Metabolic Engineering of Wheat Provitamin A by Simultaneously

Article pubs.acs.org/JAFC

Metabolic Engineering of Wheat Provitamin A by Simultaneously Overexpressing CrtB and Silencing Carotenoid Hydroxylase (TaHYD) Jian Zeng,† Xiatian Wang,† Yingjie Miao,†,‡ Cheng Wang, Mingli Zang, Xi Chen, Miao Li, Xiaoyan Li, Qiong Wang, Kexiu Li, Junli Chang, Yuesheng Wang, Guangxiao Yang,* and Guangyuan He* The Genetic Engineering International Cooperation Base of the Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of the Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China S Supporting Information *

ABSTRACT: Increasing the provitamin A content in staple crops via carotenoid metabolic engineering is one way to address vitamin A deficiency. In this work a combination of methods was applied to specifically increase β-carotene content in wheat by metabolic engineering. Endosperm-specific silencing of the carotenoid hydroxylase gene (TaHYD) increased β-carotene content 10.5-fold to 1.76 μg g−1 in wheat endosperm. Overexpression of CrtB introduced an additional flux into wheat, accompanied by a β-carotene increase of 14.6-fold to 2.45 μg g−1. When the “push strategy” (overexpressing CrtB) and “block strategy” (silencing TaHYD) were combined in wheat metabolic engineering, significant levels of β-carotene accumulation were obtained, corresponding to an increase of up to 31-fold to 5.06 μg g−1. This is the first example of successful metabolic engineering to specifically improve β-carotene content in wheat endosperm through a combination of methods and demonstrates the potential of genetic engineering for specific nutritional enhancement of wheat. KEYWORDS: CrtB, carotene hydroxylase, RNA interference, β-carotene, provitamin A, wheat

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lycopene β-cyclase (LCYB) to introduce ε- and β-end groups and produces α- and β-carotene, respectively.20,21 Carotenes are further converted into lutein by β-ring hydroxylase (HYDB) and ε-ring hydroxylase (CYP) from α-carotene or converted into β-cryptoxanthin and zeaxanthin by HYDB2 (Figure 1). βCarotene with two β-end groups is the most potent dietary source of vitamin A, whereas α-carotene or β-cryptoxanthin has only one β-end group and therefore half the provitamin A potential compared to β-carotene.22 Carotene hydroxylase catalyzes the key steps of depleting provitamin A function by converting provitamin A compounds into non-provitamin A xanthophylls. In higher plant, two classes of structurally unrelated enzymes catalyze the hydroxylation of β-rings: heme-containing cytochrome P450 hydroxylases (CYP97A in Arabidopsis) that function predominantly in the β,ε-branch and nonheme di-iron hydroxylase (HYDB), which functions in the β,β-branch of the carotenoid biosynthesis pathway. Experimental evidence from association and linkage studies in maize demonstrates that the β-carotene hydroxylase (crtRB1) gene underlies a principal quantitative trait locus (QTL) that is associated with β-carotene accumulation in kernels and that the low expression of crtRB1 alleles accompanies higher β-carotene accumulation.23 In carrot, a natural ε-ring hydroxylase polymorphism was also correlated with higher levels of carotenoids.24 Therefore, hydroxylation is one of the key determinants in controlling provitamin A levels. To improve βcarotene content in potato and orange, the block in carotene

INTRODUCTION Carotenoids are a complex class of C40 isoprenoid pigments providing nutritional value as provitamin A and antioxidant compounds. Their varied colors attract pollinators and provide commercial value in flowers and foods.1−3 β-Carotene is the major and most effective provitamin A precursor in carotenoids, and an inadequate intake of vitamin A can cause many serious diseases, such as cataracts, cardiovascular disease, and certain cancers.4−7 According to data from the World Health Organization, an estimated 250 million preschool children are vitamin A-deficient. Vitamin A deficiency (VAD) remains a challenging and prevalent problem in developing countries (http://www.who.int/nutrition/topics/vad/en/). Therefore, metabolic engineering of carotenoid biosynthetic pathway in staple crops to specifically improve provitamin A content is an economical way to address the VAD problem. Wheat is one of the most important cereal crops worldwide.8 Given the huge daily consumption of wheat-based products in populations worldwide, the content of β-carotene in wheat grain could significantly affect VAD. However, owing to the complexity of the wheat genome and the inefficiency of wheat transformation, there are limited reports on carotenoid metabolic engineering to improve carotenoid content in wheat.9,10 In higher plants, the main pathway of carotenoid biosynthesis has been studied.11−15 Phytoene synthase (PSY) is the first committed enzyme and is regarded as the rate-controlling enzyme for endosperm carotenoid biosynthesis9,16−19 (Figure 1). After the desaturation of phytoene, pro-lycopene was isomerized to lycopene. Lycopene is the immediate precursor of provitamin A carotenes, and its cyclization represents the first branch point in the carotenoid biosynthetic pathway; lycopene is then cyclized by lycopene ε-cyclase (LCYE) and/or © 2015 American Chemical Society

