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Agricultural and Environmental Chemistry

Comparative evaluation on vitamin E and carotenoids accumulation in sweet corn (Zea mays L.) seedlings under temperature stress Xinbo Guo, Nan Xiang, Chunyan Li, Gaoke Li, Yongtao Yu, and Jianguang Hu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04452 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Journal of Agricultural and Food Chemistry

Comparative evaluation on vitamin E and carotenoids accumulation in sweet corn (Zea mays L.) seedlings under temperature stress

Authors: Nan Xiang†, Chunyan Li‡, Gaoke Li‡, Yongtao Yu‡, Jianguang Hu‡, *, Xinbo Guo†, *

Affiliations: † School of Food Science and Engineering, South China University of Technology, Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), Guangzhou, 510640, China ‡ Crop Research Institute, Guangdong Academy of Agricultural Sciences; Key Laboratory of Crops Genetics Improvement of Guangdong Province, Guangzhou, 510640, China.

Corresponding authors: * Xinbo Guo, Tel & Fax: (+86) 20-87113848; E-mail: [email protected] * Jianguang Hu, Tel & Fax: (+86) 20-85514234; Email: [email protected]

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1

ABSTRACT

2

This study aims to investigate the response profiles of vitamin E and carotenoids on

3

transcription and metabolic levels of sweet corn seedlings under temperature stress. The

4

treated temperatures were set as 10°C (low temperature, LT), 25°C (control, CK) and

5

40°C (high temperature, HT) for sweet corn seedlings. The gene expression profiles of

6

vitamin E and carotenoids biosynthesis pathways were analyzed by RT-qPCR, and the

7

composition profiles were analyzed by high performance liquid chromatography

8

(HPLC). Results showed that vitamin E gradually accumulated in response to LT stress

9

but was limited by HT stress. The increase of carotenoids was suppressed by LT stress

10

whereas HT stress promoted it. The existed results elaborated the interactive and

11

competitive relationships of vitamin E and carotenoids in sweet corn seedlings to

12

respond extreme temperature stress at transcriptional and metabolic levels. The present

13

study would improve sweet corn temperature resilience with integrative knowledge in

14

the future.

15

KEYWORDS

16

Sweet corn, temperature stress, vitamin E, carotenoids, biosynthesis

17


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INTRODUCTION

19

Abiotic stress generally triggers plenty physiological changes in plants thus alters

20

crop development and reduces yield. Among the series stresses, high and low

21

temperature (HT and LT) turn to be critical factors on limiting plants productivity

22

worldwide. LT often affects cell membrane fluidity by altering the composition of

23

membrane fatty acid thus influences the physiological and biochemical processes in

24

plants 1. On the other hand, HT stress would induce the aggregation and denaturation

25

of protein and accelerate plant senescence 2.

26

Vitamin E, as one group of lipid-soluble antioxidants, consist of eight isomers: α-,

27

β-, γ-, δ-tocopherols and tocotrienols, locate in membranes and perform in stabilizing

28

membranes. Previous study has described incredible roles of vitamin E on seeds storage,

29

germination and early seedling development by limiting nonenzymatic lipid oxidation

30

in plants under stress 3. In detail, during seeds germination and aging, tocopherols

31

protect embryo against reactive oxygen species (ROS) attack, whereas tocotrienols

32

decrease metabolic rate of seeds 4. Tocopherols have been proved with a role on

33

photoassimilate transport and structure of phloem parenchyma transfer cells in

34

Arabidopsis under LT stress 5. Besides, an increase of α-tocopherol content was crucial

35

in protecting and maintaining the function of photosynthesis apparatus under HT stress

36

6.

37

Carotenoids are synthesized in phototrophic and non-phototrophic organisms with

38

plenty functions acting in photosynthesis and photoprotection. In order to protect

39

organisms from photodamage, carotenoids raise xanthophyll cycle and regulate light



40

harvesting by scavenging free radical, limiting lipid peroxidation and stabilizing the

41

lipid bilayers of membrane 7. Besides, carotenoid-derived signals are associated with

42

plants growth and development, as well as solving external stresses by mediating 1O2

43

signaling pathway 8. Lutein and zeaxanthin, classified as xanthophylls as well as β-

44

carotene, classified as carotene are effective in radical scavenge as reported 9. They

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regularly varied under extreme temperature stress to response oxidative stress and

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scavenge ROS. The change of carotenoids profile in response to temperature stress in

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potato leaves and rice bran has been reported previously 10-11. As a model crop, maize has been extensively studied for its vitamin E and carotenoids

48 49

12.

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of sugar to starch inside endosperm of corn kernel 13, have a favorable flavor and are

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planted worldwide. During seasonal cultivation, both HT and LT stresses forced the

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growth and development of seedling stage of maize

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vitamin E and carotenoids which associated with cell metabolism and regulation were

54

varied 16-17. As described 18, both vitamin E and carotenoids biosynthesis pathways are

55

evolved with plastidic methylerythritol 4-phosphate (MEP) and shikimate pathways.

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Genetic studies have reported the significances of vitamin E 1 (vte1) and vte2 on the

57

production of tocopherols 5. Besides, HPPD (4-hydoxyphenyl pyruvate dioxygenase)

58

and vte3 (MPBQ-MT) have been decorated with roles on transformation or production

59

of tocochromanols 19-20. Existed study showed that the enhancement of gene expression

60

profiles in vitamin E biosynthesis pathway was involved with the induction of LT stress

61

in Arabidopsis thaliana

Sweet corn, derived from the mutation in the relative gene regulating the conversion

21.

14-15.

