and Biological Transformations

Coexpression of methyltransferase gene dmt50 and methylene tetrahydrofolate reductase gene increases Arabidopsis thaliana dicamba resistance Le Chen, Shigang Yao, Tao Chen, Qin Tao, Xiangting Xie, Xiang Xiao, Derong Ding, Qin He, and Jian He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04944 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Journal of Agricultural and Food Chemistry

Coexpression of methyltransferase gene dmt50 and methylene tetrahydrofolate reductase gene increases Arabidopsis thaliana dicamba resistance Le Chena, Shigang Yaoa, Tao Chena, Qin Taob, Xiangting Xieb, Xiang Xiaob, Derong Dingb, Qin Hea *, Jian Hea

a Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture,

College of Life Sciences, Nanjing Agricultural University (210095), Nanjing, Jiangsu, China. b Beijing

DBN Biotech Co., Ltd. (100080), Beijing, China.

* Address

correspondence to Qin He, [email protected];

Tel.: +86-25-84396685; Fax: +86-25-84395326.

1

ACS Paragon Plus Environment


1

Abstract

2

Dicamba, a broad-spectrum and highly efficient herbicide, is an excellent target

3

herbicide for the engineering of herbicide-resistant crops. In this study, a new

4

tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt50, was cloned

5

from the dicamba-degrading strain Rhizorhabdus dicambivorans Ndbn-20. Dmt50

6

catalyzed the methyl transfer from dicamba to THF, generating the herbicidally inactive

7

product 3,6-dichlorosalicylic acid (3,6-DCSA) and 5-methyl-THF. A dmt50 transgenic

8

Arabidopsis thaliana clearly showed dicamba resistance (560 g/ha, the normal field

9

application rate). However, Dmt50 demethylation activity was inhibited by the product

10

5-methyl-THF. Mthfr66, encoded by the 5,10-methylene-THF reductase gene mthfr66

11

could relieve the inhibition by removing 5-methyl-THF in vitro. Compared with

12

expression of dmt50 alone, simultaneous expression of dmt50 and mthfr66 further

13

improved the dicamba resistance (1120 g/ha) of transgenic A. thaliana. This study

14

provides new genes for dicamba detoxification and a strategy for the engineering of

15

dicamba-resistant crops.

16

Keywords: Dicamba; Tetrahydrofolate-dependent methyltransferase; Methylene

17

tetrahydrofolate reductase; Enzyme assays; Transgenic plants.

18 19 20 21 2


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Introduction

23

In the past decade, the global application of genetically modified (GM) herbicide-

24

resistant crops significantly increased the flexibility and efficiency of weed control and

25

resulted in great economic benefits1. In 2016, 185 million hectares of GM crops were

26

planted worldwide, and more than 80% of these crops were herbicide resistant2. In the

27

United States of America (USA), for example, the area planted with GM crops reached

28

74.0 million hectares, and nearly 90% of soybean, corn and cotton were Roundup

29

(glyphosate) resistant3. However, long-term use of glyphosate has resulted in severe

30

weed resistance. Several major weeds such as giant ragweed (Ambrosia trifida),

31

horseweed (Conyza canadensis), waterhemp (Amaranthus rudis), Palmer amaranth

32

(Amaranthus palmeri), and common ragweed (Ambrosia artemisiifolia) have

33

developed strong glyphosate resistance4. Therefore, it is urgent to find new target

34

herbicides and develop corresponding GM herbicide-resistant crops to combat

35

glyphosate-resistant weeds.

36

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used, low-cost,

37

environmentally friendly herbicide that does not persist in the environment and shows

38

little toxicity to wildlife and humans5. This herbicide is used to control more than 200

39

types of annual or biennial grass weeds, broadleaf weeds and xylophyta. In particular,

40

dicamba effectively kills many glyphosate- and bialaphos (glufosinate)-resistant

41

weeds6. Furthermore, although dicamba has been widely used for more than 50 years

42

(since it was first approved in 19627), dicamba-resistant weeds are rarely found. Thus, 3



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dicamba is considered an excellent target herbicide for the engineering of next-

44

generation herbicide-resistant GM crops.

45

Herman et al. (2005) identified a Rieske nonheme iron oxygenase (RHO)-type

46

monooxygenase, DMO (dicamba monooxygenase), from the bacterial strain

47

Stenotrophomonas maltophilia DI-68. DMO effectively demethylates dicamba to the

48

herbicidally inactive metabolite 3,6-dichlorosalicylic acid (3,6-DCSA). Recently, the

49

biotech giant Monsanto has successfully developed a GM soybean (Roundup Ready 2

50

XtendTM) that is resistant to dicamba and glyphosate and a GM cotton (Bollgard II

51

XtendFlexTM) that is resistant to dicamba and bialaphos by introducing DMO and

52

glyphosate resistance gene CP4-EPSPS or bialaphos (glufosinate) resistance gene bar.

53

These GM crops have received regulatory approval from the U.S. Department of

54

Agriculture

55

(http://www.aphis.usda.gov/newsroom/2014/08/pdf/brs_eis.pdf)

56

commercially planted since 2015. In 2016, the Roundup Ready 2 XtendTM soybean and

57

Bollgard II XtendFlexTM cotton planted area in the USA reached 8.0 million hectares

58

and 2.4 million hectares, respectively. The planted area of Roundup Ready 2 XtendTM

59

soybean is expected to reach 16.0 million hectares in 2018.

(USDA)

in

August and

2014 have

been

60

In our previous research, a dicamba-degrading strain, Rhizorhabdus dicambivorans

61

Ndbn-20, was isolated from compost samples9. A THF-dependent dicamba

62

methyltransferase gene, dmt, was cloned from this strain10. In this study, we identified

63

another THF-dependent dicamba methyltransferase gene, dmt50, and a methylene 4


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tetrahydrofolate reductase gene, mthfr66, from R. dicambivorans Ndbn-20. The

65

enzymatic characteristics of Dmt50 and its application potential in engineering

66

herbicide-resistant crops were studied.

67 68

Materials and methods

69

Chemicals and media.

70

Dicamba

71

https://en.wikipedia.org/wiki/Dicamba), THF (≥ 90% purity), and 5-methyl-THF (95%

72

purity) were purchased from Sigma-Aldrich (Shanghai, China). The compound 3,6-

73

DCSA (98% purity) was obtained from the Qingdao Chemical Reagent Co., Ltd.

