The giant diploid faba genome unlocks variation in a global protein crop.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

Stomatal morphology and physiology explain varied sensitivity to abscisic acid across vascular plant lineages.

Abscisic acid (ABA) can induce rapid stomatal closure in seed plants, but the action of this hormone on the stomata of fern and lycophyte species remains equivocal. Here, ABA-induced stomatal closure, signaling components, guard cell K+ and Ca2+ fluxes, vacuolar and actin cytoskeleton dynamics, and the permeability coefficient of guard cell protoplasts (Pf) were analyzed in species spanning the diversity of vascular land plants including 11 seed plants, 6 ferns, and 1 lycophyte. We found that all 11 seed plants exhibited ABA-induced stomatal closure, but the fern and lycophyte species did not. ABA-induced hydrogen peroxide elevation was observed in all species, but the signaling pathway downstream of nitric oxide production, including ion channel activation, was only observed in seed plants. In the angiosperm faba bean (Vicia faba), ABA application caused large vacuolar compartments to disaggregate, actin filaments to disintegrate into short fragments and Pf to increase. None of these changes was observed in the guard cells of the fern Matteuccia struthiopteris and lycophyte Selaginella moellendorffii treated with ABA, but a hypertonic osmotic solution did induce stomatal closure in fern and the lycophyte. Our results suggest that there is a major difference in the regulation of stomata between the fern and lycophyte plants and the seed plants. Importantly, these findings have uncovered the physiological and biophysical mechanisms that may have been responsible for the evolution of a stomatal response to ABA in the earliest seed plants.

Novel inflorescence architecture in gamma radiation-induced faba bean mutant populations.

Purpose: Inflorescence architecture is an important trait in the seed production of grain legumes. As several genes are responsible for this trait, any mutation, on these genes, may cause change in the inflorescence architecture. This study was conducted to evaluate inflorescence architecture in faba bean exposed to gamma radiation and to characterize the inflorescence architecture mutants phenotypically.Materials and methods: Faba bean M2 seeds (4898) generated from M1 generation of cultivars Hassawi 2 and ILB4347 were used in this study. M1 seeds were produced by irradiation treatments at two doses of gamma radiations (25 and 50 Gy). Faba bean M2 seeds were planted under field conditions. A total of 4032 mutant plants out of 4898 M2 seeds were evaluated for their inflorescence architecture.Results: A total of 20 determinate mutants were found and classified into four different types. Determinate type 1 was characterized by the formation of single terminal inflorescence on shoot apical meristem (SAM), type 2 by the formation of multiple inflorescences on SEM and generated upper branches that act as indeterminate type. Type 3 was characterized by the formation of a panicle-like inflorescence. While type 4 was characterized by the formation of primary and secondary panicle-like inflorescence. All of the determinate mutant types had shorter plant height and earlier maturity than control indeterminate type but had lower biological yield and seed yield. Among the determinate mutant types, determinate type 1 was only mutant that had a higher harvest index than the control indeterminate type. This promising mutant can be used to further breeding program to increase biological yield and seed yield.Conclusions: This study indicated potential of gamma radiation in inducing novel inflorescence architecture in faba bean. The mutants developed are valuable resources to study genes related to inflorescence architecture through forward genetics approach.

Para-crystalline membrane structures resembling prolamellar bodies in the invasion zones of indeterminate root nodules of Vicia faba L.

Novel para-crystalline structures resembling prolamellar bodies in etioplasts were found in the invasion zones of indeterminate root nodules of Vicia faba, which possess persistent meristems and exhibit sequential developmental stages. The para-crystalline structures existed in most cells in the area of the invasion zone and a hexagonal arrangement of tubular membranes was recognized. Extensive membranes, apparently procured from the structures, were often in contact with the bacteria in young infected cells. We propose that the para-crystalline structures serve as a reservoir of membranes for the formation of the numerous symbiosomes that propagate and fill the infected cells, and suggest naming them pro-symbiosome membrane bodies.

Genetic diversity in Tunisian populations of faba bean (Vicia faba L.) based on morphological traits and molecular markers.

