Identification of the Genes of the Universal Polyphenol Biosynthetic (UPB) Pathway in Plants Producing Resveratrol




Larry M. Wells

Introduction

Polyphenolic antioxidants are currently being investigated for possible health-enhancing qualities. The Universal Polyphenol Biosynthetic (UPB) pathway is important to these investigations because it is a pathway common to all plants, and provides the biosynthetic basis from which the Flavonoid Biosynthetic Pathway (FBP) stems. Thus, the UPB is at the root of the biosynthetic pathways producing anthocyanins, catechins, flavanones, flavones, flavonols and isoflavones, as well as stilbenes such as resveratrol. Some studies suggest various polyphenols have anti-inflammatory, anti-diabetic, anti-cancer, as well as anti-aging effects that may provide protection from diseases such as macular degeneration and age-related blindness (Fursova et al., 2005; Hua et al., 2011). One such anti-oxidant demonstrating these qualities, resveratrol, has been shown to slow the growth of neuroblasomas in mice (Miloso et al., 1999), and may provide protection from other types of cancers including cervical and ovarian cancer (Athar et al., 2007; Zoberi et al., 2004; Opipari et al., 2004).

The biosynthesis of polyphenols such as resveratrol begins with a 3 step reaction that produces 4-Coumaryl-CoA from phenylalanine in the UPB pathway. Beyond this pathway, a fourth step employs the enzyme stilbene synthase (STS) to produce resveratrol from 4-Coumaryl-CoA and 3 Malonyl-CoA. The overall pathway proceeds as follows:

Phenylalanine --> (1) --> Cinnamic Acid --> (2) --> 4-coumaric acid --> (3) --> 4-coumaroyl CoA --> (4) --> Resveratrol

The enzymes involved in the pathway are (1) phenylalanine ammonia-lyase (PAL), (2) cinnamate 4-hydroxylase (C4H), (3) 4-coumaroyl CoA ligase (4CL), and (4) stilbene synthase (STS). Thus, non-resveratrol producing plants lack only the STS gene in order to produce resveratrol.

While all plants express the genes in the UPB pathway, limited Genebank sequence data is available for these genes for key species producing many of the 4,000 plus polyphenolic compounds generated in biosynthetic pathways further downstream. The objectives of this project are (1) to develop degenerate primers that can be used to identify resveratrol producing plants from extracted nucleic acids, (2) to develop degenerate primers that can amplify fragments of the C4H, 4CL and STS genes in multiple plant species, and (3) to acquire sequence data for these genes that may be used in further experiments.

Materials and Methods
Vitis vinifera and Polygonum cuspidatum tissue samples were obtained from southeastern Michigan, and Vaccinium myrtillus tissue samples were collected from the New York Botanical Gardens. Plant tissue DNA was extracted using PowerPlant® Pro DNA Isolation Kit from Mo Bio Laboratories, Inc. Degenerate primers were designed to amplify gene fragments for the C4H and 4CL genes of the UPB pathway, as well as for the STS gene coding for resveratrol synthase:

C4H forward primer: ggVaactggctgcaggtBgg ---- C4H reverse primer: atgttctcSacgatgtRaggac
4CL forward primer: ggVgaRatctgcatHcgMgg --- 4CL reverse primer: gtcVccKgtgtgNagcca
STS.1 forward primer: tcKaagatcacHcaYYt ------- STS.1 reverse primer: ccBggRtgWgcaatccaRaa
STS.2 forward primer: ttYtggattgcWcaYccHgg --- STS.2 reverse primer: gcRctKgacatgttWccRta

Figure 2 reflects the corresponding homologous protein sequences from which the primer sequences were designed.

Gene fragments were PCR amplified with a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.A.), and electrophoresis was run on 1% agarose gels with ethidium bromide to visualize individual DNA bands using a Biorad UV Trans-illuminator. Each PCR product producing a band characteristic of the desired target gene fragment was purified and sent out for sequencing. When multiple bands appeared for samples with unpublished sequences, the DNA was extracted from each band in the gel, purified, and sent out for sequencing. These PCR products were purified using a QIAquick® PCR Purification Kit, or a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands). The bands of interest were sequenced at GENEWIZ, Inc. (South Plainfield, NJ, U.S.A), and were analyzed using the NCBI BLAST tool against published sequences in Genebank.

Results

The DNA extracted from leaf samples of Vitis vinifera, Polygonum cuspidatum and Vaccinium myrtillus showed a 260/280 ratio of 1.43, 1.69 and 1.23 with concentrations of 134, 275 and 224 nanograms/microliter respectively (Fig. 1).

PCR reactions that were set up using degenerate primers for C4H, 4CL and STS gene fragments yielded products showed that multiple bands were generated for some samples. Figure 3 shows the primers for C4H yielded two bands each for V. vinifera and P. cuspidatum. Primers for the 4CL gene fragments yielded similar results, producing a single band of the expected size (~230 bp) for V. vinifera and V. myrtillus and three bands for P. cuspidatum (Fig. 3). The two STS primer sets each yielded PCR products for all three plant species tested. A single band of the expected size was observed for V. vinifera, but the P. cuspidatum and V. myrtillus PCR products produced multiple bands (Fig. 3).