74

(Qingdao, China). Chromatography-grade methanol and acetonitrile and analytical-

75

grade acetic acid were purchased from the Shanghai Chemical Reagent Co., Ltd.

76

(Shanghai, China). Luria-Bertani (LB) broth and LB agar were purchased from Difco

77

Laboratories (Detroit, MI, USA). MS medium for plant culture was purchased from the

78

Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

79

Strains and vectors.

80

The bacterial strains and vectors used in this study are listed in Table 1. The dicamba-

81

degrading bacterium R. dicambivorans Ndbn-20 was grown at 30°C in LB. Escherichia

82

coli strains and Agrobacterium tumefaciens GV3101 were grown at 37°C in LB broth

83

supplemented with the appropriate antibiotics.

84

DNA and amino acid sequence analysis.

(99.3%

purity,

safety

information

and

5


environmental

impact:


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The genome of strain Ndbn-20 was acquired by Shanghai Majorbio Bio-pharm

86

Technology Co., Ltd. (Shanghai, China) using an Illumina HiSeq2000 system. Glimmer

87

software (version 3.0) (http://cbcb.umd.edu/software/glimmer) was used for de novo

88

gene prediction11. Functional annotation was accomplished by BLAST analysis of

89

protein sequences in the nonredundant protein (NR), KEGG, Swiss-Prot, and COG

90

databases12, 13. Analyses of DNA and amino acid sequences were performed using

91

OMIGA 2.0 software. DNA and amino acid sequence identity searches were conducted

92

using the BLASTN and BLASTP tools (https://blast.ncbi.nlm.nih.gov/BlaSt.cgi).

93

Expression of dmt50 and mthfr66 and purification of products.

94

The methyltransferase gene dmt50 was amplified by PCR using 2×Phanta Master Mix

95

(Vazyme Biotech Co., Ltd), the genomic DNA extracted from strain Ndbn-20 as the

96

template,

97

TAAGAAGGATATACATATGATGAGGGAGG

98

CTGAAGTGAAGTCCT-3’) and dmt50R (5’-GTGGTGGTGGTGGTGCTCGAGCT

99

ACGCCCGCGCTGCCAC-3’) (homologous arms are underlined). The methylene

100

tetrahydrofolate reductase gene mthfr66 was amplified using the primers mthfrF (5’-

101

TAAGAAGGATATACATATGATGGGCTCGCCCGTTATG-3’) and mthfrR (5’-

102

GTGGTGGTGGTGGTGCTCGAGGTGCTTTCGAGCGTAGTCAG-3’)

103

(homologous arms are underlined). The PCR products were inserted into the NdeI-XhoI

104

site of pET29 (+) using the ClonExpress® II One Step Cloning Kit (Vazyme Biotech

105

Co., Ltd). Then, the product was transformed into E. coli BL21 (DE3) for expression.

and

the

primers

6


dmt50F

(5’-

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E. coli BL21 (DE3) cells harboring pET29dmt50 or pET29mthfr66 were grown in LB

107

broth containing 50 mg/L kanamycin at 37°C. The expression of dmt50 or mthfr66 was

108

induced for 10 h by adding 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when

109

the OD600 of the culture reached 1.0. Cells were harvested by centrifugation at 12,000

110

rpm for 1 min and washed twice with 100 mM Tris-HCl buffer (pH 8.0), and the cells

111

were suspended in the same buffer. Then, the collected cells were disrupted by

112

sonication, and the cell lysate was centrifuged at 12,000 rpm for 30 min. The resulting

113

supernatant was used as the cell extract. The C-terminal His6-tagged proteins were

114

purified using Co2+-affinity chromatography following the instructions (His

115

GraviTrap™ TALON®, GE Healthcare Life Sciences Co., Ltd). Molecular weights

116

were determined by SDS-PAGE and Coomassie blue staining, and the protein

117

concentrations were quantified by the bicinchoninic acid (BCA) method using bovine

118

serum albumin as the standard (BCA Protein Assay Kit, Sangon Biotech Shanghai Co.,

119

Ltd.).

120

Enzyme assays.

121

The dicamba methyltransferase activity of the purified Dmt50 was determined in a 300

122

μL mixture containing 0.1 mg of Dmt50, 2.0 mM THF, 1.0 mM dicamba and 100 mM

123

Tris-HCl buffer (pH 8.0). The mixture was incubated at 45°C for a certain time

124

(according to the assay needs), and then the reaction was terminated by boiling at 100°C

125

for 1 min. The dicamba conversion and metabolite generation were determined by high-

126

performance liquid chromatography (HPLC). One unit of methyltransferase activity 7



127

was defined as the amount of enzyme that catalyzed the conversion of 1.0 nmol of

128

substrate per min. The optimal reaction temperature was determined under standard

129

conditions at pH 8.0 (100 mM Tris-HCl buffer) and different temperatures (20 to 45°C).

130

For an optimal pH test, three different buffering systems, 20 mM HAc-NaAc buffer

131

(pH 3.6 to 5.8), 20 mM citric acid-Na2HPO4 buffer (pH 5.5 to 8.5), and 20 mM glycine-

132

NaOH buffer (pH 8.6 to 10.0), were used. Each value was the average from three

133

independent experiments.

134

Analytical method.

135

The enzyme reaction mixture was filtered through a 0.22 μm Millipore membrane to

136

remove protein precipitate produced during boiling. HPLC analysis was performed on

137

an UltiMate 3000 Titanium system (Thermo Fisher Scientific) equipped with a C18

138

reversed-phase column (4.6 by 250 mm, 5 μm; Agilent Technologies). The mobile

139

phase was a mixture of ultrapure water (58.4%), acetonitrile (31.7%), methanol (7.5%),

140

and acetic acid (2.4%). The flow rate was 1.0 mL/min, and the injection volume was

141

20 μL. The detection wavelengths were 275 nm for dicamba and 319 nm for 3,6-DCSA.

142

The reaction mixtures were analyzed by tandem mass spectrometry (Agilent G6410B)

143

according to a reported method10. Metabolites were separated, and their identities were

144

confirmed by electrospray ionization (ESI)-MS spectra, which were recorded in

145

negative ionization mode on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher

146

Scientific) equipped with an ESI probe14.