Received: Revised: Accepted: Published: 9083

September 2, 2015 September 29, 2015 October 1, 2015 October 1, 2015 DOI: 10.1021/acs.jafc.5b04279 J. Agric. Food Chem. 2015, 63, 9083−9092

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Journal of Agricultural and Food Chemistry

Figure 1. Carotenoid biosynthetic pathway. Enzymatic conversions are indicated with arrows, with the enzymes in bold. PSY/CrtB, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerases; LCYB, lycopene β-cyclase; LCYE, lycopene εcyclase; HYDB, β-carotene hydroxylase; CYP, carotenoid ε-hydroxylase (cytochrome P450 type).

the provitamin A content in the wheat endosperm, “push strategies” and “block strategies” were combined in wheat metabolic engineering. The expected result of this engineering was that the content of β-carotene would increase in the endosperm of transgenic wheat compared with that in the wild type.

hydroxylase expression was implemented and accumulated a high ratio of β-carotene in total carotenoid content, indicating that it is a practicable and effective approach to improve provitamin A.25,26 Up to now, there are two strategies to accumulate carotenoids in plant metabolic engineering. The first strategies are “push strategies”, which are dependent on the overexpression of a rate-limiting enzyme in the pathway, such as successful reports in rice and maize with overexpression of PSY or bacterial phytoene synthase gene (CrtB).27−31 The second strategies are “block strategies”, which rely on the silencing of a downstream biosynthetic step. Besides the examples of potato and orange, other researches of “block strategies” include the silencing of lycopene β-cyclases to increase lycopene in tomato fruits or the silencing of zeaxanthin epoxidase to accumulate zeaxanthin in potato tubers.32,33 Although carotenoid is one of the major pigments and determines the nutritional value of wheat,34 wheat grains have very low carotenoid content and mainly accumulate lutein, which lacks provitamin A activity. In our previous study, the β-carotene content was increased by introducing CrtB and CrtI genes, whereas the high ratio of βcarotene accumulation could not be previously estimated, which also unveiled three important regulation nodes to be regulated in the carotenoid biosynthetic pathway of hexaploid wheat: (i) phytoene biosynthesis; (ii) lycopene cyclization; and (iii) carotene hydroxylation.9 There are two types of HYD genes (TaHYD1 and TaHYD2) in common wheat, which encode proteins that are 72% identical to each other over 90% of the protein length.35 In this work, the expression of an endogenous TaHYD was blocked by RNA interference (RNAi) in transgenic wheat. To specifically and quantitatively increase

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MATERIALS AND METHODS

Plant Materials and Treatments. Wheat plants (Triticum aestivum L. cv. Bobwhite) were grown in the experimental field of Huazhong University of Science and Technology in Wuhan, China. To more favorably accumulate carotenoid, a yellow-kernel cultivar was chosen. RNA and Genomic DNA Isolation. Total RNA was extracted from different wheat tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA concentration and purity were analyzed using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The integrity of the RNA sample was assessed by nondenaturing agarose gel analysis. Genomic DNA was isolated from the wheat leaves using the cetyltrimethylammonium bromide (CTAB) extraction method.36 Plasmid Constructs. The pLRPT vector containing the endosperm-specific promoter 1Dx5 and 35S terminator was used to construct the overexpression (OE) and RNAi vector. Fragments of 225 bp corresponding to TaHYD1 and TaHYD2 were isolated by RTPCR using specific primers with incorporated restriction sites (Supporting Information Table S1). The selected fragments followed the selection strategies for RNAi in wheat, and the highly conserved fragment was chosen as the silencing target.37 The RNAi construct contained a cDNA fragment derived from TaHYD and was oriented in the sense and antisense directions at the ends of the construct, respectively, separated by an intron sequence, and the resulting 9084

