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Grasses form morphologically derived, four-celled stomata, where two dumbbell-shaped guard cells (GCs) are flanked by two lateral subsidiary cells (SCs). This innovative form enables rapid opening and closing kinetics and efficient plant-atmosphere gas exchange. The mobile bHLH transcription factor MUTE is required for SC formation in grasses. Yet whether and how MUTE also regulates GC development and whether MUTE mobility is required for SC recruitment is unclear. Here, we transgenically impaired BdMUTE mobility from GC to SC precursors in the emerging model grass Brachypodium distachyon. Our data indicate that reduced BdMUTE mobility severely affected the spatiotemporal coordination of GC and SC development. Furthermore, although BdMUTE has a cell-autonomous role in GC division orientation, complete dumbbell morphogenesis of GCs required SC recruitment. Finally, leaf-level gas exchange measurements showed that dosage-dependent complementation of the four-celled grass morphology was mirrored in a gradual physiological complementation of stomatal kinetics. Together, our work revealed a dual role of grass MUTE in regulating GC division orientation and SC recruitment, which in turn is required forGCmorphogenesis and the rapid kinetics of grass stomata. [ABSTRACT FROM AUTHOR]

The Multidrug and Toxic Compound Extrusion (MATE) proteins serve as pivotal transporters responsible for the extrusion of metabolites, thereby playing a significant role in both plant development and the detoxification of toxins. The MATE gene family within the Brachypodium distachyon, which is an important model organism of the Poaceae family, remains largely unexplored. Here, a comprehensive identification and analysis of MATE genes that complement B. distachyon were conducted. The BdMATE genes were systematically categorized into five distinct groups, predicated on an assessment of their phylogenetic affinities and protein structure. Furthermore, our investigation revealed that dispersed duplication has significantly contributed to the expansion of the BdMATE genes, with tandem and segmental duplications showing important roles, suggesting that the MATE genes in Poaceae species have embarked on divergent evolutionary trajectories. Examination of ω values demonstrated that BdMATE genes underwent purifying selection throughout the evolutionary process. Furthermore, collinearity analysis has confirmed a high conservation of MATE genes between B. distachyon and rice. The cis-regulatory elements analysis within BdMATEs promoters, coupled with expression patterns, suggests that BdMATEs play important roles during plant development and in response to phytohormones. Collectively, the findings presented establish a foundational basis for the subsequent detailed characterization of the MATE gene family members in B. distachyon. [ABSTRACT FROM AUTHOR]

Biocrusts are phototroph-driven communities inhabiting arid soil surfaces. Like plants, most photoautotrophs (largely cyanobacteria) in biocrusts are thought to exchange fixed carbon for essential nutrients like nitrogen with cyanosphere bacteria. Here, we aim to compare beneficial interactions in rhizosphere and cyanosphere environments, including finding growth-promoting strains for hosts from both environments. To examine this, we performed a retrospective analysis of 16S rRNA gene sequencing datasets, host-microbe co-culture experiments between biocrust communities/biocrust isolates and a model grass (Brachypodium distachyon) or a dominant biocrust cyanobacterium (Microcoleus vaginatus), and metabolomic analysis. All 18 microbial phyla in the cyanosphere were also present in the rhizosphere, with additional 17 phyla uniquely found in the rhizosphere. The biocrust microbes promoted the growth of the model grass, and three biocrust isolates (Bosea sp._L1B56, Pseudarthrobacter sp._L1D14 and Pseudarthrobacter picheli_L1D33) significantly promoted the growth of both hosts. Moreover, pantothenic acid was produced by Pseudarthrobacter sp._L1D14 when grown on B. distachyon exudates, and supplementation of plant growth medium with this metabolite increased B. distachyon biomass by over 60%. These findings suggest that cyanobacteria and other diverse photoautotrophic hosts can be a source for new plant growth-promoting microbes and metabolites.

