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.

Combination of three null mutations affecting seed protein accumulation in pea (Pisum sativum L.) impacts positively on digestibility.

The presence of so-called anti-nutritional factors can reduce the bioavailability of nutrients following consumption of seeds which are otherwise an excellent source of proteins, carbohydrates and micronutrients. Among the proteins associated with negative effects on quality in pea (Pisum sativum L.) seeds are lectin, pea albumin 2 (PA2) and trypsin inhibitors (TI). Here we have investigated the impact of these proteins on protein digestibility and amino acid availability, using naturally occurring and derived mutant lines of pea lacking these proteins. The mutations were stacked to generate a triple mutant which was compared with a wild-type progenitor and a line lacking the major seed trypsin inhibitors alone. In vitro digestions following the INFOGEST protocol revealed significant differences in the degree of hydrolysis, protein profile and apparent amino acid availability among the pea variants. Proteins resistant to digestion were identified by MALDI-TOF mass spectrometry and amino acid profiles of digested samples determined. The results indicate that pea seeds lacking certain proteins can be used in the development of novel foods which have improved protein digestibility, and without negative impact on seed protein concentration or yield.

An allelic series of starch-branching enzyme mutants in pea (Pisum sativum L.) reveals complex relationships with seed starch phenotypes.

A set of mutant pea lines carrying induced mutations within the major seed-expressed starch-branching enzyme gene has been characterised at the molecular, chemical and agronomic levels. Eight of the induced mutations, three of which predicted a premature stop codon, were compared with the naturally occurring starch-branching enzyme mutation within the same genetic background. Starch, amylose and sugar measurements, coupled with analysis by ultra-high performance liquid chromatography-size exclusion chromatography of starches, identified a range of phenotypes which were grouped according to the nature of the mutation. Homology modelling of proteins supported the differences in phenotypes observed. Differences in field performance were evident for selected mutants, particularly in seed yield and mean seed weight traits for early compared with late spring sowings. The data show the potential of an allelic series of mutants at this locus for nutritional studies. CHEMICAL COMPOUNDS: starch, amylose, amylopectin, raffinose, stachyose, verbascose.

An Integrated Linkage Map of Three Recombinant Inbred Populations of Pea (Pisum sativum L.).

Biparental recombinant inbred line (RIL) populations are sets of genetically stable lines and have a simple population structure that facilitates the dissection of the genetics of interesting traits. On the other hand, populations derived from multiparent intercrosses combine both greater diversity and higher numbers of recombination events than RILs. Here, we describe a simple population structure: a three-way recombinant inbred population combination. This structure was easy to produce and was a compromise between biparental and multiparent populations. We show that this structure had advantages when analyzing cultivar crosses, and could achieve a mapping resolution of a few genes.

Real-time monitoring of rhizosphere nitrate fluctuations under crops following defoliation.

BACKGROUND: Management regime can hugely influence the efficiency of crop production but measuring real-time below-ground responses is difficult. The combination of fertiliser application and mowing or grazing may have a major impact on roots and on the soil nutrient profile and leaching. RESULTS: A novel approach was developed using low-cost ion-selective sensors to track nitrate (NO3-) movement through soil column profiles sown with the forage crops, Lolium perenne and Medicago sativa. Applications of fertiliser, defoliation of crops and intercropping of the grass and the legume were tested. Sensor measurements were compared with conventional testing of lysimeter and leachate samples. There was little leaching of NO3- through soil profiles with current management practices, as monitored by both methods. After defoliation, the measurements detected a striking increase in soil NO3- in the middle of the column where the greatest density of roots was found. This phenomenon was not detected when no NO3- was applied, and when there was no defoliation, or during intercropping with Medicago. CONCLUSION: Mowing or grazing may increase rhizodeposition of carbon that stimulates soil mineralization to release NO3- that is acquired by roots without leaching from the profile. The soil columns and sensors provided a dynamic insight into rhizosphere responses to changes in above-ground management practices.