147

Construction of transgenic A. thaliana. 8


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The codons of the genes were optimized based on plant codon characteristics by the

149

GenScript Optimum-Gene Codon Optimization system to ensure that the genes were

150

efficiently expressed in plants. The optimized genes were synthesized by GenScript Co.,

151

Ltd. (Nanjing, China) and ligated into the clone vector pGEM-T (Promega Corporation

152

Co., Ltd.). Then, the genes were cleaved from cloning vectors and ligated into the

153

corresponding site of the pDBNBC-01 vector (Table 1), constitutive promoter

154

(AtUbi10) was used to make exogenous gene express10.Then, expression vectors were

155

introduced into A. tumefaciens GV3101 by the liquid nitrogen method as previously

156

described15. A. thaliana ecotype Columbia was transfected with A. tumefaciens

157

GV3101 cells bearing expression vectors by the floral dip method16. Resistant T1

158

generation transgenic plants were identified by glufosinate screening and used in

159

subsequent experiments.

160

Analysis of the transcription level of dmt50 and mthfr66 in transgenic A. thaliana

161

by Real time-qPCR (RT-qPCR).

162

RT-qPCR was used for the quantitative analysis of the transcription of dmt50 and

163

mthfr66 genes in wild type A. thaliana (WT) and transgenic A. thaliana. Total RNA

164

was isolated from the leaves of 14 days old A. thaliana using RNAiso Plus (Takara,

165

Dalian, China). The isolated RNA was purified with the RT reagent PrimeScrip Kit

166

with gDNA Eraser (Takara, Dalian, China) to remove DNA contamination, and then

167

cDNA was synthesized following the manufacturer’s instructions. All cDNA from

168

different sample were quantified by NanoDrop2000 (Thermo Scientific, USA) and 9



169

diluted in 200 ng/μL. The primer pairs RT-50 F: 5’-CACGGCGTGGAACTCAGTGG-

170

3’ / RT-50 R: 5’-CAGCCGTTTGCTGGCAACCA-3’ and RT-66 F: 5’-

171

GAGGCGGCCGGCATAGAT-3’ / RT-66 R: 5’-CTTGGCGCGAAGGACTGC-3’

172

were used for the amplification of dmt50 and mthfr66 (codons optimized), respectively.

173

RT-qPCR was performed in the IntelliQube (LGC, England) with TB Green™

174

Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Dalian, China). The PCR reaction

175

mixture contain 0.8μL TB Green Premix Ex Taq II (Tli RNaseH Plus), 0.8 μL template

176

(cDNA), 0.032 μL forward/reverse primer respectively and 0.016 μL ROX Reference

177

Dye. All components were added in reaction mixtures automatically and the analyses

178

were performed in double. PCR conditions were as follows: 95 °C for 30 s, followed

179

by 35 cycles at 95 °C for 10 s, 60 °C for 30 s. The pMD™ 19-T vector (Takara, Japan)

180

contain codon-optimized dmt50 and mthfr66 genes were used for creation of standard

181

curve. The standard curves were generated by the amplification of serial 10-fold

182

dilutions of plasmid solutions.

183

Dicamba resistance assays.

184

Two different assays were used to estimate plant dicamba resistance: 1) Seeds were

185

germinated vertically on 1/2 MS plates containing different concentrations of dicamba

186

(0, 1, 2 and 3 mg/L), and 2) seeds were first germinated vertically on 1/2 MS plates (not

187

containing dicamba). Then, 14-day-old seedlings were transferred into soil (nutritive

188

soil:vermiculite=1:3). After 3 days, dicamba (500 mg/L, dissolved in water) was

189

sprayed to make final concentrations of 0, 560 and 1120 g/ha, respectively. The 10


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190

seedlings (both assays) were incubated in a plant growth chamber with a 16 h light/8 h

191

dark photoperiod at 22°C/18°C. Fourteen days after seedlings were transferred, the root

192

length and fresh weight were measured.

193

Accession numbers.

194

The GenBank accession numbers of the genome of strain Ndbn-20, dmt50 gene,

195

mthfr66 gene, codon-optimized dmt50 and mthfr66 genes are LGVA01000000,

196

MH784615, MH835449, MK258186 and MK258187, respectively.

197 198

Results and discussion

199

Screening of the THF-dependent methyltransferase gene dmt50 from the genome

200

of R. dicambivorans Ndbn-20.

201

In a previous study, we sequenced the draft genome of R. dicambivorans Ndbn-20

202

and cloned the THF-dependent methyltransferase gene dmt from the strain10. Recently,

203

the complete genome of R. dicambivorans Ndbn-20 was sequenced by single-molecule

204

real-time (SMRT) technology14. In this study, we used BLASTP to search the complete

205

genome of R. dicambivorans Ndbn-20 using the sequences of reported THF-dependent

206

methyltransferases such as DesA, LigM and Dmt (Table S1). This search resulted in

207

the discovery of another putative methyltransferase gene, orf0805 (designated dmt50)

208

(Fig. 1). Dmt50 shared its highest sequence identity (49%) with the dicamba

209

methyltransferase Dmt from R. dicambivorans Ndbn-2010 and shared 46% and 47%

210

identity with the vanillate methyltransferases DesA17 and LigM18 from Sphingomonas 11



211

paucimobilis SYK-6, respectively. In addition, six other genes were found in the

212

vicinity of dmt50. Upstream of dmt50, there were two genes, orf0806 and orf0802,

213

encoding proteins that shared 53% and 58% identity with a MFS transporter and a

214

TonB-dependent receptor, respectively. Downstream of dmt50, there were three genes,

215

orf0803, orf0801 and orf0804, that were similar to mthfr (encoding 5,10-methylene-

216

THF reductase), dhc (encoding the bifunctional enzyme 5,10-methylene-THF

217

dehydrogenase/5,10-methylene-THF cyclohydrolase), and purU (encoding formyl-

218

THF deformylase), respectively (their amino acid sequences shared 52%, 75%, and 68%

219

identity). The genes mthfr, dhc and purU have been reported to be involved in THF

220

metabolism. These analyses suggested that dmt50 might encode a THF-dependent

221

methyltransferase gene.

222

Dmt50 shows THF-dependent dicamba methyltransferase activity in vitro.

223

The dmt50 gene was cloned into pET29a (+) and expressed in E. coli BL21 (DE3).