Among these influences,

Key genes in carotenoids biosynthesis pathway were


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described with important roles on synthesis 22. And recently, some of their profiles were

63

analyzed under extreme temperature stress in Satsuma mandarin

64

regulation mechanism especially in sweet corn remained merely investigated.

65

Therefore, this study aims at the metabolic profile response to temperature stress, rather

66

than functional traits related to the development and phenotype, in order to provide a

67

deeper understanding on regulative mechanism of sweet corn seedlings under extreme

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temperature stress for improving agricultural production.

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MATERIALS AND METHODS

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Plant materials and treatments

23.

However, the

71

The sweet corn seeds (YT28: a single cross hybrid that widely planted for products

72

development), obtained from Crop Research Institute, Guangdong Academy of

73

Agricultural Sciences (Guangzhou, China), were sterilized with 75% alcohol for 1 min

74

and washed with distilled water for at least 7 times before use. The culturing condition

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of sweet corn seeds was 25°C temperature with 95% humidity in darkness. Seeds were

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evenly placed in 9 identical trays and grew to seedlings. Each of the 3 trays were then

77

transported into three chambers with different temperature as 10°C, 25°C and 40°C,

78

other settings were the same. Sweet corn seedlings were cultivated in three different

79

temperatures for 30 hours. Samples were collected at 0, 15 and 30 hours after treatment

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(HAT) with diverse names as CK (control, grew in 25°C), LT (low temperature, grew

81

in 10°C) and HT (high temperature, grew in 40°C) with three biological replicates.

82

Same samples at 0 HAT were used for all three treatments.

83

Reagent and chemicals



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Methanol (MeOH), methyl tert-butyl ether (MTBE), ethanol (EtOH), n-hexane,

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isopropyl alcohol, ethyl acetate and acetic acid (HPLC grade) were purchased from

86

ANPEL Scientific Instrument Co. (Shanghai, China). Sodium chloride (NaCl),

87

pyrogallol, potassium hydroxide (KOH), sodium hydroxide (NaOH) and ammonium

88

acetate (analytical grade) were purchased from Sangon Biotech Co. Ltd. (Shanghai,

89

China). L-ascorbic acid (AsA) and 2,6-Di-tert-butyl-4-methylphenol (BHT) were

90

purchased from Sigma Aldrich (St. Louis, Mo, USA).

91

RNA extraction, reverse transcription and RT-PCR analysis

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The total RNA of sweet corn seedlings was extracted with Plant RNA Kit (Tiangen,

93

Beijing, China) and reversed to cDNA by PrimeScriptTM RT Reagent Kit with gDNA

94

Eraser (Takara Biotechnology, Dalian, China), respectively. Real-time qPCR was

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operated with SYBP® Premix Ex TaqTM Kit (Tiangen, Beijing, China) by LightCycler®

96

480 Real-Time PCR System (F.Hoffmann-La Roche Ltd, Switzerland).

97

103644252) was selected as reference gene and the primer sequences of selected genes

98

in this study were listed in Supplementary Table S1. Ct values were processed with 2-

99

△△Ct

ZmADF

(ID:

method for calculating relative expression. Results were performed as means ±

100

standard deviation (SD).

101

Extraction of vitamin E and carotenoids

102

The extraction methods of vitamin E and carotenoids in sweet corn seedlings referred

103

to previous report in our lab 24. Briefly, sweet corn seedlings were mixed with EtOH

104

(95%), NaCl (17.52 g/L), pyrogallol in EtOH (63.055 g/L), AsA (176 mg/mL) and

105

KOH (600 g/L) and saponified at 75°C for 45 minutes before being extracted with n-


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hexane/ethyl acetate (9:1 v/v). The supernatant was collected and dried with nitrogen

107

gas. The residues were dissolved in n-hexane solution with isopropyl alcohol (1%) for

108

vitamin E analysis, while the residues were dissolved in MTBE with 1% BHT for

109

carotenoids analysis, respectively.

110

Quantification of Vitamin E compositions The quantification of vitamin E were implemented by HPLC method used previously

111

. The NP-HPLC method were completed with Waters 2475 Multi λ fluorescence

112

24

113

detector and Waters 515 HPLC pump (Waters Corporation, Milford, MA, USA). An

114

Agilent ZORBAX RX-SIL column (250 mm×4.6 mm, and 5µm particle size) was used.

115

The affluent phase consisting of n-hexane/isopropyl alcohol/acetic acid (99.05:0.85:0.1,

116

v/v/v) with flow rate of 1 mL/min. Detections were carried out with an excitation

117

wavelength of 290 nm and an emission wavelength of 330 nm. The vitamin E isomers

118

content were quantitated by contrasting with eight external standards on the basis of

119

retention time. Four tocopherol standards were obtained from Wako Pure Chemical

120

Industries (Tokyo, Japan) while four tocotrienol standards were acquired from

121

Chromadex, Ltd. (Irvine, CA, USA). The results were expressed as µg/g FW (means ±

122

SD).

123

Quantification of carotenoids compositions

124

The quantifications of carotenoids were carried out with improvement of former 25.

125

method

A Waters HPLC system (Waters Corporation, Milford, MA, USA), a

126

YMC™ carotenoid 30, 5μm packing, 4.5 × 250 mm column with 25°C temperature and

127

a photodiode array detector were applied in measurements. A) A’/B’ (9:1) and B) A’/B’



128

(1:9) (A’: 97% MeOH – water and 0.05M ammonium acetate; B’: 100% MTBE; 0.1%

129

BHT in both A’ and B’, w/v) were used as mobile phases. The gradient elution was as

130

follows: from 0 to 18 min, 0% to 20% B; from 18 to 20 min, 20% to 50% B; from 20

131

to 25 min, 50% to 90% B; from 29 to 29.5 min, 90% to 10% B; from 29.5 to 40 min,

132

10% to 0% B. Carotenoids, detected at 450nm, were calculated according to standards

133

purchased from CaroteNature (Switzerland) and showed as µg/100g FW (means ± SD).