Genetic diversity within Vicia faba L. is key to the genetic improvement of this important species. In this study, morphological traits and RAPD molecular markers were used to assess the levels of polymorphism across 12 Tunisian populations, three major and nine minor from different locations. Analysis of morphological traits indicated that the three major populations showed significant differences and the nine minor populations exhibited considerable variation for most traits. The grain yield of the Alia population could be increased by inoculation. Of the seven primers tested, it was clear that the Cs12 primer would be recommend for genetic diversity analysis of V. faba.Within population genetic diversity exhibited 94% of total diversity. Intra-population genetic diversity (HS) was 0.16, which was clearly higher than between population genetic diversity (DST = 0.06) UPG-MA showed a high level of genetic variation between major and minor populations of V. faba L. Particularly the minor populations showed a high level of diversity and was divided into two subclusters. Ltaifia was separated from the other populations. In addition to a high grain yield, these populations showed the lowest Nei and Shannon indices (H = 0.08 and I = 0.13) justifying their homogeneity. For these reasons, these cultivars can be considered a selected population. However, the Takelsa population showed the highest Nei and Shannon indices (H = 0.13 and I = 0.21), indicating that this population was the most heterogeneous, which is interesting for breeding programs.

Use of synteny to identify candidate genes underlying QTL controlling stomatal traits in faba bean (Vicia faba L.).

KEY MESSAGE: We have identified QTLs for stomatal characteristics on chromosome II of faba bean by applying SNPs derived from M. truncatula , and have identified candidate genes within these QTLs using synteny between the two species. Faba bean (Vicia faba L.) is a valuable food and feed crop worldwide, but drought often limits its production, and its genome is large and poorly mapped. No information is available on the effects of genomic regions and genes on drought adaptation characters such as stomatal characteristics in this species, but the synteny between the sequenced model legume, Medicago truncatula, and faba bean can be used to identify candidate genes. A mapping population of 211 F5 recombinant inbred lines (Mélodie/2 × ILB 938/2) were phenotyped to identify quantitative trait loci (QTL) affecting stomatal morphology and function, along with seed weight, under well-watered conditions in a climate-controlled glasshouse in 2013 and 2014. Canopy temperature (CT) was evaluated in 2013 under water-deficit (CTd). In total, 188 polymorphic single nucleotide polymorphisms (SNPs), developed from M. truncatula genome data, were assigned to nine linkage groups that covered ~928 cM of the faba bean genome with an average inter-marker distance of 5.8 cM. 15 putative QTLs were detected, of which eight (affecting stomatal density, length and conductance and CT) co-located on chromosome II, in the vicinity of a possible candidate gene-a receptor-like protein kinase found in the syntenic interval of M. truncatula chromosome IV. A ribose-phosphate pyrophosphokinase from M. truncatula chromosome V, postulated as a possible candidate gene for the QTL for CTd, was found some distance away in the same chromosome. These results demonstrate that genomic information from M. truncatula can successfully be translated to the faba bean genome.

Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling?

Exposing plants to low VPD reduces leaf capacity to maintain adequate water status thereafter. To find the impact of VPD on functioning of stomata, stomatal morphology and leaf anatomy, fava bean plants were grown at low (L, 0.23 kPa) or moderate (M, 1.17 kPa) VPDs and some plants that developed their leaves at moderate VPD were then transferred for 4 days to low VPD (M→L). Part of the M→L-plants were sprayed with ABA (abscisic acid) during exposure to L. L-plants showed bigger stomata, larger pore area, thinner leaves and less spongy cells compared with M-plants. Stomatal morphology (except aperture) and leaf anatomy of the M→L-plants were almost similar to the M-plants, while their transpiration rate and stomatal conductance were identical to that of L-plants. The stomatal response to ABA was lost in L-plants, but also after 1-day exposure of M-plants to low VPD. The level of foliar ABA sharply decreased within 1-day exposure to L, while the level of ABA-GE (ABA-glucose ester) was not affected. Spraying ABA during the exposure to L prevented loss of stomatal closing response thereafter. The effect of low VPD was largely depending on exposure time: the stomatal responsiveness to ABA was lost after 1-day exposure to low VPD, while the responsiveness to desiccation was gradually lost during 4-day exposure to low VPD. Leaf anatomical and stomatal morphological alterations due to low VPD were not the main cause of loss of stomatal closure response to closing stimuli.

