SlMYC2 Involved in Methyl Jasmonate-Induced Tomato Fruit Chilling

Mar 12, 2018 - pTRV1, pTRV2, and pTRV2-SlMYC2 were transformed into competent cells E. coli with heat shock method and grown on select Luria-Bertani (...
6 downloads 9 Views 580KB Size
Subscriber access provided by UNIV OF DURHAM

Agricultural and Environmental Chemistry

SlMYC2 Involved in MeJA-induced Tomato Fruit Chilling Tolerance Dedong Min, Fujun Li, Xinhua Zhang, Xixi Cui, Pan Shu, Lulu Dong, and Chuntao Ren J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00299 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

1

SlMYC2 Involved in MeJA-induced Tomato Fruit Chilling Tolerance

2

Dedong Min, Fujun Li, Xinhua Zhang*, Xixi Cui, Pan Shu, Lulu Dong, Chuntao Ren

3

School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo

4

255049, Shandong, PR China

5 6

* Corresponding author

7

Tel: +86-533-2786398; Email: [email protected] (Xinhua Zhang)

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

8

ABSTRACT

9

MYC2, a basic helix-loop-helix transcription factor, is a master regulator in Jasmonic acid (JA)

10

signaling pathway. However, the functions of SlMYC2 in MeJA-mediated fruit chilling tolerance are

11

far from being clearly understood. Thus, in present work, we constructed SlMYC2-silenced tomato

12

fruit by virus-induced gene silencing (VIGS) and investigated the function of SlMYC2 in

13

MeJA-induced tomato fruit chilling tolerance. The results showed that MeJA treatment markedly

14

induced the SlMYC2 expression, increased proline content, lycopene content and antioxidant enzyme

15

activities, including superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, inhibited

16

the increase of electrical conductivity and malondialdehyde content, and effectively reduced the

17

chilling injury (CI) incidence and CI index. However, these effects of MeJA treatment were partially

18

counteracted in SlMYC2-silenced tomato fruit, and the CI incidence and CI index in

19

(SlMYC2-silenced + MeJA)-treated fruit were higher than those in MeJA-treated fruit. Our results

20

indicated that SlMYC2 might be involved in MeJA-induced chilling tolerance, possibly by

21

ameliorating the antioxidant enzyme system of fruit and increasing proline and lycopene levels.

22

KEYWORDS: MYC2 transcription factor, methyl jasmonate, virus-induced gene silencing, chilling

23

injury, tomato fruit

2

ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

Journal of Agricultural and Food Chemistry

24

INTRODUCTION

25

Cold storage is one of the main methods used to prolong the storage time and maintain postharvest

26

quality of horticultural crops. However, low temperature could result in chilling injury (CI) for many

27

cold-sensitive fruit and vegetables.1,2 The main symptoms of CI are discoloration, surface lesion and

28

abnormally ripening, which further shorten the storage time and lead to quality deterioration.3

29

Therefore, it is urgent to understand the physiological mechanism of CI in fruit and optimize

30

methods that alleviate the CI symptoms.

31

Jasmonic acid (JA) and methyl jasmonate (MeJA), an endogenous regulator, play important roles

32

in the development and defense responses of many plant,4 such as Chinese bayberry,5 bean6 and

33

cotton.7 Recently, most literature suggested that MeJA could enhance the chilling tolerance in many

34

fruit and vegetables, including cucumber,8 cowpea,9 loquat,10 peach,11 tomato,12 etc. In addition,

35

MeJA, an important plant hormone, not only has no side effects on the health, but also could

36

promote the biosynthesis of natural products with healthy characteristics in many plant, then

37

improving their beneficial on human health.13 Thus, the application of MeJA is greatly promising in

38

alleviating CI and maintaining fruit quality during postharvest cold storage periods. The induced

39

chilling tolerance of these fruit and vegetables by MeJA might be due to reduced the H2O2 levels,

40

enhanced antioxidant enzyme activity, improved proline and γ-aminobutyric acid contents, higher

41

level of energy charge and cooperation with catabolism of arginine.8-12 Nevertheless, the molecular

42

mechanisms of MeJA-induced fruit chilling tolerance remain poorly understood.

