Plant Foods Hum Nutr (2014) 69:379–385 DOI 10.1007/s11130-014-0450-9
ORIGINAL PAPER
Overexpression of Folate Biosynthesis Genes in Rice (Oryza sativa L.) and Evaluation of Their Impact on Seed Folate Content Wei Dong & Zhi-jun Cheng & Cai-lin Lei & Xiao-le Wang & Jiu-lin Wang & Jie Wang & Fu-qing Wu & Xin Zhang & Xiu-ping Guo & Hu-qu Zhai & Jian-min Wan
Published online: 29 November 2014 # Springer Science+Business Media New York 2014
Abstract Folate (vitamin B9) deficiency is a global health problem especially in developing countries where the major staple foods such as rice contain extremely low folates. Biofortification of rice could be an alternative complement way to fight folate deficiency. In this study, we evaluated the availability of the genes in each step of folate biosynthesis pathway for rice folate enhancement in the japonica variety kitaake genetic background. The first enzymes GTP cyclohydrolase I (GTPCHI) and aminodeoxychorismate synthase (ADCS) in the pterin and para-aminobenzoate branches resulted in significant increase in seed folate content, respectively (P<0.01). Overexpression of two closely related enzymes dihydrofolate synthase (DHFS) and folypolyglutamate synthase (FPGS), which perform the first and further additions of glutamates, produced slightly increase in seed folate content separately. The GTPCHI transgene was combined with each of the other transgenes except ADCS to investigate the effects of gene stacking on seed folate accumulation. Seed folate contents in the gene-stacked plants were higher than the individual low-folate transgenic parents, but lower than the high-folate GTPCHI transgenic lines, pointing to an inadequate supply of para-aminobenzoic acid (PABA) precursor
Wei Dong, Zhi-jun Cheng and Cai-lin Lei contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11130-014-0450-9) contains supplementary material, which is available to authorized users. W. Dong : Z.
initiated by ADCS in constraining folate overproduction in gene-stacked plants. Keywords Staple food-folate biofortification-metabolic engineering-gene stacking
Introduction Folates (vitamin B9) are essential micronutrients in human diet, participating as enzyme co-factors in nucleotide biosynthesis, methylation cycle and amino acid metabolism [1]. Human depend on plants as a major source of dietary folates. Rice as a major staple food contains low levels of folates (http://www.nal.usda.gov/fnic/foodcomp/search/) [2]. Upon milling, most micronutrients are removed, only 5 % of the folate present in brown rice [3]. Therefore, folate deficiency is prevalent in rice-eating populations mainly in developing countries, which can lead to neural tube defects [4], coronary heart diseases [5], impaired cognitive functions [6], and cancer [7]. Folates are tripartite molecules consisting of a pterin moiety, PABA and one or several glutamate residues. In plants, the pterin moiety is produced from GTP in cytosol, PABA is synthesized from chorismate in plastids, and the two precursors are transported into mitochondria to form polyglutamate tetrahydrofolate (Fig. 1) [8, 9]. Folate biofortification of staple foodstuff by approaches of metabolic engineering to manipulate folate biosynthesis pathway is an alternative complementary strategy to the current interventions, viz., folic acid capsule intake, fortification with synthetic folic acid, and diet diversification [10]. In previous reports, folate contents were increased by overexpression of both GTPCHI and ADCS genes up to 25- and 100-fold in
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Fig. 1 Diagram of the plant folate biosynthesis pathway ① GTP cyclohydrolase I, AT3G07270, Cytoplasm; ② dihydroneopterin aldolase, AT3G11750, Cytoplasm; ③ aminodeoxychorismate synthase, AT2G28880, Plastids; ④ aminodeoxy chorismate lyase, AT5G57850, Plastids; ⑤ hydroxymethyldihydropterin pyrophosphokinase/ dihydropteroate synthase, AT4G30000, Mitochondria; ⑥ dihydrofolate
synthase, AT3G10160, Mitochondria; ⑦ dihydrofolatere ductase, AT4G34570, Mitochondria; ⑧ folylpolyglutamate synthase, AT 5 G 4 1 4 8 0 , M i t o c h o n d r i a . A b b r e v i a t i o n s : A D C aminodeoxychorismate, DHN dihydroneopterin, HM DHP hydroxymethyldihydropterin, THF tetrahydrofolate. Putative localization of folate biosynthesis genes predicted by Target P
