Genetic Engineering of Maize (Zea mays L.) with Improved Grain

Subscriber access provided by READING UNIV

Article

Genetic Engineering of Maize (Zea mays L.) with Improved Grain Nutrients Xiaotong Guo, Xiaoguang Duan, Yongzhen Wu, Jieshan Cheng, Juan Zhang, Hongxia Zhang, and Bei Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05390 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 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.

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

Page 1 of 37

Journal of Agricultural and Food Chemistry

Title: Genetic Engineering of Maize (Zea mays L.) with Improved Grain Nutrients

Xiaotong Guo,1,# Xiaoguang Duan,2,# Yongzhen Wu,1 Jieshan Cheng,1 Juan Zhang,1 Hongxia Zhang,1 and Bei Li,1,*

1

College of Agriculture,

Ludong University, 186 Hongqizhong Road, Yantai, China 264025

2

School of Life Science and Technology,

ShanghaiTech University, 393 Middle Huaxia Road, Pudong, Shanghai, 201210

*Corresponding author: Bei Li Email: [email protected] Phone: 86-0535-6664662;

#

These authors contribute equally to this work.

1

1

ACS Paragon Plus Environment


2

ABSTRACT

3 4

Cell wall invertase plays important roles in the grain filling of crop

5

plants. However, its functions in the improvement of grain nutrients are

6

not investigated. In this work, the stable expression of cell wall invertase

7

encoding genes from different plant species, and the contents of total

8

starch, protein, amino acid, nitrogen, lipid and phosphorus were

9

examined in transgenic maize plants. High expression of cell wall

10

invertase gene conferred enhanced invertase activity and sugar content

11

on transgenic plants, leading to increased grain yield and improved grain

12

nutrients. Transgenic plants with high expression of transgene produced

13

more total starch, protein, nitrogen, and essential amino acids in the

14

seeds. Overall, the results indicate that cell wall invertase gene can be

15

used as a potential candidate for the genetic breeding of grain crops with

16

both improved grain yield and quality.

17 18

KEYWORDS: invertase, transgenic plants, grain, nutrient, maize

19

2


Page 2 of 37

Page 3 of 37


20

INTRODUCTION

21

Maize (Zea mays L.) is an important crop plant grown worldwide for

22

food, forage, energy and industrial materials. For grain crops, the

23

agronomic traits of grain number and weight, which determine the grain

24

yield per plant, are determined by genetic, physiological and

25

environmental factors.1 In addition to the conventional breeding

26

approach to improve the yield of crop plants, genetic engineering is a

27

more efficient way to generate new cultivars with improved agronomic

28

traits for a wider range of applications.

29

Cell wall invertase plays a crucial role in plant growth and

30

development, especially in the grain filling of crop plants.2-5 In rice

31

(Oryza sativa), four cell wall invertase encoding genes were observed to

32

be expressed in immature seeds and were proposed to have an important

33

function during the early developmental stages of the grain filling

34

phase.6 Overexpression of the cell wall invertase gene OsGIF1, under

35

the control of its own promoter, increased grain size and weight.7 Further

36

study revealed that OsGIF1 also functions in pre-existing and induced

37

defence.8 In soybean, suppressing the expression of invertase inhibitor

38

genes improved the weight and accumulations of hexoses, starch, and

39

protein of mature seeds in transgenic plants.9 In bamboo, three invertase

40

encoding genes were found to have individual roles in the adaption of

41

the plant to environmental changes.10 In tomato, silencing inhibitory 3



42

protein of the endogenous cell wall invertase gene significantly

43

increased seed weight and fruit sugar hexoses.11 In maize, constitutive

44

expression of the cell wall invertase encoding genes from Arabidopsis,

45

rice and maize, under the control of the cauliflower mosaic virus (CaMV)

46

35S promoter, all improved grain yield in transgenic plants.12 However,

47

to improve the grain’s nutritional quality, genetic improvement in both

48

grain yield and nitrogen concentration is necessary.13 Biofortified staple

49

crops such as rice, maize and wheat harboring essential micronutrients,

50

as well as new crop varieties having ability to combat chronic disease,

51

are under development.14 As a major source of food and animal feed, as

52

well as one of the world’s three major cereal crops (along with rice and

53

wheat), maize is deficient in lysine,15 a necessary amino acid for health

54

maintenance.16 Therefore, breeding high yield cultivar with high grain

55

nitrogen concentration is important for improving the nutritional quality

56

of maize.13 Recent advances in plant genetic engineering have provided

57

new opportunities to improve lysine content in maize.17-20

58

Although the function of cell wall invertase in increasing grain size

59

and weight has been well studied, its possible role in grain quality

60

improvement still remains unknown. To assess the potential of cell wall

61

invertase in the breeding of new crop varieties with increased grain

62

nutrients, transgenic maize plants constitutively expressing the cell wall

63

invertase gene from Arabidopsis, rice and maize, generated in our 4


Page 4 of 37

Page 5 of 37


12

64

previous study,

were continuously grown in the field for six

65

generations, and the contents of starch, lipid and protein in the seeds of

66

transgenic plants were investigated in this study.