DOI: 10.1021/acs.jafc.5b04279 J. Agric. Food Chem. 2015, 63, 9083−9092

Article


Analysis of Carotenoid Composition by HPLC. Carotenoid extracts from mature grain were prepared according to the method of Rodrı ́guez-Suaŕ ez et al. with modifications.42 Seed samples were ground into fine powder and placed in a 50 mL volumetric centrifuge tube. Pigments were extracted with 4 mL of acetone (containing 0.1% BHT) during 2 min by vortexing, following sonication for 1 min. The mixture was centrifuged at 5000 rpm for 5 min at 4 °C, and the extracted carotenoids in the acetone phase were collected. The extraction operation was repeated three times, and the acetone fractions were pooled. The solvent was evaporated under a nitrogen stream, and the pigments were dissolved in 0.3 mL of methyl tert-butyl ether containing 0.01% (w/v) BHT. After centrifuging at 12000 rpm at 4 °C for 30 min, the sample was filtered through a 0.22 μm filter before high-performance liquid chromatography (HPLC) analysis. For quantitative purposes, β-apo-8′-carotenal was added to each sample as an internal standard prior to extraction (10 μg g−1 of freeze-dried sample). Analyses were performed in quadruplicate. All of the operations were performed under dimmed light to prevent the isomerization and photodegradation of carotenoids. The sample was injected into the HPLC system. The HPLC system included a model 2996 photodiode array detection (DAD) system, a 1525 solvent delivery system, and a Breeze2 Chromatography Manager (Waters Corp., Milford, MA, USA). The carotenoids were separated on a YMC C30 carotenoid column (150 × 4.6 mm, packing 3 μm) (Wilmington, NC, USA) at 25 °C. All of the eluate was under 200−700 nm monitoring. Solvent A was acetonitrile/methanol (3:1, v/v), containing 0.01% BHT and 0.05% triethylamine (TEA, Sigma, Shanghai, China), and solvent B was 100% MTBE, containing 0.01% BHT. The parameters of mobile phase gradient were programmed as follows: 0−10 min, A/B (95:5); 10−19 min, A/B (86:14); 19−29 min, A/B (75:25); 29−54 min, A/B (50:50); 54−66 min, A/B (26:74), and back to the initial condition for re-equilibration. All of the solvents were of HPLC grade (J. T. Baker, Phillipsburg, NJ, USA). Carotenoid standards for lutein, zeaxanthin, β-cryptoxanthin, α-carotene, trans-βcarotene, β-apo-8′-carotenal, and trans-lycopene calibration were purchased from Sigma-Aldrich (Shanghai, China); 9-cis-β-carotene was purchased from Carotenature (Lupsingen, Switzerland) (Supporting Information Figure S1). These standards and the β-apo-8′carotenal internal standard were used to generate standard calibration curves.43−46 Quantitative PCR (qPCR) Analysis. The qPCR analysis was performed with a Real-Time PCR Detection System (CFX Connect Optics Module, Bio-Rad, Hercules, CA, USA) using SuperReal PreMix Plus (SYBR Green) (FP205, Tiangen, Beijing, China). The amplification was performed with the following program: 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Fluorescence was acquired at 60 °C. The specificity of the unique amplification product was determined by a melt curve analysis from 55 to 99 °C. The data were analyzed using Lightcycler software version 4 and were normalized to the expression of the wheat β-actin gene due to its relatively constitutive expression level throughout the wheat developmental process. The quality of the cDNA templates and PCR amplifications was verified by an analysis of negative controls without template and no-reverse transcription for each primer pair. A dissociation curve analysis was performed following qPCR, and a single peak was observed for each primer pair. Some of the qPCR products were separated on agarose gels, and single bands at expected sizes were detected.

plasmid was named pLRPT-HYD-RNAi (Figure 2). The intron was derived from the wheat TAK14 gene (AF325198). pLRPT-CrtB

Figure 2. Structures of the three transformation plasmids (pLRPTHYD-RNAi, pLRPT-CrtB, and pAHC20) used in this study. 1Dx5, endosperm-specific promoter of the high-molecular-mass glutenin subunit gene 1Dx5 from wheat; CaMV35S, cauliflower mosaic virus (CaMV) 35S terminator; Ubi, constitutive maize ubiquitin-1 promoter; TP, transit peptide from pea Rubisco small subunit (rbcS); CrtB, phytoene synthase gene from E. uredovora; Bar, bialaphos resistance gene; NOS, Agrobacterium tumefaciens nopaline synthase (NOS) terminator.