Background Like all plant cells, the guard cells of stomatal complexes are encased in cell walls that are composed of diverse, interacting networks of polysaccharide polymers. The properties of these cell walls underpin the dynamic deformations that occur in guard cells as they expand and contract to drive the opening and closing of the stomatal pore, the regulation of which is crucial for photosynthesis and water transport in plants. Scope Our understanding of how cell wall mechanics are influenced by the nanoscale assembly of cell wall polymers in guard cell walls, how this architecture changes over stomatal development, maturation and ageing and how the cell walls of stomatal guard cells might be tuned to optimize stomatal responses to dynamic environmental stimuli is still in its infancy. Conclusion In this review, we discuss advances in our ability to probe experimentally and to model the structure and dynamics of guard cell walls quantitatively across a range of plant species, highlighting new ideas and exciting opportunities for further research into these actively moving plant cells. [ABSTRACT FROM AUTHOR]

In higher plants, the shift from vegetative to reproductive development is governed by complex interplay of internal and external signals. TERMINALFLOWER1 (TFL1) plays a crucial role in the regulation of flowering time and inflorescence architecture in Arabidopsis thaliana. This study aimed to explore the function of BdRCN4, a homolog of TFL1 in Brachypodium distachyon, through functional analyses in mutant and transgenic plants. The results revealed that overexpression of BdRCN4 in B. distachyon leads to an extended vegetative phase and reduced production of spikelets. Similar results were found in A. thaliana, where constitutive expression of BdRCN4 promoted a delay in flowering time, followed by the development of hypervegetative shoots, with no flowers or siliques produced. Our results suggest that BdRCN4 acts as a flowering repressor analogous to TFL1, negatively regulating AP1, but no LFY expression. To further validate this hypothesis, a 35S::LFY-GR co-transformation approach on 35::BdRCN4 lines was performed. Remarkably, AP1 expression levels and flower formation were restored to normal in co-transformed plants when treated with dexamethasone. Although further molecular studies will be necessary, the evidence in B. distachyon support the idea that a balance between LFY and BdRCN4/TFL1 seems to be essential for activating AP1 expression and initiating floral organ identity gene expression. This study also demonstrates interesting conservation through the molecular pathways that regulate flowering meristem transition and identity across the evolution of monocot and dicot plants.Key message: BdRCN4 acts as an ortholog of TFL1 in B. distachyon, promoting the indeterminate state of meristems and repressing floral transition, by downregulating AP1 through changes in LFY/BdRCN4(TFL1) ratio. [ABSTRACT FROM AUTHOR]

The model organism Neurospora crassa has been cultivated in laboratories since the 1920s and its saprotrophic lifestyle has been established for decades. However, beyond their role as saprotrophs, fungi engage in intricate relationships with plants, showcasing diverse connections ranging from mutualistic to pathogenic. Although N. crassa has been extensively investigated under laboratory conditions, its ecological characteristics remain largely unknown. In contrast, Brachypodium distachyon, a sweet grass closely related to significant crops, demonstrates remarkable ecological flexibility and participates in a variety of fungal interactions, encompassing both mutualistic and harmful associations. Through a comprehensive microscopic analysis using electron, fluorescence, and confocal laser scanning microscopy, we discovered a novel endophytic interaction between N. crassa and B. distachyon roots, where fungal hyphae not only thrive in the apoplastic space and vascular bundle but also may colonize plant root cells. This new and so far hidden trait of one of the most important fungal model organisms greatly enhances our view of N. crassa, opening new perspectives concerning the fungus' ecological role. In addition, we present a new tool for studying plant–fungus interspecies communication, combining two well-established model systems, which improves our possibilities of experimental design on the molecular level. [ABSTRACT FROM AUTHOR]

Homoserine dehydrogenase (HSD) is a key enzyme in the biosynthesis of aspartate-family amino acids such as L-homoserine and L-threonine. But HSD exhibited low activity and is feedback inhibited by L-threonine, which severely restricts the biosynthesis level of L-homoserine and L-threonine. In this study, eight HSDs from different species were mined through database search. Among of them, BdHSD derived from Brachypodium distachyon had the highest catalytic activity with 7. 6 U/ mg, and was not feedback-inhibited by L-threonine. The optimal catalytic pH of BdHSD was 10.5, and the optimal catalytic temperature was 38 °C . To improve the catalytic activity of BdHSD, this study further performed the directed evolution of BdHSD, and three BdHSD mutants T186A, N283K, and A137T/ I188V with higher catalytic activity were obtained through multiple rounds of screening. The enzyme activity of the mutant T186A reached 10.3 U/ mg, which was 35. 6% higher than that of the wild type. The L-homoserine fermentation analysis suggested that the BdHSD mutant could effectively enhance the synthesis level of L-homoserine. In summary, this study had mined and evolved the BdHSD with high efficient catalytic, which provided a powerful catalytic element for the efficient biosynthesis of L-homoserine, L-threonine, L-methionine, and other aspartate-family amino acids. [ABSTRACT FROM AUTHOR]