Perspectives on the genetic improvement of health- and nutrition-related traits in pea.

Pea (Pisum sativum L.) is a widely grown pulse crop that is a source of protein, starch and micronutrients in both human diets and livestock feeds. There is currently a strong global focus on making agriculture and food production systems more sustainable, and pea has one of the smallest carbon footprints of all crops. Multiple genetic loci have been identified that influence pea seed protein content, but protein composition is also important nutritionally. Studies have previously identified gene families encoding individual seed protein classes, now documented in a reference pea genome assembly. Much is also known about loci affecting starch metabolism in pea, with research especially focusing on improving concentrations of resistant starch, which has a positive effect on maintaining blood glucose homeostasis. Diversity in natural germplasm for micronutrient concentrations and mineral hyperaccumulation mutants have been discovered, with quantitative trait loci on multiple linkage groups identified for seed micronutrient concentrations. Antinutrients, which affect nutrient bioavailability, must also be considered; mutants in which the concentrations of important antinutrients including phytate and trypsin inhibitors are reduced have already been discovered. Current knowledge on the genetics of nutritional traits in pea will greatly assist with crop improvement for specific end uses, and further identification of genes involved will help advance our knowledge of the control of the synthesis of seed compounds.

Fulvic acid increases forage legume growth inducing preferential up-regulation of nodulation and signalling-related genes.

The use of potential biostimulants is of broad interest in plant science for improving yields. The application of a humic derivative called fulvic acid (FA) may improve forage crop production. FA is an uncharacterized mixture of chemicals and, although it has been reported to increase growth parameters in many species including legumes, its mode of action remains unclear. Previous studies of the action of FA have lacked appropriate controls, and few have included field trials. Here we report yield increases due to FA application in three European Medicago sativa cultivars, in studies which include the appropriate nutritional controls which hitherto have not been used. No significant growth stimulation was seen after FA treatment in grass species in this study at the treatment rate tested. Direct application to bacteria increased Rhizobium growth and, in M. sativa trials, root nodulation was stimulated. RNA transcriptional analysis of FA-treated plants revealed up-regulation of many important early nodulation signalling genes after only 3 d. Experiments in plate, glasshouse, and field environments showed yield increases, providing substantial evidence for the use of FA to benefit M. sativa forage production.

A natural mutation in Pisum sativum L. (pea) alters starch assembly and improves glucose homeostasis in humans.

Elevated postprandial glucose (PPG) is a significant risk factor for non-communicable diseases globally. Currently, there is a limited understanding of how starch structures within a carbohydrate-rich food matrix interact with the gut luminal environment to control PPG. Here, we use pea seeds (Pisum sativum) and pea flour, derived from two near-identical pea genotypes (BC1/19RR and BC1/19rr) differing primarily in the type of starch accumulated, to explore the contribution of starch structure, food matrix and intestinal environment to PPG. Using stable isotope 13C-labelled pea seeds, coupled with synchronous gastric, duodenal and plasma sampling in vivo, we demonstrate that maintenance of cell structure and changes in starch morphology are closely related to lower glucose availability in the small intestine, resulting in acutely lower PPG and promotion of changes in the gut bacterial composition associated with long-term metabolic health improvements.

Genome-Wide Association Mapping for Agronomic and Seed Quality Traits of Field Pea (Pisum sativum L.).