224

Then, the recombinant protein was purified by Co2+-affinity chromatography. The

225

molecular weight of Dmt50 was approximately 53 kDa, which is similar to its

226

theoretical molecular weight of 53.1 kDa (Fig. S1). The dicamba methyltransferase

227

activity of Dmt50 was determined in an enzymatic reaction mixture containing 0.1 mg

228

of purified protein, 0.5 mM THF and 0.2 mM dicamba. After incubation for 120 min,

229

the disappearance of dicamba and production of metabolites were analyzed by HPLC.

230

In the absence of THF, the added dicamba was not converted, and no metabolite was

231

generated. In the presence of THF, approximately 30% of the added dicamba was 12


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transformed, and correspondingly, a new metabolite was generated (Fig. 2A). The

233

metabolite had a retention time of 8.7 min, which was equal to that of the authentic 3,6-

234

DCSA standard. MS analysis was performed to further determine the structure of the

235

metabolites. In the first-order mass spectrum, four molecular ions appeared; at m/z=205

236

(M-H)-, m/z=458 (M-H)-, m/z=219 (M-H)- and m/z=444 (M-H)-, their molecular

237

weights matched the molecular weights of 3,6-DCSA, dicamba, THF and 5-methyl-

238

THF, respectively (Fig. 2B). The second-order MS analysis of the ion at m/z=205 (M-

239

H)- showed that the metabolite had a prominent protonated molecular ion at m/z=205

240

(M-H)- and two fragment ion peaks, one at m/z=161 (loss of a -COOH group) and one

241

at m/z=187 (loss of a -OH) (Fig. 2A). Therefore, the metabolite was identified as 3,6-

242

DCSA. This metabolite had another two peaks (at m/z 207 and 209) because it

243

contained two chlorine atoms and chlorine has two isotopes,

244

natural abundances are 24% and 76%, respectively. These results demonstrated that

245

Dmt50 was a THF-dependent methyltransferase that catalyzed the demethylation of

246

dicamba to 3,6-DCSA.

247

Biochemical properties of Dmt50.

37Cl

and

35Cl,

whose

248

Dmt50 displayed dicamba methyltransferase activity only in the presence of THF,

249

indicating that Dmt50 was THF dependent. Dmt50 could not catalyze the methyl

250

transfer of vanillate, syringate, isoproturon and alachlor (Fig. S2). The optimum

251

conditions for Dmt50 activity were sought at temperatures from 4 to 75°C and at pH

252

values ranging from 3.6 to 10.0, with the highest activity being found at 45°C and pH 13



253

8.0 (Fig. S3A, Fig. S3B). The incubation of 0.1 mg of purified Dmt50 at optimal

254

conditions for 5 min resulted in a specific activity of 146 nmol/min/mg toward dicamba,

255

which was higher than those of Dmt (114 nmol/min/mg) and DMO (134 nmol/min/mg).

256

Dmt50 was feedback-inhibited by the product 5-methyl-THF.

257

In the time course of dicamba conversion by purified Dmt50, dicamba was quickly

258

converted to 3,6-DCSA at the beginning of the reaction; however, the conversion rate

259

gradually slowed over time (Fig. 3). In the first 5 min of incubation, approximately 19%

260

of dicamba was converted to 3,6-DCSA; in the next 15 min, only another 10% of the

261

added dicamba was converted, and in the 30 min of incubation after that, only another

262

5% was converted. At 60 min, the conversion rate was reduced to nearly zero, and no

263

more dicamba was converted. The possible reason might be that the dicamba

264

methyltransferase activity of Dmt50 was inhibited by the product 5-methyl-THF or the

265

product 3,6-DCSA. To determine whether the two products inhibit Dmt50, different

266

concentrations of 5-methyl-THF or 3,6-DCSA were exogenously added to the

267

enzymatic reaction mixture. The results showed that the addition of 3,6-DCSA had no

268

effect on the methyltransferase activity of Dmt50; however, the addition of 5-methyl-

269

THF clearly inhibited Dmt50, and the higher the concentration of 5-methyl-THF was,

270

the more serious its inhibitory effect on Dmt50 (Fig. 4). Interestingly, Dmt and LigM

271

were also inhibited by 5-methyl-THF10,

272

might be a common property of THF-dependent methyltransferases.

273

Inhibition of Dmt50 by 5-methyl-THF was relieved by Mthfr66.

20.

Therefore, inhibition by 5-methyl-THF

14


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274

Although Dmt50 presented higher enzyme activity than Dmt and DMO at the

275

beginning of their reactions, the inhibition by the product 5-methyl-THF limited the

276

average Dmt50 conversion rate over the whole reaction. Theoretically, removal of the

277

product 5-methyl-THF from the enzymatic reaction may relieve its feedback inhibition

278

on Dmt50 and thus improve the overall conversion rate. According to previous DNA

279

and amino acid sequence analysis, three genes involved in THF metabolism located

280

downstream of dmt50. Therefore, we speculated that these enzymes may play important

281

role to overcoming the feedback inhibition of 5-methyl-THF on Dmt50 in strain Ndbn-

282

20. Among these genes (mthfr, dhc and purU), mthfr encodes the methylene

283

tetrahydrofolate reductase (Mthfr). Mthfr catalyzes the NAD+-dependent oxidation of

284

5-methyl-THF to 5,10-methylene-THF or the reverse reaction using flavin adenine

285

dinucleotide (FAD) as a cofactor (NAD+: 5-methyl-THF oxidase activity/NADH: 5,10-

286

methylene-THF reductase activity)20.

