Claire Domoney

Can We Improve the Nutritional Quality of Legume Seeds

The Food and Agriculture Organization statistics for 2001 ( ) show that 274 million metric tons of grain legumes were produced across the world, of which 177 million were soybeans ( Glycine max ; one-half of which were produced in the U.S.)

Responses of the pea (Pisum sativum L.) leaf metabolome to drought stress assessed by nuclear magnetic resonance spectroscopy

While many compounds have been reported to change in laboratory based drought-stress experiments, little is known about how such compounds change, and are significant, under field conditions. The Pisum sativum L. (pea) leaf metabolome has been profiled, using 1D and 2D NMR spectroscopy, to monitor the changes induced by drought-stress, under both glasshouse and simulated field conditions. Significant changes in resonances were attributed to a range of compounds, identified as both primary and secondary metabolites, highlighting metabolic pathways that are stress-responsive. Importantly, these effects were largely consistent among different experiments with highly diverse conditions. The metabolites that were present at significantly higher concentrations in drought-stressed plants under all growth conditions included proline, valine, threonine, homoserine, myoinositol, γ-aminobutyrate (GABA) and trigonelline (nicotinic acid betaine). Metabolites that were altered in relative amounts in different experiments, but not specifically associated with drought-stress, were also identified. These included glutamate, asparagine and malate, with the last being present at up to 5-fold higher concentrations in plants grown in field experiments. Such changes may be expected to impact both on plant performance and crop end-use.

Achievements and Challenges in Improving the Nutritional Quality of Food Legumes

Quality aspects of food crops have globally important market economic and health repercussions in the current climate of food security. Grain legumes have high potential for the nutritional quality improvement of foods, but limited data on manipulating seed quality is available as the primary focus has been hitherto on phenotypic and agronomic trait improvement. This has resulted in a lack of innovation and low attractiveness of legume food products that, with the emergence of novel food habits, have together contributed to reduced legume food consumption. This trend now needs to be challenged and circumvented. In this review we have assessed the key factors affecting the nutritional quality of legume seeds such as protein, starch, dietary fiber, natural antioxidant compounds and anti-nutritional factors. All have been reviewed with emphasis on how these components might influence consumer acceptance and functional properties of legume based food products. Biofortification approaches and technological processing are discussed as ways in which the nutritional value of legumes and their consumption might be enhanced. In order to increase consumption of grain legumes, we propose that efforts should concentrate on identifying nutritionally enhanced and genetically diverse germplasm, and on linking genetics with sensorial and processing quality. This will assist the development of breeding/selection tools for traits that determine consumer demand, facilitating the implementation of quality breeding objectives in legume breeding programs. Equally important, efforts should focus on developing attractive, convenient ready-to-eat and tasty legume-based food formulations, contributing to the diversification of healthier and more nutritional diets. As a result of such a targeted effort, legume cultivation and consumption could be enhanced leading to a reduction in both the global economic burden caused by malnutrition and associated chronic diseases, and the environmental impact of agriculture.

The cytotoxic effect of Bowman–Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.

Combined metabolomic and genetic approaches reveal a link between the polyamine pathway and albumin 2 in developing pea seeds

Several legume seed proteins that are potentially allergenic, poorly digested by farm animals, and/or have undesirable functional properties, have been described. One of these is the albumin protein in pea (Pisum sativum) called PA2. A naturally occurring mutant line that lacks PA2 has been exploited in studies to determine the biological function of this nonstorage protein in seed development. The mutant, which has a small seed, a tall plant phenotype, and lacks most of the PA2-encoding genes, has been crossed with a standard cultivar, ‘Birte,’ which contains PA2 to give rise to a recombinant inbred (RI) population. An F3 line carrying the mutation and having a short plant phenotype has been used to generate backcross (BC) lines with ‘Birte.’ Despite having a lower albumin content, seeds from the mutant parent and RI lines lacking PA2 have an equivalent or higher seed nitrogen content. Metabolite profiling of seeds revealed major differences in amino acid composition and polyamine content in the two parent lines. This was investigated further in BC lines, where the effects of differences in seed size and plant height between the two parents were eliminated. Here, differences in polyamine synthesis were maintained as was a difference in total seed protein between the BC line lacking PA2 and ‘Birte.’ Analysis of enzyme activities in the pathways of polyamine synthesis revealed that the differences in spermidine content were attributable to changes in the overall activities of spermidine synthase and arginine decarboxylase. Although the genes encoding spermidine synthase and PA2 both localized to the pea linkage group I, the two loci were shown not to be closely linked and to have recombined in the BC lines. A distinct locus on linkage group III contains a gene that is related to PA2 but expressed predominantly in flowers. The results provide evidence for a role of PA2 in regulating polyamine metabolism, which has important functions in development, metabolism, and stress responses in plants.