43

MYC2, a basic helix-loop-helix transcription factor, is the master regulator in JA signaling

44

pathway and induces JA-mediated responses such as wounding, water deficit stress and oxidative

45

stress adaptation.14-16 Recently, zhao et al.17 pointed out that MeJA treatment could induce the 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

46

expression of MaMYC2a, MaMYC2b and cold-responsive pathway genes, thus, MaMYC2 might

47

participate in MeJA-induced banana chilling tolerance. However, the functions of SlMYC2 in

48

MeJA-mediated fruit chilling tolerance are far from being clearly understood. Hence, it is urgent to

49

find an effective method that can be used to study the function of SlMYC2 in MeJA-mediated fruit

50

chilling tolerance. Virus-induced gene silencing (VIGS) is an effective, rapid and simple reverse

51

genetics approach for studying plant genes function18, 19 and has been widely used in several plant

52

species, such as tomato,20 spinach,21 peach,22 litchi23 and strawberry.24

53

In addition, tomato fruit is a typical representative of the respiratory climacteric fruit, and is a

54

model material for studying chilling injury of fruits. Thus, in present work, we firstly constructed

55

SlMYC2-silenced tomato fruit by VIGS and treated with MeJA before cold storage. The indexes

56

correlated with fruit chilling tolerance such as CI index, electrical conductivity, malondialdehyde

57

(MDA) content and antioxidant enzyme activities were measured. Meanwhile, the expression levels

58

of SlMYC2 in tomato fruit exposed to chilling were also investigated by quantitative real-time

59

polymerase chain reaction (qRT-PCR). The aim of this work was to study the role of SlMYC2 in

60

MeJA-induced chilling tolerance of tomato fruit.

61

MATERIALS AND METHODS

62

Construction of SlMYC2-Silenced Tomato Fruit. Firstly, Total RNA was isolated from frozen

63

tomato tissue (1.5 g) according to the method of Zhang et al.25 The first strand complementary DNA

64

(cDNA) was obtained with 2 µg of total RNA, Oligo(dT18) and M-MLV reverse transcriptase.

65

Then, a 454-bp fragment of SlMYC2 gene (GenBank Accession No. KF428776) was amplified by

66

PCR from tomato cDNA sources using gene specific primes (forward: 5’-GGG GTA CCC CTG

67

GTC AGG CGT TGT ATA GTT C-3’ with a KpnI restriction site and reverse: 5’-CCG CTC GAG 4

ACS Paragon Plus Environment

Page 4 of 30

Page 5 of 30

Journal of Agricultural and Food Chemistry

68

GAG GAG GAT TCT TCT GTT GTT GC-3’ with a XhoI restriction site). The production of PCR

69

was cloned into pTRV2 via a KpnI/XhoI digestion to form pTRV2-SlMYC2.

70

pTRV1, pTRV2 and pTRV2-SlMYC2 were transformed into competent cells E.coli with heat

71

shock method and grown on select Luria- Bertani (LB) media containing appropriate antibiotics

72

overnight at 37 °C. All constructs were then purified by Fast-Plasmid Mini kit and the identity of the

73

final constructs was assayed by sequencing.

74

Finally, the purified plasmid of pTRV1, pTRV2 and pTRV2-SlMYC2 were transformed into

75

Agrobacterium strain GV3101. A 5 mL agrobacterium culture with 50 µg mL-1 kanamycin (Kan), 50

76

µg mL-1 gentamicin (Gen) and 20 µg mL-1 rifampicin (Rif) was grown overnight at 28 °C. The next

77

day, the culture was transformed into 50 mL yeast extract and beef (YEB) medium that containing

78

50 µg mL-1 Kan, 50 µg mL-1 Gen, 20 µg mL-1 Rif, 10 mM 2-N-morpholino ethanesulfonic acid