carries the bacterial phytoene synthase gene under the control of 1Dx5 promoter, which had been used in previous research and was stored in our laboratory.9 A sequence encoding a transit peptide from the pea Rubisco small subunit (rbcS) was fused to the 1Dx5 promoter in the plasmid pLRPT-CrtB. Plasmid pAHC20 carrying a selectable bar gene, which confers resistance to the herbicide phosphinothricin, was also used. Wheat Transformation and Regeneration. Transgenic wheat lines were generated according to the bombardment method as reported by Sparks et al.38 Wheat immature scutella (14 days after pollination, DAP) from the Bobwhite cultivar were transformed with the plasmids of pLRPT-HYD-RNAi/pLRPT-CrtB/pAHC20 at a 2:2:1 molar ratio, pLRPT-HYD-RNAi/pAHC20 at a 3:1 molar ratio, and pLRPT-CrtB/pAHC20 at a 3:1 molar ratio. The regenerated plants were screened using the herbicide phosphinotricin (3 mg L−1). The surviving plants were transferred to soil and grown to maturity under growth chamber conditions (22/16 °C day/night, 16/8 h light/dark cycle, and 300 μmol m−2 s−1 photosynthetic photon flux density). The regenerated plants were confirmed by PCR amplification using genespecific primers, and then the products were sequenced (Supporting Information Tables S1 and S2). The immature seed from PCRpositive transgenic plants was also confirmed by Western blotting. Then, the transgenic plants were self-pollinated, and the nonsegregant lines were selected for analyses of the carotenoid composition and carotenoid biosynthetic gene expression levels. Western Blotting. The bacterial expression vectors pET-32a and pET-28 were used to express CrtB and HYD proteins in Escherichia coli, respectively. The primers were designed to amplify only the coding regions (CrtB and HYD) (Supporting Information Table S1). The construction of the expression vector pET-32a-CrtB and pET-28HYD and the expression and purification of the recombinant proteins were performed as described by Miao et al.39 The CrtB and HYD proteins were purified from a heterologous system. Polyclonal antibodies against the CrtB and HYD proteins were generated by immunizing Japanese white rabbits with CrtB and HYD proteins. Total wheat seed proteins were extracted from the transgenic and control lines according to the method of He et al.40 Western blotting was performed following the method of Ma et al.41 An antibody against the plant housekeeping protein actin was used to normalize for equal amounts of proteins and to calculate the relative loading volume for each sample. The amounts of CrtB and HYD proteins were determined by a densitometry analysis of the Western blotting results in three biological replicates using Bio-Rad Quantity One 1-D software version 4.6.2 (Bio-Rad, Hercules, CA, USA).

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RESULTS Different Metabolic Strategies Generate Different Effects with Distinct Carotenoid Profiles. The immature scutella from the Bobwhite cultivar were transformed with three constructs containing unlinked transgenes. The pLRPT-HYDRNAi and pLRPT-CrtB were controlled by the endospermspecific 1Dx5 promoter. The selectable marker bialaphos resistance gene (bar) gene was expressed constitutively (Figure 2). The hypothesis is that the successful transformations would 9085


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Figure 3. Propagation of transgenic wheat and selection of nonsegregant lines. OB is the line with expression of CrtB; HYDi is the line with silencing of HYD; OBI is the line with expression of CrtB and silencing of HYD.

2.24 μg g−1 DW (HYDi) to 5.55 μg g−1 DW (OBI). The βcarotene contents of the HYDi-9, OB-8, and OBI-5 lines significantly increased by 10.5-, 14.6-, and 31-fold, respectively (Figure 5B). Thus, the significantly increased carotenoid and provitamin A contents in the transgenic lines were due primarily to the increased β-carotene levels. The different transgenic lines showed diverse changes under the effects of different transgenes. The content of β-carotene in the transgenic line HYDi-9 increased 10.5-fold (1.76 μg g−1 DW) by silencing HYD compared with the wild-type (0.167 μg g−1 DW), accompanied by decreased lutein and zeaxanthin contents. The overexpression of CrtB in the wheat endosperm, introducing more flux into the carotenoid biosynthetic pathway, increased the total carotenoid content, particularly the content of lutein and β-carotene. The combination of overexpressing CrtB and silencing HYD resulted in the highest accumulation of β-carotene, with levels up to 31-fold (5.06 μg g−1 DW) higher than that of the wild type. The contents of lutein and zeaxanthin in the OBI lines were lower than those in the OB lines but higher than those in the HYDi lines. Notably, lycopene appeared and accumulated in all three of the transgenic lines. By contrast, the first committed carotenoid product, phytoene, appeared only in the transgenic lines that expressed CrtB. Different geometric isomers of carotenoid were also detected in the three classes of transgenic lines (Figure 4). 9-cis-β-carotene was the major component of β-carotene isomers. In addition, trans-lycopene and cis-lycopene were also detected in the three transgenic lines. An undefined carotenoid appeared in the carotenoid profiles of three transgenic lines, the absorbance spectrum of which was similar to that of a lycopene isomer (Figure 4). The phenotype of the transgenic lines was stably inherited (Supporting Information Table S4). Profiles of Endogenous Carotenoid Biosynthetic Genes in Different Transgenic Wheat Lines. Expression of both transgenes, CrtB and HYD, was determined by qRTPCR in the endosperms of transgenic and control wheat lines (Table 1). As anticipated, transcript levels of CrtB and HYD