67 68

MATERIALS AND METHODS

69

Plant Materials. Previously, we isolated the cell wall invertase genes

70

from Arabidopsis, rice and maize, and respectively introduced them into

71

the maize inbred line Ye478.12 Four individual transgenic lines, which

72

showed different expression levels of each transgene, were selected (A1,

73

A3, A4 and A5, transgenic lines AtGIF1-1, AtGIF1-3, AtGIF1-4 and

74

AtGIF1-5 for transgene AtGIF1 from Arabidopsis; O1, O3, O4 and O6,

75

transgenic lines OstGIF1-1, OsGIF1-3, OsGIF1-4 and OsGIF1-6 for

76

transgene OstGIF1 from rice; Zm1, Zm2, Zm3 and Zm4, transgenic

77

lines ZmGIF1-1, ZmGIF1-2, ZmGIF1-3 and ZmGIF1-4 for transgene

78

ZmGIF1 from maize). In the present study, two transgenic lines, which

79

showed the highest expression level of each transgene, were selected for

80

grain nutrient examinations (A3, A4, O1, O4, Zm1 and Zm3).

81

Field Trials. Wild type (WT) Ye478 and different transgenic lines (T6

82

generation) were grown in field on Wuse Farm of Shanghai (Shanghai,

83

China) in May 2016. Trial plots were arranged in a random complete

84

block design with three replications. Forty seeds of WT and each

85

transgenic line were sown in a double row plot. The plot was 2.5 m in 5



86

length and 1.2 m in width, with an interval of 0.25 m between plants.

87

Plants were thinned at five-leaf-stage, leaving 20 plants in each plot (a

88

population density of approximately 66700 plants/ha). Plants at

89

flowering stage were self-pollinated. Mature ears were harvested, dried

90

and weighed to determine the yield (dry weight). Cobs and kernels were

91

collected to take pictures. Ear length and weight, grain number per row,

92

100-grain-weight, total protein, N, amino acid content, as well as total

93

phosphorus (P), lipid and starch content of thirty randomly selected ears

94

from each plot were measured.

95

RNA Isolation and Quantitative Real-Time PCR Analyses. To

96

confirm the stable expression of transgenes, quantitative real-time PCR

97

(qRT-PCR) was performed with RNA samples isolated from the 4th fully

98

expanded leaves of field grown WT and transgenic plants at

99

five-leaf-stage. Wild type plants segregated from transgenic lines

100

AtGIF1-1, OsGIF1-1 and ZmGIF1-1, respectively, were used as control.

101

Total RNA was extracted with Trizol reagent (Takara, Kyoto, Japan) and

102

the first-strand cDNA was synthesized with HiScriptIIQ RT SuperMix

103

for qPCR (+gDNA wiper) (Vazyme, Shanghai, China). PCR was

104

performed with 96-well optical reaction plates by iQ5 (Bio-Rad,

105

Richmond, CA, USA) after pre-incubation for 3 min at 95°C, followed

106

by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s,

107

and extension at 72°C for 20 s. Amplification of the detected genes was 6


Page 6 of 37

Page 7 of 37


108

monitored every cycle using SYBR green fluorescence. All experiments

109

were carried out using the iQ5 real-time PCR detection system and

110

AceQ qPCR SYBR Green Master Mix (Vazyme, Shanghai, China). Fold

111

changes of RNA transcripts were calculated by the ∆Ct method [Ct

112

(GIF1) – Ct (actin1)] with maize actin1 as an internal control. Each

113

experiment was repeated three times. Gene specific primers used in this

114

study are

AtGIF1-F

115

AtGIF1-R

(5’-CGGATCAGTCCAACTTGGTT-3’)

116

AtGIF1,

117

OsGIF1-R

118

OsGIF1,

119

ZmGIF1-R

120

ZmGIF1, actin1-F (5’-ATCACCATTGGGTCAGAAAGG-3’ ） and

121

actin1-R (5’-GTGCTGAGAGAAGCCAAAATAGAG-3’) for actin1.

122

Sugar Content Determination and Invertase Activity Assays. Seeds

123

of wild type (segregated from transgenic line ZmGIF1-1) and

124

homozygous transgenic lines (T6) were sown in 28×28 cm (diameter

125

×depth) pots filled with homogeneous loam, and grown under 14 h of

126

light/10 h of dark cycles in green house at 32°C (light) or 22°C (dark)

127

with a photosynthetic photon flux density of 700 µmol/m2/s, and a

128

relative humidity of 50-60%. Shoots were thinned to two plants per pot

129

at three-leaf-stage. At five-leaf-stage, the 4th fully expanded leaves were

OsGIF1-F

(5’-TAATTCAGTGGCCGGTTAGG-3’)

transgene

(5’-CTCTGAGGAGCCTGATCGAC-3’)

(5’-AGGCTCCATTCATCATGACC-3’) ZmGIF1-F

for

for

7


for

and

transgene

(5’-GAACGGCAAGATATCCCTGA-3’)

(5’-CATGACCGGCTTCTTCATCT-3’)

and

and

transgene


130

collected for sugar contents determination and invertase activity assays.

