CRISPR/Cas9-mediated lipoxygenase gene-editing in yellow pea leads to major changes in fatty acid and flavor profiles.

Introduction: Although pulses are nutritious foods containing high amounts of protein, fiber and phytochemicals, their consumption and use in the food industry have been limited due to the formation of unappealing flavors/aromas described as beany, green, and grassy. Lipoxygenase (LOX) enzymes are prevalent among pulse seeds, and their activity can lead to the formation of specific volatile organic compounds (VOCs) from certain polyunsaturated fatty acids (PUFAs). As a widespread issue in legumes, including soybean, these VOCs have been linked to certain unappealing taste perception of foods containing processed pulse seeds. Methods: To address this problem in pea and as proof of principle to promote the wider use of pulses, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) construct was designed to create null alleles (knockouts) of PsLOX2 which had been implicated in the generation of VOCs in peas. Results and discussion: Successful CRISPR/Cas9-mediated LOX gene editing of stable transgenic pea lines (TGP) was confirmed by DNA sequencing of the wild type (WT) and TGP pslox2 mutant lines. These lines were also assessed for LOX activity, PUFA levels, and VOCs. Compared to WT peas, the TGP lines showed a significant reduction (p < 0.05) in LOX activity and in the concentration of key VOCs, including hexanal, 2-hexenal, heptanal, (E)-2-heptenal, (E,E)-2,4-heptadienal, 1-octen-3-ol, octanal, (E)-2-octenal (E,E)-2,4-nonadienal and furan-2-pentyl. The content of two essential PUFAs, linoleic and α-linolenic acids, the known substrates of LOX in plants, was higher in TGP flours, indicating the efficacy of the CRISPR-mediated gene editing in minimizing their oxidation and the further modification of PUFAs and their products. The collection of VOCs from the headspace of ground pea seeds, using a portable eNose also distinguished the TGP and WT lines. Multiple regression analysis showed that LOX activity correlated with the two VOCs, heptanal and (E,E)-2,4-heptadienal in pea flours. Partial Least Squares Regression (PLS-R) plot for selected PUFAs, VOCs, and sensor responses in WT and TGP lines showed distinct clusters for WT and TGP lines. Together this data demonstrates the utility of CRISPR mediated mutagenesis of PsLOX2 to quickly improve aroma and fatty acid (FA) profiles of pea seeds of an elite Canadian variety.

The SoyaGen Project: Putting Genomics to Work for Soybean Breeders.

The SoyaGen project was a collaborative endeavor involving Canadian soybean researchers and breeders from academia and the private sector as well as international collaborators. Its aims were to develop genomics-derived solutions to real-world challenges faced by breeders. Based on the needs expressed by the stakeholders, the research efforts were focused on maximizing realized yield through optimization of maturity and improved disease resistance. The main deliverables related to molecular breeding in soybean will be reviewed here. These include: (1) SNP datasets capturing the genetic diversity within cultivated soybean (both within a worldwide collection of > 1,000 soybean accessions and a subset of 102 short-season accessions (MG0 and earlier) directly relevant to this group); (2) SNP markers for selecting favorable alleles at key maturity genes as well as loci associated with increased resistance to key pathogens and pests (Phytophthora sojae, Heterodera glycines, Sclerotinia sclerotiorum); (3) diagnostic tools to facilitate the identification and mapping of specific pathotypes of P. sojae; and (4) a genomic prediction approach to identify the most promising combinations of parents. As a result of this fruitful collaboration, breeders have gained new tools and approaches to implement molecular, genomics-informed breeding strategies. We believe these tools and approaches are broadly applicable to soybean breeding efforts around the world.

Tissue specific changes in elements and organic compounds of alfalfa (Medicago sativa L.) cultivars differing in salt tolerance under salt stress.

Soil salinity is a global concern and often the primary factor contributing to land degradation, limiting crop growth and production. Alfalfa (Medicago sativa L.) is a low input high value forage legume with a wide adaptation. Examining the tissue-specific responses to salt stress will be important to understanding physiological changes of alfalfa. The responses of two alfalfa cultivars (salt tolerant 'Halo', salt intolerant 'Vernal') were studied for 12 weeks in five gradients of salt stress in a sand based hydroponic system in the greenhouse. The accumulation and localization of elements and organic compounds in different tissues of alfalfa under salt stress were evaluated using synchrotron beamlines. The pattern of chlorine accumulation for 'Halo' was: root > stem ~ leaf at 8 dSm-1, and root ~ leaf > stem at 12 dSm-1, potentially preventing toxic ion accumulation in leaf tissues. In contrast, for 'Vernal', it was leaf > stem ~ root at 8 dSm-1 and leaf > root ~ stem at 12 dSm-1. The distribution of chlorine in 'Halo' was relatively uniform in the leaf surface and vascular bundles of the stem. Amide concentration in the leaf and stem tissues was greater for 'Halo' than 'Vernal' at all salt gradients. This study determined that low ion accumulation in the shoot was a common strategy in salt tolerant alfalfa up to 8 dSm-1 of salt stress, which was then replaced by shoot tissue tolerance at 12 dSm-1.

