Article pubs.acs.org/JAFC
Concurrent Overexpression of Arabidopsis thaliana Cystathionine γ‑Synthase and Silencing of Endogenous Methionine γ‑Lyase Enhance Tuber Methionine Content in Solanum tuberosum Pavan Kumar and Georg Jander* Boyce Thompson Institute for Plant Research, 533 Tower Road, Ithaca, New York 14853, United States S Supporting Information *
ABSTRACT: Potatoes (Solanum tuberosum) are deficient in methionine, an essential amino acid in human and animal diets. Higher methionine levels increase the nutritional quality and promote the typically pleasant aroma associated with baked and fried potatoes. Several attempts have been made to elevate tuber methionine levels by genetic engineering of methionine biosynthesis and catabolism. Overexpressing Arabidopsis thaliana cystathionine γ-synthase (AtCGS) in S. tuberosum up-regulates a rate-limiting step of methionine biosynthesis and increases tuber methionine levels. Alternatively, silencing S. tuberosum methionine γ-lyase (StMGL), which causes decreased degradation of methionine into 2-ketobutyrate, also increases methionine levels. Concurrently enhancing biosynthesis and reducing degradation were predicted to provide further increases in tuber methionine content. Here we report that S. tuberosum cv. Désirée plants with AtCGS overexpression and StMGL silenced by RNA interference are morphologically normal and accumulate higher free methionine levels than either single-transgenic line. KEYWORDS: methionine, potato, tuber, essential amino acids, cystathionine γ-synthase, methionine γ-lyase
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source and sink tissue.7,8 Finally, methionine can be catabolized to 2-ketobutyrate by methionine γ-lyase (MGL) and thereby serves as a precursor for isoleucine biosynthesis (Figure 1).9−11 Methionine abundance in Arabidopsis can be regulated by increasing biosynthesis or decreasing catabolism. Overexpression studies show that CGS, rather cystathionine β-lyase or methionine synthase, is a rate-limiting step of methionine synthesis from O-phosphohomoserine.12,13 Arabidopsis CGS activity is feedback-regulated by SAM at the level of mRNA stability.14 However, if the first 90 nucleotides of Arabidopsis CGS are truncated (mto1-CGS), the enzyme is insensitive to feedback inhibition by SAM,15 and Arabidopsis overexpressing mto1-CGS accumulates higher methionine levels than CGSoverexpressing plants.16 It is interesting to note that this regulation of CGS activity is not conserved among all plant species. For instance, methionine feeding experiments indicate that potato CGS is insensitive to feedback inhibition.17,18 Mutations in threonine synthase, which competes with CGS for a common substrate, also increase Arabidopsis methionine content.19 On the catabolic side, Arabidopsis mgl knockout mutations increase seed methionine content without having significant effects on plant morphology.9 Increasing the free, rather than protein-bound, methionine content of potato tubers is generally considered to be more feasible. Free methionine constitutes up to 60% of the total methionine in the tubers of cultivated potato varieties,1 and 93% of the natural variation in total tuber methionine content in tested potato varieties was due to differences in the free
INTRODUCTION The potato (Solanum tuberosum) is one of the world’s most important food crops and a major source of nutrients in human diets. However, the essential amino acid methionine has relatively low abundance in potato tubers and is limiting in the diet when compared to the daily uptake values recommended by the World Health Organization.1 In addition to its role as an essential nutrient, methionine breakdown to methional by the Strecker degradation reaction contributes to the pleasant aroma of baked and fried potatoes.2 Although supplementing free methionine during potato processing to improve the aroma is possible, this is not economically feasible. Thus, elevating the endogenous tuber methionine levels is desirable for increasing both the nutrient content and the market value of potatoes. Methionine biosynthesis from aspartate via a pathway shared with lysine, threonine, and isoleucine (Figure 1) has been studied extensively in Arabidopsis thaliana (Arabidopsis).3−5 OPhosphohomoserine, which is produced by homoserine kinase, is a common precursor for both methionine and threonine biosynthesis. Threonine synthase (TS) catalyzes the formation of threonine, whereas cystathionine γ-synthase (CGS) forms cystathionine from O-phosphohomoserine and cysteine. The sequential activity of cystathionine β-lyase and methionine synthase leads to methionine synthesis from cystathionine. Methionine not only serves as an essential protein building block but also is a precursor for other plant metabolites (Figure 1). S-Adenosylmethionine synthase forms S-adenosylmethionine (SAM), which is a primary methyl group donor for DNA replication, cell wall development, and other biological processes, as well as being a precursor for the synthesis of ethylene, vitamin B1, and polyamines.6 Methionine methyltransferase catalyzes the formation of S-methylmethionine, which is involved in the transport of reduced sulfur between © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
January 19, 2017 March 11, 2017 March 15, 2017 March 15, 2017 DOI: 10.1021/acs.jafc.7b00272 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
both of these transgenic approaches had an even greater lysine content.28 Similar approaches have been used to increase lysine in maize seeds, where this is a limiting essential amino acid.29,30 The success with increasing lysine content through increased synthesis and decreased catabolism28 suggested that a similar approach could be used to increase methionine content in potatoes. Here we show that concomitant AtCGS overexpression25 and StMGL silencing31 increase tuber methionine content in potato variety Désirée.
