Accelerated Growth Rate and Increased Drought Stress Resilience of the Model Grass Brachypodium distachyon Colonized by Bacillus subtilis B26.
ABSTRACT: Plant growth-promoting bacteria (PGB) induce positive effects in plants, for instance, increased growth and reduced abiotic stresses susceptibility. The mechanisms by which these bacteria impact the host plant are numerous, diverse and often specific. Here, we studied the agronomical, molecular and biochemical effects of the endophytic PGB Bacillus subtilis B26 on the full life cycle of Brachypodium distachyon Bd21, an established model species for functional genomics in cereal crops and temperate grasses. Inoculation of Brachypodium with B. subtilis strain B26 increased root and shoot weights, accelerated growth rate and seed yield as compared to control plants. B. subtilis strain B26 efficiently colonized the plant and was recovered from roots, stems and blades as well as seeds of Brachypodium, indicating that the bacterium is able to migrate, spread systemically inside the plant, establish itself in the aerial plant tissues and organs, and is vertically transmitted to seeds. The presence of B. subtilis strain B26 in the seed led to systemic colonization of the next generation of Brachypodium plants. Inoculated Brachypodium seedlings and mature plants exposed to acute and chronic drought stress minimized the phenotypic effect of drought compared to plants not harbouring the bacterium. Protection from the inhibitory effects of drought by the bacterium was linked to upregulation of the drought-response genes, DREB2B-like, DHN3-like and LEA-14-A-like and modulation of the DNA methylation genes, MET1B-like, CMT3-like and DRM2-like, that regulate the process. Additionally, total soluble sugars and starch contents increased in stressed inoculated plants, a biochemical indication of drought tolerance. In conclusion, we show a single inoculation of Brachypodium with a PGB affected the whole growth cycle of the plant, accelerating its growth rates, shortening its vegetative period, and alleviating drought stress effects. These effects are relevant to grasses and cereal crops.
Project description:Drought is a major limiting factor of crop productivity worldwide and its incidence is predicted to increase under climate change. Drought adaptation of cool-season grasses is thus a major challenge to secure the agricultural productivity under current and future climate conditions. Endophytes are non-pathogenic plant-associated bacteria that can play an important role in conferring resistance and improving plant tolerance to drought. In this study, the effect of inoculation of the bacterial endophyte Bacillus subtilis strain B26 on growth, water status, photosynthetic activity and metabolism of timothy (Phleum pratense L.) subjected to drought stress was investigated under controlled conditions. Under both drought-stress and non-stressed conditions, strain B26 successfully colonized the internal tissues of timothy and had a positive impact on plant growth. Exposure of inoculated plant to a 8-week drought-stress led to significant increase in shoot and root biomass by 26.6 and 63.8%, and in photosynthesis and stomatal conductance by 55.2 and 214.9% respectively, compared to non-inoculated plants grown under similar conditions. There was a significant effect of the endophyte on plant metabolism; higher levels of several sugars, notably sucrose and fructans and an increase of key amino acids such as, asparagine, glutamic acid and glutamine were recorded in shoots and roots of colonized plants compared to non-colonized ones. The accumulation of the non-protein amino acid GABA in shoots of stressed plants and in roots of stressed and unstressed plants was increased in the presence of the endophyte. Taken together, our results indicate that B. subtilis B26 improves timothy growth under drought stress through the modification of osmolyte accumulation in roots and shoots. These results will contribute to the development of a microbial agent to improve the yield of grass species including forage crops and cereals exposed to environmental stresses.
Project description:Drought is an important environmental stress limiting the productivity of major crops worldwide. Understanding drought tolerance and possible mechanisms for improving drought resistance is therefore a prerequisite to develop drought-tolerant crops that produce significant yields with reduced amounts of water. <i>Brachypodium distachyon</i> (Brachypodium) is a key model species for cereals, forage grasses, and energy grasses. In this study, initial screening of a Brachypodium germplasm collection consisting of 138 different ecotypes exposed to progressive drought, highlighted the natural variation in morphology, biomass accumulation, and responses to drought stress. A core set of ten ecotypes, classified as being either tolerant, susceptible or intermediate, in response to drought stress, were exposed to mild or severe (respectively, 15 and 0% soil water content) drought stress and phenomic parameters linked to growth and color changes were assessed. When exposed to severe drought stress, phenotypic data and metabolite profiling combined with multivariate analysis revealed a remarkable consistency in separating the selected ecotypes into their different pre-defined drought tolerance groups. Increases in several metabolites, including for the phytohormones jasmonic acid and salicylic acid, and TCA-cycle intermediates, were positively correlated with biomass yield and with reduced yellow pixel counts; suggestive of delayed senescence, both key target traits for crop improvement to drought stress. While metabolite analysis also separated ecotypes into the distinct tolerance groupings after exposure to mild drought stress, similar analysis of the phenotypic data failed to do so, confirming the value of metabolomics to investigate early responses to drought stress. The results highlight the potential of combining the analyses of phenotypic and metabolic responses to identify key mechanisms and markers associated with drought tolerance in both the Brachypodium model plant as well as agronomically important crops.