SUMMARY: Photoperiod employs complicated networks to regulate various developmental processes in plants, including flowering transition. However, the specific mechanisms by which photoperiod affects epigenetic modifications and gene expression variations in plants remain elusive. In this study, we conducted a comprehensive analysis of DNA methylation, small RNA (sRNA) accumulation, and gene expressions under different daylengths in facultative long‐day (LD) grass Brachypodium distachyon and short‐day (SD) grass rice. Our results showed that while overall DNA methylation levels were minimally affected by different photoperiods, CHH methylation levels were repressed under their favorable light conditions, particularly in rice. We identified numerous differentially methylated regions (DMRs) that were influenced by photoperiod in both plant species. Apart from differential sRNA clusters, we observed alterations in the expression of key components of the RNA‐directed DNA methylation pathway, DNA methyltransferases, and demethylases, which may contribute to the identified photoperiod‐influenced CHH DMRs. Furthermore, we identified many differentially expressed genes in response to different daylengths, some of which were associated with the DMRs. Notably, we discovered a photoperiod‐responsive gene MYB11 in the transcriptome of B. distachyon, and further demonstrated its role as a flowering inhibitor by repressing FT1 transcription. Together, our comparative and functional analysis sheds light on the effects of daylength on DNA methylation, sRNA accumulation, and gene expression variations in LD and SD plants, thereby facilitating better designing breeding programs aimed at developing high‐yield crops that can adapt to local growing seasons. Significance Statement: Our comprehensive analysis of DNA methylation, small RNA accumulation, and gene expressions combined with functional analysis of long‐day (LD) photoperiod‐induced MYB11 in Brachypodium distachyon offers insights into the impact of daylength on epigenetic modifications, and gene expression variations in LD and short‐day plants. Our research fosters the potential for more calculated scheming of breeding programs that strive to produce high‐yielding crops capable of adapting to their respective local growing seasons. [ABSTRACT FROM AUTHOR]

Key message BdDREB-39 is a DREB/CBF transcription factor, localized in the nucleus with transactivation activity, and BdDREB-39-overexpressing transgenic yeasts and tobacco enhanced the tolerance to oxidative stress. Abstract The DREB/CBF transcription factors are generally recognized to play an important factor in plant growth, development and response to various abiotic stresses. However, the mechanism of DREB/CBFs in oxidative stress response is largely unknown. This study isolated a DREB/CBF gene BdDREB-39 from Brachypodium distachyon (B. distachyon). Multiple sequence alignment and phylogenetic analysis showed that BdDREB-39 was closely related to the DREB proteins of oats, barley, wheat and rye and therefore its study can provide a reference for the excavation and genetic improvement of BdDREB-39 or its homologs in its closely related species. The transcript levels of BdDREB-39 were significantly up-regulated under H2O2 stress. BdDREB-39 was localised in the nucleus and functioned as a transcriptional activator. Overexpression of BdDREB-39 enhanced H2O2 tolerance in yeast. Transgenic tobaccos with BdDREB-39 had higher germination rates, longer root, better growth status, lesser reactive oxygen species (ROS) and malondialdehyde (MDA), and higher superoxide dismutase (SOD) and peroxidase (POD) activities than wild type (WT). The expression levels of ROS-related and stress-related genes were improved by BdDREB-39. In summary, these results revealed that BdDREB-39 can improve the viability of tobacco by regulating the expression of ROS and stress-related genes, allowing transgenic tobacco to accumulate lower levels of ROS and reducing the damage caused by ROS to cells. The BdDREB-39 gene has the potential for developing plant varieties tolerant to stress. [ABSTRACT FROM AUTHOR]

Summary: The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types.We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo‐SEM, subcritical water extraction and ssNMR were used to study wall architecture.BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan‐xylan and/or xylan–lignin interactions.Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials. [ABSTRACT FROM AUTHOR]