287

Thus, the generated 5-methyl-THF could be further converted to 5,10-methylene-

288

THF by Mthfr. To verify this assumption, we designed an experiment to remove 5-

289

methyl-THF from the methyltransferase enzyme reaction by addition of Mthfr. In strain

290

Ndbn-20, four Mthfr genes were found, but only the Mthfr genes in scaffold 66 (mthfr66)

291

was successfully expressed in E. coli BL21 (DE3). The gene mthfr66 was amplified

292

from the genome of R. dicambivorans Ndbn-20, ligated into pET29a (+) and expressed

293

in E. coli BL21 (DE3). Mthfr66 was purified by Co2+-affinity chromatography as

294

previously described. The molecular mass of the purified protein was approximately 30 15



295

kDa, which matched the predicted molecular mass of 31.8 kDa (Fig. S4). The purified

296

Mthfr66 was canary yellow because it contained a FAD cofactor. The purified Mthfr66

297

displayed 5-methyl-THF oxidase activity and convert 5-methyl-THF to 5,10-

298

methylene-THF, which was unstable and quickly spontaneously converted to THF in

299

vitro21 (Fig. S5, S6). The specific activity of Mthfr66 was 42 nmol/min/mg toward 5-

300

methyl-THF. When both Mthfr66 and Dmt50 were added to the reaction mixture,

301

dicamba was nearly completely transformed to 3,6-DCSA (Fig. 5). The reaction can

302

approach the maximum dicamba degradation efficiency when 0.12 mg Mthfr66 was

303

added, an amount of Mthfr66 over 0.12 mg did not further increase the reaction rate

304

(Fig. S7). These results showed that in vitro addition of Mthfr66 relieved the inhibition

305

of 5-methyl-THF on Dmt50 and improve the overall catalytic rate of Dmt50.

306

A. thaliana carrying a codon-optimized dmt50 gene clearly exhibited resistance to

307

dicamba.

308

To assess whether Dmt50 could detoxicates dicamba in plants, gene dmt50 was

309

optimized according to the codon usage bias and GC content of plants. The optimized

310

dmt50 was introduced into A. Thaliana by liquid nitrogen method. RT-qPCR results

311

showed that dmt50 was expressed in the transgenic A. Thaliana (Fig. S8). The dicamba

312

resistance of the transgenic A. thaliana were evaluated. As shown in Fig. 6, after 14

313

days of seeding in plates, all of the nontransgenic A. thaliana plants were sensitive to

314

dicamba; their roots were not developed, and their leaves were dry. Whereas the

315

transgenic A. thaliana plants carrying dmt50 developed long roots and green but curly 16


Page 16 of 37

Page 17 of 37


316

leaves under 1.0 mg/L dicamba treatment. The seedlings fresh weight of A. thaliana

317

carrying dmt50 was also significantly higher than that of the nontransgenic A. thaliana

318

under 1.0 mg/L dicamba treatment (P < 0.05). Under 2.0 and 3.0 mg/L dicamba

319

treatment, the growth of transgenic plant seedlings was seriously restrained, and there

320

was no obvious difference between the nontransgenic A. thaliana and A. thaliana

321

carrying dmt50. These results indicated that A. thaliana carrying dmt50 could not

322

tolerate 2.0 mg/L (or above) dicamba on plates.

323

The dicamba resistance of transgenic A. thaliana grown in soil was also evaluated.

324

Transgenic A. thaliana plants were sprayed with dicamba at 560 g/ha (the normal field

325

application rate) and 1120 g/ha. As shown in Fig. 7, although there was a growth delay

326

at 560 g/ha dicamba, A. thaliana carrying dmt50 were alive and developed green leaves.

327

The root length and fresh weight were significantly higher than those in nontransgenic

328

A. thaliana (P < 0.05). At 1120 g/ha dicamba, A. thaliana carrying dmt50 and

329

nontransgenic A. thaliana were all dead. These results suggested that A. thaliana

330

carrying dmt50 could tolerate 560 g/ha dicamba but not 1120 g/ha dicamba. Although

331

Dmt50 presented relatively higher dicamba methyltransferase activity than Dmt in vitro,

332

A. thaliana carrying dmt50 did not display higher dicamba resistance than A. thaliana

333

carrying dmt, which might be due to the relative lower dicamba methyltransferase

334

activity of Dmt50 at 20-30 °C (the normal growth temperature of plants). The relative

335

demethylase activity of crude lysates from dmt50 transgenic Arabidopsis thaliana also

336

be tested under a series of temperatures, the result is similar to that of the purified 17



337

Dmt50 (Fig. S9).

338

Simultaneous expression of dmt50 and mthfr66 relieved the 5-methyl-THF

339

inhibition of Dmt50 in A. thaliana.

340

To evaluate the possibility of relieving the 5-methyl-THF inhibition on Dmt50 in A.

341

thaliana by simultaneously introducing dmt50 and mthfr66 into A. thaliana. A

342

bitransgenic A. thaliana carrying dmt50 and mthfr66 was successfully constructed. RT-

343

qPCR analysis showed that these two genes have similar expression level in the

344

bitransgenic A. thaliana (Fig. S8). The dicamba resistance of the bitransgenic A.

345

thaliana were evaluated. As shown in Fig. 6, the growth vigor of the bitransgenic A.

346

thaliana plants under 1.0 mg/L dicamba was nearly as strong as those of nontransgenic

347

A. thaliana without dicamba treatment. Under 2.0 or 3.0 mg/L dicamba treatment, the

348

root length and fresh weight were significantly higher in the bitransgenic plants than

349

those in the nontransgenic A. thaliana (P < 0.01). Green curly leaves and roots also

350

developed better than single dmt50 transgenic plants under 2.0 mg/L and 3.0 mg/L

351

dicamba. In addition, under the 3 mg/L dicamba treatment, the root length was

352

significantly longer in the bitransgenic A. thaliana than that in the A. thaliana carrying

353

dmt50 (P < 0.05). These results indicated that bitransgenic A. thaliana carrying dmt50

354

and mthfr66exhibited higher dicamba resistance than A. thaliana carrying dmt50.

355

Similar results were obtained in soil cultivation assays (Fig. 7). At 560 g/ha dicamba

356

treatment, the root length and fresh weight were significantly higher in bitransgenic A.

357

thaliana than those in A. thaliana carrying dmt50 (P < 0.05) and nontransgenic A. 18


Page 18 of 37

Page 19 of 37


358

thaliana (P < 0.01). Even under 1120 g/ha treatment (double the normal field

359

application rate), the fresh weight of the bitransgenic A. thaliana was significantly

360

higher than that of the nontransgenic A. thaliana (P < 0.05). These results suggested

361

that the bitransgenic A. thaliana carrying dmt50 and mthfr66 could tolerate up to 1120

362

g/ha dicamba.

363

Our study showed that introduction of dmt50 obviously improved the dicamba

364

resistance of A. thaliana. However, Dmt50 has two disadvantages that limit its

365

application. One disadvantage is that its dicamba methyltransferase activity was

366

seriously inhibited by product 5-methyl-THF which led to a much slow overall activity.

