Molecular bases for drought tolerance in common vetch: designing new molecular breeding tools.

BACKGROUND: Common vetch (Vicia sativa L.) is a forage grain legume of high protein content and high nitrogen fixation, relevant in sustainable agriculture systems. Drought is the main limiting factor of this crop yield. Genetic resources collections are essential to provide genetic variability for breeding. The analysis of drought associated parameters has allowed us to identify drought tolerant and sensitive ecotypes in a vetch core collection. RESULTS: To understand the mechanisms involved in drought response we analysed transcriptomic differences between tolerant and sensitive accessions. Polymorphic variants (SNPs and SSRs) in these differential expressed genes (DEGs) have also been analysed for the design of drought-associated markers. A total of 1332 transcripts were commonly deregulated in both genotypes under drought. To know the drought adaptive response, we also analysed DEGs between accessions. A total of 2646 transcripts are DEG between sensitive and tolerant ecotypes, in watered and drought conditions, including important genes involved in redox homeostasis, cell wall modifications and stress-response. The integration of this functional and genetic information will contribute to understand the molecular mechanisms of drought response and the adaptive mechanisms of drought tolerance in common vetch. The identification of polymorphic variants in these DEGs has also been screened for the design of drought-associated markers that could be used in future breeding program strategies. CONCLUSIONS: Our studies shed light for the first time in common vetch about the genes and pathways associated with drought tolerance. In addition, we identify over 100 potential drought associated polymorphism, as SNPs or SSRs, which are differently present in drought and tolerant genotypes. The use of these molecular markers for trait prediction would enable the development of genomic tools for future engineering strategies by screening of germplasm crop collections for traits related with crop drought resilience, adaptability or yield in vetch.

Impact of priming on seed germination, seedling growth and gene expression in common vetch under salinity stress.

Salinity is one of the most important abiotic stress factors that is expanding its influence because of global climate change and global warming. It causes gene expression changes, a reduction in seed germination and related characteristics, and poor seedling establishment in many crop plants by creating a lower osmotic potential in the seedbed and/or toxic ion effects in germinated seeds. In recent years, seed priming has been considered a promising strategy in modern stress management to protect plants against stress conditions. This study was conducted to elucidate the effects of osmopriming with polyethylene glycol 6000 (PEG-6000) on seed germination, seedling growth and gene expression in the common vetch (Vicia sativa L.) in different saline conditions. Common vetch seeds were primed with PEG-6000 solutions having different osmotic potentials (0.00, -0.50,  -0.75, -1.00, -1.25, and -1.50 MPa) for 12 hours. Control (un-primed) and primed seeds were germinated and seedlings were grown in different saline conditions (EC= zero, 4, 8 and 16 dS m-1). Furthermore, gene expression was compared in the primed seedlings in two different osmotic potentials (0.00 and -1.50 MPa) by microarray technology. Results demonstrated that germination percentage of common vetch seeds and seedling growth were diminished by high salinity. However, several priming treatments alleviated the adverse effects of high salinity on germination and early seedling growth of common vetch. The microarray showed that the expression of many genes in both stress and normal conditions was not significantly different.

Transcriptome-Wide Characterization and Functional Identification of the Aquaporin Gene Family During Drought Stress in Common Vetch.

Aquaporins (AQPs) are transmembrane channels that are essential for the movement of water and other small molecules between biofilms in various physiological processes in plants. In this study, based on transcriptome-wide data, we identified and described a total of 21 AQP genes in common vetch (Vicia sativa subsp. sativa), which is an economically important pasture legume worldwide. Based on phylogenetic analyses, the VsAQPs were sorted into four subfamilies, including four plasma membrane intrinsic proteins (PIPs), six tonoplast intrinsic proteins (TIPs), seven NOD26-like intrinsic proteins, and four small basic intrinsic proteins. Furthermore, chemical and physical properties of these VsAQPs, including the isoelectric point and theoretical molecular weight, were analyzed. Analyses of the AQP signature sequences and key residues indicated the substrate specificity of each VsAQP. A set of VsAQPs was selected for gene expression analysis in a number of tissues and after drought stress treatments using real-time quantitative reverse transcription/polymerase chain reaction assays. Most of the PIPs and TIPs were proposed to have critical roles in regulating the flow of water during drought stress. Heterologous expression experiments in yeast indicated that VsPIP1;2 and VsPIP2;2 are key candidate genes for improving drought stress tolerance. The results reported in this study could be a crucial resource for further practical analyses and for genetic improvement of drought stress tolerance in common vetch.