result in a yellower kernel appearance in transgenic lines. The differences in the external coloration of the grains were confirmed by measuring the color index by Chroma Meter.47 Colorimetric results of grains demonstrated that transgenic lines had higher values of b* (yellowness), which showed significant correlation with lutein and β-carotene content, in contrast to two controls (Supporting Information Figure S2). The differences of the color index indicate that different transgenic lines accumulated different levels of carotenoid in grains. The most intense red/yellow grains from the positive lines were chosen to generate the transgenic progeny by selfpollination for further analyses (Figure 3). To examine carotenoid accumulation in the seeds of different transgenic lines, a HPLC analysis was conducted to compare the profiles of carotenoid content and composition in wheat seeds. There were no significant differences in carotenoid composition between the wild type and vector control (VC). The carotenoid profiles of the mature seeds from three T2 transgenic lines were different from those of the wild-type controls. Several novel carotenes were detected in the transgenic wheat lines including lycopene, β-cryptoxanthin, αcarotene, and phytoene (Supporting Information Table S3). Meanwhile, the HPLC results revealed that, consistent with the silencing of the HYD gene, the levels of β-carotene were significantly increased in seeds from the HYD RNAi (HYDi) lines compared with that of the VC lines. Detailed HPLC analyses were conducted to determine the carotenoid content and composition of seeds from different transgenic lines of the T3 generation (Figure 4). Total carotenoid and provitamin A contents in three T3 transgenic lines were significantly improved, and the content also showed the difference between the same generation from the same T2 transgenic lines (Figure 5A). The total carotenoid content increased to 3.7 μg g−1 dry weight (DW) in the HYDi-9 plants, to 7.35 μg g−1 DW in the OB-8 plants, and to 9.3 μg g−1 DW in the OBI-5 plants compared with the wild type (1.22 μg g−1 DW). Furthermore, the provitamin A content significantly increased in all three of the transgenic wheat lines, ranging from 9086


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Figure 5. Carotenoid and provitamin A contents in the grains from transgenic wheat. (A) Total carotenoid and provitamin A contents in the wheat whole grains of T3 transgenic lines (OBI-5, HYDi-9, and OB-8) and their untransformed control, Bobwhite. The amount of total carotenoid is equal to the sum of all of the carotenoids, and the provitamin A carotenoids were calculated as the sum of α-carotene, βcryptoxanthin, and β-carotene contents in the extracted samples. (B) Carotenoid compositions in the wheat grains from transgenic and control lines in the T3 generation. The average of each carotenoid species was determined from five individual plant heads per line. The data are presented as the means ± SD.

Figure 4. HPLC characterization of carotenoids that were extracted from the grains of T3 transgenic and control wheat: (A) OBI-5; (B) OB-8; (C) HYDi-9; (D) VC-10 (transgenic vector control); (E) Bobwhite (wild-type). Peaks: 1, lutein; 2, zeaxanthin; 3, β-cryptoxanthin; 4, α-carotene; 5, trans-β-carotene; 6, 9-cis-β-carotene; 7, undefined carotene; 8,9, cis-lycopene; 10, trans-lycopene; 11, isomer of lycopene; 12, phytoene.

transgenic plants, expression of the endogenous carotenogenic gene was measured by qRT-PCR. As shown in Figure 7, the expression of the endogenous carotenogenic genes was different in the transgenic, VC, and wild-type lines, whereas that in the VC and wild-type lines showed no difference in the wheat endosperm. In the transgenic line OB with the exogenous gene CrtB, the expression levels of Phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), LCYE, and LCYB were up-regulated, whereas in the HYDi and OBI lines, PSY was up-regulated. Because of the down-regulation of HYD in the transgenic line HYDi, the transcript levels of PDS, LCYE, and LCYB showed the same tendencies and presented greater changes, except for ZDS expression. The combination of overexpressing CrtB and silencing HYD efficiently improved provitamin A levels by increasing the β-carotene content in the transgenic line OBI. Thus, PDS, ZDS, LCYE, and LCYB expression levels had superimposed effects accompanied by the highest expression levels in the three classes of transgenic lines.