Genome-wide association study (GWAS) was conducted to identify loci associated with agronomic (days to flowering, days to maturity, plant height, seed yield and seed weight), seed morphology (shape and dimpling), and seed quality (protein, starch, and fiber concentrations) traits of field pea (Pisum sativum L.). A collection of 135 pea accessions from 23 different breeding programs in Africa (Ethiopia), Asia (India), Australia, Europe (Belarus, Czech Republic, Denmark, France, Lithuania, Netherlands, Russia, Sweden, Ukraine and United Kingdom), and North America (Canada and USA), was used for the GWAS. The accessions were genotyped using genotyping-by-sequencing (GBS). After filtering for a minimum read depth of five, and minor allele frequency of 0.05, 16,877 high quality SNPs were selected to determine marker-trait associations (MTA). The LD decay (LD1/2max,90) across the chromosomes varied from 20 to 80 kb. Population structure analysis grouped the accessions into nine subpopulations. The accessions were evaluated in multi-year, multi-location trials in Olomouc (Czech Republic), Fargo, North Dakota (USA), and Rosthern and Sutherland, Saskatchewan (Canada) from 2013 to 2017. Each trait was phenotyped in at least five location-years. MTAs that were consistent across multiple trials were identified. Chr5LG3_566189651 and Chr5LG3_572899434 for plant height, Chr2LG1_409403647 for lodging resistance, Chr1LG6_57305683 and Chr1LG6_366513463 for grain yield, Chr1LG6_176606388, Chr2LG1_457185, Chr3LG5_234519042 and Chr7LG7_8229439 for seed starch concentration, and Chr3LG5_194530376 for seed protein concentration were identified from different locations and years. This research identified SNP markers associated with important traits in pea that have potential for marker-assisted selection towards rapid cultivar improvement.

Variation in Pea (Pisum sativum L.) Seed Quality Traits Defined by Physicochemical Functional Properties.

Pea is one of the most produced and consumed pulse crops around the world. The study of genetic variability within pea germplasm is an important tool to identify outstanding accessions with optimal functional and nutritional qualities. In the present study, a collection of 105 pea accessions was analysed for physicochemical properties, pasting viscosity, and basic composition parameters. While pasting viscosities were negatively correlated to hydration capacity, cooking time, and basic composition, a positive correlation was found between the hydration capacity and the basic composition parameters. Basic composition (protein, fibre, fat, and resistant starch) parameters were further evaluated regarding seed trait morphology, namely, seed shape, colour, and surface. Allelic characterisation at the r and rb genetic loci was performed in a subgroup of 32 accessions (3 phenotyped as smooth and 29 as rough seeded), revealing that none of the initially classified rough-seeded accessions were rb mutants, 19 were r mutants, and 13 were neither r nor rb. Despite their initial phenotypic classification, the 13 accessions genetically classified as smooth behaved differently (p < 0.05) to the 19 r mutants in terms of physicochemical properties, pasting viscosity, and basic composition parameters. Using multivariate analysis of the most discriminatory parameters for the food-related traits studied, the best-performing accessions at functional and nutritional levels were identified for future plant breeding to improve field pea production and consumption.

Speed breeding in growth chambers and glasshouses for crop breeding and model plant research.

'Speed breeding' (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants' daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber-based SB approaches with supporting data from experimentation with several crops. We describe the conditions that promote the rapid growth of bread wheat, durum wheat, barley, oat, various Brassica species, chickpea, pea, grass pea, quinoa and Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale up plant numbers for single-seed descent (SSD). In addition, instructions are provided on how to perform SB on a small scale in a benchtop growth cabinet, enabling optimization of parameters at a low cost.

NMR Metabolomics Defining Genetic Variation in Pea Seed Metabolites.