79

(MES) and 20 µM acetosyringone (AS) and grown overnight at 28 °C. Subsequently, the

80

agrobacterium cells were harvested and resuspended with infiltration media (containing 10mM

81

MgCl2, 10 mM MES, 200 µM AS), adjusted to OD600≥1.0. The agrobacterium soluble containing

82

pTRV1 and pTRV2 (as a control) or pTRV1 and pTRV2-SlMYC2 (as SlMYC2-silenced fruit) in a 1:1

83

ratio added and cultured at room temperature for 2 h. After 2 h, the agrobacterium soluble was

84

infiltrated into the carpopodium of tomato fruit about 10 d after pollination from a green house in

85

Zibo, Shandong province, China.

86

Fruit and Treatment. Tomato fruit (Solanum lycopersicum L. cv. Badun) containing control and

87

SlMYC2-silenced fruit were hand-harvested at mature-green stage. After transported immediately to

88

our laboratory, both control and SlMYC2-silenced tomato fruit were randomly divided into two

89

groups. One group of control or SlMYC2-silenced fruit were treated with 0.05 mM MeJA as 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

90

described in our previous work,12 the other were treated with air as control. Each treatment was

91

replicated 3 times (60 fruit /replicated).

92

After treatment, the container was opened and ventilated for 1 h. Then, all fruit were stored at

93

2±1 °C and 80-90 % relative humidity. The mesocarp of fruit were cut at 0, 1, 3, 7, 14, 28 d, frozen

94

immediately in liquid nitrogen and stored at -80 °C until further analysis of physiological indicators.

95

The fifteen fruits were sampled at 14 and 28 d of storage and stored at room temperature with

96

80-90 % relative humidity for 7 d to assay the CI symptom and color change index.

97

CI Incidence and CI Index. CI incidence and CI index were measured that used to evaluate the

98

CI degree of tomato fruit during cold storage. According to the method of Zhang et al.26 with little

99

modification, the CI symptom was measured visually and CI index was calculated using following

100

scale: 0=no CI symptom, 1=less than 5% of surface area, 2=from 5% to 25%; 3=from 25% to 50%,

101

4=more than 50% of surface area. CI index=Σ(scale×the number of fruit within this scale)/(total

102

fruit×4).

103 104

The CI incidence (%) was calculated as the number of CI fruit divided by the total number of fruit recorded and multiplied by 100.

105

Color Change Index. According to the method of Cao et al.,27 the color change index was

106

assayed. Color change index=Σ(color scale value×number of the fruits within each scale)/(5×total

107

number of fruits).

108

Electrical Conductivity and Malondialdehyde Content. The electrical conductivity was

109

measured by DDS-307A conductivity meter (Leici Inc., Shanghai, China) as described in our

110

previous work.28 The MDA content was assayed with UV-2102 PCS spectrophotometer (Unico Inc.,

111

Shanghai, China) according to the method of Jin et al.29 The amount of MDA expressed as nanomole 6

ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30

Journal of Agricultural and Food Chemistry

112

MDA per gram of fresh weight (FW).

113

Extraction and Measurement of Enzyme Activities. Frozen tomato tissue (1.0 g) was

114

homogenized with 5 mL of sodium phosphate buffer (SPB, pH=7.0) containing 1% (w/v)

115

polyvinylpyrrolidone and centrifuged at 10000g for 15 min at 4 °C. Then, the supernatant was

116

collected and used for measurement of enzymes activities including superoxide dismutase (SOD),

117

peroxidase (POD) and catalase (CAT) and ascorbate peroxidase (APX).

118

SOD activity was assayed according to the method of Zeng et al.30 One unit of SOD activity was

119

defined as the amount of enzyme causing 50% inhibition of photochemical reduction of nitroblue

120

tetrazolium.

121 122

POD activity was measured as described by Luo et al.31 One unit of POD activity was defined as the absorbance increase of 0.01 units per minute under assay condition.