High concentrations of Na+ and Cl- ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress.

Despite the fact that most plants accumulate both sodium (Na(+)) and chloride (Cl(-)) ions to high concentration in their shoot tissues when grown in saline soils, most research on salt tolerance in annual plants has focused on the toxic effects of Na(+) accumulation. There have also been some recent concerns about the ability of hydroponic systems to predict the responses of plants to salinity in soil. To address these two issues, an experiment was conducted to compare the responses to Na(+) and to Cl(-) separately in comparison with the response to NaCl in a soil-based system using two varieties of faba bean (Vicia faba), that differed in salinity tolerance. The variety Nura is a salt-sensitive variety that accumulates Na(+) and Cl(-) to high concentrations while the line 1487/7 is salt tolerant which accumulates lower concentrations of Na(+) and Cl(-). Soils were prepared which were treated with Na(+) or Cl(-) by using a combination of different Na(+) salts and Cl(-) salts, respectively, or with NaCl. While this method produced Na(+)-dominant and Cl(-)-dominant soils, it unavoidably led to changes in the availability of other anions and cations, but tissue analysis of the plants did not indicate any nutritional deficiencies or toxicities other than those targeted by the salt treatments. The growth, water use, ionic composition, photosynthesis, and chlorophyll fluorescence were measured. Both high Na(+) and high Cl(-) reduced growth of faba bean but plants were more sensitive to Cl(-) than to Na(+). The reductions in growth and photosynthesis were greater under NaCl stress and the effect was mainly additive. An important difference to previous hydroponic studies was that increasing the concentrations of NaCl in the soil increased the concentration of Cl(-) more than the concentration of Na(+). The data showed that salinity caused by high concentrations of NaCl can reduce growth by the accumulation of high concentrations of both Na(+) and Cl(-) simultaneously, but the effects of the two ions may differ. High Cl(-) concentration reduces the photosynthetic capacity and quantum yield due to chlorophyll degradation which may result from a structural impact of high Cl(-) concentration on PSII. High Na(+) interferes with K(+) and Ca(2+) nutrition and disturbs efficient stomatal regulation which results in a depression of photosynthesis and growth. These results suggest that the importance of Cl(-) toxicity as a cause of reductions in growth and yield under salinity stress may have been underestimated.

Exogenously applied ascorbic acid ameliorates detrimental effects of NaCl and mannitol stress in Vicia faba seedlings.

The adverse effects of either NaCl or mannitol on growth, nitrogen content, and antioxidant system in Vicia faba seedlings were investigated. The role of exogenous ascorbic acid in increasing resistance to these stressors was also evaluated. Thus, with an increase in concentration of either NaCl or mannitol in culture media, a progressively greater significant decrease in percentage germination, in growth parameters, and in nitrogen constituents of the germinating beans, was observed. On the other hand, amide-, nitrate-, and total soluble-N contents appeared to show a progressive significant increase. Exogenous addition of ascorbic acid (4 mM) to the stressful media induced a pronounced significantly increased percentage germination and the growth attributes, whereas nitrogen constituents were variably changed in relation to values maintained in beans treated with either NaCl or mannitol. Furthermore, exogenous addition of ascorbic acid to NaCl or mannitol media induced a significant increase in the contents of ascorbate and glutathione and enzymatic antioxidant activities, in particular, in beans treated with the three highest concentrations of NaCl or mannitol, throughout the period of the experiments (12 days). Thus, ascorbic acid ameliorates the adverse effects of the stressful media; the magnitude of amelioration being a function of the type and the concentration of the stressful agent as well as of the duration of treatment. The importance of the above-mentioned changes in growth and metabolism to stress tolerance in broad bean is discussed.