Low Phytate Peas (Pisum sativum L.) Improve Iron Status, Gut Microbiome, and Brush Border Membrane Functionality In Vivo (Gallus gallus).

The inclusion of pulses in traditional wheat-based food products is increasing as the food industry and consumers are recognizing the nutritional benefits due to the high protein, antioxidant activity, and good source of dietary fiber of pulses. Iron deficiency is a significant global health challenge, affecting approximately 30% of the world's population. Dietary iron deficiency is the foremost cause of anemia, a condition that harms cognitive development and increases maternal and infant mortality. This study intended to demonstrate the potential efficacy of low-phytate biofortified pea varieties on dietary iron (Fe) bioavailability, as well as on intestinal microbiome, energetic status, and brush border membrane (BBM) functionality in vivo (Gallus gallus). We hypothesized that the low-phytate biofortified peas would significantly improve Fe bioavailability, BBM functionality, and the prevalence of beneficial bacterial populations. A six-week efficacy feeding (n = 12) was conducted to compare four low-phytate biofortified pea diets with control pea diet (CDC Bronco), as well as a no-pea diet. During the feeding trial, hemoglobin (Hb), body-Hb Fe, feed intake, and body weight were monitored. Upon the completion of the study, hepatic Fe and ferritin, pectoral glycogen, duodenal gene expression, and cecum bacterial population analyses were conducted. The results indicated that certain low-phytate pea varieties provided greater Fe bioavailability and moderately improved Fe status, while they also had significant effects on gut microbiota and duodenal brush border membrane functionality. Our findings provide further evidence that the low-phytate pea varieties appear to improve Fe physiological status and gut microbiota in vivo, and they highlight the likelihood that this strategy can further improve the efficacy and safety of the crop biofortification and mineral bioavailability approach.

Effect of stage of maturity at harvest for forage pea (Pisum sativum L.) on eating behavior, ruminal fermentation, and digestibility when fed as hay to yearling beef heifers.

The objective of this study was to evaluate the stage of maturity at harvest for pea hay (Pisum sativum L., c.v. CDC Horizon) on dry matter intake (DMI), eating behavior, ruminal fermentation, and digestibility when fed to beef heifers. Pea hay was cut at EARLY (defined to occur when flat pods were on one or more nodes), MID (when seeds filled the pods at one or more nodes and the leaves were changing from green to gold), and LATE (yellow dry seeds filled pods on most or all of the nodes and the pods and leaves had a yellow color) phases, and was cured in the field and baled. Six ruminally-cannulated Speckle Park heifers were used in a replicated 3 × 3 Latin square design with three 18-d periods including 12 d for adaptation, 2 d for measurement of ruminal pool sizes, and 4 d for the collection of eating behavior, ruminal pH, ruminal digesta, and feces. For all treatments, the respective pea hay was included at 40% of the dietary DM. Stage of maturity at harvest for pea hay did not affect total DMI, pea hay DMI, or the total short-chain fatty acid concentration in ruminal fluid with averages of 8.6 kg/d, 3.2 kg/d, and 96.55 mM, respectively. The duration of time spent ruminating decreased with advancing pea hay maturity when reported as min/d, min/kg DMI, and min/kg neutral detergent fiber (NDF) (P ≤ 0.01). Mean ruminal pH also decreased with advancing pea maturity (P < 0.01). The ruminal DM and undigested NDF corrected for OM pools were not affected by stage of maturity (P ≥ 0.55) nor was the rate of digestion for NDF. However, NDF passage rate decreased by 0.21%/h with advancing pea hay maturity (P = 0.02). Apparent total tract digestibility of NDF (average = 16.30%, P = 0.41) was not affected, but starch digestibility decreased from 96.10% to 93.08% with advancing pea hay maturity (P = 0.07). Overall, stage of maturity at harvest for pea hay does not appear to affect DMI or NDF digestibililty but decreases chewing activity, apparent total tract starch digestibility, ruminal pH, and ruminal NDF passage rate.

Development of a Sequence-Based Reference Physical Map of Pea (Pisum sativum L.).

Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (∼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

Genotypic and heat stress effects on leaf cuticles of field pea using ATR-FTIR spectroscopy.

MAIN CONCLUSION: ATR-FTIR spectroscopy in combination with uni- and multivariate analysis was used to quantify the spectral-chemical composition of the leaf cuticle of pea, investigating the effects of variety and heat stress. Field pea (Pisum sativum L.) is sensitive to heat stress and our goal was to improve canopy cooling and flower retention by investigating the protective role of lipid-related compounds in leaf cuticle, and to use results in the future to identify heat resistant genotypes. The objective was to use Attenuated Total Reflection (ATR)-Fourier Transform Infrared (FTIR) spectroscopy, a non-invasive technique, to investigate and quantify changes in adaxial cuticles of fresh leaves of pea varieties that were subjected to heat stress. Eleven varieties were grown under control (24/18 °C day/night) and heat stress conditions (35/18 °C day/night, for 5 days at the early flowering stage). These 11 had significant spectral differences in the integrated area of the main lipid region, CH2 region, CH3 peak, asymmetric and symmetric CH2 peaks, ester carbonyl peak, and the peak area ratio of CH2 to CH3 and ester carbonyl to CH2 asymmetric peak, indicating that cuticles had spectral-chemical diversity of waxes, cutin, and polysaccharides. Results indicated considerable diversity in spectral-chemical makeup of leaf cuticles within commercially available field pea varieties and they responded differently to high growth temperature, revealing their diverse potential to resist heat stress. The ATR-FTIR spectral technique can, therefore, be further used as a medium-throughput approach for rapid screening of superior cultivars for heat tolerance.

