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

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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

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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


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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

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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

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

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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

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both improved grain yield and quality.

17 18

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

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2


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INTRODUCTION

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

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

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

24

yield per plant, are determined by genetic, physiological and

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environmental factors.1 In addition to the conventional breeding

26

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

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more efficient way to generate new cultivars with improved agronomic

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traits for a wider range of applications.

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Cell wall invertase plays a crucial role in plant growth and

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

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(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

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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

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

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

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

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

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



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protein of the endogenous cell wall invertase gene significantly

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

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expression of the cell wall invertase encoding genes from Arabidopsis,

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

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

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

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grain yield and nitrogen concentration is necessary.13 Biofortified staple

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

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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

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

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

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

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nitrogen concentration is important for improving the nutritional quality

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

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

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Although the function of cell wall invertase in increasing grain size

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

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

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invertase in the breeding of new crop varieties with increased grain

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nutrients, transgenic maize plants constitutively expressing the cell wall

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


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12

64

previous study,

were continuously grown in the field for six

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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from each plot were measured.

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RNA Isolation and Quantitative Real-Time PCR Analyses. To

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

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

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expanded leaves of field grown WT and transgenic plants at

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

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

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

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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

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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


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108

monitored every cycle using SYBR green fluorescence. All experiments

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

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

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

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

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

114

study are

AtGIF1-F

115

AtGIF1-R

(5’-CGGATCAGTCCAACTTGGTT-3’)

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AtGIF1,

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OsGIF1-R

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OsGIF1,

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ZmGIF1-R

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

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

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Sugar Content Determination and Invertase Activity Assays. Seeds

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

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

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

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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

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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.

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Sugar contents were determined as described previously.21 For invertase

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activity analyses, leaves were ground in extraction buffer and

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

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re-suspended in extraction buffer as described previously.22

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Total Starch and Amylose Content Analyses. Thirty dried kernels of

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

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transgenic lines harvested from Wuse Farm in Shanghai (Shanghai,

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

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

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

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

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

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content was determined with the total starch kit following the

144

manufacturer’s instruction (Megazyme International Ireland Limited,

145

Wicklow, Ireland).

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Amylose content in starch was determined using a col-orimetric

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

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

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

150

curves. The iodine affinity of starches was determined through

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


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

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

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The dried kernels of wild type (segregated from transgenic line

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

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

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

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

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

160

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

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

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

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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

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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

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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



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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

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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

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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

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196

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

197

(Bruker, Karlsruhe, Germany).32

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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

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

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