Photosynthesis in lightfleck areas of homobaric and heterobaric leaves.

Leaves within a canopy are exposed to a spatially and temporally fluctuating light environment which may cause lateral gradients in leaf internal CO(2) concentration and diffusion between shaded and illuminated areas. In previous studies it was hypothesized that lateral CO(2) diffusion may support leaf photosynthesis, but the magnitude of this effect is still not well understood. In the present study homobaric leaves of Vicia faba or heterobaric leaves of Glycine max were illuminated with lightflecks of different sizes, mimicking sunflecks. Photosynthetic properties of the lightfleck areas were assessed with combined gas exchange measurements and chlorophyll fluorescence imaging. Lateral diffusion in homobaric leaves with an interconnected intercellular air space stimulated photosynthesis and the effect was largest in small lightfleck areas, in particular when plants were under drought stress. Such effects were not observed in the heterobaric leaves with strongly compartmented intercellular gas spaces. It is concluded that lateral diffusion may significantly contribute to photosynthesis of lightfleck areas of homobaric leaves depending on lightfleck size, lateral diffusivity, and stomatal conductance. Since homobaric leaf structures have been reported for many plant species, it is hypothesized that leaf homobary may have an impact on overall plant performance under conditions with a highly heterogeneous light environment.

Quantitative genetic analysis of embryo heterosis in faba bean (Vicia faba L.).

Seeds, i.e. embryos, may be genetically different from either of their parents and moreover may express their own heterosis. The objective was to genetically analyse embryo heterosis for their own weight (i.e. seed weight) in comparison with their seedlings' heterosis, taking the large-seeded crop (Vicia faba L.) as model. A specific diallel mating scheme was used, based on four parental lines, creating 76 seed genotypes in generations P, F(1), F(2) and BC. Mature seed weight was assessed for these embryo genotypes in 3 years at one German location, and young plant biomass yield of seedlings emerging from these seeds in two greenhouse experiments. The quantitative genetic analysis showed an average of 10.6% mid-parent heterosis for mature seed weight and 14.5% mid-parent heterosis for juvenile biomass. In both traits, the embryos contributed markedly and significantly via their own genes to the genetic variation. For mature embryo weight heterosis, apparently the parental difference in seed weight was decisive, whereas for juvenile biomass heterosis, genetic unrelatedness of parents had priority.

Mechano-sensitive channels regulate the stomatal aperture in Vicia faba.

In the bright fields, stomata of the plants are fully opened to raise the transpiration rate and CO(2) uptake required for photosynthesis. Stomatal opening is driven by the activation of plasma membrane H(+)-ATPase and K(+)(in) channels, and the Ca(2+)-dependent inactivation and blockage of both components were supposed to be inevitable function to regulate the stomatal aperture. Although, it is still obscure how these activities are regulated at the open state. Application of an amphipathic membrane creator, trinitrophenol (TNP), instantly generates the convex curvature in the plasma membrane, which occurs in the phases of stomatal opening and closure. TNP surely activates mechanosensitive Ca(2+)-permeable channels and attenuates the promotion of stomatal opening, but does not inhibit and promote stomatal closure. These results suggest that activation of mechanosensitive Ca(2+)-permeable channels regulates the opening phase of stomata in plants.

Protective action of salicylic acid against bean yellow mosaic virus infection in Vicia faba leaves.