were detected only in the corresponding transgenic lines. Then, the expression of CrtB and HYD proteins in the transgenic and control lines was also analyzed by Western blot. The levels of CrtB and HYD proteins in the transgenic and control lines are shown in Figure 6, in which a clear band of CrtB with expected molecular weight was detected only in the transgenic OB and OBI lines. HYD expression was detected in the transgenic, wild-type, and VC lines. However, significant differences were observed between the transgenic and control lines, and all of the transgenic lines exhibited decreased levels of HYD in comparison with wild type, which meant that the translational level of HYD was down-regulated by RNAi. To further understand changes of carotenoid content in the transgenic wheat lines, the expression of endogenous carotenoid biosynthetic genes was analyzed to determine whether they were regulated in response to transgene expressing. In the present study, the 1Dx5 endosperm-specific promoter was employed to avoid influencing the expression levels in other tissues. As shown in Figure 6, the expression of CrtB was only detected in the OB and OBI lines and most of the lines showed similar expression, indicating the stability driven by the 1Dx5 promoter. To further examine the effect of the transgenes on the expression of carotenogenic genes in the

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DISCUSSION Vitamin A deficiency is a global health problem and accounts for increased childhood mortality and disease.7,48−50 Due to the widespread effects of vitamin A deficiency and effective benefits of carotenoid, there are attempts to improve carotenoid content 9087


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Journal of Agricultural and Food Chemistry Table 1. Transgene Expression Level and Carotenoid Content in Transgenic Linesa relative transcript levelsb transgenic line (T3 generation)

crtB

HYDi

Bobwhite OB-8 HYDi-9 OBI-5

0 0.124 ± 0.005 0 0.098 ± 0.004

0 0 0.046 ± 0.003 0.062 ± 0.004

total carotenoidsc (μg g−1 dry seed wt) 1.22 7.37 3.70 9.31

± ± ± ±

0.12 0.59 0.29 0.74

provitamin Ac (μg g−1 dry seed wt) 0.16 2.56 2.25 5.55

± ± ± ±

0.04 0.23 0.17 0.46

a The data represent mean values ± SD and are derived from at least three independent plants per line. bRelative transpcript levels (normalized with respect to the β-actin transcript) were determined by qRT-PCR. cTotal carotenoids (sum of all carotenoid content measured) and provitamin A content (sum of α-carotene, β-cryptoxanthin, β-carotene) were determined by HPLC analysis.

Figure 6. Levels of CrtB and HYD proteins in the endosperms from transgenic and control wheat lines as indicated by Western blotting: (A) CrtB proteins in the OB-8 and control lines; (B) HYD proteins in the HYDi-9 and control lines; (C) CrtB proteins in the OBI-5 and control lines; (D) HYD proteins in the OBI-5 and control lines. The double asterisk indicates significant differences between the control Bobwhite and transgenic lines at a P = 0.01 probability level. The relative amounts of the CrtB and HYD proteins in the transgenic lines were densitometrically quantified with respect to the nontransformed control line (cv. Bobwhite).

(7.37 μg g−1 DW) and 14.6-fold (2.45 μg g−1 DW) in transgenic lines OB as compared with wild type (Figure 5). Although CrtB expression varied, the carotenoid contents in the different transgenic lines were not significantly different, suggesting that the enzyme activity of CrtB was sufficient for product synthesis. This may be due to the limitation of isoprenoid precursors in the carotenoid pathway.28,30 Meanwhile, it is interesting to note that when the CrtB gene was overexpressed in a low-carotenoid wheat phenotype, of which the kernel was light yellow, the accumulations of total carotenoid and β-carotene were less than those in the present study with a yellow kernel.9 This phenomenon might be explained by the observation that storage capacity also appears to be responsible for the enhanced accumulation of carotenoids, such as high-pigment (hp) mutants of tomato

in staple crops by metabolic engineering, especially improving β-carotene content.27,28 In our study, improving the content of β-carotene was the first goal. A combination of “push strategy” and “block strategy” holds good prospect to further increase of the provitamin A content in wheat. Endosperm-Specific Expression of CrtB Introduces More Substrate Flux into Carotenoid Biosynthetic Pathway. In this study, our results demonstrate that CrtB expression in the wheat endosperm significantly increased the carotenoid content, suggesting that the expression of CrtB can provide an additional carotenoid flux into carotenoid biosynthesis. The results of Western blotting detected only the expression of CrtB in transgenic lines (Figure 6), which was also consolidated this conclusion. The total carotenoid and provitamin A contents were significant improved, up to 6.5-fold 9088