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MATERIALS AND METHODS
Plant Material. Wild type S. tuberosum cv. Désirée and previously described lines with silenced MGL expression (MGL6 and MGL34)31 were vegetatively propagated by tissue culture on CM medium (4.3 g/ L Murashige and Skoog basal salt mixture, 0.1 g/L myo-inositol, 0.4 mg/L thiamin-HCl, 20 g/L sucrose, 8 g/L agar, adjusted to pH 5.7) at 22 °C under a 16 h light/8 h dark regimen.31 Single-node stem segments of MGL6 and MGL34 were used as starting material for tissue culture and AtCGS transformation. Gene Cloning and Transformation. An Arabidopsis CGS gene clone (AtCGS: ATU43709) was obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/), and a 1.7 kb AtCGS gene fragment (Figure S1A) was amplified by Phusion (Thermo Scientific, Waltham, MA, USA) using AtCGS primers containing Gateway cloning sites (Table S1). The AtCGS gene was cloned into the pDONR207 entry vector by BP reaction and subsequently transferred behind the 2X 35S promoter in the pMDC32 destination vector (Figure S1B)32 by LR reaction using the Gateway cloning kit (Invitrogen, Carlsbad, CA, USA). The presence of the intact 1.7 kb AtCGS fragment was confirmed by PCR and DNA sequencing. The pMDC32 plasmid containing AtCGS was moved into wild type Désirée and the MGL-silenced MGL6 and MGL34 potato lines using leaf transformation, as described previously.33 The pMDC32 vector alone was also used to generate empty vector control plants. Positive transformants were selected on the basis of their ability to grow on medium with 5 mg/L hygromycin, and the presence of the hygromycin phosphotransferase (HPT) gene was detected by PCR (Figure S1C). Amino Acid Assays. Freshly frozen and lyophilized tuber and leaf material from 3-month-old plants was used for amino acid extraction. All of the steps of extraction, separation, and analysis of amino acids followed a previously described protocol.26 Total Protein Quantification. Total soluble proteins from lyophilized tubers were extracted in 20 mM HCl, and the total protein content was measured using the Bradford reagent (Bio-Rad, Hercules, CA, USA).34 Bovine serum albumin was used for preparing a standard curve to determine protein abundance. Real-Time Quantitative PCR (qRT-PCR). Total RNA isolation, cDNA synthesis, and analysis of transcript abundance were carried out as explained previously.35 Transcript abundance of S. tuberosum, methionine γ-lyase, eEF1a and AtCGS were measured by quantitative PCR using the primers listed in Table S1. Statistical Analysis. All statistical analyses were conducted using JMP Pro 12 (www.jmp.com).
Figure 1. Aspartate-derived amino acid biosynthesis pathway in plants. Key pathway intermediates and enzymes are shown. Enzymes that were overexpressed (cystathionine γ-synthase, CGS) or silenced (methionine γ-lyase, MGL) to increase potato tuber methionine content are circled by thick lines and broken lines, respectively. AK, aspartate kinase; ALS, acetolactate synthase; BCAT, branched-chain amino acid aminotransferase; CBL, cystathionine β-lyase; DHDPS, dihydrodipicolinate synthase; HSD, homoserine dehydrogenase; LKR, lysine ketoglutarate reductase; MMT, methionine methyltransferase; MS, methionine synthase; SAMS, S-adenosylmethionine synthase; TA, threonine aldolase; TD, threonine deaminase; TS, threonine synthase.
