Different combinations of three rate-limiting enzymes in phytosterol biosynthesis, the Arabidopsis thaliana hydroxyl methylglutaryl CoA1 (HMGR1) catalytic subunit linked to either constitutive or seed-specific β-conglycinin promoter, and the Glycine max sterol methyltransferase1 (SMT1) and sterol methyltransferase2-2 (SMT2-2) genes, under the control of seed-specific Glycinin-1 and Beta-phaseolin promoters, respectively, were engineered in soybean plants. Mature seeds of transgenic plants displayed modest increases in total sterol content, which points towards a tight control of phytosterol biosynthesis. However, in contrast to wild-type seeds that accumulated about 35% of the total sterol in the form of intermediates, in the engineered seeds driven by a seed-specific promoter, metabolic flux was directed to Δ5-24-alkyl sterol formation (99% of total sterol). The engineered effect of end-product sterol (sitosterol, campesterol, and stigmasterol) over-production in soybean seeds resulted in an approximately 30% increase in overall sitosterol synthesis, a desirable trait for oilseeds and human health. In contradistinction, increased accumulation of cycloartenol and 24(28)-methylencylartanol (55% of the total sterol) was detected in plants harbouring the constitutive t-HMGR1 gene, consistent with the previous studies. Our results support the possibility that metabolic flux of the phytosterol family pathway is differentially regulated in leaves and seeds.
Phytosterols, or C-24 alkyl sterols, are a family of natural products that occur in higher plants at all stages of development. These compounds, notably sitosterol and its Δ22 analogue stigmasterol, have gained special attention over the past few decades owing to their recently discovered ‘protective’ properties that can reduce cholesterol levels thereby benefiting human health (Normen et al., 2000). Consequently, there is much interest in increasing the natural phytosterol content in seed oils by genetic engineering the phytosterol pathway. It is worth noting that the medical literature continues to use the name β-sitosterol for 24α-ethyl cholesterol. In this case, the Greek letter was not meant to imply configuration, but to distinguish it from α- and γ- sitosterol. Because the latter two compounds were shown to be mixtures, the modern convention is to drop the β (Nes, 2000).
Sitosterol and other phytosterols are lipids derived from the mevalonate pathway of isoprenoid biosynthesis, which occurs in the cytoplasm/ER (Hartmann and Benveniste, 1987), and their formation is controlled at the level of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (HMGR) activity. HMGR (EC 220.127.116.11) and its corresponding gene(s) have been studied extensively, presumably providing a ‘coarse’ control on the production of cytosolic isoprenoids, and more specifically, on phytosterol levels. Initial clues for the regulatory role of this enzyme in plants came from the identification of a tobacco mutant (LAB14) that over-produces sterols because of increased HMGR activity (Maillot-Vernier et al., 1989; Gondet et al., 1994). Subsequent analysis of the Arabidopsis hmgr1 mutant, which exhibits early senescence, male sterility, and reduced sterol levels, confirmed the pivotal role of this enzyme in affecting phytosterol biosynthesis (Suzuki et al., 2004).
The transgenic over-expression of the catalytic domain of the HMGR1 gene was found to be more efficient in phytosterol accumulation, suggesting a possible role of the N-terminal membrane-spanning domain in sterol sensing and regulation by feedback inhibition, analogous to animal systems (Chappell et al., 1995; Harker et al., 2003a; Muñoz-Bertomeu et al., 2007). Recent studies of transgenic plants engineered with foreign HMGR genes showed that phytosterol levels can be increased up to twofold (Hey et al., 2006) and even greater increases can be generated (up to 10-fold changes in total sterols), depending on the plant species tested. Compared with wild-type plants, these engineering studies reveal a tight regulation of phytosterol biosynthesis in plant leaves and seeds (Table 1 and Table S2). Consequently, potential bottlenecks in metabolite flux may exist complicating efforts to engineer plants with high levels of sitosterol in seed oils.