tomatoes and rice, respectively [11, 12]. Ectopic expression of wheat HPPK/DHPS slightly increased folate levels in rice leaves and grains [13]. Increased FPGS activity in rice was associated with higher intracellular folate levels [14]. Overexpression of the other folate biosynthesis genes originated from bacterial such as folKE (homolog of plant genes encoding HMDHP pyrophosphokinase and dihydropteroate synthase, HPPK/DHPS and GTPCHI) and folA (homolog of the plant gene encoding DHF reductase and thymidylate synthase, DHFR/TS) led to 3-fold increase and 2-fold increase in folates in Lactococcus lactis, respectively [15]. However, the availability of ectopic expression folate biosynthesis genes for rice folate enhancement is still incomplete and the effects of stacking these genes on rice folate content require further study. This study aims to evaluate the impacts on rice seed content by ectopic expression of individual folate biosynthesis genes from A. thaliana, and stacking two-transgene combinations.
ligated by T4 DNA ligase (NEB), subsequently introduced into Agrobacterium tumefaciens strain EHA105 by electroporation, and further transformed into japonica variety Kitaake, respectively, according the protocol with minor modification [16]. DNA and RNA Analysis Genomic DNA was extracted from leaves using 2 M cetyltrimethyl ammonium bromide (CTAB) extraction buffer. Genotyping was performed according to standard amplification parameters with primers (Table S1). Seed RNAs were extracted using a hot phenol method [17]. Reverse transcription was performed using reverse transcriptase kit (Takara). Gene expression levels were determined by real-time PCR on an ABI 7900 HT System (Table S2) (Takara). Expression levels were calculated using the 2-ΔΔCt method [18]. Folate Assays
Material and Methods Molecular Cloning and Transformation Coding DNA sequences (CDSs) of the eight genes (Fig. 1) were amplified from A. thaliana using a RT-PCR kit (Takara) with the primers shown in Table S1, and subcloned into the PMD-18 T vector (Takara). The resulting plasmids and pCUbi1390 vector were digested with appropriate enzymes,
Grain samples were collected from at least six plants per line at 25 days post anthesis (DPA) and then frozen in liquid nitrogen. Each sample (200 mg grain) was incubated for 15 min at 95 °C in 1 ml of phosphate buffer (50 mM, pH 7, with 1 % ascorbic acid and 0.1 % 2-mercaptoethanol). After cooling, the samples were homogenized with a grinder (Geno 2000, SPEX CertiPrep, 300 rps, 4 min), and sequentially treated with amylase, protease and conjugase enzymes to free folate for bacterial growth. A microbiological assay based on
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Fig. 2 Schematic representation of T-DNA regions in rice transformation Gray pointers depict promoters, filled pointers depict CDSs of A. thaliana folate biosynthesis genes, and gray bars represent transcriptional terminators. 35S, cauliflower mosaic virus 35S promoter, and Ubi, rice ubiquitin; hptII, gene encoding hygromycin phosphotransferase; T35S and Tn, transcriptional terminators of the 35S transcript of cauliflower mosaic virus and nopaline synthase gene, respectively; LB and RB, left and right T-DNA borders, respectively
Fig. 3 qPCR analysis in single gene transgenic rice plants a GTPCHI, Wt and three G+-lines b ADCS, Wt and three A+-lines c DHNA, Wt and three N+-lines d ADCL, Wt and three D+-lines e HPPK, Wt and three H+-lines f DHFS, Wt and three S+-lines g DHFR, Wt and three R+-lines h FPGS, Wt and three F+-lines. Values for wild type (Wt, nontransgenic plants) are shown for comparison. Levels of mRNA relative to rice ubiquitin in 25 DPA seeds were determined by real-time PCR. Transgenic seeds were sampled from T0 transgenic plants
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growth of Lactobacillus rhamnosus (ATCC 7469) on exogenous folate was used to measure total folate content [19, 20]. The certified reference material (CRM 485, from Community Bureau of Reference, BCR) was analyzed with the samples as a quality control [19]. Folate content varies due to varieties, storage conditions, processing methods, and decreases with seed development. Each line together with its control was analyzed in triplicate, and all manipulations were carried out under subdued light.