Project description:Dehydration proteins (dehydrins, DHNs) confer tolerance to water-stress deficit in plants. We performed a comparative genomics and evolutionary study of DHN genes in four model <i>Brachypodium</i> grass species. Due to limited knowledge on dehydrin expression under water deprivation stress in <i>Brachypodium,</i> we also performed a drought-induced gene expression analysis in 32 ecotypes of the genus' flagship species <i>B. distachyon</i> showing different hydric requirements. Genomic sequence analysis detected 10 types of dehydrin genes (<i>Bdhn</i>) across the <i>Brachypodium</i> species. Domain and conserved motif contents of peptides encoded by <i>Bdhn</i> genes revealed eight protein architectures. <i>Bdhn</i> genes were spread across several chromosomes. Selection analysis indicated that all the <i>Bdhn</i> genes were constrained by purifying selection. Three upstream <i>cis</i>-regulatory motifs (BES1, MYB124, ZAT) were detected in several <i>Bdhn</i> genes. Gene expression analysis demonstrated that only four <i>Bdhn</i>1-<i>Bdhn</i>2, <i>Bdhn</i>3, and <i>Bdhn</i>7 genes, orthologs of wheat, barley, rice, sorghum, and maize genes, were expressed in mature leaves of <i>B. distachyon</i> and that all of them were more highly expressed in plants under drought conditions. <i>Brachypodium</i> dehydrin expression was significantly correlated with drought-response phenotypic traits (plant biomass, leaf carbon and proline contents and water use efficiency increases, and leaf water and nitrogen content decreases) being more pronounced in drought-tolerant ecotypes. Our results indicate that dehydrin type and regulation could be a key factor determining the acquisition of water-stress tolerance in grasses.
Project description:<h4>Background and aims</h4>Brachypodium distachyon (Brachypodium) is a model system for studying cereal, bioenergy, forage and turf grasses. The genetic and evolutionary basis of the adaptation of this wild grass species to drought stress is largely unknown. Peroxidase (POD) may play a role in plant drought tolerance, but whether the allelic variations of genes encoding the specific POD isoenzymes are associated with plant response to drought stress is not well understood. The objectives of this study were to examine natural variation of POD isoenzyme patterns, to identify nucleotide diversity of POD genes and to relate the allelic variation of genes to drought tolerance traits of diverse Brachypodium accessions.<h4>Methods</h4>Whole-plant drought tolerance and POD activity were examined in contrasting ecotypes. Non-denaturing PAGE and liquid chromatography-mass spectrometry were performed to detect distinct isozymes of POD in 34 accessions. Single nucleotide polymorphisms (SNPs) were identified by comparing DNA sequences of these accessions. Associations of POD genes encoding specific POD isoenzymes with drought tolerance traits were analysed using TASSEL software.<h4>Key results</h4>Variations of POD isoenzymes were found among accessions with contrasting drought tolerance, while the most tolerant and susceptible accessions each had their own unique POD isoenzyme band. Eight POD genes were identified and a total of 90 SNPs were found among these genes across 34 accessions. After controlling population structure, significant associations of Bradi3g41340.1 and Bradi1g26870.1 with leaf water content or leaf wilting were identified.<h4>Conclusions</h4>Brachypodium ecotypes have distinct specific POD isozymes. This may contribute to natural variations of drought tolerance of this species. The role of specific POD genes in differentiating Brachypodium accessions with contrasting drought tolerance could be associated with the general fitness of Brachypodium during evolution.
Project description:MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKC(c)-type, 7 MIKC*-type, 9 M?, 7 M? and 2 M? MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.
Project description:<h4>Background</h4>Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor composed of NF-YA, NF-YB and NF-YC proteins. Using the dicot plant model system Arabidopsis thaliana (Arabidopsis), NF-Y were previously shown to control a variety of agronomically important traits, including drought tolerance, flowering time, and seed development. The aim of the current research was to identify and characterize NF-Y families in the emerging monocot model plant Brachypodium distachyon (Brachypodium) with the long term goal of assisting in the translation of known dicot NF-Y functions to the grasses.<h4>Methodology/principal findings</h4>We identified, annotated, and further characterized 7 NF-YA, 17 NF-YB, and 12 NF-YC proteins in Brachypodium (BdNF-Y). By examining phylogenetic relationships, orthology predictions, and tissue-specific expression patterns for all 36 BdNF-Y, we proposed numerous examples of likely functional conservation between dicots and monocots. To test one of these orthology predictions, we demonstrated that a BdNF-YB with predicted orthology to Arabidopsis floral-promoting NF-Y proteins can rescue a late flowering Arabidopsis mutant.<h4>Conclusions/significance</h4>The Brachypodium genome encodes a similar complement of NF-Y to other sequenced angiosperms. Information regarding NF-Y phylogenetic relationships, predicted orthologies, and expression patterns can facilitate their study in the grasses. The current data serves as an entry point for translating many NF-Y functions from dicots to the genetically tractable monocot model system Brachypodium. In turn, studies of NF-Y function in Brachypodium promise to be more readily translatable to the agriculturally important grasses.