Table 1. Bioengineering efforts to improve seed sterol content and composition
In plants, phytosterols are present in amounts varying from approximately 0.01%–0.1% wet weight, which translates to 100–1000 mg of total sterol in a kilogram of fresh weight material (Nes, 1977). Soybean leaves synthesize sterols, mostly Δ5-phytosterols (Grunwald, 1981). Yet, in mature soybean seeds, approximately 35% of the total sterols are present as intermediates (Nes, 1990; Marshall et al., 2001). Soybean plants have recently been reported to synthesize two isoforms of SMT, SMT1 and SMT2, with distinct substrate specificities and product outcomes (Nes et al., 2003; Neelakandan et al., 2009; Shi et al., 1996). The reaction catalysed by C-24 sterol methyl transferase (SMT) 1 is considered the first committed step in phytosterol biosynthesis (Nes et al., 1991). It has been suggested to be ‘slow’ in terms of the catalytic turnover and therefore rate limiting in phytosterol biosynthesis (Nes and Venkatramesh, 1999), thus offering avenues for ‘fine’ control of sterol composition (Schaeffer et al., 2000; Sitbon and Jonsson, 2001; Holmberg et al., 2002). Engineered modifications in cholesterol and sitosterol metabolite levels can lead to reduced toxic glycoalkaloids in potato (Arnqvist et al., 2003). The SMT2 gene product catalyses a second methyl transfer to 24(28)-methylene lophenol substrate and it acts at the branch point directing carbon flux towards C-24 ethyl sterol (sitosterol and stigmasterol) biosynthesis and away from brassinosteroid biosynthesis. Previous studies have clearly elucidated the function of SMT2 genes in balancing the C-24 methyl sterol (campesterol)/ethyl (sitosterol) ratio, which in turn affects plant growth and development (Schaller et al., 1998; Schaeffer et al., 2001; Carland et al., 2002; Hase et al., 2005). In view of these coarse and fine control points in phytosterol biosynthesis, we considered the HMGR and SMT biosynthesis steps as two crucial bottlenecks that might impact our efforts to generate high levels of sitosterol in soybean seeds. To this end, we successfully over-expressed the catalytic subunit of HMGR1 alone and in combination with SMT1 in a seed-specific manner. Furthermore, to explore the possible rate-limiting role of the SMT2 gene in controlling sterol accumulation and composition in soybean seed tissues, we over-expressed the soybean SMT2-2 gene (Neelakandan et al., 2009, 2010), in a seed-specific manner.
Analysis of transgenic plants
As part of this study, several transgenic soybean plants were developed that over-expressed the candidate genes believed to be rate-limiting in soybean phytosterol synthesis (Figure S3). The overview of transgenic plant development and screening has been outlined in the Table 2. Putative transgenic plants were first screened by leaf painting assays (Zeng et al., 2004), analysed by PCR for the presence of the transgene (Figure S4), and finally followed by Southern hybridization for final confirmation and analysis of copy number (Figure 1). Table 2 details the number of independent events generated and analysed at the molecular and biochemical level, for each transgenic construct. At least two independent events with 1–3 transgene insertions, showing promising expression levels, were selected for each gene construct (Figure 2) and checked for transgene inheritance and segregation in the subsequent generations.
Table 2. Details of transgenic soybean development for modified sterol biosynthesis
The seed storage gene promoter activity is expected to peak at the mid- to late stages of seed maturation (Naito et al., 1988). Therefore, the expression of the engineered foreign gene was assayed by RT-PCR analysis in mid-mature cotyledons, and also in different vegetative tissues to confirm the tissue specificity of the seed-specific promoters and to compare the efficiency of these tissue-specific promoters to that of the CaMV 35S promoter in seeds. Glycinin-1 (Iida et al., 1995), the α′ subunit of β-conglycinin (Lessard et al., 1991) gene promoters from soybean, and the β-phaseolin promoter (phas) from Phaseolus vulgaris (Chandrasekharan et al., 2003) were used in this study. The seed-specific promoters were found to direct a low basal level of transgene expression in leaf tissues as compared with the wild type, but expression was induced in a seed-specific manner in the different constructs and events tested (Figure 2). In general, transgenic plants with a single-copy gene (especially B events of SH plants) showed better expression as compared with those with multiple copies (C event in Figures 1 and 2). The seed-specific promoters, both native and heterologous, were found to drive a high level of transgene expression in soybean seeds. Transgene expression was checked in the T2 and T3 generation seeds by RT-PCR. The CaMV35S promoter driving t-HMGR1 gene expression was also found to be active in seeds, although to a significantly lesser extent than that of the seed-specific β-conglycinin promoter (Figure 2), at the specific developmental stage sampled.