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Results and Discussions Overexpression of the AtGTPCHI, AtADCS, AtDHFS, and AtFPGS Genes Enhanced Seed Folate Content To avoid possible negative feedback regulated by endogenous folate biosynthesis genes, we independently overexpressed folate biosynthesis genes (Fig. 1) from A. thaliana. Four single-copy genes AtGTPCHI, AtADCS, AtADCL and AtDHFS, one of AtDHNA genes, which is most homologous to rice DHNA, and mitochondrially localized AtHPPK/DHPS, AtDHFR, and AtFPGS [21, 22] were chosen as the targeted genes. Eight transformation vectors were constructed (Fig. 2) and introduced into the genome of japonica rice variety Kitaake. Expression of each transgene was confirmed by real-time PCR (Fig. 3).
Fig. 4 Total seed folate contents in the T1 and T2 generations of transgenic rice a GTPCHI, Wt and three G+-lines b ADCS, Wt and three A+-lines c DHNA, Wt and three N+-lines d ADCL, Wt and three D+-lines e HPPK, Wt and three H+-lines f DHFS, Wt and three S+-lines g DHFR, Wt and three R+-lines h FPGS, Wt and three F+-lines. Values for wild type (Wt, nontransgenic plants) are shown for comparison. Black bars and gray bars represent folate contents measured in seeds of T1 and T2 plants, respectively. * and ** denote significantly differences at P<0.05 and P<0.01 level
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Among transgenic lines, the potential of folate biofortification was clearly displayed by overexpression of AtGTPCHI (G+-lines), showing 3.7–6.1-fold and 3.3– 3.7-fold higher folate contents in two generations, respectively, than the wild type (P<0.01) (Fig. 4a). Previously, the capability of GTPCHI from mammalian and A. thaliana for folate enhancement has been revealed in different plants [12, 23–26]. However, the folate content of GTPCHI transgenic rice was unchanged, which is different from the other reports [11]. Also, distinguished with negative effect of ADCS on folate biofortification [11], our AtADCS lines (A+-lines) had 1.5–1.8-fold increase of folate contents, relative to wild type (P<0.01) (Fig. 4b). The inconsistence of our results from previous studies could be contributed to the difference in promoters used or the transformed varieties. We detected seeds folate content in
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kitaake was 3.2 times higher than that in Nippon Bare japonica, which also demonstrated nearly 4 times higher than the reported Nippon Bare japonica [11]. Similar to the result of overexpressing bacterial origin of DHFS/ FPGS (FolC) increased folate content by 19.0 to 42.6 % in Arabidopsis [24], engineering of AtDHFS here resulted in 14.5–27.2 % increase of total folate content in T1 generation (P>0.05) (Fig. 4f). And overexpression of AtFPGS increased seed folate contents by 7.5 to 19.9 % and 4.3 to 45.5 %, compared to wild type, in the T1 and T2 generations, respectively (Fig. 4h). Conversely, folate contents in AtDHNA (N+-) and AtADCL (D+-) lines were lower in folate content than wild type. AtHPPK (H+-lines) and AtDHFR (R+-lines) plants showed insignificantly change of folate levels (Fig. 4c, d, e, and g). While overexpressing bacterial- or wheat-origin of HPPK/DHPS genes in Arabidopsis and rice caused folate content increase ranging from 20.6 to 29.4 % and from 40 to 75 %, respectively [13].