134

Statistical analysis

135

Data analysis were operated by SPSS 18.0 (SPSS Inc., Chicago, IL, USA) using

136

Duncan’s and Pearson’s multiple comparison post-test according to time and

137

temperature, respectively. All measurements were performed in triplicates and results

138

were expressed as means ± SD (n = 3).

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RESULTS

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In order to investigate the response profiles of vitamin E and carotenoids on

141

transcriptional and metabolic levels of sweet corn seedlings under temperature stress,

142

sweet corn seedlings were treated with three different temperatures (10°C, 25°C and

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40°C) for 0, 15 and 30 HATs. The vitamin E and carotenoids profiles in sweet corn

144

seedlings grew in CK, LT and HT groups were studied by NP-HPLC and RP-HPLC

145

methods, respectively. RT-qPCR was operated for the examinations of relative

146

expression levels of genes in vitamin E and carotenoids biosynthesis pathways.

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Variations of vitamin E contents and biosynthesis pathway

148

The composition profiles and contents of vitamin E isomers in sweet corn seedlings

149

were shown in Figure 1 and Table 1. The four tocopherol contents reached the


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maximum levels at 15 HAT in control samples. Tocotrienols expressed different

151

profiles as δ-tocotrienol had the highest content at 15 HAT, α-tocotrienol kept

152

accumulating from 0.80 ± 0.06 μg/g FW to 1.02 ± 0.04 μg/g FW and γ-tocotrienol

153

remained a stable level (p < 0.05) during growth. In the process of growth, total vitamin

154

E contents raised to the highest at 15 HAT as 28.72 ± 0.28 μg/g FW. The seedlings

155

grew in LT group gradually accumulated vitamin E isomers during HAT as comparing

156

to CK group, among which, α-tocopherol, γ -tocopherol, γ-tocotrienol and δ-

157

tocotrienol increased more than 30%. Therefore, the total vitamin E content presented

158

an increase tendency from 22.24 ± 1.55 μg/g FW to 29.95 ± 1.76 μg/g FW (p < 0.05).

159

Applying HT reduced the contents of γ- tocopherol and γ- tocotrienol, meanwhile, the

160

accumulations of β-tocopherol and δ-tocopherol at 15 HAT were impeded comparing

161

to CK group. The fluctuation trends of α- tocopherol and δ- tocotrienol in HT group

162

were similar with LT group after treatment. HT merely increased the total vitamin E

163

content as compared to LT group. In general, the vitamin E in sweet corn seedlings

164

gradually accumulated in response to LT treatment but its yield was impeded while

165

treated with HT.

166

The relative expression levels of selected genes in tocopherols and tocotrienols

167

biosynthesis pathway were shown in Figure 2. With the elongation of time, the relative

168

expression levels of genes in CK group were varied, as DXPR, GGPPS, HGGT, MPBQ-

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MT and TC achieved the maximum expression levels at 15 HAT. HPPD was down-

170

regulated whereas HPT was up-regulated. TMT had the lowest relative expression level

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at 15 HAT which was opposite to TC. The relative expression levels of HPPD, HPT



172

and GGPPS in LT group were lowest at 15 HAT. Besides, the relative expression levels

173

of DXPR, HGGT, MPBQ-MT, TC and TMT were induced by 15.5, 135.3, 8.0, 10.1 and

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2.1 folds under 30 hours LT treatment comparing to untreated samples. The relative

175

expression levels of GGPPS, HPT, HGGT, MPBQ-MT and TC were the maximum at

176

15 HAT and were respectively folded as 2.0, 4.3, 3.0, 15.6 and 12.7 comparing to the

177

initial levels in HT group. Besides, the relative expressions of DXPR and TMT were

178

induced while weakened expression of HPPD was detected during HAT in HT group.

179

Variations of carotenoids contents and biosynthesis pathway

180

The three carotenoids compounds, lutein, β-carotene and zeaxanthin were detected

181

and results were shown in Figure 3 and Table 2. Growing in 25°C, β-carotene slightly

182

accumulated at 15 HAT and then declined. Comparing to CK group, the increase of β-

183

carotene was repressed under LT stress while it obviously folded from 7.04 ± 0.67

184

μg/100 g FW to 13.64 ± 2.01 μg/100 g FW at 30 HAT in HT group. The content of

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zeaxanthin reached the maximum level at 15 HAT in CK group. However, with LT

186

treatment, the seedlings lost zeaxanthin as they could not be detected under the same

187

test conditions. On the contrary, zeaxanthin accumulated under HT stress from 7.20 ±

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0.40 μg/100 g FW to 9.16 ± 0.59 μg/100 g FW. Lutein content raised at 15 HAT and

189

then reduced in CK group. The increase of lutein was impeded under LT stress as well

190

thus its value remained a stable level. In particular, lutein was stimulated and varied

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from 74.78 ± 4.49 μg/100g FW to 160.1 ± 12.9 μg/100g FW in HT group. In general,

192

the total carotenoids content was the highest at 15 HAT in CK group. A constant value

193

of carotenoids content was exhibited in LT group whereas it was promoted during HAT


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194


under HT stress.

195

The varied relative expression of selected genes in carotenoids biosynthesis pathway

196

were shown in Figure 2. As presented, the upstream genes PSY, ZDS and CRTISO in

197

the synthesis pathway were subject to positive expression during HAT in CK group.