De Novo Assembly and Discovery of Genes That Involved in Drought Tolerance in the Common Vetch.

The common vetch (Vicia sativa) is often used as feed for livestock because of its high nutritional value. However, drought stress reduces forage production through plant damage. Here, we studied the transcriptional profiles of common vetch exposed to drought in order to understand the molecular mechanisms of drought tolerance in this species. The genome of the common vetch has not been sequenced, therefore we used Illumina sequencing to generate de novo transcriptomes. Nearly 500 million clean reads were used to generate 174,636 transcripts, including 122,299 unigenes. In addition, 5313 transcription factors were identified and these transcription factors were classified into 79 different gene families. We also identified 11,181 SSR loci from di- to hexa-nucleotides whose repeat number was greater than five. On the basis of differentially expressed genes, Gene Ontology analysis identified many drought-relevant categories, including "oxidation-reduction process", "lipid metabolic process" and "oxidoreductase activity". In addition to these, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified pathways, such as "Plant hormone signal transduction", "Glycolysis/Gluconeogenesis" and "Phenylpropanoid biosynthesis", as differentially expressed in the plants exposed to drought. The expression results in this study will be useful for further extending our knowledge on the drought tolerance of common vetch.

Effect of Instant Controlled Pressure-Drop (DIC), Cooking and Germination on Non-Nutritional Factors of Common Vetch (Vicia sativa spp.).

Legumes are widely consumed by humans, being an important source of nutrients; however, they contain non-nutritional factors (NNFs), such as phytic acid (IP6), raffinose, stachyose, total phenolic compounds, condensed tannins, and flavonoids, that have negative effects on human health. Although vetches (Vicia sativa) are widely cultivated, they are not intended for human feeding due to their contents of NNF. Usually, the NNF are removed by cooking or germinating; however, germination is a process that requires extended time, and cooking may compromise the viability of some nutrients. To promote vetches for human consumption, the effect of the Instant Controlled Pressure Drop (DIC) process was studied as an alternative to cooking and germinating to decrease NNF contents. Results showed that compared to raw vetches, DIC treatment reduced total phenolic compounds (48%), condensed tannins (28%), flavonoids (65%), IP6 (92%), raffinose (77%), and stachyose (92%). These results are very similar to the ones achieved by traditional ways of removing NNF.

Potential value of the common vetch (Vicia sativa L.) as an animal feedstuff: a review.

The objective of this review was to systematically evaluate common vetch seeds as a potential feedstuff for animals, by summarizing and discussing the available published literature covering their nutritional composition as well as their content of antinutritional factors and potential techniques for their reduction. In addition, animal feeding studies that have investigated the effect of inclusion of common vetch seeds on animal growth and performance were identified and evaluated to stimulate interest in their use as a good source of nutrients for inclusion in animal diets. The collective literature shows that common vetch seeds are a less costly (in comparison with alternatives) and rich source of protein and minerals for farmed animals, are of high digestibility and have a high energy content, and can be used to partially or totally replace soya bean meal and/or to replace a large proportion of cereals in the diet. Furthermore, the literature shows that common vetch seeds contain a range of antinutritional factors which, if they are to be utilized in non-ruminant diets and to increase their utilizing efficiency, need to be removed or inactivated. This can be achieved via certain pre-processing methods, the combination of which may deliver better results.

Effects of copper on germination and reserve mobilization in Vicia sativa L. seeds.

The effects of different copper concentrations on percentage germination, increase in fresh weight and radicle growth of Vicia sativa L. seeds were studied. Physiological studies showed that the germination rate was not affected up to a concentration of 5 × 10⁻³ M, but already at 10⁻³ M the copper stopped root elongation. Structural and ultrastructural observations of embryo and cotyledon reserve mobilization showed that inhibition of radicle growth at 10⁻³ M Cu concentration cannot be ascribed to nutrient shortage but probably to an effect of copper on radicle cell division and elongation. In seeds treated with 5 × 10⁻³ M CuBr2, the copper completely inhibited cotyledon protein mobilization, so that embryo protein mobilization supported normal growth of the radicle up to 30 h after imbibition. The particular protein content of adjacent cotyledon cells is also discussed.

Nutrient limitation of native and invasive N2-fixing plants in northwest prairies.