SUMMARY: The demand for agricultural production is becoming more challenging as climate change increases global temperature and the frequency of extreme weather events. This study examines the phenotypic variation of 149 accessions of Brachypodium distachyon under drought, heat, and the combination of stresses. Heat alone causes the largest amounts of tissue damage while the combination of stresses causes the largest decrease in biomass compared to other treatments. Notably, Bd21‐0, the reference line for B. distachyon, did not have robust growth under stress conditions, especially the heat and combined drought and heat treatments. The climate of origin was significantly associated with B. distachyon responses to the assessed stress conditions. Additionally, a GWAS found loci associated with changes in plant height and the amount of damaged tissue under stress. Some of these SNPs were closely located to genes known to be involved in responses to abiotic stresses and point to potential causative loci in plant stress response. However, SNPs found to be significantly associated with a response to heat or drought individually are not also significantly associated with the combination of stresses. This, with the phenotypic data, suggests that the effects of these abiotic stresses are not simply additive, and the responses to the combined stresses differ from drought and heat alone. Significance Statement: 149 accessions of Brachypodium were phenotyped across time under control, drought, heat, and the combination of drought and heat. Unexpectedly, heat alone resulted in the most amount of tissue damage in comparison to drought alone or the combined stress treatment. Climate of origin was significantly correlated with changes in height and plant tissue damage under stress and several genetic loci were also found to be associated with phenotypic changes under stress. [ABSTRACT FROM AUTHOR]

• Seven novel CAMTA genes with CG-1 domains were identified in Brachypodium distachyon , a model cereal plant. • Most BdCAMTA proteins were found to be acidic and localized in the nucleus, suggesting their potential roles in transcriptional regulation. • The BdCAMTA genes exhibited diverse responses to cold, drought, and salinity stresses without specificity, indicating versatile roles in plant stress responses. Calmodulin-binding transcription activators (CAMTA s) is a family of transcriptional factors, which are highly sensitive to various stressors and hormone signals. They are involved in regulating plant growth, development, stress response, and have distinct biological roles in different plant compartments. Although the gene families coding the CAMTA transcription factors have been identified and functionally characterized in many plant species, it has not been previously reported in Brachypodium distachyon , which is a model organism for genomic research in cereals and grasses. In the present study, seven novel CAMTA genes were identified in the B. distachyon genome, all of which contain the CG-1 (pfam03859) domain. Their sequence details were provided with exon numbers ranging from 10 to 13 and protein length varying from 836 to 1034 amino acid residues. All BdCAMTA proteins, except BdCAMTA1, were found to be acidic and localized to the nucleus. The BdCAMTA genes exhibit diverse responses to cold, drought, and salinity stresses, without being specific to any stress. Therefore, upcoming studies should prioritize the investigation of molecular mechanisms governing functional specificity and redundancy among individual members of CAMTA. These findings establish a valuable scientific foundation for future research concerning the roles of the CAMTA gene family in plants. [ABSTRACT FROM AUTHOR]

Styrene is an important industrial chemical. Although several studies have reported microbial styrene production, the amount of styrene produced in batch cultures can be increased. In this study, styrene was produced using genetically engineered Escherichia coli. First, we evaluated five types of phenylalanine ammonia lyases (PALs) from Arabidopsis thaliana (AtPAL) and Brachypodium distachyon (BdPAL) for their ability to produce trans-cinnamic acid (Cin), a styrene precursor. AtPAL2-expressing E. coli produced approximately 700 mg/L of Cin and we found that BdPALs could convert Cin into styrene. To assess styrene production, we constructed an E. coli strain that co-expressed AtPAL2 and ferulic acid decarboxylase from Saccharomyces cerevisiae. After a biphasic culture with oleyl alcohol, styrene production and yield from glucose were 3.1 g/L and 26.7% (mol/mol), respectively, which, to the best of our knowledge, are the highest values obtained in batch cultivation. Thus, this strain can be applied to the large–scale industrial production of styrene. [ABSTRACT FROM AUTHOR]