methionine levels.20 Additionally, increasing the abundance of protein-bound methionine in potato tuber proteins is challenging, because it requires either expressing non-potato storage proteins with high methionine content or re-engineering endogenous potato proteins to increase the number of methionine residues without compromising other protein functions. Several groups have attempted to increase the free methionine content in potato tubers by genetic engineering. It has been demonstrated that homoserine kinase is not the rate-limiting enzyme for potato methionine biosynthesis,21 and ectopic expression of Escherichia coli homoserine kinase failed to increase the tuber methionine levels.22 RNAi-mediated silencing of threonine synthase increased tuber methionine levels without affecting threonine levels, but negatively affected yield and plant growth.23 Overexpressing potato CGS did not increase methionine levels, possibly due to failure of this enzyme to compete with the threonine synthase for Ophosphohomoserine.17 Although potato plants overexpressing feedback-insensitive Arabidopsis mto1-CGS accumulated higher methionine levels, the transgenic plants were morphologically abnormal, showed altered physiology, and had reduced tuber yield.24 However, overexpression of AtCGS, which is sensitive to SAM inhibition in Arabidopsis, increased tuber methionine levels and did not cause an abnormal growth phenotype in potatoes.25 Similar overexpression of AtCGS in other plant species also increased free methionine content.16,26,27 In another approach, Huang et al. demonstrated that potato methionine levels could be increased by silencing MGL expression, without affecting the plant growth and tuber yield.31 Although the lysine content in Arabidopsis can be elevated by either increasing dihydrodipicolinate synthase or decreasing lysine ketoglutarate reductase (Figure 1), plants subjected to
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RESULTS Generation of AtCGS Overexpressing Transgenic Plants. Wild type potato variety Désirée and two independent MGL-silenced lines, MGL6 and MGL34,31 were transformed with AtCGS expressed from the 35S promoter. As a control, the pMDC32 empty vector (EV) alone also was transformed into the three potato lines. Five independent transformed lines growing on rooting medium were tested for the presence of the transgene insertion by PCR amplification of the hygromycin phosphotransferase (hpt) gene (Figure S1C). Except for a single MGL34-EV line, all of the lines were found to possess the inserted transgene. Two independent lines of AtCGSB
DOI: 10.1021/acs.jafc.7b00272 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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nor MGL silencing had a significant effect on these leaf and tuber traits (P > 0.05, ANOVA). Overexpression of AtCGS Increases Tuber Free Methionine Content. Free methionine content in AtCGSoverexpressing and control tubers was measured in the first and second generations after transformation. There was a significant effect of AtCGS expression on free methionine levels relative to wild type Désirée (P < 0.01) (Figure 4A,B). When compared to wild type Désirée, AtCGS overexpression had no significant effect on the abundance of threonine (Figure 4C,D), which is metabolically linked to methionine (Figure 1). Isoleucine, however, showed a significant (P = 0.03) decrease in the first but not the second generation after transformation (Figure 4E,F). We also measured the abundance of other amino acids of these tubers (Table S2), which showed no consistent effect of AtCGS overexpression on amino acid abundance relative to that of wild type relative Désirée tubers. Simultaneous Overexpression of AtCGS and Silencing of StMGL Further Increase Free Methionine Levels. We previously reported that reducing StMGL expression by RNA interference increases tuber methionine levels by decreasing methionine degradation.31 Therefore, we hypothesized that simultaneous overexpression of AtCGS and silencing of StMGL would further increase free methionine levels. There was a significant increase in free methionine content in MGL-silenced lines in response to AtCGS overexpression in both the first and second generations after transformation (P < 0.01) (Figure 4A,B). The genetic background (MGL6 vs MGL34) had a significant effect on methionine accumulation in the first generation (P = 0.03, two-way ANOVA, with the MGL34 line having more methionine), but not in the second generation (P = 0.19, two-way ANOVA). Threonine and isoleucine contents of first- and second-generation tubers were unaffected by AtCGS overexpression (Figure 4B,C,E,F). Relative to wild type Désirée, there was a significant increase in the tuber methionine content in the double-transgenic lines in both the first and second generations (P < 0.05, ANOVA; Figure 4A,B). We also measured the effect of AtCGS overexpression on the abundance of other amino acids in first- and second-generation tubers, compared to those that had only StMGL expression silenced (Table S3). In addition to methionine, the abundance of leucine, valine, and phenylalanine was changed by AtCGS overexpression in the first generation (P < 0.05, two-way ANOVA). However, the effect on these amino acids was not replicated in the second generation. Other than methionine, only tyrosine was influenced by the genetic background (MGL6 and MGL34) in the first generation, and no amino acids were affected in the second generation.