Sterol content and composition in the engineered seeds
The biochemical phenotypes of transformed plant tissues, along with wild-type plants, were evaluated by analysis of phytosterol content and composition of T2 and homozygous T3 seeds. Sterol compositions in leaf and seed tissues of wild-type soybeans were strikingly different as evident from the analysis of sterol profiles by GC-MS (see Figure S5). Leaf tissues were enriched in Δ5-sterols, occupying around 80% of the total sterol pool (data not shown), whereas the proportion of Δ5-sterols in seed tissue is only around 65% (Figure 3). Among the sterol end products, the proportion of campesterol was found to be higher than stigmasterol in seeds. The main intermediate sterols, cycloartenol and C24-methylene cycloartenol, were detected in nearly equal proportions in the seeds of untransformed plants (Figures 3 and 4).
Both leaf and seed sterol profiles were examined in transgenic plants obtained by constitutive over-expression of the HMGR1 catalytic subunit in a constitutive manner (HC). We found that the sterol composition, especially the proportion of sitosterol, in leaf tissues of transgenic plants remained more or less the same as compared with the wild-type leaf (data not shown). However, HC seeds exhibited dramatically elevated proportions of C24-methylene cycloartenol and lowered amounts of stigmasterol and sitosterol, whereas campesterol levels remained unaffected (Figures 3 and 4). Overall, the proportion of end-product sterols was reduced to 40% as compared with wild-type seeds with 65%, and the percentage of intermediates rose to 55% as compared with 35% in wild type (Figure 3). Furthermore, we found up to a 42% increase in total sterols per gram of leaf tissue [a fold change (transgenic/wild-type ratio) of 1.4], in agreement with previous studies (data not shown), but a similar increase was not observed in the seeds of soybean.
We observed a correlation between transgene expression at the transcriptional level and the level of sterol alteration in seeds. In the engineered seeds, developed with transgene expression driven by seed-specific promoters, sterol composition was found to be profoundly modified with elevated amounts of sterol end products, mainly sitosterol, observed as high as 90%–95% of the total sterol pool (Figure 3). Moreover, intermediates like cycloartenol and C-24 methylene cycloartenol were undetectable in HM, SH, and SM2 transgenic seeds (Figure 4). Notably, the proportions of campesterol were not affected in SM2, (also in HC and HM seeds) and in SH seeds, a negligible increase was observed. In spite of the considerable changes in seed sterol composition, the total sterol levels remained more or less unaltered, although a small increase of 6% was observed in SH construct and up to a 3% increase in HM homozygous seeds (Table 2).
Based on previous studies summarized in Table 1, and Table S2, it would appear that transcriptional regulation of HMGR and SMT genes is crucial in the regulation of the sterol biosynthesis pathway. To further exploit the potential regulatory points in phytosterol biosynthesis, we engineered the soybean SMT2-2 gene, which exhibits temporally regulated transcript abundance in soybean seed developmental stages, along with the HMGR1 and SMT1 genes, which are known to catalyse committed steps in the pathway.
Constitutive over-expression of t-HMGR1 results in high-level intermediates in seeds
Transgene over-expression under the control of constitutive promoters is the most prevalent and routine strategy to test gene function, but is not devoid of unintended pleiotropic effects (Hey et al., 2006). In our study, we used the constitutive CaMV35S promoter to drive the catalytic domain of the Arabidopsis HMGR1 (t-HMGR1) gene in soybean. The transgenic plants were morphologically indistinguishable from wild-type plants, which is in agreement with previous studies (Muñoz-Bertomeu et al., 2007; Re et al., 1995). It is interesting to note that enhanced metabolic flux, as a result of t-HMGR1 over-expression, led to an increase in the total sterol levels in transgenic leaves (data not shown) without much alteration in the composition of sterol component, whereas sterol composition was significantly affected in seeds from these same plants. These observations clearly indicate the differential nature of regulation of metabolic fluxes of the sterol biosynthesis pathway in leaf and seed tissues as previously reported (Holmberg et al., 2002). The possible translocation of sterols from source to sink tissues as previously seen in other crops (Nes et al., 1982) also cannot be ruled out. In this context, the actual proportion of seed sterols synthesized de novo versus those derived from leaf tissues in these transgenic plants needs to be investigated in soybean. The results obtained in this study further suggest that constitutive promoters might be useful for alterations of sterol levels in vegetative tissues, but might be of limited success in reproductive tissues.