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that tuber-specific silencing of LCYE and HYD in potato resulted in the up-regulation of PSY.26,55 It also suggested that the feedback induction of endogeneous genes was correlated with increased levels of carotenoids or intermediates.56 In the transgenic line OB-8, the additional carotenoid flux that was added into metabolic flux in the pathway coincided with the upregulation of genes encoding downstream enzymes, whereas HYD down-regulation led to β-carotene accumulation accompanied by the up-regulation of upstream genes. These results suggest that carotenoid biosynthesis is a complicated system and that metabolic feedback regulation generally exists in the carotenoid biosynthetic pathway. Combination of “Push Strategy” and “Block Strategy” Significantly Enhances Provitamin A Content in Wheat Endosperm. From the results of transgenic line HYDi-9, βcarotene content was significantly improved compared with wild type. However, wheat grains have a very low carotenoid content, which does not have sufficient flux to accumulate abundant specific products by blocking downstream genes; therefore, the possibility thus exists to further improve βcarotene content by adding upstream flux. In our transgenic line OB-8, the carotenoid content significantly increased, indicating that the additional flux could be introduced by CrtB expression. The highest levels of β-carotene accumulation to date were obtained using a combination of “push” and “block” strategies in wheat. Several carotenoid isomers were not calculated into the total carotenoid content; thus, the actual total carotenoid content was even higher than the calculated content. The most famous and successful exploitation case of carotenoid metabolic engineering is “Golden Rice”, which can supply sufficient provitamin A through the daily diet. The U.S. recommended dietary allowance (RDA) for 1−3-year-old children is 300 μg of vitamin A.57 Transgenic staple crops such as maize and potato also reached this RDA standard.28,58 Although the best wheat transgenic lines were not yet up to the standard, phytoene and lycopene accumulation also showed that there still existed huge potentials for more β-carotene accumulation. Although desaturation and cyclase gene expression levels were highest in the OBI transgenic lines, phytoene and lycopene accumulation showed that changing the expression levels of other carotenogenic genes would shift the rate-limiting action to another step in the wheat carotenoid pathway. These results also showed that integration of another gene, the carotene desaturase gene (CrtI), could further improve the content of β-carotene and other carotenoids by increasing the desaturation activity in wheat carotenoid biosynthetic pathway. For instance, in the transgenic wheat with introduced CrtB and CrtI, the accumulation of β-carotene and total carotenoid was more abundant than sole expression of CrtB in transgenic wheat.9 In the transgenic line OBI-5, the contents of lutein and zeaxanthin showed a decrease compared with the OB-8 lines, which suggests that the HYDi could exert the effect simultaneously on both β,ε-carotenoid and β,β-carotenoid to accumulate β-carotene. Meanwhile, the higher accumulation of lutein than zeaxanthin in the OB and OBI transgenic lines suggests that the transgenic lines also exhibited a preference for carotenoid metabolic flux toward the β,ε-carotene branch when additional flux was added into the pathway, similar to the wildtype accumulation of lutein. CrtB and HYDi Regulate β-Carotene Accumulation in Wheat Endosperm through Different Mechanisms. In the present study, overexpression of CrtB and silencing of HYD

Figure 7. Expression levels of the endogenous carotenoid biosynthetic genes in the endosperm from transgenic and control wheat lines. The gene expression levels were measured by quantitative reverse transcription PCR and are determined relative to the transcript levels of the constitutively expressed β-actin gene in the same samples. The expression levels of these genes for the transformed lines are given as the expression levels relative to the values for the nontransformed control line Bobwhite. The qRT-PCR results for each gene were generated from three biological replicates with three technical replicates each, and the data are shown as the means ± SD.