overexpressing and EV controls, in the wild type, MGL6, and MGL34 genetic backgrounds, were selected for the further experiments. Quantitative reverse transcriptase PCR (qRT-PCR) showed that all of the plant lines overexpressing AtCGS accumulated AtCGS transcripts (Figure 2A). Two-way ANOVA showed that
Figure 2. Transcript levels in tubers of wild type and MGL-silenced potato lines, with and without AtCGS overexpression: AtCGS (A) and StMGL (B) transcript levels in tubers of plants transformed with either the empty vector (EV, solid fill) or AtCGS expressed from the 35S promoter (AtCGS, hatched fill) in wild type Désirée (white background) and each of two independent MGL-silenced lines, MGL6 (light gray background) and MGL34 (dark gray background). Data are the mean ± SE of N = 3. P values were calculated by two-way ANOVA to determine the significant difference in (A) AtCGS expression with the transformation constructs EV and AtCGS as factors and (B) StMGL expression in plants with and without AtCGS expression.
there was a significant effect of the AtCGS construct on AtCGS transcripts (P < 0.0001) and that there was no significant effect of the potato line that was transformed (P = 0.63) (Figure 2B). As reported previously,31 StMGL transcripts were downregulated in the MGL6 and MGL34 lines compared to wild type Désirée (P < 0.001, two-way ANOVA; Figure 2B). We further tested whether AtCGS overexpression affected StMGL transcript accumulation. StMGL transcripts were less abundant (P = 0.03, two-factor ANOVA) in AtCGS-overexpressing MGL34 compared to EV-transformed MGL34 (Figure 2B), but this was not the case for MGL6 (P = 0.41) and wild type (P = 0.23). Two-way ANOVA comparing StMGL expression in all 18 EV and 18 AtCGS transgenic replicates in the three genetic backgrounds (wild type, MGL6, and MGL34) also did not demonstrate an effect of the AtCGS transgene on StMGL expression (P = 0.076). No Significant Phenotypic Alterations of AtCGS Transformation. We did not observe any visible morphological changes in leaf and tuber architecture in response to AtCGS overexpression. Representative leaves and tubers from wild type and MGL-silenced plants, with and without AtCGS overexpression, are shown in panels A and B of Figure 3. Furthermore, we measured total dry leaf mass, tuber fresh mass, the number of tubers per plant, and tuber protein content in all transgenic lines (Figure 3C−F). Neither AtCGS overexpression
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DISCUSSION Successful approaches to increasing the free methionine content in plants include genetic engineering of the biosynthetic pathway, classical breeding, ectopic expression of methionine-rich proteins, and addition of methionine residues to abundant plant proteins (reviewed in ref 36). On the basis of knowledge gained from studying methionine biosynthesis and regulation in Arabidopsis, several attempts have been made to increase the methionine levels in potatoes. Among known pathway enzymes, CGS is the most widely explored for increasing methionine levels. Overexpression of StCGS did not increase methionine levels in potato.17 In contrast, overexpression of an N-terminal-deleted form of Arabidopsis CGS (D-AtCGS), which is insensitive to the feedback inhibition by C
DOI: 10.1021/acs.jafc.7b00272 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. Phenotype of AtCgS-overexpressing wild type and StMGL-silenced potato plants. (A, B) Representative leaves and tubers from wild type (WT) and MGL-silenced plants, with and without AtCgS overexpression. (C) Leaf mass (dry), mean ± SE of N = 3. (D) Tuber fresh mass, mean ± SE of N = 3. (E) Number of tubers per plant, mean ± SE of N = 3. (F) Tuber protein content, mean ± SE of N = 5 or 6. Potato plants were transformed with either the empty vector (EV, solid fill) or AtCGS expressed from the 35S promoter (AtCGS, hatched fill). Two independent transformants (labeled 1 and 2) were used for wild type Désirée (white background) and each of two independent MGL-silenced lines, MGL6 (light gray background) and MGL34 (dark gray background). No significant differences between plant lines or constructs were detected by ANOVA.
Figure 4. Amino acid levels in tubers of wild type and MGL-silenced potato lines, with and without AtCGS overexpression. Free methionine (A, B), threonine (C, D), and isoleucine levels (E, F) were measured in tubers in the first (A, C, E) and second (B, D, F) generationd after transformation with either the empty vector (EV, solid fill) or AtCGS expressed from the 35S promoter (CGS, hatched fill). Two independent transformants (labeled 1 and 2) were used for wild type (WT, white background) and each of two independent MGL-silenced lines, MGL6 (light gray background) and MGL34 (dark gray background). Data are the mean ± SE of N = 4−6. P values to determine the effects of the AtCGS expression on tuber amino acid levels were calculated by two-way ANOVA with the plant lines and transformation constructs (EV and CGS) as factors.