198

Negative regulations of these genes were achieved through LT stress. On the contrary,

199

HT improved the expressions of ZDS and CRTISO, as well as PSY and PDS at 15 HAT.

200

The expression level of β-LCY increased at 30 HAT in both CK and LT groups but

201

showed maximum level at 15 HAT in HT group. β-CHY similarly expressed in CK and

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LT groups while it was apparently up-regulated in HT group. LT hindered the

203

expression of LUT5 at 15 HAT while HT elevated it. Except β-CHY and LUT5, the

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formation of lutein needs the participations of the enzymes encoded by ε-CHY and ε-

205

LCY. The expressions of ε-CHY and ε-LCY were restricted until 30 HAT in LT group.

206

However, both of them were stimulated by HT at 15 HAT. ZEP, which encoded the

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enzyme conversed zeaxanthin to antheraxanthin, was repressed in LT in contrast to CK

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group. In HT group, the expression of ZEP was obviously promoted. Generally, the

209

expression levels of most of the genes in pathway was down regulated under LT stress

210

whereas they were activated under HT stress.

211

Correlations among gene expressions and compositions

212

The correlations of relative expressions of genes and components were analyzed by

213

Origin 2018 and results were shown in Figure 4 and Figure 5. Genes that corelated to

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contents were varied according to different cultivation temperature. In CK group, most

215

of the relative expressions of genes showed correlations with vitamin E isomers and



216

carotenoids components. In LT group, the expression of HPPD and ZDS, differently

217

from other genes, oppositely correlated to contents. In HT group, the expression of

218

DXPR was correlated with most of compositions. Besides, genes that might be regulated

219

by temperature such as HPT, ZEP, β-CHY and LUT5 were shown with higher correlate

220

values in Figure 4D and 5D.

221

DISCUSSION

222

Under abiotic stress, plants induced enzymatic and non-enzymatic resistance for

223

protecting themselves from oxidative stress raised by over-generated ROS, among

224

which, antioxidants such as vitamin E were referred to non-enzymatic defend system 3.

225

α- and γ-tocopherols, as the main isomers of vitamin E in plants, were identified with

226

antioxidant capability under oxidative stress

227

photomechanism and free radical scavengers 8, raising resistance for oxidative damage.

228

Maize has gone through extreme temperature stress which affected its development.

229

The stressed seedlings were forced to the failure of growth 14-15. Therefore, the present

230

study may support the interactive and competitive relationship of vitamin E and

231

carotenoids in stressed sweet corn seedlings.

232

The alterations of vitamin E content under temperature stress

17.

Carotenoids, functioned in

233

The variation of total vitamin E content in sweet corn seedlings grew in appropriate

234

temperature was shown in results. As reported 26, total tocopherol content presented a

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net increase in seedlings growing under illumination while no changes were detected in

236

dark. Different tocopherol profiles were discussed previously as they were influenced

237

by the presence or absence of light 27. Therefore, sweet corn seedlings were supposed


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to accumulate vitamin E during growth but then consume vitamin E especially

239

tocopherols due to the absence of light for 30 hours as vitamin E was associated with

240

photosynthesis.

241

Tocopherols were promoted under LT stress. To the date, tocopherols were proved

242

with abilities on protecting cells from oxidative damage by limiting the fracture of

243

double bonds in polyunsaturated fatty acid (PUFA) 28. The block biosynthesis of PUFA

244

resulted in decreasing fluidity of plasma membrane under LT showed significant role

245

of PUFA in resisting stress. Therefore, the present study speculated that the increased

246

tocopherols in sweet corn seedlings were combing with PUFA to protect the integrity

247

of plasma membrane under LT stress. At the same time, LT was supposed to slow down

248

the metabolic reactions

249

tocopherols and resulted in the gradual increase tendency of vitamin E. On the contrary,

250

the less tocopherols in sweet corn seedlings in HT group might be the aim to avoid the

251

increase fluidity of plasma membrane 2. Similar to tocopherols, tocotrienols efficiently

252

perform in resistance to oxidative stress. For example, α-tocotrienol is regarded as a

253

more effective compound in scavenging peroxyl radical than α-tocopherol, besides, δ-

254

tocotrienol has strong ability on limiting lipid peroxidation 29. Moreover, tocotrienols

255

were identified with a higher ROS scavenge ability than tocopherols in vitro

256

However, the tocotrienol profiles in plants have seldom been discussed thus the reasons

257

of varied tocotrienols in this study would be figured out with deeper studies.

258

Relative gene expressions in vitamin E biosynthesis pathway were related to the

259

content variations

14,

together with darkness, influenced the accumulation of


30.


260

In CK group, the overexpression of HGGT might in favor of the production of 31.

261

tocotrienols according to previous report

Besides, TC, as an enzyme catalyzed

262

MPBQ and DMPBQ to δ-/γ-tocopherols, its corresponding gene up-regulated at 15

263

HAT and was supposed to change the accumulation of tocopherols. Although TMT

264

expressed contrarily to the variations of its products, the activity of its encoded enzyme

265

merely affects the accumulation of α-tocopherol as studied 32. The correlations among

266

gene expressions and contents were shown in Figure 4A. MPBQ-MT was supposed to

267

regulate the accumulation of α-tocopherol as r = 0.997. Concerning the function of

268

HPPD reported in transgenic Brassica napus plants

269

regulated during HAT in our study might play a relatively minor role on contents

270

alteration. α-tocopherol, as the major composition of vitamin E isomers in seedlings,

271

was related to the accumulation of total vitamin E as r = 0.988.