367

This study provides a strategy for relieve the feedback inhibition by coexpression of

368

dmt50 and mthfr66. Compared with transgenic A. thaliana carrying dmt50, the dicamba

369

resistance of the bitransgenic A. thaliana carrying dmt50 and mthfr66 was significantly

370

improved. Another disadvantage is that the dicamba methyltransferase activity of

371

Dmt50 is very low at 20-30 °C (the normal growth temperature of plants),

372

approximately only one fifth of the activity under optimum temperature (45 °C), which

373

might be an important reason for the low dicamba resistance of A. thaliana carrying

374

dmt50. In the future, we will improve the activity of Dmt50 (especially the activity

375

under 20-30 °C) and relieve the feedback inhibition of 5-methyl-THF through directed

376

evolution technology and rational protein design.

377 378

Abbreviations used 19



379

Page 20 of 37

GM: genetically modified; THF: tetrahydrofolate; dicamba: 3,6-dichloro-2-

380

methoxybenzoic acid; 3,6-DCSA: 3,6-dichlorosalicylic

acid;

Dmt/dmt:

dicamba

381

methyltransferase; Mthfr/mthfr: methylene tetrahydrofolate reductase; DMO: dicamba

382

monooxygenase.

383 384

Acknowledgements

385

This work was supported by the National Natural Science Foundation of China (No.

386

31770117 and 31500041), National Science and Technology Major Project

387

(2018ZX0800907B-002), the Science and Technology Project of Jiangsu province

388

(BE2016374), the Fundamental Research Funds for the Central Universities

389

(KYZ201861 and KJQN201645) and Postgraduate Research & Practice Innovation

390

Program of Jiangsu Province (KYCX17_0653).

391 392

Supporting information description

393

Figure S1. SDS-PAGE of Dmt50 heterogeneously expressed in E. coli BL21 (DE3).

394

Figure S2. HPLC analysis of vanillate (A), syringate (B), isoproturon (C) and alachlor

395

(D) degradation by Dmt50. Figure S3. The relative dicamba methyltransferase activity

396

of Dmt50 under a series of temperatures (A) and pH (B). Figure S4. SDS-PAGE of

397

Mthfr66 heterogenous expressed in E. coli BL21 (DE3). Figure S5. The Mthfr66

398

relative activity of 5-methyl-THF conversion under a series of temperatures (A) and pH

399

(B). Figure S6. HPLC analysis of the products generated during 5-methyl-THF 20


Page 21 of 37


400

conversion by Mthfr66. Figure S7. Effect of exogenously added Mthfr66 on the

401

conversion of dicamba. Figure S8. RT-qPCR analysis of transcription of dmt50 and

402

mthfr66 in transgenic A. thaliana. Figure S9. Effects of temperature on the dicamba

403

demethylase activities of the crude lysates from dmt50 transgenic A. thaliana.

404

Table S1. Deduced function of each ORF within the scaffold 50 of the strain Ndbn-20.

405 406

References

407

(1)

Barrows, G.; Sexton, S.; Zilberman, D. Agricultural Biotechnology: The

408

Promise and Prospects of Genetically Modified Crops. J. Econ. Perspect. 2014,

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28 (1), 99–120.

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(2)

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ISAAA Br. No. 52 No. ISAAA: Ithaca, NY. (3)

413 414

ISAAA, 2016. Global Status of Commercialized Biotech/GM Crops: 2016.

Cerdeira, A. L.; Duke, S. O. The Current Status and Environmental Impacts of Glyphosate-Resistant Crops: A Review. J. Environ. Qual. 2006, 35 (5), 1633.

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Walker, S.; Widderick, M.; Storrie, A.; Osten, V. Preventing Glyphosate

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Resistance in Weeds of the Northern Grain Region. Weed Manag. Balanc.

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people, planet, profit. 14th Aust. Weeds Conf. Wagga Wagga, New South

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Wales, Aust. 6-9 Sept. 2004 Pap. Proc. 2004, 428–431.

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Vencill, W. K. Herbicide Handbook., 8th ed.; Weed Science Society of America: Lawrence, Kansas, 2002.

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Behrens, M. R.; Mutlu, N.; Chakraborty, S.; Dumitru, R.; Jiang, W. Z.; 21



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LaVallee, B. J.; Herman, P. L.; Clemente, T. E.; Weeks, D. P. Dicamba

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Resistance: Enlarging and Preserving Biotechnolog-Based Weed Management

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Strategies. Science. 2007, 316 (5828), 1185–1188.

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Harrison, S. K. The Pesticide Manual: A World Compendium, 8th Edition. J. Environ. Qual. 1988, 17 (2), 345.

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Herman, P. L.; Behrens, M.; Chakraborty, S.; Chrastil, B. M.; Barycki, J.;

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Weeks, D. P. A Three-Component Dicamba O-Demethylase from

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Pseudomonas Maltophilia, Strain DI-6: Gene Isolation, Characterization, and

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Heterologous Expression. J. Biol. Chem. 2005, 280 (26), 24759–24767.

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Yao, L.; Jia, X.; Zhao, J.; Cao, Q.; Xie, X.; Yu, L.; He, J.; Tao, Q. Degradation

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of the Herbicide Dicamba by Two Sphingomonads via Different O-

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Demethylation Mechanisms. Int. Biodeterior. Biodegrad. 2015, 104, 324–332.

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Yao, L.; Yu, L.; Zhang, J.; Xie, X.; Tao, Q.; Yan, X.; Hong, Q.; Qiu, J.; He, J.;

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Ding, D. A Tetrahydrofolate-Dependent Methyltransferase Catalyzing the

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Demethylation of Dicamba in Sphingomonas Sp. Strain Ndbn-20. Appl.

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Environ. Microbiol. 2016, 82 (18), 5621–5630.

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Li, R.; Zhu, H.; Ruan, J.; Qian, W.; Fang, X.; Shi, Z.; Li, Y.; Li, S.; Shan, G.;

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Kristiansen, K.; et al. De Novo Assembly of Human Genomes with Massively

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Parallel Short Read Sequencing. Genome Res 2010, 20 (2), 265–272.