The Multidrug and Toxic Compound Extrusion (MATE) proteins serve as pivotal transporters responsible for the extrusion of metabolites, thereby playing a significant role in both plant development and the detoxification of toxins. The MATE gene family within the Brachypodium distachyon, which is an important model organism of the Poaceae family, remains largely unexplored. Here, a comprehensive identification and analysis of MATE genes that complement B. distachyon were conducted. The BdMATE genes were systematically categorized into five distinct groups, predicated on an assessment of their phylogenetic affinities and protein structure. Furthermore, our investigation revealed that dispersed duplication has significantly contributed to the expansion of the BdMATE genes, with tandem and segmental duplications showing important roles, suggesting that the MATE genes in Poaceae species have embarked on divergent evolutionary trajectories. Examination of ω values demonstrated that BdMATE genes underwent purifying selection throughout the evolutionary process. Furthermore, collinearity analysis has confirmed a high conservation of MATE genes between B. distachyon and rice. The cis-regulatory elements analysis within BdMATEs promoters, coupled with expression patterns, suggests that BdMATEs play important roles during plant development and in response to phytohormones. Collectively, the findings presented establish a foundational basis for the subsequent detailed characterization of the MATE gene family members in B. distachyon.

Gibberellins are diterpenoid phytohormones that regulate plant growth, and are biosynthesized from a diterpene intermediate, ent - kaurene, which is produced from geranylgeranyl diphosphate via ent -copalyl diphosphate (ent -CDP). The successive 2 cyclization reactions are catalyzed by 2 distinct diterpene synthases, ent -CDP synthase (ent -CPS) and ent -kaurene synthase (KS). Various diterpene synthase genes involved in specialized metabolism were likely created through duplication and neofunctionalization of gibberellinbiosynthetic ent -CPS and KS genes in crops. Brachypodium distachyon is a monocotyledonous species that is a model plant in grasses. We herein found 1 ent-CPS gene homolog BdCPS and 4 tandemly arrayed KS-like genes BdKS1, KSL2, KSL3, and KSL4 in the B. distachyon genome, a simpler collection of paralogs than in crops. Phylogenetic and biochemical analyses showed that BdCPS and BdKS1 are responsible for gibberellin biosynthesis. BdKSL2 and BdKSL3 are suggested to be involved in specialized diterpenoid metabolism. Moreover, we restored KS activity of BdKSL2 through amino acid substitution. [ABSTRACT FROM AUTHOR]

Methionine sulfoxide reductases (MSRs) serve an important role in maintaining the redox balance in plant cells, but the function of the Brachypodium (Brachypodium distachyon (L.) P. Beauv.) homologs have not been reported to date. Previous studies have demonstrated that Brachypodium distachyon MSRB1.1 gene belongs to the MSRB subfamily and is induced by salinity or osmotic stress. In the present study, we found that the BdMSRB1.1 protein localized to the chloroplast. In our experiments, we used transgenic Arabidopsis thaliana with constitutive expression of BdMSRB1.1 and a loss-of-function msrb1 mutant, verified by MSR enzyme activity assays and quantitative reverse transcription-PCR. When treated with NaCl, mannitol, H2O2 or abscisic acid, the phenotype of the BdMSRB1.1 overexpression line exhibited higher tolerance than that of the wild type, whereas the msrb1 mutant was more sensitive. Under conditions of salt stress, constitutive expression of BdMSRB1.1 in Arabidopsis promoted reactive oxygen species (ROS)-scavenging capacity, supported by increased activity of ROS-removal enzymes, increased transcription levels of several ROS-clearance genes, and decreased malondialdehyde content; the msrb1 mutant plants exhibited the opposite results. Furthermore, BdMSRB1.1 overexpression led to increased soluble sugar content, in accordance with the observed upregulation of the sucrose synthase and sucrose phosphate synthase genes, which are key in the soluble sugar synthesis pathway. These findings suggest that BdMSRB1.1 plays a positive role in responses to salinity and osmotic stress. [ABSTRACT FROM AUTHOR]

Understanding how plants adapt to changing environments and the potential contribution of transposable elements (TEs) to this process is a key question in evolutionary genomics. While TEs have recently been put forward as active players in the context of adaptation, few studies have thoroughly investigated their precise role in plant evolution. Here, we used the wild Mediterranean grass Brachypodium distachyon as a model species to identify and quantify the forces acting on TEs during the adaptation of this species to various conditions, across its entire geographic range. Using sequencing data from more than 320 natural B. distachyon accessions and a suite of population genomics approaches, we reveal that putatively adaptive TE polymorphisms are rare in wild B. distachyon populations. After accounting for changes in past TE activity, we show that only a small proportion of TE polymorphisms evolved neutrally (