In this study, morphological, ultrastructural and physiological modifications of faba bean (Vicia faba cv Giza 461) leaves in response to bean yellow mosaic virus (BYMV) infection and salicylic acid (SA) treatments were examined. Under BYMV stress, leaves showed symptoms including severe mosaic, mottling, crinkling, size reduction and deformations. Three weeks after virus inoculation, photosynthetic rate, pigment contents and transpiration rate were significantly reduced in response to BYMV infection. Ultrastructural investigations of BYMV-infected leaves demonstrated that most chloroplasts with increased stromal area became spherical in shape and some lost their envelopes, either partially or totally. The internal structures of chloroplast, grana and thylakoids were dilated. Two kinds of inclusions were detected in BYMV-infected leaves: straight or slightly curved bands sometimes coiled or looped at the end, and electron opaque crystals with varied shapes. BYMV-infected cells showed lower chloroplast number in comparison to the control. Spraying of SA on faba bean leaves helped to reduce or prevent the harmful effects produced after virus infection. Application of 100 microM SA three days before inoculation restored the metabolism of infected leaves to the levels of healthy controls. SA treatment improved plant health by increasing the photosynthesis rates, pigment contents and levels of other parameters studied similar to control values. Moreover, SA treatment increased plant resistance against BYMV. This was observed through induction of chloroplast number, reduction in percentage of infected plants, decrease in disease severity and virus concentration of plants treated with SA prior to BYMV inoculation. Cells of SA-treated samples showed well-developed chloroplasts with many starch grains and well-organized cell organelles. The present results provide an overview of the negative effects on faba bean leaves due to BYMV infection from physiological and subcellular perspectives. Also, a role of SA involved in induction of resistance against BYMV infection in bean plants is discussed.

Arbuscular mycorrhizal fungi reduce the construction of extrafloral nectaries in Vicia faba.

Arbuscular mycorrhizal fungi (AMF) can alter the physiology and morphology of their host plant, and therefore may have indirect effects on insect herbivores and pollinators. We conducted this study to test the hypothesis that AMF can also affect insects involved in protection-for-food mutualisms. We examined the constitutive and inducible production of food rewards [extrafloral (EF) nectaries] in Vicia faba plants by manipulating the presence/absence of AMF and by simulating various levels of herbivory. Plants inoculated with AMF produced significantly fewer EF nectaries than uninoculated plants, even after accounting for differences in plant growth. In contrast to earlier studies, EF nectaries were not inducible: damaged plants produced significantly fewer EF nectaries than undamaged plants. Moreover, the effects of mycorrhizal and damage status on EF nectary production were additive. The reduction in EF nectaries in mycorrhizal plants potentially represents a mechanism for indirect effects of AMF on the protective insects that exploit EF nectaries as a food source (e.g., ants). Reduced reward size should result in reduced protection by ants, and could therefore be a previously unappreciated cost of the mycorrhizal symbiosis to host plants. However, the overall effect of AMF will depend upon the extent to which the reduction of EF nectaries affects the number and activity of ants and the extent to which AMF alter other aspects of host plant physiology. Our results emphasize the complexity of multitrophic interactions, particularly those that span belowground and aboveground ecology.

Transfer of phloem-mobile substances from the host plants to the holoparasite Cuscuta sp.

During the development of the haustorium, searching hyphae of the parasite and the host parenchyma cells are connected by plasmodesmata. Using transgenic tobacco plants expressing a GFP-labelled movement protein of the tobacco mosaic virus, it was demonstrated that the interspecific plasmodesmata are open. The transfer of substances in the phloem from host to the parasite is not selective. After simultaneous application of (3)H-sucrose and (14)C-labelled phloem-mobile amino acids, phytohormones, and xenobiotica to the host, corresponding percentages of the translocated compounds are found in the parasite. An open continuity between the host phloem and the Cuscuta phloem via the haustorium was demonstrated in CLSM pictures after application of the phloem-mobile fluorescent probes, carboxyfluorescein (CF) and hydroxypyrene trisulphonic acid (HPTS), to the host. Using a Cuscuta bridge (14)C-sucrose and the virus PVY(N) were transferred from one host plant to the another. The results of translocation experiments with labelled compounds, phloem-mobile dyes and the virus should be considered as unequivocal evidence for a symplastic transfer of phloem solutes between Cuscuta species and their compatible hosts.

ALAMEDA, a structural-functional model for faba bean crops: morphological parameterization and verification.