Fig. 5 Total seed folate content in double-gene transgenic rice plants a G+ × D+, GTPCHI+ × ADCL+ double transgenic plants; b G+ × N+, GTPCHI + × DHNA+ double transgenic plants; c G+ × H+, GTPCHI + × HPPK+ double transgenic plants; d G+ × R+, GTPCHI + × DHFR+ double transgenic plants; e G+ × S+, GTPCHI + × DHFS+ double transgenic plants; f G+ × F+, GTPCHI +×FPGS+ double transgenic plants. Values for single GTPCHI +, ADCL+, DHNA+, HPPK+, DHFR+, DHFS+, FPGS+ transgenic lines are shown for comparison. All numbers on the x axis correspond to the different crosses from single parent lines. The T1 individuals used for crossing were genotyped positive with singlecopy tranformants, either hemizygous or homozygous
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Folates in Gene Stacked Rice Plants and Possible Strategies for Improving Folate Contents To test whether the effect of AtGTPCHI on folate accumulation was enhanced by combination with other folate biosynthesis transgenes, we crossed the T1 plants of G+lines with other single transgene plants and obtained six derivatives with combined two transgenes (Fig. 5). Three double-gene progeny lines were obtained from each cross except for ‘G+ × F+’ lines for which there were two double-gene progeny lines (Fig. 5). Compared to wild type plants, double-transgene plants had indistinguishable phenotypes. Total folate contents of five double-gene progeny lines were higher than the low-folate parents (including D+-, N+-, H+-, R+-, and S+-types), but similar to, or lower than the high folate parent G+-lines (Fig. 5a-e). Stacking of transgenes in the cross ‘G+ × F+’ resulted in higher folate contents than both parents, but the differences were not significant (P>0.05) (Fig. 5f). Thus, folate
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contents in double-gene transgenic lines, derived from the crosses of G+ −lines with the other single gene (Fig. 1) transgenic lines, were not improved much higher than both parents as expected. Previous results revealed that combining expressing of GTPCHI and ADCS in rice and tomato have increased grains and fruits folate content, respectively [9, 10]. But in our study, co-expression of GTPCHI and ADCL, the gene catalyzing the step after ADCS, did not increase folate content, demonstrating that ADCL cannot form the flux to PABA individually. GTPCHI plants, which enhanced folate content by pterins accumulation [11], have increased folates content in the other single transgenic plants by crossing. Therefore, folate biosynthesis by possible high pterins concentration in double gene transgenic lines limited folate production is probably due to the absence of elevated levels of PABA. Hence, overexpression of ADCS in our existing double transgenic plants might compensate the absence of PABA precursor, and potentially improve folate content. It was reported that pterin and PABA levels in GTPCHI+ or GTPCHI+/ADCS+ transgenic fruits were much higher than those in the wild type, but the intermediates, including dihydropteroate and dihydrofolate, did not accumulate, indicating a flux constraint during dihydropteroate synthesis [12]. In the present study, the HPPK/DHPS gene controlling this step was overexpressed to address the limiting step, but failed to increase folate content, demonstrating that shortage of pterin and PABA precursors in HPPK lines. Further study could include simultaneous overexpression of genes for the first enzymes of the three branches of the folate pathway, i.e., GTPCHI, ADCS and HPPK. To eliminate the expression of the γ-glutamyl hydrolase (GGH) in our transgenic lines may increase the composition of polyglutamate tetrahydrofolate [14]. Moreover, our previous study showing a QTL locus underlying rice folate content did not include folate biosynthesis genes [27], and rice biofortified seeds did not show a significant effect on expression of endogenous folate biosynthesis genes [28]. Therefore, proposed genes related to homeostasis, catabolism, membrane transport, de novo synthesis of folate-binding protein (FBP) and vacuolar storage of folates could be crucial in achieving rice folate biofortification to address folate malnutrition [29–33].
Acknowledgments This work was supported by grants from the National Basic Research Program of China (Grant no. 2007CB10880-1, 2013CB127000), and Transgenic Science and Technology Program (2013ZX08001-006) Conflict of Interest The authors have declared that there were no competing interests. The manuscript does not contain any studies with human or animal subjects.
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