Speed breeding is a powerful tool to accelerate crop research and breeding

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand1. This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called ‘speed breeding’, which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2–3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.Fully enclosed, controlled-environment growth chambers can accelerate plant development. Such ‘speed breeding’ reduces generation times to accelerate crop breeding and research programmes, and can integrate with other modern crop breeding technologies.

Bowman-Birk Inhibitors from Legumes and Human Gastrointestinal Health: Current Status and Perspectives

Bowman-Birk inhibitors and their variants (BBI) from legumes, such as soybean, pea, lentil and chickpea, are a class of naturally-occurring protease inhibitors which have potential health-promoting properties within the gastrointestinal tract. BBI can resist both acidic conditions and the action of proteolytic enzymes, and transit through the stomach and small intestine without major degradation, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. These potential pharmacological benefits have been linked recently to the intrinsic ability of BBI to inhibit serine proteases, and the data suggest that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI. However, the therapeutic targets and the action mechanisms of BBI remain unknown. Their elucidation will provide insights into the properties of these plant protease inhibitors as colorectal chemopreventive agents, providing a strong base for the development of legume crops and their products as pro-nutritional, health-promoting food. The deployment of modern genomic tools and genome sequence information are underpinning studies of natural and induced polymorphism in BBI. Genetic markers for BBI variants with improved properties can be exploited ultimately in legume breeding programmes to assist the introgression of such variant genes and the development of superior genotypes for human nutrition.

Combinatorial variation in coding and promoter sequences of genes at the Tri locus in Pisum sativum accounts for variation in trypsin inhibitor activity in seeds

Abstract. Cultivars of Pisum sativum that differ with respect to the quantitative expression of trypsin/chymotrypsin inhibitor proteins in seeds have been examined in terms of the structure of the corresponding genes. The patterns of divergence in the promoter and coding sequences are described, and the divergence among these exploited for the development of facile DNA-based assays to distinguish genotypes. Quantitative effects on gene expression may be attributed to the overall gene complement and to particular promoter/coding sequence combinations, as well as to the existence of distinct active-site variants that ultimately influence protein activity. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00438-002-0667-4.

Temporal and spatial activity of a promoter from a pea enzyme inhibitor gene and its exploitation for seed quality improvement

The promoter from one of the two seed-expressed genes encoding trypsin/chymotrypsin inhibitors (TI) has been isolated and characterised in transgenic pea lines, following its re-introduction by Agrobacterium-mediated transformation, as a TI promoter-beta-glucuronidase (GUS) gene fusion. The promoter from this gene (TI1) directed expression of GUS enzyme at late stages of embryogenesis, comparable to those determined for activity of the homologous native TI genes. GUS expression was detected in roots of plants subjected to drought stress conditions, indicating that the TI1 gene, normally seed-specific in its expression, can be induced under these conditions. A second gene construct utilised the TI1 gene promoter to direct expression of an antisense TI gene. Seed TI activities in some lines transformed with this construct were reduced significantly. A limitation of the pea transformation methodology for antisense manipulations, in particular, is the observed frequency of non-transmission of transgenes from primary transformants (up to 80%).

The anti-proliferative effect of TI1B, a major Bowman-Birk isoinhibitor from pea (Pisum sativum L.), on HT29 colon cancer cells is mediated through protease inhibition

Bowman-Birk inhibitors (BBI) from legumes, such as soyabean, pea, lentil and chickpea, are naturally occurring plant protease inhibitors which have potential health-promoting properties within the mammalian gastrointestinal tract. BBI can survive both acidic conditions and the action of proteolytic enzymes within the stomach and small intestine, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. In a previous study, we reported the ability of a recombinant form of TI1B (rTI1B), representing a major BBI isoinhibitor from pea, to influence negatively the growth of human colorectal adenocarcinoma HT29 cells in vitro. In the present study, we investigate if this effect is related directly to the intrinsic ability of BBI to inhibit serine proteases. rTI1B and a novel engineered mutant, having amino acid substitutions at the P1 positions in the two inhibitory domains, were expressed in the yeast Pichia pastoris. The rTI1B proved to be active against trypsin and chymotrypsin, showing K i values at nanomolar concentrations, whereas the related mutant protein was inactive against both serine proteases. The proliferation of HT29 colon cancer cells was significantly affected by rTI1B in a dose-dependent manner (IC50 = 31 (sd 7) μm), whereas the inactive mutant did not show any significant effect on colon cancer cell growth. In addition, neither recombinant protein affected the growth of non-malignant colonic fibroblast CCD-18Co cells. These findings suggest that serine proteases should be considered as important targets in investigating the potential chemopreventive role of BBI during the early stages of colorectal carcinogenesis.