Seed-specific transgene over-expression accumulates end-product sterols at the expense of intermediates in seeds
Native and heterologous seed-specific promoters were used to drive the respective candidate transgenes. Seed-specific promoters were found to drive high level of expression of the t-HMGR1 gene, as compared to the constitutive promoter in seeds of the specific developmental stage analysed in our study (Figure 2). Earlier studies have proposed a coordinated regulation of enzymes involved in sterol biosynthesis, whose activity is low in the initial stages of seed development, but tends to peak at the mid-mature stages (Harker et al., 2003b). This coincides with the pattern of expression expected of seed storage protein gene promoters. The intermediate sterols were not detected in transgenic seeds with seed-specific over-expression. This is consistent with previous studies in other crops such as tobacco (Holmberg et al., 2002, 2003), where the accumulation of end products was enhanced and intermediates were less abundant with the use of seed-specific promoters. Because intermediates, like cycloartenol, were well below detectable limits in HM seeds, it could be postulated that the increased flux generated by HMGR over-expression might have a positive stimulatory effect on downstream steps leading to sitosterol biosynthesis in seeds. The coordinated transcriptional up-regulation of the t-HMGR1 and SMT1 genes was found to be more efficient in terms of overall increases in sterol levels, which is consistent with previous studies (Holmberg et al., 2003). The fact that no intermediates could be detected in seeds further suggests that there is an efficient conversion of intermediates to end products, leading to a beneficial accumulation of end-product sterols to approximately 90%–95% of the total sterol pool.
The present study demonstrates that the regulation of 4-desmethyl sterol synthesis in soybean seeds can be successfully modified by targeted engineering of committed steps in the isoprenoid and sterol biosynthesis pathways, without affecting germination and viability. Based on our findings, the total sterol content seems to be tightly regulated in soybean seeds with only modest increases in total sterols under all conditions examined in this study. Attempts to increase seed sterol content have met with only limited success in the past (see Table 1 for details). This warrants a thorough study of the true control points in seed sterol synthesis and regulation, the extent of endogenous flux in seeds in terms of substrate availability, and the nature and proportion of metabolite translocation from the leaves.
Earlier studies on transcriptional regulation of sterol methyltransferase genes in soybean seeds (Neelakandan et al., 2009) have indicated that the mRNA expression peaks at early developmental stages. Based on the information available on the Arabidopsis microarray database, the endogenous expression profile of the genes, HMGR1, SMT1, and SMT2-1, also reveals a peak expression at the early seed developmental stages with a progressive decline as the seed matures, except for the HMGR1 gene that shows more or less static expression in all stages (Figure S6). Taken together, it is tempting to speculate that the SMT1 and SMT2 genes (of the post-cycloartenol pathway) show coordinate transcriptional regulation, whereas HMGR1 expression appears to be unrelated with respect to seed development. It would be worthwhile to test the effect of transgene expression under the control of seed-specific promoters with altered temporal regulation, coinciding with early stages of seed development.
We hypothesized that by molecular engineering of genes coding for rate-limiting enzymes in the isoprenoid/sterol pathway, we could increase the total sterols in soybean seeds. Contrary to our expectations, we observed only limited variation in sterol content. The existing sterol flux within the developing seeds was mobilized towards the synthesis of sterol end products, which are beneficial for human health. The potential bottlenecks in seed sterol synthesis, availability of the precursor molecule, cytosolic acetyl CoA, and the possibility of additional rate-limiting enzymes between HMGR and sitosterol synthesis cannot be ruled out. Alternatively, the levels of free sitosterol that is generated for esterification in seeds might possibly be under strict developmental control.