abnormal phenotype not only in potato but also in tobacco, suggesting that D-AtCGS will not be an ideal gene for enhancing potato methionine accumulation.15,16,37 In addition, D-AtCGS overexpression did not produce similar results in beans, leaving total methionine content unchanged in soybean
methionine-derived SAM, caused similar increases in methionine content (2−6-fold) compared to the overexpression of native AtCGS (4−6-fold).24,25 However, D-AtCGS potato plants had abnormal leaf architecture and a 40−60% reduction in tuber yield.24 The D-AtCGS transformation resulted in the D
DOI: 10.1021/acs.jafc.7b00272 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry and decreasing it in azuki bean.38 In an independent study, DAtCGS overexpression did increase the methionine levels of soybean seeds, but with altered lipid levels.39 In contrast, overexpression of a native form of AtCGS increased the methionine levels in potato and Arabidopsis without reported abnormalities.12,13,25 Thus, we selected AtCGS rather than DAtCGS or StCGS for the current study. In addition to CGS, MGL, which catalyzes methionine degradation, was another target enzyme.40 Silencing MGL expression increases methionine levels in potato by decreasing methionine catabolism.31 Whereas previous studies targeted a single gene to increase tuber methionine levels, we engineered two genes to achieve a synergistic effect on methionine accumulation. The simultaneous increase in methionine biosynthesis and decrease in catabolism increased methionine levels to a significant extent. However, it appears that methionine levels are being tightly regulated, because the fold change in the AtCGS transcript level was not reflected in a similar fold change in tuber methionine content. As feedback inhibition of AtCGS transcription appears to be absent in potato,17 this suggests the presence of additional, as yet unknown, mechanisms that regulate free methionine levels in potato tubers. Methionine is an important amino acid involved in multiple cellular processes. Thus, the cytosolic methionine level is tightly regulated.5 It is noticeable that a very high level of methionine is associated with growth and developmental abnormalities at the expense of lowered yield and/or titer of other amino acids. Six-fold or greater increases in methionine abundance,23,24 plasmid vector-specific effects, or the location of gene overexpression in the host could cause the observed effects on plant growth and tuber yield. These negative phenotypic effects are one of the main reasons for failure in the commercialization of crop plants having increased levels of methionine. We did not notice any plant or tuber abnormalities in our assays, which showed up to 3-fold increases in free methionine abundance relative to wild type controls. A 3-fold increase in free methionine content roughly doubles the total methionine content in potato tubers. Relative to human dietary guidelines suggested by the World Health Organization,1 methionine is about half as abundant as other essential amino acids found in potato tubers. Thus, a doubling of total tuber methionine content can have a significant effect on making the potato tuber amino acid content more balanced. Further increases in tuber free methionine content are nevertheless desirable for promoting the aroma of baked and fried potatoes.2 In addition to CGS and MGL, threonine synthase expression was previously targeted to increase the flow of carbon toward methionine (Figure 1). Antisense silencing of potato threonine synthase resulted in a >200-fold increase in methionine levels, a 45% of reduction in threonine abundance, and negative effects on plant growth.23 Perhaps more subtle knockdown of threonine synthase expression, or regulation in a more tuberspecific manner, would increase tuber methionine content without negative growth and yield effects. Such constructs could be combined with MGL knockout mutations to enable further methionine increases. Rather than RNA interference, the technology that was available when the current experiments were conducted, gene knockout or expression modification using CRISPER/Cas9 would be better for such potato metabolic engineering, due to the likely more general
acceptance of crop plants with point mutations induced by CRISPER/Cas9 compared to those containing transgenes.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00272. Figure S1: Cloning of AtCGS. Table S1: Primer pairs used for cloning and transcript analysis. Table S2: Amino acid levels in tubers of AtCGS overexpressing potato lines, with and without AtCGS overexpression. Table S3: Amino acids levels in tubers of MGL-silenced potato lines, with and without AtCGS overexpression (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(G.J.) E-mail:
[email protected]. Phone: (607) 254-1365. Fax: (607) 254-1242. ORCID
Pavan Kumar: 0000-0003-4718-3601 Georg Jander: 0000-0002-9675-934X Funding
This research was funded by the U.S. Department of Agriculture − National Institute of Food and Agriculture Award 2014-67013-21659 to G.J. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Joyce Van Eck, Weihua Wang, Kaitlin Pidgeon, and Michelle Tjahjadi for assistance with plant transformation. REFERENCES
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DOI: 10.1021/acs.jafc.7b00272 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