Project description:The temperate wild grass <i>Brachypodium distachyon</i> (Brachypodium) serves as model system for studying turf and forage grasses. Brachypodium collections show diverse responses to drought stress, but little is known about the genetic mechanisms of drought tolerance of this species. The objective of this study was to identify quantitative trait loci (QTLs) associated with drought tolerance traits in Brachypodium. We assessed leaf fresh weight (LFW), leaf dry weight (LDW), leaf water content (LWC), leaf wilting (WT), and chlorophyll fluorescence (Fv/Fm) under well-watered and drought conditions on a recombinant inbred line (RIL) population from two parents (Bd3-1 and Bd1-1) known to differ in their drought adaptation. A linkage map of the RIL population was constructed using 467 single nucleotide polymorphism (SNP) markers obtained from genotyping-by-sequencing. The Bd3-1/Bd1-1 map spanned 1,618 cM and had an average distance of 3.5 cM between adjacent single nucleotide polymorphisms (SNPs). Twenty-six QTLs were identified in chromosome 1, 2, and 3 in two experiments, with 14 of the QTLs under well-watered conditions and 12 QTLs under drought stress. In Experiment 1, a QTL located on chromosome 2 with a peak at 182 cM appeared to simultaneously control WT, LWC, and Fv/Fm under drought stress, accounting for 11-18.7% of the phenotypic variation. Allelic diversity of candidate genes <i>DREB2B, MYB</i>, and <i>SPK</i>, which reside in one multi-QTL region, may play a role in the natural variation in whole plant drought tolerance in Brachypodium. Co-localization of QTLs for multiple drought-related traits suggest that the gene(s) involved are important regulators of drought tolerance in Brachypodium.
Project description:BACKGROUND: Adverse environmental conditions severely influence various aspects of plant growth and developmental processes, causing worldwide reduction of crop yields. The C-repeat binding factors (CBFs) are critical transcription factors constituting the gene regulatory network that mediates the acclimation process to low temperatures. They regulate a large number of cold-responsive genes, including COLD-REGULATED (COR) genes, via the CBF-COR regulon. Recent studies have shown that the CBF transcription factors also play a role in plant responses to drought and salt stresses. Putative CBF gene homologues and their downstream genes are also present in the genome of Brachypodium distachyon, which is perceived as a monocot model in recent years. However, they have not been functionally characterized at the molecular level. RESULTS: Three CBF genes that are responsive to cold were identified from Brachypodium, designated BdCBF1, BdCBF2, and BdCBF3, and they were functionally characterized by molecular biological and transgenic approaches in Brachypodium and Arabidopsis thaliana. Our results demonstrate that the BdCBF genes contribute to the tolerance response of Brachypodium to cold, drought, and salt stresses by regulating downstream targets, such as DEHYDRIN5.1 (Dhn5.1) and COR genes. The BdCBF genes are induced under the environmental stress conditions. The BdCBF proteins possess transcriptional activation activity and bind directly to the promoters of the target genes. Transgenic Brachypodium plants overexpressing the BdCBF genes exhibited enhanced resistance to drought and salt stresses as well as low temperatures, and accordingly endogenous contents of proline and soluble sugars were significantly elevated in the transgenic plants. The BdCBF transcription factors are also functional in the heterologous system Arabidopsis. Transgenic Arabidopsis plants overexpressing the BdCBF genes were also tolerant to freezing, drought, and salt stresses, and a set of stress-responsive genes was upregulated in the transgenic Arabidopsis plants. CONCLUSIONS: Taken together, our results strongly support that the BdCBF transcription factors are key regulators of cold stress responses in Brachypodium and the CBF-mediated cold stress signaling pathway is conserved in this plant species. We believe that this study would confer great impact on stress biology in monocot species and could be applied to engineer abiotic stress tolerance of bioenergy grass species.
Project description:BACKGROUND:Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice), the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar). To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model) and Sorghum bicolor (sorghum). We then compared the glycoside hydrolases across species, at the levels of the whole genome and individual glycoside hydrolase families. RESULTS:We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51), we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. CONCLUSIONS:This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets the stage for Brachypodium to be a grass model for investigations of these enzymes and their diverse roles in planta. Insights gained from Brachypodium will inform translational research studies, with applications for the improvement of cereal crops and bioenergy grasses.
Project description:<h4>Background</h4>The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses.<h4>Results</h4>We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis.<h4>Conclusion</h4>In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.