The present study was designed to generate nutritionally enhanced soybean oils with increased phytosterol levels. Notably, the resultant engineered seeds of altered sterol composition afford plasticity in the intermediate to end-product ratios. Equally important, this study shows for the first time that engineered modifications in seed phytosterol homeostasis can produce enriched levels of beneficial and desirable sterols for human consumption. This is highly significant given the low natural genetic variability in seed sterol levels especially among soybean genotypes and crops in general, thus imposing a serious limitation over conventional and molecular plant breeding approaches.
The seeds of soybean cv Jack and the transgenic plants were sown in three gallon pots containing promix in the Sears greenhouse facility, University of Missouri (MU), and grown under controlled conditions (27/18 °C day/night temperature and a photoperiod of 14/10) until maturity. Tissues were collected at vegetative and reproductive stages (Fehr and Caviness, 1977), frozen immediately, and stored at −80 °C for molecular and biochemical studies.
Development of recombinant gene constructs
Four different recombinant constructs were generated to over-express the catalytic domain of AtHMGR1 in a constitutive (HC) and seed-specific (HM) manner, in combination with seed-specific up-regulation of GmSMT1 (SH), and another, wherein the GmSMT2-2 gene was over-expressed in a seed-specific manner (SM2). Figure S3 illustrates the cloning strategy and details of the organization of gene cassettes generated in the binary vector pZY101Asc for soybean transformation. The primer sequences used for amplification and cloning are detailed in Table S3. The plant selection was conferred by the bar gene driven by a duplicated 35S promoter sequence and NOS polyadenylation signal. For generating the β-conglycinin: At t-HMGR1 vector, the C-terminal catalytic domain along with a part of the linker region (amino acids 164–592) of Arabidopsis HMGR-1 gene (GenBank accession number P14891), denoted as t-HMGR1, was amplified from leaf cDNA and subcloned into pBetaConSoyHyg, which harbours the seed-specific β-conglycinin promoter and phaseolin terminator (abbreviated as HM) and then checked for correct orientation. The whole-gene cassette was subsequently cloned into the binary vector at the Hind III site. The Glycinin:GmSMT1 construct was developed by amplification of the soybean SMT1 (GenBank accession number U43683) gene from the subcloning vector (GmSMTpET23a), cloned into the intermediate vector pKMS3, downstream to the seed-specific glycinin-1 promoter, and subsequent insertion of the whole-gene expression cassette (Glycinin-1 promoter-SMT1gene-Glycinin-1 terminator) into the binary vector, pZY101-Asc (SM1) at the Asc I independently, and along with the t-HMGR-1 gene expression cassette (SH). Another vector CaMV35S:At t-HMGR1 was generated by cloning the Arabidopsis HMGR1 cDNA C-terminal into the pART7Asc vector (Gleave, 1992; modified vector from Dr. Ed Cahoon, University of Nebraska) containing the CaMV35S promoter and ocs terminator, followed by insertion of the expression cassette into the soybean transformation vector at the Asc I site (abbreviated as HC). The recombinant vector β-phaseolin: GmSMT2-2 was developed by amplifying the phaseolin promoter from the pPhas vector, sub-cloning it into the pKMS3 vector replacing the glycinin-1 promoter followed by cloning of the GmSMT2-2 cDNA sequences downstream of the phaseolin promoter. The whole-gene cassette, together with the phaseolin terminator was then introduced into the destination vector at the Sma I site.
All of the plant gene expression vectors were used to transform the soybean cultivar Jack at the MU Plant Transformation Core Facility, by using Agrobacterium-based transformation of cotyledonary explants as described previously (Zhang et al., 1999). Primary transformants were selected on plates by the herbicide (basta) selection (Zeng et al., 2004) and screened in green house conditions by leaf painting assays.
Molecular characterization of transgenic plants
The putative transgenic plants were screened by PCR for the presence of the transgene. Genomic DNA was isolated from young leaf tissue by the CTAB method (Saghai-Maroof et al., 1984), checked for integrity by electrophoresis on 0.8% agarose gels, and quantified using nanodrop model ND-1000 UV-Vis spectrophotometer (NanoDropTechnologies, Wilmington, DE). PCR primers designed for the transgene coding sequences are listed in the Table S3. Amplifications were performed with 50 ng genomic DNA using Taq 2X mastermix (New England Biolabs, Ipswich, MA), using the respective primers at the corresponding annealing temperatures.