19,

the detected HPPD down-

272

In contrast to CK group, γ-tocopherol and δ-tocopherol in LT group accumulated at

273

30 HAT which supposed to be related with the up-expression of TC. Previously, vte1

274

(TC) transgenic plants possessed higher tocopherol contents as well as more integral

275

membrane under drought stress have been proved 33. According to existed study 20, we

276

supposed that the stimulated expression of MPBQ-MT under LT might accelerate the

277

addition of CH3 to form γ-isomers, as δ-isomers unchanged whereas γ-isomers yielded

278

at 30 HAT. Evidently, the enhancement of γ- tocopherol was detected in 5 days’ cold

279

stress (4°C) Arabidopsis 34. Concerning the correlations between gene expressions and

280

contents in Figure 4B, a negative value of correlation was expressed between HPPD

281

and vitamin E, which was different from others and needed further study. DXPR, HGGT


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and TC were supposed with positive roles on regulations of γ-tocotrienol (r = 1.000),

283

meanwhile, the positive correlation values among gene expressions and total vitamin E

284

contents reflected the stimulations of LT stress.

285

Compared with 25°C, TC was subject to positive regulation in 40°C as its relative

286

expression increased by 28 folds at 15 HAT in comparison with the initial level, and

287

thus might result in the increases of δ-tocochromanols. TMT in HT samples performed

288

a more slightly rising trend during growth time than LT group, thus the products (α-

289

tocopherol, β-tocopherol and α-tocotrienol) were supposed to be consumed for

290

oxidative stress tolerance 35. The relationships between contents and relative expression

291

levels of genes were shown in Figure 4C. DXPR was up-regulated under HT stress and

292

was correlated with the increases of β-tocopherol and δ-tocotrienol as r = 1.000. Besides,

293

the variation of vitamin E was correlated with most variations of the isomers especially

294

tocopherols.

295

The alterations of carotenoid content under temperature stress

296

During the growth from sprouts to seedlings under light, carotenoids were 36.

297

significantly raised in rapeseed

Besides, light-grown cabbage seedlings possessed

298

higher quantity of carotenoids than the dark-grown one

299

increased the carotenoids content whereas negative effects on carotenoids accumulation

300

were performed through darkness. All the detected components raised to their

301

maximum levels at 15 HAT, among which, lutein, occupied the majority of carotenoids

302

in sweet corn seedlings, contributed to the variation of total carotenoids as r = 1.000

303

(Figure 5A).


37.

Thus, plants growth


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304

LT was supposed to destroy the fluidity of membrane 1. However, lutein had its

305

ability in reducing the fluidity of membrane 38 and kept a stable level under LT stress

306

in sweet corn seedlings. On the other hand, HT was suggested with the role on

307

accelerating aging of plants thus might force to accumulate lutein as it was corelated

308

with plant senescence 18. The production of lutein was enhanced under HT condition as

309

reported in a potato specie 39. Zeaxanthin, plays an indispensable role in photoprotection

310

as it derives from violaxanthin under the catalyzation of violaxanthin de-epoxidase

311

known as xanthophyll cycle with enhanced dissipation of energy as heat when

312

encounters excessive light 40. Therefore, the production of zeaxanthin was limited in

313

darkness. On the other hand, zeaxanthin took part in protecting lipid peroxidation

314

under oxidative stress such as LT thus it decreased in the detected data. As reported,

315

3°C treated potato leaves yielded less zeaxanthin than the control one in 23°C

316

Oppositely, zeaxanthin accumulated in dark treated leaves under HT stress 42. Besides,

317

the varied profiles of β-carotene was identified as it bounded to reaction center and took

318

part in scavenging 1O2 under temperature stress 43. In summary, LT stress influenced

319

the accumulation of carotenoids, conversely, HT stress improved its production.

320

Recently, the increased carotenoids in bananas ripening at HT was reported 44. Overall,

321

the variations of carotenoids compounds were the combinative effects of darkness and

322

temperature.

323

Relative gene expressions in carotenoid biosynthesis pathway were connected to

324

the content variations

325

41

11.

The increase of total carotenoids content in CK group at 15 HAT might achieve


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326

through the up-regulation of upstream genes PSY, PDS, ZDS and CRTISO as they were

327

reviewed with important roles in carotenoids accumulation

328

Figure 5A showed lutein, as the most abundant component detected among carotenoids

329

in sweet corn seedlings in this study, was supposed to regulate the variation of total

330

carotenoids as r = 1.000. Besides, the changes of expression level of LUT5 and lutein,

331

zeaxanthin content were similar which showed important role of LUT5 on the

332

accumulation of carotenoids 22.

22.

The correlations in

333

The influences of LT on carotenoids biosynthesis were shown in Figure 5B as

334

relative expression levels of most of the genes were contrarily changed to total

335

carotenoid content. ZDS was the only gene down-regulated during the treatment thus

336

was supposed with an indispensable role in content reduction. However, the regulative

337

profile of ZDS has been seldomly studied 45. The present study had described the role

338

of ZDS under abiotic stress. Besides, the over-expression of genes in pathway, together

339

with the limitation on carotenoid contents, provided evidences for the function of

340

carotenoids when encounter stress. Similarly, the up-regulation profiles of PSY, PDS,

341

ε-LCY, ε-CHY and β-CHY have been detected in 10°C matured Satsuma mandarin as

342

compared with control 23. However, as the study supposed, different species responded

343

differently towards stress 23. Therefore, the regulation of temperature stress remains a

344

long way off.