Nuclear magnetic resonance (NMR) spectroscopy profiling was used to provide an unbiased assessment of changes to the metabolite composition of seeds and to define genetic variation for a range of pea seed metabolites. Mature seeds from recombinant inbred lines, derived from three mapping populations for which there is substantial genetic marker linkage information, were grown in two environments/years and analyzed by non-targeted NMR. Adaptive binning of the NMR metabolite data, followed by analysis of quantitative variation among lines for individual bins, identified the main genomic regions determining this metabolic variability and the variability for selected compounds was investigated. Analysis by t-tests identified a set of bins with highly significant associations to genetic map regions, based on probability (p) values that were appreciably lower than those determined for randomized data. The correlation between bins showing high mean absolute deviation and those showing low p-values for marker association provided an indication of the extent to which the genetics of bin variation might be explained by one or a few loci. Variation in compounds related to aromatic amino acids, branched-chain amino acids, sucrose-derived metabolites, secondary metabolites and some unidentified compounds was associated with one or more genetic loci. The combined analysis shows that there are multiple loci throughout the genome that together impact on the abundance of many compounds through a network of interactions, where individual loci may affect more than one compound and vice versa. This work therefore provides a framework for the genetic analysis of the seed metabolome, and the use of genetic marker data in the breeding and selection of seeds for specific seed quality traits and compounds that have high commercial value.

Ultra-high performance liquid chromatography-size exclusion chromatography (UPLC-SEC) as an efficient tool for the rapid and highly informative characterisation of biopolymers.

Starch has a complex molecular structure, with properties dependent on the relative chain lengths and branching structure of its constituent molecules, which varies due to polymorphisms in starch biosynthetic genes, as well as environmental factors. Here we present the application of ultra-high performance size exclusion chromatography to the separation of starch chains from plant seeds. Several methods, have been used to analyse chain length distributions in starch, all with limitations in terms of analysis time, sample preparation and molecular weight range. Here we demonstrate that chain length distributions can be obtained with dramatically reduced analysis time using ultra-high performance size exclusion chromatography. The method may also show improvements in resolution of some fine structural features. Understanding links between starch fine structure and biosynthetic genes will allow bioengineering of starches with tailored properties. This technique may have application to the size separation and resolution of a range of biopolymers of value to the food, drink and pharmaceutical industries.

The stage of seed development influences iron bioavailability in pea (Pisum sativum L.).

Pea seeds are widely consumed in their immature form, known as garden peas and petit pois, mostly after preservation by freezing or canning. Mature dry peas are rich in iron in the form of ferritin, but little is known about the content, form or bioavailability of iron in immature stages of seed development. Using specific antibodies and in-gel iron staining, we show that ferritin loaded with iron accumulated gradually during seed development. Immunolocalization and high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that iron-loaded ferritin was located at the surface of starch-containing plastids. Standard cooking procedures destabilized monomeric ferritin and the iron-loaded form. Iron uptake studies using Caco-2 cells showed that the iron in microwaved immature peas was more bioavailable than in boiled mature peas, despite similar levels of soluble iron in the digestates. By manipulating the levels of phytic acid in the digestates we demonstrate that phytic acid is the main inhibitor of iron uptake from mature peas in vitro. Taken together, our data show that immature peas and mature dry peas contain similar levels of ferritin-iron, which is destabilized during cooking. However, iron from immature peas is more bioavailable because of lower phytic acid levels compared to mature peas.

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 demand 1 . 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.

Genetic Variation Controlling Wrinkled Seed Phenotypes in Pisum: How Lucky Was Mendel?

One of the traits studied by Mendel in pea (Pisum sativum L.) was the wrinkled-seeded phenotype, and the molecular basis for a mutation underlying this phenotype was discovered in the 1990s. Although the starch-branching enzyme gene mutation identified at the genetic locus r is most likely to be that in seeds available to Mendel in the mid-1800s, it has remained an open question as to whether or not additional natural mutations in this gene exist within Pisum germplasm collections. Here, we explore this question and show that all but two wrinkled-seeded variants in one such collection correspond to either the mutant allele described previously for the r locus or a mutation at a second genetic locus, rb, affecting the gene encoding the large subunit of Adenosine diphosphoglucose (ADP-glucose) pyrophosphorylase; the molecular basis for the rb mutation is described here. The genetic basis for the phenotype of one (JI 2110) of the two lines which are neither r nor rb has been studied in crosses with a round-seeded variant (JI 281); for which extensive genetic marker data were expected. In marked contrast to the trait studied by Mendel and the rb phenotype; the data suggest that the wrinkled-seeded phenotype in JI 2110 is maternally determined, controlled by two genetic loci, and the extent to which it is manifested is very sensitive to the environment. Metabolite analysis of the cotyledons of JI 2110 revealed a profile for sucrose and sucrose-derived compounds that was more similar to that of wild-type round-seeded, than that of wrinkled-seeded r, pea lines. However, the metabolite profile of the seed coat (testa) of JI 2110 was distinct from that of other round-seeded genotypes tested which, together with analysis of recombinant inbred progeny lines, suggests an explanation for the seed phenotype.