Southern blot analysis
Southern analysis was performed on the transgenic plants at the T0 and T1 generation for confirming the presence and inheritance of the transgene and ascertaining the copy number. Approximately 20 μg of genomic DNA was restricted with Hind III or Xba I enzymes, respectively, electrophoresed on 0.9% agarose gels, and blotted on to Hybond-N+ nylon transfer membranes (Amersham Pharmacia Biotech Inc, NJ). The entire coding sequence of the bar gene 552-bp fragment was amplified using the forward primer, BarF: 5′-ATGAGCCCAGAACGACGCCC-3′; reverse primer, BarR: 5′-TCAGATCTCGGT GACGGGCA-3′, and gels were purified using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), labelled with α32P dCTP using Prime-It II Random Primer Labeling Kit (Stratagene, Santa Clara, CA, USA) and used as a probe in Southern hybridization. The membranes were incubated in hybridization buffer consisting of 0.5 m sodium phosphate pH 7.2, 1 mm EDTA pH 8.0, 7% SDS, and 1% BSA, overnight at 65 °C after the addition of freshly denatured radio-labelled probe (Sambrook and Russell, 2001). Subsequent incubations in wash buffers containing 40 mm sodium phosphate pH 7.2, 1 mm EDTA pH 8.0, 5% SDS, and 0.5% BSA (wash buffer I) and 40 mm sodium phosphate pH 7.2, 1 mm EDTA pH 8.0, 1% SDS (wash buffer II) at 65 °C for 15 min each were performed and the membranes were subjected to autoradiography. The blots were later stripped and re-probed with the transgene specific probe for confirmation of transgene integration.
Transgene expression by RT-PCR
Total RNA was isolated from leaf, flower, and seed tissues with RNEasy Plant Mini Kit (Qiagen) and cDNA synthesis was performed as per Neelakandan et al. (2009). The transgene expression was verified initially by semi-quantitative RT-PCR using transgene-specific primer sequences. The reaction conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 26–28 cycles of 94 °C for 15 s, 55–60 °C for 30 s, and 72 °C for 1–2 min, followed by a final annealing at 72 °C for 5 min. The amplified products were run on 1% agarose gels and visualized by ethidium bromide staining and photographed. The relative transcript abundance was also estimated by quantitative real-time polymerase chain reaction (RT-PCR) performed on an ABI PRISM 7000 SDS instrument (ABI, Applied Biosystems, Carlsbad, CA) using SYBR green fluorescent dye as described in Neelakandan et al. (2009). Gene-specific primers were designed using Primer Express 2.0 software (ABI); their sequences and the respective amplicon sizes are reported in the Table S4.
The sterol analyses of soybean samples were conducted in triplicate as described in Neelakandan et al. (2009), from three independent plants of each event. The analytes were identified by comparison with retention times and mass spectra to those of authentic standards.
The support of the Missouri Soybean Merchandising Council (Project #04-259) to H.T.N. and National Science Foundation (MCB-0920212) to W.D.N. is gratefully acknowledged. We are greatly indebted to Jenny Su, Liwen Zhou, and Dr. Zhanyuan Zhang at the MU Plant Transformation Core Facility, for developing the transgenic soybean plants used in this study. We thank Dr. Edgar Cahoon (University of Nebraska, Lincoln) for kindly providing the sub-cloning vectors, BetaConsoyHyg, pKMS3, and the binary vector, pZY101Asc, and Dr. Gary Stacey (University of Missouri, Columbia) for the pPhas-Gly vector. Thanks are also due to Dr. Satish Guttikonda and Dr. Rajesh Kumar (University of Missouri, Columbia) for helpful discussions and support. Special thanks to Ms. Theresa Musket (University of Missouri, Columbia) for careful editing of the manuscript. We also thank the anonymous reviewers for the comments and suggestions which helped to improve the quality of the manuscript.