345

The expression levels of PSY, PDS, β-LCY, ε-LCY and LUT5 were activated under

346

HT stress at 15 HAT and played positive roles on the accumulation of β-carotene,

347

zeaxanthin and lutein. The high expression levels of LUT5 and ZEP under HT stress



Page 18 of 39

348

would maintain active transformations among xanthophyll cycle which played

349

protective roles under oxidative stress such as heat

350

expression level of β-CHY was correlated with lutein, zeaxanthin, β-carotene and total

351

carotenoid contents as r = 1.00. As reported, the overexpression of β-CHY elevated

352

zeaxanthin contents as well as enhanced stress tolerance in transgenic tobacco

353

Moreover, overexpression of β-CHY resulted the accumulation of β-carotene in

354

Arabidopsis under high-light and HT environment

355

CHY was associated with reduced ZEP expression thus caused zeaxanthin accumulation

356

49.

357

the expressions of β-CHY and ZEP were stimulated which needed further study. Besides,

358

lutein was supposed to positively regulate the total carotenoid contents as it accounted

359

for the majority (Figure 5C, r = 1.000).

360

Relationship between vitamin E and carotenoids

48.

46.

In particular, the varied

47.

However, the expression of β-

The present study, treated seedlings with HT, supposed a different regulation as both

361

As Figure 2 shown, the formations of vitamin E and carotenoids were shared with

362

GGDP, which not only combined with HGA to yield GGMB but also took part in the

363

formation of PPDP under the catalyzations of HGGT and PSY. Measured databases

364

showed an accumulation in total vitamin E content and a limitation on increasing

365

carotenoids under LT stress. Meanwhile, the expression of HGGT significantly

366

increased by 135 folds at 30 HAT as compared with the initial, while PSY was slightly

367

up-regulated. On the contrary, the accumulation of vitamin E was repressed whereas

368

total carotenoid content raised when seedlings gone through HT stress. Different from

369

the down-regulation of HGGT at 30 HAT, PSY was highly expressed after treatment.


Page 19 of 39


370

Therefore, the present study suggested a relationship between vitamin E and

371

carotenoids under temperature stress. Besides, both the two compositions performed in

372

oxidative stress tolerance, which influenced membrane fluidity and other cell

373

regulations, would be identified with other relationships after further investigation.

374

Differences between the effects of extreme temperature

375

For vitamin E, LT stress stimulated relative genes’ expression which might be related

376

to the yield of them to maintain membrane fluidity. HT stress limited the up-regulation

377

of some genes as well as the accumulation of vitamin E in order to maintain cell

378

morphology under harsh environment. As shown in Figure 4D, HPT was relatively

379

expressed with varied temperature both at 15 HAT and 30 HAT indicated that it might

380

be regulated by temperature and would play important roles in stress response in sweet

381

corn seedlings.

382

For carotenoids, seedlings under LT stress were supposed to consume more

383

carotenoids for regulating membrane state and resisting stress. And most of the

384

expression levels of relative genes declined. Carotenoids apparently raised as well as

385

relative expressions of genes were induced in response to HT stress, which supposed

386

more tolerant profiles of sweet corn seedlings. Besides, the yield of lutein indicated

387

aging in sweet corn seedlings in HT environment. As shown in Figure 5D, LUT5, β-

388

CHY and ZEP relatively expressed with varied temperature both at 15 HAT and 30

389

HAT. The detected profiles might indicate regulations of temperature on those genes

390

which were supposed to be related with stress response in sweet corn seedlings.

391

In summary, owing to the functions in protection and regulation under temperature



Page 20 of 39

392

stress, vitamin E and carotenoids varied under treatments and were correlated to relative

393

genes in biosynthesis pathways. Vitamin E accumulated under LT stress probably

394

owing to its function on membrane fluidity while carotenoids yielded under HT stress

395

as probably the results of senescence and ROS production in sweet corn seedlings.

396

Therefore, this study investigated the variations of vitamin E and carotenoids under

397

extreme temperature stress for promoting theoretical process on improving sweet corn

398

resistance in extreme environment.

399

ABBREVIATIONS USED

400

HAT, hours after treatment; CK, control; LT, low temperature; HT, high temperature;

401

ROS, reactive oxygen species; PUFA, polyunsaturated fatty acid; DXP, 1-deoxy-D-

402

xylulose-5-phosphate;

403

diphosphate; DPP, dimethylallyl diphosphate; GGDP, geranylgeranyl diphosphate;

404

PDP, phytyl diphosphate; HPP, 4-hydoxyphenyl pyruvate; HGA, homogentisic acid;

405

MPBQ, 2-methyl-6-phytylquinol; DMPBQ, 2,3-dimethyl-5-phytylquinol; GGMB, 6-

406

geranylgeranyl-2-methylbenzene-1,4-diol;

407

dimethylbenzene-1,4-diol;

408

GGPPS, geranylgeranyl diphosphate synthase; GGDR, geranylgeranyl diphosphate

409

reductase; TAT, tyrosine aminotransferase; HPPD, 4-hydoxyphenyl pyruvate

410

dioxygenase; HPT, homogentisic acid phytyl transferase; HGGT, homogentisate

411

geranylgeranyl

412

methyltransferase; TC, tocopherol cyclase; TMT, tocopherol methyltransferase; PSY,

413

phytoene synthase; PDS, phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS, ζ-

MEP,

transferase;

methylerythritol

DXPR,

4-phosphate;

GGDMB,

IPP,

6-geranylgeranyl-2,3-

1-deoxy-D-xylulose-5-phosphate

MPBQ-MT,

isopentenyl

reductase;

2-methyl-6-phytyl-1,4-benzoquinolither


Page 21 of 39


414

carotene desaturase; CRTISO, carotenoid isomerase; β-LCY, lycopene β-cyclase; ε-

415

LCY, lycopene ε-cyclase; LUT5, β-ring hydroxylase; β-CHY, β-carotene hydroxylase;

416

ε-CHY, ε-carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-

417

epoxidase; NXS, neoxanthin synthase.