123

CAT and APX activity was determined according to the method of Jiang et al.32 One unit was

124

defined as the amount of enzyme causing an absorbance change of 0.1 per minute under assay

125

conditions. One unit of APX activity was defined as the absorbance change of 0.001 units per

126

minute.

127

Protein content was measured according to the method of Bradford33 with bovine serum albumin

128

as a standard. All the activities of antioxidant enzymes were expressed as unit per milligram protein

129

(U mg−1 protein).

130

Proline and Lycopene content. Proline content was determined according to the method of Li et

131

al.34 and Liu et al.35 Fruit tissue (1.5 g) was homogenized with 5 mL 3% (w/v) sulfosalicylic acid and

132

incubated at 100 °C for 10 min. After incubation, the supernatant (0.25mL) mixed with glacial acetic

133

acid (0.25 mL) and ninhydrin (0.5 mL), then boiled at 100 °C for 40 min. After cooled to room 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

134

temperature, the reaction mixture was added 0.5 mL of toluene and the absorbance of organic phase

135

was measured at 520 nm. The resulting values were compared to a standard curve constructed with

136

known amounts of proline. The proline content was expressed as µg g-1 FW.

137

Lycopene content was determined according to the method of Kuti and Konuru.36 Tomato fruit

138

tissue (1.5 g) was homogenized in 10 mL of hexane/ methanol/ acetone (2:2:1) and centrifuged at

139

10000g for 10 min. The upper hexane layer was diluted 10 times and measured at 505 nm using an

140

UV-2102 PCS spectrophotometer against a hexane black. The concentration of total lycopene was

141

calculated from the data using a specific extinction coefficient of 3400. The results were expressed

142

as g kg−1 FW.

143

Semi-quantitative Reverse Transcription PCR. Semi-quantitative reverse transcription PCR

144

(SqRT-PCR) was performed as described of Chi et al.37 The specific primer was designed outside the

145

region targeted for silencing, the SlUbi3 gene was used as reference gene and the results were as

146

follows:

147

SlMYC2-forward: 5’-CAG TTT TGC CTT CTT CGG GC-3’

148

SlMYC2-reverse: 5’-TTC GCT GGC TTT CTA CCT CG-3’

149

SlUbi3-forward: 5’-TCCATCTCGTGCTCCGTCT-3’

150

SlUbi3-reverse: 5’-CTGAACCTTTCCAGTGTCATCAA-3’

151

The SqRT-PCR program was processed with denaturation step at 95 °C for 4 min, followed by 18,

152

21, 24, 27, 30 and 35 cycles of 30 s at 94 °C, 30 s at 60 °C and 45 s at 72 °C, and extension at 72 °C

153

for 10 min. Finally, the PCR production was run on a 1 % agarose gel with 0.5 µg ml-1 ethidium

154

bromide.

155

qRT-PCR assay. qRT-PCR was performed according to our previous method.38 The relative 8

ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30

Journal of Agricultural and Food Chemistry

156

expression of SlMYC2 was measured by qRT-PCR using the SYBR Green I Master Mix

157

(Toyobo, Osaka, Japan) on a LineGene 9600 detection system (Bioer, HangZhou, China). The

158

specific primers of qRT-PCR were same with primes that used in SqRT-PCR.

159

And, the qRT-PCR program was processed with preliminary step of 2 min at 95 °C, followed by

160

40 cycles 15 s at 95 °C and 20 s at 60 °C, and 45 s at 72 °C. The melting curves were measured from

161

55 °C to 95 °C at 0.5 °C increments. The gene expression level of SlMYC2 was calculated using the

162

method of 2-△△Ct.

163

Data Analysis. The experiments were performed with absolutely randomized design. All the data

164

were analyzed with SPSS 19.0 (SPSS Inc., chicago, LC, USA). One-way analysis of variance

165

(ANOVA) was used for assay of date and the significant differences were conducted by Ducan’s

166

multiple comparisons. Differences at P