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Tatusov, R. L. The COG Database: A Tool for Genome-Scale Analysis of Protein Functions and Evolution. Nucleic Acids Res. 2000, 28 (1), 33–36. 22


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Kanehisa, M.; Goto, S.; Furumichi, M.; Tanabe, M.; Hirakawa, M. KEGG for

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Representation and Analysis of Molecular Networks Involving Diseases and

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Drugs. Nucleic Acids Res. 2010, 38 (Database issue), 355–360.

445

(14)

Li, N.; Yao, L.; He, Q.; Qiu, J.; Cheng, D.; Ding, D.; Tao, Q.; He, J.; Jiang, J.

446

3,6-Dichlorosalicylate Catabolism Is Initiated by the DsmABC Cytochrome

447

P450 Monooxygenase System in Rhizorhabdus Dicambivorans Ndbn-20. Appl.

448

Environ. Microbiol. 2018, 84 (4), e02133-17.

449

(15)

Chen, H.; Nelson, R. S.; Sherwood, J. L. Enhanced Recovery of Transformants

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of Agrobacterium Tumefaciens after Freeze-Thaw Transformation and Drug

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Selection. Biotechniques 1994, 16 (4), 664–668,670.

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Zhang, X.; Henriques, R.; Lin, S. S.; Niu, Q. W.; Chua, N. H. Agrobacterium-

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Mediated Transformation of Arabidopsis Thaliana Using the Floral Dip

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Method. Nat. Protoc. 2006, 1 (2), 641.

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Masai, E.; Sasaki, M.; Minakawa, Y.; Abe, T.; Sonoki, T.; Miyauchi, K.;

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Katayama, Y.; Fukuda, M. A Novel Tetrahydrofolate-Dependent O-

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Demethylase Gene Is Essential for Growth of Sphingomonas Paucimobilis

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SYK-6 with Syringate. 2004, 186 (9), 2757–2765.

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Abe, T.; Masai, E.; Miyauchi, K.; Katayama, Y.; Fukuda, M. A

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Tetrahydrofolate-Dependent O-Demethylase , LigM , Is Crucial for Catabolism

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of Vanillate and Syringate in Sphingomonas Paucimobilis SYK-6. 2005, 187

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Kohler, A. C.; Mills, M. J. L.; Adams, P. D.; Simmons, B. A.; Sale, K. L.

464

Structure of Aryl O-Demethylase Offers Molecular Insight into a Catalytic

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Tyrosine-Dependent Mechanism. 2017, 1–10.

466

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Maria A. Vanoni, David P. Ballou, R. G. M. Methylenetetrahydrofolate Reductase. J. Biol. Chem. 1983, 258 (10), 11510–11514.

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Igari, S.; Ohtaki, A.; Yamanaka, Y.; Sato, Y.; Yohda, M.; Odaka, M.; Noguchi,

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K.; Yamada, K. Properties and Crystal Structure of Methylenetetrahydrofolate

470

Reductase from Thermus Thermophilus HB8. PLoS One 2011, 6 (8), e23716.

24


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Figure captions

Figure 1. A, Organization of the methyltransferase gene cluster in scaffold 50. The arrows indicate the sizes and transcriptional direction of each gene. The gene dmt50 encodes the dicamba methyltransferase; mthfr encodes the 5,10-methylene-THF reductase;

dhc

encodes

the

bifunctional

enzyme

5,10-methylene-THF

dehydrogenase/5,10-methylene-THF cyclohydrolase; and purU encodes formyl-THF deformylase. The open reading frames orf0806 and orf0802 encode a MFS transporter and a TonB-dependent receptor, respectively. The two white arrows represent genes encoding hypothetical proteins. B, Proposed THF-dependent dicamba demethylation system in strain Ndbn-20 and the mechanism by which the addition of Mthfr increases the overall Dmt50 conversion rate. The product 5-methyl-THF was a feedback inhibitor of Dmt50, and Mthfr removed 5-methyl-THF from the reaction, thus relieving the inhibition of 5-methyl-THF on Dmt50. Figure 2. HPLC and mass spectrometry analysis of the products generated during dicamba conversion by Dmt50. A, HPLC spectra of the reaction mixture obtained at 120 min, the mixture containing 0.1 mg of purified Dmt50, 2.0 mM THF, 1.0 mM dicamba and 100 mM Tris-HCl buffer (pH 8.0) in a 300 μL volume and the reaction was incubated at 45°C for 120 min, the detection wavelengths were 279nm and 319nm for dicamba and 3,6-DCSA, respectively. The “mAU” mean “milli absorbance unit”, a unit of HPLC signal. B, The first-order mass spectral analysis of the reaction mixture 25



obtained at 120 min; C, The second-order mass analysis of the m/z=205 (M-H)- ion, which corresponded to 3,6-DCSA. Figure 3. Time course of dicamba conversion by purified Dmt50. The mixture containing 0.1 mg of purified Dmt50, 2.0 mM THF, 1.0 mM dicamba and 100 mM Tris-HCl buffer (pH 8.0) in a 300 μL volume and the reaction was incubated at 45°C. Figure 4. Effect of exogenously added 3,6-DCSA or 5-methyl-THF on the conversion of dicamba. The mixture containing 0.1 mg of purified Dmt50, 2.0 mM THF, 1.0 mM dicamba and different concentration of 3,6-DCSA or 5-methyl-THF was added. The total volume was 300 μL and the reaction was incubated at pH 8.0, 45°C for 120 min. Figure 5. Dicamba conversion rate. The methyltransferase reaction mixture (300 μL) contained 2.0 mM THF, 1.0 mM dicamba, 1.0 mM NAD+, 1.0 mM menadione in 100 mM Tris-HCl buffer (pH 8.0). The reaction was incubated at 30°C for 120 min. A, 0.1 mg of Dmt50 was added to the reaction mixture; B, 0.1 mg of Dmt50 and 0.1 mg of Mthfr66 were added to the reaction mixture. Figure 6. The difference in growth of transgenic A. thaliana at different dicamba concentrations. A, The growth of transgenic plants on plates. (“dmt50”: transgenic A. thaliana carrying dmt50; “dmt50+mthfr”: bitransgenic A. thaliana carrying dmt50 and mthfr66, “CK”: nontransgenic A. thaliana); B and C, The root length and fresh weight of transgenic A. thaliana on plates, respectively. Data are presented as the means ± SE of 25 independent determinations. Significant differences were determined by Fisher’s test and are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01). 26


Page 26 of 37

Page 27 of 37


Figure 7. The difference in growth of transgenic A. thaliana seedlings in soil sprayed with different dicamba concentrations. A, The growth conditions of transgenic plant seedlings. (“dmt50”: single dmt50 gene transgenic A. thaliana; “dmt50+mthfr”: dmt50 and mthfr66 bitransgenic A. thaliana; “CK”: nontransgenic A. thaliana, a negative control; 0 g/ha dicamba treatment as a positive control); B and C, The root length and fresh weight of transgenic A. thaliana seedlings in soil. Data are presented as the means ± SE of 4 independent determinations. Significant differences were determined by Fisher’s test and are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01).