The results of the DNA sequencing confirmed the designed degenerate primers were successful in amplifying targeted gene fragments for an 867 bp P. cuspidatum C4H gene, a 547 bp V. myrtillus 4CL gene, a 526 bp V. vinifera STS gene and a 227 bp v. vinifera STS gene. The previously unpublished sequence for a P. cuspidatum C4H gene fragment is shown in Fig. 4.

Discussion

The objective to design degenerate primers to pick up desired gene fraggments for the C4H, 4CL and STS genes met limited success because resulting PCR productes sometimes yielded multiple bands for targeted fragments. This probably means the degenerate primers were not specific enough, although some usable sequence data was gleaned for the C4H, 4CL and STS gene fragments for select plant samples.

Adjusting the annealing temperatures may help produce better PCR results, but more investigation is needed to be conclusive. It is possible the primers are picking up conserved regions in a family of related genes for which there are multiple copies of various sized fragments. Each band that returned sequencing data was analyzed using NCBI's BLAST search or sequence alignment tool as needed.

The sequence data obtained confirmed the presence of the STS gene in V. vinifera. Further sequence data was also obtained for previously unpublished fragments of the C4H gene for P. cuspidatum, demonstrating the C4H primer set may be effective under certain conditions. To better determine the efficacy of primer sets designed to pick up the C4H, 4CL and STS genes, it may be useful to increase the variety of resveratrol producing plant samples in the study.

Literature cited

Athar M, Back JH, Tang X, Kim KH, Kopelovich L, Bickers DR, et al. Resveratrol: a review of preclinical studies for human cancer prevention. Toxicol Appl Pharmacol. 2007;224:274–83.

Fursova AZh, Gesarevich OG, Gonchar AM, Trofimova NA, Kolosova NG. [Dietary supplementation with bilberry extract prevents macular degeneration and cataracts in senesce-accelerated OXYS rats]. Adv Gerontol. 2005;16:76-9. Russian. PubMed PMID: 16075680.

Hua J, Guerin KI, Chen J, Michan S, Stahl A, et al. Resveratrol inhibits pathologic retinal neovascularization in Vldlr(−/−) mice. Invest Ophthalmol Vis Sci. 2011;52:2809–2816.

Miloso, M, Bertelli, AA, Nicolini, G, and Tredici, G Resveratrol-induced activation of the mitogen-activated protein kinases, ERK1 and ERK2, in human neuroblastoma SH-SY5Y cells. Neurosci Lett, 264: 141-144, 1999.

Opipari AW, Tan L, Boitano AE, Sorenson DR, Aurora A, Liu JR. Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer Research. 2004;64(2):696–703.

Zoberi I, Bradbury CM, Curry HA, Bisht KS, Goswami PC, Roti JL, Gius D. Radiosensitizing and antiproliferative effects of resveratrol in two human cervical tumor cell lines. Cancer Lett. 2004;175:165–173.

Figures


Figure 1-Isolation of Plant DNA: V. vinifera, P. cuspidatum and V. myrtillus


Figure 2- Sequence alignments were performed using NCBI's sequence alignment tool to determine homologous regions of the C4H, 4CL and STS genes from which primers were designed for PCR.


Figure 3- PCR results using degenerate primers show bands indicating target gene fragments were amplified, (A) Cinnamate 4-hydroxylase (C4H), (B) 4-coumaroyl CoA ligase (4CL), (C) Stilbene Synthase (STS)


Figure 4-C4H Gene Fragment Sequence Analysis For Polygonum cuspidatum


Abstract
In excess of 4,000 polyphenolic compounds have been identified in nature, and there is a growing body of evidence that suggests hosts of these phytochemicals possess many beneficial qualities. Key to the production of these compounds is the Universal Polyphenol Biosynthetic (UPB) pathway which generates the precursors needed to produce the vast diversity of polyphenols being investigated in industry and medicine. In this study, degenerate primer sets were developed to amplify gene fragments involved in the UPB pathway, as well as primers that can be used to identify plants producing the polyphenol resveratrol. Gene fragments from several resveratrol-producing plants were amplified for cinnamate 4-hydroxylase (C4H), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS). These fragments were isolated, sequenced and analyzed to confirm the identity of the expected PCR products. Results yielded mitigated success, producing primers able to pick up multiple fragments of target genes for V. vinifera, P. cuspidatum and V. myrtillus. Limited gene fragment sequence data gleaned includes one previously unpublished gene fragment sequence for a P. cuspidatum C4H gene. Fragments of the STS gene were detected for P. cuspidatum and V. vinifera. Further, STS primer sets appear helpful in identifying resveratrol producers, but a greater variety of plant samples need to be tested to determine primer efficacy.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin for his instruction and help with this project. Thank you to Dr. Dubrovsky for the use of the lab equipment needed for DNA extraction. Thanks also to Bo Liu and Xie Xie for their assistance and patience when the project required hours of extra time. Especially, I would like to thank God for providing the opportunity to work in the labs at Fordham University.


This document was last modified 05/24/2012.
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