Bean Phenylpropanoid Pathway Genomics
Increasingly, North American consumers are choosing foods for the healthful qualities they have, in addition to their nutritive value. The Pulse Canada Nutrition and Health fact sheets emphasize the importance of beans in a healthy diet. Antioxidant phytochemicals, particularly polyphenols, in beans are considered one of the major contributors to these health benefits (Sato et al. 2005 J Nutr Biochem 16: 547). This includes evidence that beans and bean extracts can reduce cancer incidence. For example, cooked beans and bean extract attenuate colon carcinogenesis in azoxymethane-Induced Ob/Ob mice (Bobe et al.2008 Nutr Cancer 60: 373) and a dry bean- containing diet exerts an inhibitory effect on mammary carcinogenesis in a well-characterized rodent model for breast cancer (Thompson et al., 2008 J Nutrition 138:2091). Epidemiological and lab studies show that increased bean consumption is associated with reduced cancer rates. A 20 of year study detected a significant inverse relationship between the frequency of legume intake and colon cancer incidence (Singh & Fraser 1998 Am J Epidemiology 148:761; Fraser 1999 American J Clin Nutrition 70:532S). Other research (Kolonel et al. 2000 Cancer Epidemiol Biomark & Prevention 9:795) found an inverse relationship between non-soy legume consumption and prostate cancer. Data from 41 countries revealed that countries with the greatest consumption of beans had the lowest death rates due to breast, prostate, and colon cancer (Correa 1981 Cancer Res 41:3685). Rat studies found that feeding pinto beans reduced the number of rats with colon cancer by 50% compared to casein-fed rats (Hughes et al. 1997 J Nutrition 127:2328) and feeding either black beans or navy beans reduced the number of animals that had colon cancer by over 50% (Hangen & Bennik 2003 Nutrition Cancer 44:60). In addition, because dry beans are low in fat and a source of low glycemic index carbohydrates there is the potential to develop a consumer understanding of beans as a preferred source of these compounds in their diet. To make this case more compelling efforts need to be made to increase the levels of these components in the bean through conventional or molecular-assisted selection.
Selection efforts are hampered by the lack of molecular information about the genes that code for the enzymes and regulatory proteins in the phenylpropanoid pathway. The phenylpropanoid pathway in plants is complex and leads to the synthesis of hundreds of different compounds that play roles in many plant functions including: defense, signalling to symbiotic organisms,and environmental stress responses (Dixon et al. 1995 Plant Cell 7:1085; Soleka 2007 Acta Physiol Plantarum 19:257). The major classes of compounds have been identified and include lignins and lignans, anthocyanins and condensed tannins and isoflavones and pterocarpans. In addition, many of the enzymes that catalyse the reactions in the phenylpropanoid pathway have been identified and genes that encode them have been isolated in a number of plant species. However, the information about the genes for this important pathway in Phaseolus is fragmentary. To allow assessments of the expression of genes in the phenylpropanoid pathway we have cloned and sequenced gene tags for 35 separate steps in various branches of the pathway (#CV670734-CV670751,#CV670752-CV670763, CW652094-CW652090,CW652103-CW652105 ). Preliminary results from microarray expression studies of these genes in various bean varieties indicate that there are positive and negative correlations between gene expression levels and the levels of phenylpropanoid compounds in seed tissue (Reinprecht et al. 2004 5th Canadian Pulse Research Workshop, London, p8; Reinprecht and Pauls 2009 Food Res International, submitted). However, interpretation of the results is limited by the fact that many of the genes exist as multiple copies in many plants, including bean (Clough et al. 2004 Genome 47: 819; Tsai et al. 2006 New Phytol 172: 47; Velasco et al. 2007 PLoS ONE 2:e1326). The limited information that is available for Phaseolus and our data (Reinprecht & Pauls 2009 Food Res International, submitted) indicates that this is true for Phaseolus as well.