27



Page 28 of 37

Tables

Table 1. Strains and vectors used in this study. Strain or Relative characteristics vector R. Dicamba utilizer; streptomycinr (50 μg/mL) dicambivora ns Ndbn-20 (CCTCC M 2014550) E. coli BL21 Host strain for expression vector (DE3)

Source or reference 9

Expression vector; kanamycinr (100 μg/mL)

Lab stock

pET29a (+)

Vazyme Biotech

pET29admt5 pET29a (+) derivative carrying dmt50 gene 0 pET29amthf pET29a (+) derivative carrying mthfr gene r pGEM-T Clone vector; ampicillinr (100 μg/mL)

This study

pDBNBCBinary expression vector derived from 01 pCAMBIA2301; kanamycinr (100 μg/mL) pDBN10985 pDBNBC-01 derivative carrying dmt50 gene with a chloroplast transit peptide-coding region; kanamycinr (100 μg/mL) pDBN11098 pDBNBC-01 derivative carrying dmt50 and mthfr genes with a chloroplast transit peptide-coding region; kanamycinr (100 μg/mL)

Lab stock

28


This study Promega

This study

This study

Page 29 of 37


Figures

Figure 1. A, Organization of the methyltransferase gene cluster in scaffold 50. The arrows indicate the sizes and transcriptional direction of each gene. The gene dmt50 encodes the dicamba methyltransferase; mthfr encodes the 5,10-methylene-THF reductase; dhc encodes the bifunctional enzyme 5,10-methylene-THF dehydrogenase/5,10-methylene-THF cyclohydrolase; and purU encodes formyl-THF deformylase. The open reading frames orf0806 and orf0802 encode a MFS transporter and a TonB-dependent receptor, respectively. The two white arrows represent genes encoding hypothetical proteins. B, Proposed THF-dependent dicamba demethylation system in strain Ndbn-20 and the mechanism by which the addition of Mthfr increases the overall Dmt50 conversion rate. The product 5-methyl-THF was a feedback inhibitor of Dmt50, and Mthfr removed 5-methyl-THF from the reaction, thus relieving the inhibition of 5-methyl-THF on Dmt50. 29



30


Page 30 of 37

Page 31 of 37


Figure 2. HPLC and mass spectrometry analysis of the products generated during dicamba conversion by Dmt50. A, HPLC spectra of the reaction mixture obtained at 120 min, the mixture containing 0.1 mg of purified Dmt50, 2.0 mM THF, 1.0 mM dicamba and 100 mM Tris-HCl buffer (pH 8.0) in a 300 μL volume and the reaction was incubated at 45°C for 120 min, the detection wavelengths were 279nm and 319nm for dicamba and 3,6-DCSA, respectively. The “mAU” mean “milli absorbance unit”, a unit of HPLC signal. B, The first-order mass spectral analysis of the reaction mixture obtained at 120 min; C, The second-order mass analysis of the m/z=205 (M-H)- ion, which corresponded to 3,6-DCSA.

31



Figure 3. Time course of dicamba conversion by purified Dmt50. The mixture containing 0.1 mg of purified Dmt50, 2.0 mM THF, 1.0 mM dicamba and 100 mM Tris-HCl buffer (pH 8.0) in a 300 μL volume and the reaction was incubated at 45°C.

32


Page 32 of 37

Page 33 of 37


Figure 4. Effect of exogenously added 3,6-DCSA or 5-methyl-THF on the conversion of dicamba. The mixture containing 0.1 mg of purified Dmt50, 2.0 mM THF, 1.0 mM dicamba and different concentration of 3,6-DCSA or 5-methyl-THF was added. The total volume was 300 μL and the reaction was incubated at pH 8.0, 45°C for 120 min.

33



Figure 5. Dicamba conversion rate. The methyltransferase reaction mixture (300 μL) contained 2.0 mM THF, 1.0 mM dicamba, 1.0 mM NAD+, 1.0 mM menadione in 100 mM Tris-HCl buffer (pH 8.0). The reaction was incubated at 30°C for 120 min. A, 0.1 mg of Dmt50 was added to the reaction mixture; B, 0.1 mg of Dmt50 and 0.1 mg of Mthfr66 were added to the reaction mixture.

34


Page 34 of 37

Page 35 of 37


Figure 6. The difference in growth of transgenic A. thaliana at different dicamba concentrations. A, The growth of transgenic plants on plates. (“dmt50”: transgenic A. thaliana carrying dmt50; “dmt50+mthfr”: bitransgenic A. thaliana carrying dmt50 and mthfr66, “CK”: nontransgenic A. thaliana); B and C, The root length and fresh weight of transgenic A. thaliana on plates, respectively. Data are presented as the means ± SE of 25 independent determinations. Significant differences were determined by Fisher’s test and are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01).

35



Figure 7. The difference in growth of transgenic A. thaliana seedlings in soil sprayed with different dicamba concentrations. A, The growth conditions of transgenic plant seedlings. (“dmt50”: single dmt50 gene transgenic A. thaliana; “dmt50+mthfr”: dmt50 and mthfr66 bitransgenic A. thaliana; “CK”: nontransgenic A. thaliana, a negative control; 0 g/ha dicamba treatment as a positive control); B and C, The root length and fresh weight of transgenic A. thaliana seedlings in soil. Data are presented as the means ± SE of 4 independent determinations. Significant differences were determined by Fisher’s test and are indicated by one asterisk (P < 0.05) or two asterisks (P < 0.01).

36


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Page 37 of 37


Graphic for table of contents

37


Coexpression of methyltransferase gene dmt50 and methylene

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