From Mendel's discovery on pea to today's plant genetics and breeding : Commemorating the 150th anniversary of the reading of Mendel's discovery.

KEY MESSAGE: This work discusses several selected topics of plant genetics and breeding in relation to the 150th anniversary of the seminal work of Gregor Johann Mendel. In 2015, we celebrated the 150th anniversary of the presentation of the seminal work of Gregor Johann Mendel. While Darwin's theory of evolution was based on differential survival and differential reproductive success, Mendel's theory of heredity relies on equality and stability throughout all stages of the life cycle. Darwin's concepts were continuous variation and "soft" heredity; Mendel espoused discontinuous variation and "hard" heredity. Thus, the combination of Mendelian genetics with Darwin's theory of natural selection was the process that resulted in the modern synthesis of evolutionary biology. Although biology, genetics, and genomics have been revolutionized in recent years, modern genetics will forever rely on simple principles founded on pea breeding using seven single gene characters. Purposeful use of mutants to study gene function is one of the essential tools of modern genetics. Today, over 100 plant species genomes have been sequenced. Mapping populations and their use in segregation of molecular markers and marker-trait association to map and isolate genes, were developed on the basis of Mendel's work. Genome-wide or genomic selection is a recent approach for the development of improved breeding lines. The analysis of complex traits has been enhanced by high-throughput phenotyping and developments in statistical and modeling methods for the analysis of phenotypic data. Introgression of novel alleles from landraces and wild relatives widens genetic diversity and improves traits; transgenic methodologies allow for the introduction of novel genes from diverse sources, and gene editing approaches offer possibilities to manipulate gene in a precise manner.

SGRL can regulate chlorophyll metabolism and contributes to normal plant growth and development in Pisum sativum L.

Among a set of genes in pea (Pisum sativum L.) that were induced under drought-stress growth conditions, one encoded a protein with significant similarity to a regulator of chlorophyll catabolism, SGR. This gene, SGRL, is distinct from SGR in genomic location, encoded carboxy-terminal motif, and expression through plant and seed development. Divergence of the two encoded proteins is associated with a loss of similarity in intron/exon gene structure. Transient expression of SGRL in leaves of Nicotiana benthamiana promoted the degradation of chlorophyll, in a manner that was distinct from that shown by SGR. Removal of a predicted transmembrane domain from SGRL reduced its activity in transient expression assays, although variants with and without this domain reduced SGR-induced chlorophyll degradation, indicating that the effects of the two proteins are not additive. The combined data suggest that the function of SGRL during growth and development is in chlorophyll re-cycling, and its mode of action is distinct from that of SGR. Studies of pea sgrL mutants revealed that plants had significantly lower stature and yield, a likely consequence of reduced photosynthetic efficiencies in mutant compared with control plants under conditions of high light intensity.

Eliminating anti-nutritional plant food proteins: the case of seed protease inhibitors in pea.

Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving or abolishing the corresponding inhibitory activity, along with associated molecular markers for breeding programmes. The potential for making large changes to plant protein profiles for improved and sustainable food production through diversity is illustrated. The strategy employed here to reduce anti-nutritional proteins in seeds may be extended to allergens and other seed proteins with negative nutritional effects. Additionally, the novel variants described for pea will assist future studies of the biological role and health-related properties of so-called anti-nutrients.