131

Sugar contents were determined as described previously.21 For invertase

132

activity analyses, leaves were ground in extraction buffer and

133

centrifuged at 12 000 g for 10 min. The pellet was washed twice and

134

re-suspended in extraction buffer as described previously.22

135

Total Starch and Amylose Content Analyses. Thirty dried kernels of

136

wild type (segregated from transgenic line ZmGIF1-1) and each

137

transgenic lines harvested from Wuse Farm in Shanghai (Shanghai,

138

China) in 2016 (T7 generation) were boiled for 3 min to peel off the seed

139

coat. Embryo was removed from endosperm, and the remaining parts of

140

seeds were dried to constant weight at 45°C. Then, the dried endosperm

141

was ground into fine powder. The precipitate was collected after soluble

142

sugar extraction and was washed three times with 80% EtOH. Starch

143

content was determined with the total starch kit following the

144

manufacturer’s instruction (Megazyme International Ireland Limited,

145

Wicklow, Ireland).

146

Amylose content in starch was determined using a col-orimetric

147

amylose content assay as described previously.23 Potato amylose (A0512;

148

sigma-aldrich, St. Louis, MO, USA) and amylopectin (10118;

149

sigma-aldrich) were used as standard samples to establish the standard

150

curves. The iodine affinity of starches was determined through

151

potentiometric autotitrator (Metrohm 907Titrando, Herisau, Switzerland) 8


Page 8 of 37

Page 9 of 37


152

as described by Schoch.24 Amylose content was also analyzed using the

153

GPC-RI system developed by Park and his colleagues.25

154

The dried kernels of wild type (segregated from transgenic line

155

ZmGIF1-1) and each transgenic lines harvested in 2016 (T7 generation)

156

were steeped in 0.14% NaHSO3 for 36 h at 52°C, and then ground and

157

dried for the assay. Each starch sample (4 mg) was mixed with 4 ml

158

DMSO and stirred in a boiling water bath for 24 h. The sample was

159

filtered through 2 µm pore size filter and then injected into a PL-GPC

160

220 instrument (Polymer Laboratories, Inc., Amherst, MA, USA) with

161

three Phenogel columns (Phenomenex, Inc., Tor-rance, CA, USA), a

162

guard column (Phenomenex, Inc., Torrance, CA, USA) and a differential

163

refractive index detector. The eluent system with DMSO containing 0.5

164

mM NaNO3 was used at a flow rate of 0.8 ml min−1. The column oven

165

temperature was controlled at 80°C. For molecular weight (MW)

166

calibration, standard dextrans (American Polymer Standards Co., Mentor,

167

OH, USA) with different MW were used.26

168

Total Protein and Nitrogen Content Analyses. Endosperm and all

169

samples were treated at 105°C for 30 min, dried at 70°C to a constant

170

weight, and then weighed to obtain the dry weight (DW).27 Dried

171

samples were then ground into fine powder. Total N content was

172

determined as described previously using an amount of 0.5 g powder.28

173

For total protein content analyses, an amount of 0.5 g ground power 9



Page 10 of 37

174

were measured as described previously.29

175

Amino Acid Assays. For amino acid analyses, a total amount of 20 mg

176

fine powder (same batch as used for total protein and nitrogen content

177

analyses) was hyrolyzed in 1 ml of 6 M HCl for 24h at 105°C. After

178

centrifugation at 12000 rpm for 10 min, 100 µl liquid supernatant was

179

decanted and dried in vacuum, and then dissolved in 1 ml of 0.1 N HCl.

180

After centrifugation as described previously, 1 µl supernatant was

181

separated by analytical column ZORBAX Eclipse-AAA (Agilent, 5 µm,

182

4.6 × 250 mm) and analyzed using HPLC-UV System (1260, Agilent

183

Technologies, USA) which was detected with ultraviolet detector at

184

262nm and 338nm. And the limit of detection is 1 pmol. The A phase is

185

40 mM Na2HPO4 (PH 7.8) and the B phase is acetonitrile: methanol:

186

water (45:45:10, v/v). The flow rate was set at 2 ml/min and the column

187

temperature was set at 40°C. Standard samples were purchased from

188

sigma-aldrich (LAA21).

189

Determination of Phosphorus and Lipid Contents. Dried kernels were

190

heated at 105°C for 30 min, dried to a constant weight at 75°C and

191

ground into fine powder. An amount of 0.5 g fine powder was digested

192

with a modified Kjeldahl procedure using H2SO4+ H2O2 as catalyst,

193

and analyzed for phosphorus concentration by automated colorimetry

194

using the molybdovanadate method.31

195

Lipid

content

was

analyzed

by

near-infrared

10


30

reflectance

Page 11 of 37


196

spectroscopy (NIRS) on a VECTOR22/N as described previously

197

(Bruker, Karlsruhe, Germany).32

198

Statistical Analysis. ANOVA methodology or Student’s t-test was used

199

for data analyses. Differences in value means were compared according

200

to Duncan’s multiple comparison tests. Values of 0.01 < P < 0.05 or P

Genetic Engineering of Maize (Zea mays L.) with Improved Grain

Recommend Documents