418

SUPPLEMENTARY INFORMATION

419

Table S1. The Gene ID and primer sequences of selected genes in vitamin E,

420

carotenoids biosynthesis pathways and reference gene of ZmADF used in this study.

421

ACKNOWLEDGMENTS

422

Authors are very thankful to Analytical and Testing Center of SCUT for laboratory

423

analyses.

424

FUNDING SORRCES

425

Authors are greatly thankful to Science and Technology Planning Project of

426

Guangzhou-China (201804020081); Guangdong Academy Team Project of Fresh Corn

427

Breeding and Industrialization (201610TD); Science and Technology Planning Project

428

of Guangdong Province-China (2016B02033004); National Natural Science

429

Foundation of China (31501765) for financial support, valuable suggestions and

430

guidelines.

431

CONFLICT OF INTERSET

432 433

The authors declare that they have no conflict of interest. AUTHOR CONTRIBUTIONS

434

N.X., C.Y.L. and X.B.G. conceived and designed the experiments; Y.T.Y. and J.G.H

435

provided the materials; N.X. performed the experiments; N.X. and X.B.G. analyzed the



436

data; N.X. and X.B.G. wrote the paper; N.X., G.K.L and X.B.G. interpreted the data

437

and revised the manuscript.


Page 22 of 39

Page 23 of 39


438

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Figure captions Figure 1. Vitamin E results. A: HPLC profiles of Vitamin E isomers. a: α-tocopherol; b: α-tocotrienol; c: β-tocopherol; d: γ-tocopherol; e: γ-tocotrienol; f: δ-tocopherol; g: δtocotrienol. B: Vitamin E contents. No letters in common means significant difference in each column (p < 0.05). Figure 2. Gene relative expression levels of vitamin E and carotenoids biosynthesis pathways. Data presented were as mean ± SD of triplicates. CK: control group (25°C); LT: low temperature (10°C); HT: high temperature (40°C). ‘*’ means significant differences comparing to CK according to time (p < 0.05, **: p < 0.01); ‘#’ means significant differences comparing to CK according to temperature (p < 0.05, ##: p < 0.01). The expressions of corresponding gene of enzymes showed in pink ellipses were measured whereas in orange ellipses were not. Both the syntheses are conducted in plastid. The figure shows syntheses in chloroplast. A: HGA can be synthesize in cytosol and chloroplast, it with its precursors can be transported through membrane of plastid; B: Xanthophyll cycle is connected to ABA production; C: PUFA act on membrane; D: Yielded tocopherols and tocotrienols influence PUFA synthesis; E: Yielded carotenoids take part in photoprotection in PSII in thylakoid. Figure 3. Carotenoids results. A: HPLC profiles of carotenoid compositions. B: Carotenoids contents. No letters in common means significant difference in each column (p < 0.05). Figure 4. Pearson’s analyses of vitamin E biosynthesis pathway and their composition profiles. A, B, C: Pearson’s correlation of between the variation of gene relative



expression and vitamin E content during HAT. A: CK group; B: LT group; C: HT group. D: Pearson’s correlation between the variation of relative gene expression and applied temperature. The ‘T’ in ‘α-T’, ‘β-T’, ‘γ-T’ and ‘δ-T’ stands for ‘tocopherol’ while ‘T3’ stands for ‘tocotrienol’; ‘Vit E’ stands for ‘total vitamin E’. Figure 5. Pearson’s analyses of carotenoids biosynthesis pathway and their composition profiles. A, B, C: Pearson’s correlation between the variation of relative gene expression and content during HAT. A: CK group; B: LT group; C: HT group. D: Pearson’s correlation between the variation of relative gene expression and applied temperature. ‘Lut.’ stands for ‘Lutein’; ‘β-Car.’ stands for ‘β-carotene’; ‘Zea.’ stands for ‘Zeaxanthin’; ‘Car.’ stands for ‘total carotenoids’.


Page 32 of 39

Page 33 of 39


Table 1 The variation of vitamin E content of sweet corn seedlings a: CK: control group (25°C); LT: low temperature (10°C); HT: high temperature (40°C). Groups