BACKGROUND: Plant structural (i.e. architectural) models explicitly describe plant morphology by providing detailed descriptions of the display of leaf and stem surfaces within heterogeneous canopies and thus provide the opportunity for modelling the functioning of plant organs in their microenvironments. The outcome is a class of structural-functional crop models that combines advantages of current structural and process approaches to crop modelling. ALAMEDA is such a model. METHODS: The formalism of Lindenmayer systems (L-systems) was chosen for the development of a structural model of the faba bean canopy, providing both numerical and dynamic graphical outputs. It was parameterized according to the results obtained through detailed morphological and phenological descriptions that capture the detailed geometry and topology of the crop. The analysis distinguishes between relationships of general application for all sowing dates and stem ranks and others valid only for all stems of a single crop cycle. RESULTS AND CONCLUSIONS: The results reveal that in faba bean, structural parameterization valid for the entire plant may be drawn from a single stem. ALAMEDA was formed by linking the structural model to the growth model 'Simulation d'Allongement des Feuilles' (SAF) with the ability to simulate approx. 3500 crop organs and components of a group of nine plants. Model performance was verified for organ length, plant height and leaf area. The L-system formalism was able to capture the complex architecture of canopy leaf area of this indeterminate crop and, with the growth relationships, generate a 3D dynamic crop simulation. Future development and improvement of the model are discussed.

Lateral diffusion of CO2 from shaded to illuminated leaf parts affects photosynthesis inside homobaric leaves.

Gas exchange is generally regarded to occur between the leaf interior and ambient air, i.e. in vertical (anticlinal) directions of leaf blades. However, inside homobaric leaves, gas movement occurs also in lateral directions. The aim of the present study was to ascertain whether lateral CO2 diffusion affects leaf photosynthesis when illuminated leaves are partially shaded. Measurements using gas exchange and chlorophyll fluorescence imaging techniques were performed on homobaric leaves of Vicia faba and Nicotiana tabacum or on heterobaric leaves of Glycine max and Phaseolus vulgaris. For homobaric leaves, gas exchange inside a clamp-on leaf chamber was affected by shading the leaf outside the chamber. The quantum yield of photosystem II (Phi(PSII)) was highest directly adjacent to a light/shade border (LSB). Phi(PSII) decreased in the illuminated leaf parts with distance from the LSB, while the opposite was observed for nonphotochemical quenching. These effects became most pronounced at low stomatal conductance. They were not observed in heterobaric leaves. The results suggest that plants with homobaric leaves can benefit from lateral CO2 flux, in particular when stomata are closed (e.g. under drought stress). This may enhance photosynthetic, instead of nonphotochemical, processes near LSBs in such leaves and reduce the photoinhibitory effects of excess light.

Size selectivity of aqueous pores in stomatous cuticles of Vicia faba leaves.

Size selectivity of aqueous pores in Vicia leaf cuticles was investigated by measuring the penetration of calcium salts into the abaxial surface of detached leaves. Molecular weights of salts ranged from 111 g mol(-1) to 755 g mol(-1). Penetration in light at 20 degrees C and 100% humidity was a first order process and rate constants of penetration ranged from 0.39 h(-1) (CaCl2) to 0.058 h(-1) (Ca-lactobionate). Penetration was a first order process in the dark as well, but the rate constants were smaller by a factor of 1.82. Plotting logarithmatised rate constants versus anhydrous molecular weights resulted in straight lines both in light and in the dark. The slopes per hour were very similar and the average slope was -1.2 x 10(-3) mol g(-1). Hence, size selectivity was not affected by stomatal opening, and in light or darkness permeability of Vicia cuticles decreased by a factor of 2.9 when molecular weight increased from 100 g mol(-1) to 500 g mol(-1). Silver nitrate was preferentially precipitated as silver chloride in guard cells, glandular trichomes and at the base of trichomes. It was concluded that these precipitates mark the location of aqueous pores in Vicia leaf cuticles. The size selectivity of aqueous pores in Vicia leaf cuticles is small compared to that observed in poplar leaf cuticles, in which permeability decreased by a factor of 7-13 for the same range of molecular weights. It is also much smaller than size selectivity of the lipophilic pathway in cuticles. These findings suggest that active ingredients of pesticides, growth regulators and chemical inducers with high molecular weights penetrate leaves at higher rates when formulated as ions.