accumulated more carotenoid due to the increased plastid volume per cell.51,52 This result indicates that storage capacity for carotenoid accumulation is a potential target to improve carotenoid content in metabolic engineering.53 In wheat grain, lutein existed in whole grains, not just in the seed endosperm. Experimental evidence from Qin showed that the content of zeaxanthin and lutein in mature wheat embryos was very low, and other compounds were hardly detected.35 This implies that most of the carotenoid content is stored in wheat endosperm, including β-carotene. In addition, the up-regulation of carotenogenic gene expression levels explains the lutein and β-carotene increase, and the phytoene accumulation suggests that desaturation enzymes are insufficient in carotenoid biosynthesis, indicating that the carotenoid pathway can be further manipulated at the levels of PDS and ZDS in these transgenic lines. HYD enzymes play a significant role in the downstream carotenoid pathway, which influences the accumulation of provitamin A in plants. In Arabidopsis chy1chy2lut2 mutants, βcarotene is the main carotenoid in leaves due to the loss of hydroxylase function.54 In potato carotenoid metabolic engineering, the accumulation of β-carotene was enhanced by silencing of β-carotene hydroxylases (CHY1 and CHY2) in potato tuber.26 The transcriptional and translational levels of carotene hydroxylase were simultaneously down-regulated by HYD silencing, accompanied by the β-carotene content increasing 10-fold and the lutein and zeaxanthin decreasing in the HYDi-9 transgenic line compared with that of the wild type, suggesting that silencing of HYD prevented a part of β-carotene from hydroxylating into lutein or zeaxanthin. Furthermore, although LCYE and LCYB expression levels were up-regulated in HYDi-9, lycopene accumulation revealed insufficient cyclases in carotene biosynthesis. However, lycopene was not detected in wild-type, suggesting that the activity of cyclases is enough for carotene biosynthesis. One of the possible reasons for this observed phenotype could be that the down-regulation of HYD might lead to a feedback regulation to up-regulate PSY expression and add more flux into the pathway. This phenomenon was also reported in other studies, which showed 9089


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Doctoral Program of Higher Education of China (Grant 2012014211075).

regulate different endogenous carotenoid biosynthesis genes with different variation, suggesting the accumulation of βcarotene through different mechanisms. In OB lines, the expressions of PDS, ZDS, LCYE, LCYB, and HYD2 were upregulated, and only PSY and HYD1 were unaffected (Figure 7). In contrast, in HYDi lines, the expression of PSY was upregulated, but ZDS was unaffected. In both transgenic lines, βcarotene and total carotenoid were enriched by the transgenes. In transgenic line OB expressing CrtB, the greater upstream flux introduced into the pathway forward the corresponding downstream gene up-regulation. In contrast, in the HYDi line silencing HYD, the accumulation of β-carotene resulted in feedback regulation accompanied by the up-regulation of upstream genes. In addition, the up-regulation levels in HYDi lines were higher than in OB lines. These results suggest that the flux added into OB and HYDi lines may be through different mechanisms, in one case due to the additional flux introduced and in another through the feedback regulation to up-regulate PSY. The presented results indicate that the block strategy to silence HYD in the wheat carotenoid pathway seems to be feasible to accumulate β-carotene content specifically. Indeed, the combination of these two strategies is effective in accumulating β-carotene in the wheat endosperm. Our results show that this strategy leads to the highest accumulation of βcarotene in the wheat endosperm; the results also demonstrate the feasibility of utilizing this strategy in carotenoid metabolic engineering and reveal the different mechanisms of β-carotene accumulation.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Dr. Sandmann from the Institute of Molecular Bioscience, J. W. Goethe University, Frankfurt am Main, Germany, for providing the plasmid pACCRT-EB. We thank Dandan Li for editorial assistance and helpful discussions.

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ABBREVIATIONS USED CrtB, bacterial phytoene synthase gene; CYP, ε-ring hydroxylase; DAP, days after pollination; HPLC, high-performance liquid chromatography; HYD, carotenoid hydroxylase; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; OE, overexpression; PDS, phytoene desaturase; PSY, phytoene synthase; QTL, quantitative trait locus; RNAi, RNA interference; VAD, vitamin A deficiency; ZDS, ζ-carotene desaturase

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04279. Primer sequences used in this study; generation of transgenic wheat plants; carotenoid content and composition in T2 and T3 seeds from transgenic and control wheat plants; absorption spectra of carotenoids; color characteristics of grains from transgenic and control lines of wheat, CIE b* values (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(G.Y.) E-mail: [email protected]. Phone: 0086-(0)2787792271. Fax: 0086-(0)27-87792272. *(G.H.) E-mail: [email protected]. Phone: 0086-(0)2787792271. Fax: 0086-(0)27-87792272. Present Address ‡

Department of Food Science and Engineering, School of Marine Science, Ningbo University, No. 818 Fenghua Road, Ningbo 315211, Zhejiang Province, China. Author Contributions †

J.Z., X.W., and Y.M. contributed equally to this work.

Funding

This work was supported by the International S&T Cooperation Key Projects of the Chinese Ministry of Science and Technology (Grant 2009DFB30340), the National Genetically Modified New Varieties of Major Projects of China (Grant 2015ZX08002004-007 and 2015ZX08010004), the National Natural Science Foundation of China (No. 31071403 and 31371614), and the Research Fund for the 9090


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Metabolic Engineering of Wheat Provitamin A by Simultaneously

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