CK

LT

HT

a

HAT

α-tocopherol

α-tocotrienol

β-tocopherol

γ-tocopherol

γ-tocotrienol

δ-tocopherol

δ-tocotrienol

Total

0

12.43 ± 1.03a

0.80 ± 0.06a

1.08 ± 0.14ab

4.58 ± 0.13b

2.38 ± 0.21b

0.44 ± 0.01a

0.53 ± 0.01a

22.24 ± 1.55a

15

17.22 ± 0.13b

0.91 ± 0.02ab

1.28 ± 0.02c

5.41 ± 0.43c

2.62 ± 0.05b

0.57 ± 0.01c

0.72 ± 0.02c

28.72 ±0.28c

30

13.30 ± 0.90a

1.02 ± 0.04b

1.00 ± 0.06a

3.46 ± 0.40a

2.54 ± 0.13b

0.44 ± 0.04a

0.59 ± 0.05b

22.36 ± 1.49a

0

12.43 ± 1.03a

0.80 ± 0.06a

1.08 ± 0.14ab

4.58 ± 0.13b

2.38 ± 0.21b

0.44 ± 0.01a

0.53 ± 0.01a

22.24 ± 1.55a

15

13.56 ± 1.04a

1.00 ± 0.08b

1.20 ± 0.05bc

5.42 ± 0.73c

2.54 ± 0.14b

0.58 ± 0.01c

0.67 ± 0.03c

24.97 ± 1.01b

30

16.69 ± 1.24b

1.03 ± 0.04b

1.32 ± 0.10c

6.40 ± 0.12d

3.22 ± 0.21c

0.51 ± 0.03c

0.70 ± 0.04c

29.95 ± 1.76c

0

12.43 ± 1.03a

0.80 ± 0.06a

1.08 ± 0.14ab

4.58 ± 0.13b

2.38 ± 0.21b

0.44 ± 0.01a

0.53 ± 0.01a

22.24 ± 1.54a

15

15.73 ± 0.56b

1.19 ± 0.12c

1.13 ± 0.06bc

3.75 ± 0.33a

2.60 ± 0.35b

0.52 ± 0.01b

0.66 ± 0.02c

25.59 ± 0.14b

30

16.29 ± 0.60b

0.96 ± 0.05b

1.13 ± 0.03bc

3.12 ± 0.03a

1.77 ± 0.12a

0.49 ± 0.03b

0.66 ± 0.03c

24.43 ± 0.45b

Values (μg/g FW, mean ± SD, n=3). No letters in common means significant difference in each column (p < 0.05).



Table 2 The variation of carotenoids content of sweet corn seedlings a, b: CK: control group (25°C); LT: low temperature (10°C); HT: high temperature (40°C). Groups

CK

LT

HT

a b

HAT

Lutein

Zeaxanthin

β-carotene

Total

0

74.78 ± 4.49a

7.20 ± 0.40a

7.04 ± 0.67a

89.02 ± 5.18a

15

128.8 ± 15.2c

10.15 ± 0.76d

10.55 ± 0.10cd

150.5 ± 15.9c

30

102.4 ± 3.3b

7.62 ± 0.80ab

9.40 ± 1.25bc

119.4 ± 4.9b

0

74.78 ± 4.49a

7.20 ± 0.40a

7.04 ± 0.67a

89.02 ± 5.18a

15

86.33 ± 7.19ab

ND

7.97 ± 0.27ab

94.30 ± 7.37a

30

82.47 ± 2.67a

ND

8.63 ± 0.85ab

91.10 ± 3.37a

0

74.78 ± 4.49a

7.20 ± 0.40a

7.04 ± 0.67a

89.02 ± 5.18a

15

132.0 ± 12.7c

8.38 ± 0.13bc

11.83 ± 0.76d

152.2 ± 13.6c

30

160.1 ± 12.9d

9.16 ± 0.59c

13.64 ± 2.01e

182.9 ± 15.2d

Values (μg/ 100g FW, mean ± SD, n = 3). No letters in common means significant difference in each column (p < 0.05). ND, values are not quantifiable.


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Figure 1. Vitamin E results. A: HPLC profiles of Vitamin E isomers. a: α-tocopherol; b: α-tocotrienol; c: βtocopherol; d: γ-tocopherol; e: γ-tocotrienol; f: δ-tocopherol; g: δ-tocotrienol. B: Vitamin E contents. No letters in common means significant difference in each column (p < 0.05). 497x190mm (150 x 150 DPI)



Figure 2. Gene relative expression levels of vitamin E and carotenoids biosynthesis pathways. Data presented were as mean ± SD of triplicates. CK: control group (25°C); LT: low temperature (10°C); HT: high temperature (40°C). ‘*’ means significant differences comparing to CK according to time (p < 0.05, **: p < 0.01); ‘#’ means significant differences comparing to CK according to temperature (p < 0.05, ##: p < 0.01). The expressions of corresponding gene of enzymes showed in pink ellipses were measured whereas in orange ellipses were not. Both the syntheses are conducted in plastid. The figure shows syntheses in chloroplast. A: HGA can be synthesize in cytosol and chloroplast, it with its precursors can be transported through membrane of plastid; B: Xanthophyll cycle is connected to ABA production; C: PUFA act on membrane; D: Yielded tocopherols and tocotrienols influence PUFA synthesis; E: Yielded carotenoids take part in photoprotection in PSII in thylakoid. 1818x1291mm (101 x 101 DPI)


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Figure 3. Carotenoids results. A: HPLC profiles of carotenoid compositions. B: Carotenoids contents. No letters in common means significant difference in each column (p < 0.05). 494x190mm (150 x 150 DPI)



Figure 4. Pearson’s analyses of vitamin E biosynthesis pathway and their composition profiles. A, B, C: Pearson’s correlation of between the variation of gene relative expression and vitamin E content during HAT. A: CK group; B: LT group; C: HT group. D: Pearson’s correlation between the variation of relative gene expression and applied temperature. The ‘T’ in ‘α-T’, ‘β-T’, ‘γ-T’ and ‘δ-T’ stands for ‘tocopherol’ while ‘T3’ stands for ‘tocotrienol’; ‘Vit E’ stands for ‘total vitamin E’. 495x368mm (150 x 150 DPI)


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Figure 5. Pearson’s analyses of carotenoids biosynthesis pathway and their composition profiles. A, B, C: Pearson’s correlation between the variation of relative gene expression and content during HAT. A: CK group; B: LT group; C: HT group. D: Pearson’s correlation between the variation of relative gene expression and applied temperature. ‘Lut.’ stands for ‘Lutein’; ‘β-Car.’ stands for ‘β-carotene’; ‘Zea.’ stands for ‘Zeaxanthin’; ‘Car.’ stands for ‘total carotenoids’. 495x368mm (150 x 150 DPI)


Comparative Evaluation on Vitamin E and ... - ACS Publications

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