Symbiosis of selected Rhizobium leguminosarum bv. viciae strains with diverse pea genotypes: effects on biological nitrogen fixation.

Biological nitrogen fixation (BNF) can be improved by optimizing the interaction between the rhizobial inoculant and pea (Pisum sativum L.), leading to increased productivity and reduced nitrogen (N) fertilizer use. Eight Rhizobium leguminosarum bv. viciae strains were used to inoculate the super-nodulating pea mutant Rondo-nod3 (fix+), the hyper-nodulating pea mutant Frisson P88 Sym29, CDC Meadow commercial control, and the non-nodulating mutant Frisson P56 (nod-) to evaluate BNF in a greenhouse assay. Significant differences in strain × cultivar interactions were detected for shoot and root dry masses, which ranged from 1.8 to 4.7 g and from 0.27 to 0.73 g per plant, respectively; for nodule number on lateral roots, which ranged from 25 to 430 per plant; for amount of fixed N2, which ranged from 15 to 67 mg and from 4 to 15 mg per plant for shoot and root tissues, respectively; and for percentage of N derived from atmosphere (%Ndfa), which ranged from 37% to 61% and from 35% to 65% for shoot and root tissue, respectively. Strain × cultivar interactions in this study could contribute to identification of superior strains and pea breeding lines with genetic superiority in BNF. Nodule production in pea plants was not necessarily correlated with the amount of fixed N2, suggesting nodule activity is more important to BNF than is nodule number.

Molecular basis of processing-induced changes in protein structure in relation to intestinal digestion in yellow and green type pea (Pisum sativum L.): A molecular spectroscopic analysis.

The objectives of this study were (1) to quantify the protein inherent molecular structural features of green cotyledon (CDC Striker) and yellow cotyledon (CDC Meadow) pea (Pisum sativum L.) seeds using molecular spectroscopic technique (FT/IR-ATR); (2) measure the denaturation of protein molecular makeup in the two types of pea during dry roasting (120°C for 60 min), autoclaving (120°C for 60 min) or microwaving (for 5 min); and (3) correlate the heat-induced changes in protein molecular makeup to the corresponding changes in protein digestibility determined using modified three-step in vitro procedure. Compared with yellow-type, the green-type peas had higher (P<0.05) ratios of amide I to II peak height (1.698 vs. 1.805) and area (1.843 vs. 2.017). A significant correlation was observed between the amide I and II peak height (r=0.48) and peak area (r=-0.42) ratio with protein content. Compared with yellow-type, the green-type peas had lower (P<0.05) α-helix:ß-sheet ratio (1.015 vs. 0.926), indicating varietal difference in protein secondary structure makeup. All processing applications increased α-helix:ß-sheet ratio, with the largest (P<0.05) increase being observed with roasting and microwaving. The heat-induced changes in α-helix:ß-sheet ratio was strongly correlated to intestinal digestibility of protein within the green (r=-0. 86) and yellow (r=0.81) pea-types. However, across the pea types the correlation was not significant. Principal component and hierarchical cluster analyses on the entire spectral data from the amide region (ca. 1727-1480 cm(-1)) were able to visualize and discriminate the structural difference between pea varieties and processing treatments. This study shows that the molecular spectroscopy can be used as a rapid tool to screen the protein value of raw and heat-treated peas.

In vitro starch digestibility, expected glycemic index, and thermal and pasting properties of flours from pea, lentil and chickpea cultivars.

In vitro starch digestibility, expected glycemic index (eGI), and thermal and pasting properties of flours from pea, lentil and chickpea grown in Canada under identical environmental conditions were investigated. The protein content and gelatinization transition temperatures of lentil flour were higher than those of pea and chickpea flours. Chickpea flour showed a lower amylose content (10.8-13.5%) but higher free lipid content (6.5-7.1%) and amylose-lipid complex melting enthalpy (0.7-0.8J/g). Significant differences among cultivars within the same species were observed with respect to swelling power, gelatinization properties, pasting properties and in vitro starch digestibility, especially chickpea flour from desi (Myles) and kabuli type (FLIP 97-101C and 97-Indian2-11). Lentil flour was hydrolyzed more slowly and to a lesser extent than pea and chickpea flours. The amount of slowly digestible starch (SDS) in chickpea flour was the highest among the pulse flours, but the resistant starch (RS) content was the lowest. The eGI of lentil flour was the lowest among the pulse flours.