Dry edible or common beans (Phaseolus vulgaris L. Fabaceae) are divided into market classes according to their seed size, shape, colour and patterning (Singh et al. 2001 Econ Bot 45:379). Seed coat colour and patterning in common bean is determined by 15 genes (McClean et al. 2002 J Heredity 93:148). Bean seed coat pigments are flavonoids synthesized by the phenypropanoid pathway (Beninger et al. 1999 J Amer Soc Hort Sci 124:514). Our preliminary work has shown some linkage associations between the classical seed coat colour genes and genes coding for enzymes in the phenylpropanoid pathway (Zeinab & Pauls 2008 Can Society Plant Physiol Ann Meeting, Ottawa). Age-darkened cranberry and pinto beans are discounted in the market because consumers associate darkening with increased cooking time and reduced palatability.”Export markets demand a bright clean bean seed coat colour for packaging” (Steve Scholze, 2006 http://www.parheim.mb.ca/navybeansept2006comments. htm). Although the biochemical mechanism behind the post-darkening phenomena is not well understood, Beninger et al. (2005 J Ag Food Chem 53:7777) correlated decreases in proanthocyanidin and flavonoid levels with the slow-darkening in pinto bean 1533-15, relative to conventional darkening pinto germplasm. From a screen of 700 cranberry-type lines from various sources we identified a very pale cranberry-like bean [Wit-rood boontje from the Netherlands (PI439540)] that darkened significantly less than conventional cranberry beans. Significantly lower levels of proanthocyanidins were measured in Wit-rood and nondarkening F3 seed obtained from Etna x Wit-rood or Hooter x Wit-rood crosses than in Etna, Hooter or darkening F3 seeds. In addition, the activities of genes leading to the synthesis and accumulation of proanthocyanidins were lower in the nondarkening seeds compared to those that darkened. The results suggest that the nondarkening trait in Wit-rood and its nondarkening progeny is related to reduced proanthocyanidin synthesis in cranberry beans (Pauls & Wright 2008 Can Society Plant Physiol Ann Meeting, Ottawa). Beninger et al. (2004 J Ag Food Chem 52:4026) found that black beans had anthocyanin levels equivalent to grapes, apples and cranberries and beans with darker colours appeared to have higher levels of these antioxidant compounds. However, no systematic study of the phytochemical compositions (Lin et al., 2008 Chemistry 107:399) and bioactivities of extracts from different market classes of beans grown in Ontario have been carried out. In addition, the effect of including the nondarkening trait on the healthfulness of beans has not been determined.
The proposed work will provide important information for future bean breeding efforts aimed at increasing their healthfulness and their visual appeal. It will give information on the identity and bioactivity of polyphenols in beans that can be used to promote their consumption and it will provide tools and information about the genes that control their synthesis that can be used to accelerate breeding efforts towards these objectives. We will expand the initial cloning and sequencing work to develop a comprehensive catalogue of the sequence variability among phenylpropanoid pathway enzymes and regulatory genes in P. vulgaris. Information about the genes that control their synthesis may ultimately contribute to the selection of lines with enhanced levels of these compounds in beans and a long term deliverable might include new bean varieties with enhanced levels of these nutraceutical and protective compounds.
The objectives of the proposed work in the phenylpropanoid project area are to:
- To identify SNPs in the phenylpropanoid genes of a variety of bean accessions and parents of mapping populations
- Identifying correlations between phenylpropanoid gene expression and antioxidant levels and activities, and
- Determine the relationship between seed coat colour and antioxidant levels in a mapping population segregating for nondarkening seed coat colour trait
The outputs of this project will include information about the genes that code for enzymes and regulatory proteins of the phenylpropanoid pathway, which is responsible for the synthesis of phytochemicals that have important effects on human health as well as plant disease resistance, seed coat colour and nodulation.
Phenylpropanoid pathway gene SNP discovery
In order to utilize genomic information for plant breeding it is necessary to define the range of variation in the phenylpropanoid genes in germplasm that is useful for plant improvement and in the materials currently in use for genomics studies in Phaseolus. Therefore, a selection of P. vulgaris accessions, varieties and parents of mapping populations will be used for SNP discovery in the phenylpropanoid genes. The approach will be to deep sequence selected regions of genomic DNA containing the phenylpropanoid genes after direct selection of genomic DNA from the selected lines by hybridization to a high density phenypropanoid gene microarray (Albert et al. 2007 Nature Methods 4:903).