Nutrient rich conditions often promote plant invasions, yet additions of non-nitrogen (N) nutrients may provide a novel approach for conserving native symbiotic N-fixing plants in otherwise N-limited ecosystems. Lupinus oreganus is a threatened N-fixing plant endemic to prairies in western Oregon and southwest Washington (USA). We tested the effect of non-N fertilizers on the growth, reproduction, tissue N content, and stable isotope δ(15)N composition of Lupinus at three sites that differed in soil phosphorus (P) and N availability. We also examined changes in other Fabaceae (primarily Vicia sativa and V. hirsuta) and cover of all plant species. Variation in background soil P and N availability shaped patterns of nutrient limitation across sites. Where soil P and N were low, P additions increased Lupinus tissue N and altered foliar δ(15)N, suggesting P limitation of N fixation. Where soil P was low but N was high, P addition stimulated growth and reproduction in Lupinus. At a third site, with higher soil P, only micro- and macronutrient fertilization without N and P increased Lupinus growth and tissue N. Lupinus foliar δ(15)N averaged -0.010‰ across all treatments and varied little with tissue N, suggesting consistent use of fixed N. In contrast, foliar δ(15)N of Vicia spp. shifted towards 0‰ as tissue N increased, suggesting that conditions fostering N fixation may benefit these exotic species. Fertilization increased cover, N fixation, and tissue N of non-target, exotic Fabaceae, but overall plant community structure shifted at only one site, and only after the dominant Lupinus was excluded from analyses. Our finding that non-N fertilization increased the performance of Lupinus with few community effects suggests a potential strategy to aid populations of threatened legume species. The increase in exotic Fabaceae species that occurred with fertilization further suggests that monitoring and adaptive management should accompany any large scale applications.

The effect of diesel fuel on common vetch (Vicia sativa L.) plants.

When petroleum hydrocarbons contaminate soil, the carbon:nitrogen (C:N) ratio of the soil is altered. The added carbon stimulates microbial numbers but causes an imbalance in the C:N ratio which may result in immobilization of soil nitrogen by the microbial biomass, leaving none available for plant growth. As members of Leguminosae fix atmospheric nitrogen to produce their own nitrogen for growth, they may prove more successful at growing on petroleum hydrocarbon contaminated sites. During a wider study on phytoremediation of diesel fuel contaminated soil, particular attention was given to the performance of legumes versus other plant species. During harvesting of pot experiments containing leguminous plants, a recurring difference in the number and formation of root nodules present on control and contaminated Common vetch (Vicia sativa L.) plants was observed. The total number of nodules per plant was significant reduced in contaminated plants compared to control plants but nodules on contaminated plants were more developed than corresponding nodules on control plants. Plant performance of Common vetch and Westerwold's ryegrass (Lolium multiflorum L.) was compared to illustrate any difference between the ability of legumes and grasses to grow on diesel fuel contaminated soil. Common vetch was less affected by diesel fuel and performed better in low levels of diesel fuel contaminated soil than Westerwold's ryegrass. The total amount of diesel fuel remaining after 4 months in Common vetch planted soil was slightly less than in Westerwold's ryegrass planted soil.

[Regulation of fertilizer application on yield and root growth of spring wheat-faba bean intercropping system].

The effects of N and P fertilizer application on the multiple population yield and root growth of spring wheat (Triticum aestivum)--spring faba bean(Vicia sativa)intercropping system were studied by two field experiments with 0, 150 and 225 kgN.hm-2, three pot and pool experiments with 2 N applications (0, 100 mg N.kg-1 soil) and 3 P applications (0, 100 and 200 mg P.kg-1 soil). The results showed that this intercropping system had a significant advantage of yield and beneficial effects. Compared to Po supply, P fertilization increased the seed yield by 48.39% for intercropped spring wheat and 16.69% for intercropped faba bean in field experiment. Furthermore, the total seed yield of both crops was increased by 20.07% to 43.14% in pool culture, and the grain yield of intercropped faba bean was increased by 58.46% to 78.78%. In the intercropping system, the growth peak of root density of both crops was appeared alternately, the peak of wheat root growth being earlier than that of faba bean. The maximum root weight of intercropped wheat was in its early heading periods, whereas that of faba bean appeared at its maturity periods, which reduced the competition for water and nutrients between the crops, and resulted in yield increase. The weight, length and surface area of wheat roots in P supply were increased by 54.33%, 48.88% and 47.00%, and in N supply, they were increased by 15.25%, 11.61% and 11.46%, respectively. About 57.61% of wheat root weight and 69.20% of faba bean root weight were distributed at 0-30 cm soil depths in the treatments of receiving P fertilizer at various rates, which indicated that P supply increased root weight and length of both crops in the intercropping system.