DNA from varieties and inbred lines [OAC Rex (Michaels et al. 2005 Can J Plant Sci 86:733) , OAC Seaforth, Lightning (Smith et al. 2009 Can J Plant Sci 89;303), OAC Redstar (Smith et al. 2009 Can J Plant Sci 89:309), Wit-rood], the parents of the core mapping population (Bat , Jalo) and one of our mapping populations (Tar’an et al. 2001 Genome 44:1046; Tar’an et al. 2002 Crop Sci 42:544; Tar’an et al. 2003 Euphytica; Beattie et al. 2003 Genome 46:259) will be subjected to direct genomic selection using custom high-density oligonucleotide microarrays (NimbleGen) to capture genome segments, encompassing the phenylpropanoid gene sequences as described by Albert et al. (Nucleic Acids Res 31:e35 2003). In short, DNA will be size-fractionated and PCR primer sequences will be ligated to the ends of the 500-base fragments. After PCR amplification the fragments will be hybridized to the capture arrays. The non-hybridized fragments materials will be removed by washing. The retained single-stranded fragments will be eluted, amplified by PCR and shotgun sequenced as described above to identifying SNPs. Given an average size of 3 Kbp per gene covered in steps of 500 bp oligonucleotides, and allowing for a 100bp overlap between genes, the custom array would be designed to contain ~1,800 different oligo capture sequences. In total we expect to generate 50 Mbp of genomic sequence centred on phenylpropanoid genes. Alignment algorithms, maximized for SNP detection and high base calling fidelity, will be used to scrutinize sequence trace data to identify genuine SNPs (Neff et al. 1998 Plant J 14: 387)
Identifying correlations between phenylpropanoid gene expression and antioxidant levels and activities.
The identities and bioactivities of the phenylpropanoid compounds in the bean samples from various varieties and genotypes, before and after processing, will be characterized through a combination of chemical, biochemical and biological assays. They will include:
- high performance liquid chromatography coupled to diode array detection and mass spectrometry (HPLC-DAD-MS) for the analyses of extracts (Espinosa-Alonso et al. 2006 J Agric Food Chem 54:4436; Xu & Chang 2008 J Agric Food Chem 56:8365)
- ferric reducing antioxidant power (FRAP; Tsao et al. 2005 J Agric Food Chem 2005:4989) and oxygen radical absorption capacity (ORAC; Prior et al. 2003 J Agr Food Chem 51:3273 assays to determine antioxidant levels (Thaipong et al. 2006 J Food Composition Analysis 19:669; Caldero et al. 2006 Plant Foods Human Nutri 61: 161)
- in vitro screening of bean extracts (pre and post-processing) to characterize the antioxidant activity and bioavailability of the phenylpropanoid-derived compounds and their effects on biomarkers of chronic disease (i.e. inflammation, oxidative stress, and cancer growth) (Liu et al. 2004 J Agric Food Chem 52:4330; Paur et al. 2008 Food Chem Toxicol 46:1288; Power and Thompson 2003 Mol Nutr Food Res 51:845; Wolfe & Liu 2007 J Agric Food Chem 55:8896).
The cloned gene fragments will be used to construct bean phenylpropanoid pathway-specific array (Reinprecht & Pauls 2009 Food Res Internat, submitted). The usefulness of array as a screening tool for the activities of the phenylpropanoid pathway genes in different bean market classes will be tested by comparing the hybridization patterns of cDNA from developing bean seeds from a range of varieties in different market classes [pinto, cranberry, black, dark red kidney (DRK) and light red kidney (LRK)] to the array. Differences in the hybridization intensities will be correlated with antioxidant levels and activities using Pearson correlation and cluster analyses.
Examination of the relationship between seed coat colour and antioxidant levels in a mapping population segregating for nondarkening seed coat colour trait.
A segregating population will be developed from a cross between a non-darkening cranberry line and a regular darkening canberry bean (Hooter or Etna). The progeny of this cross is expected to segregate for a range of color and for the presence and absence of darkening trait. The segregating population will be phenotyped for color using a colorimeter, for presence and absence of darkening traits after exposure to UV light, and for the level of polyphenols and the anti-oxidant activity. The progeny will also be genotyped for the genes from phenylpropanoid pathway (Zeinab & Pauls, 2009, Annual Meeting CSPP, Vancouver). Genotypic and phenotypic data will be used to study the association of seed coat color and individual color compounds and genes with the level of phenolic compounds, antioxidant activity and tendency to darken after harvest. Our goal will be to develop a commercial nondarkening cranberry bean variety and to gain an understanding of the roles of phenypropanoid genes in determining the trait.
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