The model organism Neurospora crassa has been cultivated in laboratories since the 1920s and its saprotrophic lifestyle has been established for decades. However, beyond their role as saprotrophs, fungi engage in intricate relationships with plants, showcasing diverse connections ranging from mutualistic to pathogenic. Although N. crassa has been extensively investigated under laboratory conditions, its ecological characteristics remain largely unknown. In contrast, Brachypodium distachyon, a sweet grass closely related to significant crops, demonstrates remarkable ecological flexibility and participates in a variety of fungal interactions, encompassing both mutualistic and harmful associations. Through a comprehensive microscopic analysis using electron, fluorescence, and confocal laser scanning microscopy, we discovered a novel endophytic interaction between N. crassa and B. distachyon roots, where fungal hyphae not only thrive in the apoplastic space and vascular bundle but also may colonize plant root cells. This new and so far hidden trait of one of the most important fungal model organisms greatly enhances our view of N. crassa, opening new perspectives concerning the fungus‘ ecological role. In addition, we present a new tool for studying plant–fungus interspecies communication, combining two well-established model systems, which improves our possibilities of experimental design on the molecular level.

The transition to flowering is a major developmental switch in plants. In many temperate grasses, perception of indicators of seasonal change, such as changing day-length and temperature, leads to expression of FLOWERING LOCUS T1 (FT1) and FT-Like (FTL) genes that are essential for promoting the transition to flowering. However, little is known about the upstream regulators of FT1 and FTL genes in temperate grasses. Here, we characterize the monocot-specific gene INDETERMINATE1 (BdID1) in Brachypodium distachyon and demonstrate that BdID1 is a regulator of FT family genes. Mutations in ID1 impact the ability of the short-day (SD) vernalization, cold vernalization, and long-day (LD) photoperiod pathways to induce certain FTL genes. BdID1 is required for upregulation of FTL9 (FT-LIKE9) expression by the SD vernalization pathway, and overexpression of FTL9 in an id1 background can partially restore the delayed flowering phenotype of id1. We show that BdID1 binds in vitro to the promoter region of FTL genes suggesting that ID1 directly activates FTL expression. Transcriptome analysis shows that BdID1 is required for FT1, FT2, FTL12, and FTL13 expression under inductive LD photoperiods, indicating that BdID1 is a regulator of the FT gene family. Moreover, overexpression of FT1 in the id1 background results in rapid flowering similar to overexpressing FT1 in the wild type, demonstrating that BdID1 is upstream of FT family genes. Interestingly, ID1 negatively regulates a previously uncharacterized FTL gene, FTL4, and we show that FTL4 is a repressor of flowering. Thus, BdID1 is critical for proper timing of flowering in temperate grasses. [ABSTRACT FROM AUTHOR]

Abstract Background The adaptation of plants to cold stress involves changes in gene expression profiles that are associated with epigenetic regulation. Although the three-dimensional (3D) genome architecture is considered an important epigenetic regulator, the role of 3D genome organization in the cold stress response remains unclear. Results In this study, we developed high-resolution 3D genomic maps using control and cold-treated leaf tissue of the model plant Brachypodium distachyon using Hi-C to determine how cold stress affects the 3D genome architecture. We generated ~ 1.5 kb resolution chromatin interaction maps and showed that cold stress disrupts different levels of chromosome organization, including A/B compartment transition, a reduction in chromatin compartmentalization and the size of topologically associating domains (TADs), and loss of long-range chromatin loops. Integrating RNA-seq information, we identified cold-response genes and revealed that transcription was largely unaffected by the A/B compartment transition. The cold-response genes were predominantly localized in compartment A. In contrast, transcriptional changes are required for TAD reorganization. We demonstrated that dynamic TAD events were associated with H3K27me3 and H3K27ac state alterations. Moreover, a loss of chromatin looping, rather than a gain of looping, coincides with alterations in gene expression, indicating that chromatin loop disruption may play a more important role than loop formation in the cold-stress response. Conclusions Our study highlights the multiscale 3D genome reprogramming that occurs during cold stress and expands our knowledge of the mechanisms underlying transcriptional regulation in response to cold stress in plants.

Abstract Background Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. Results We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). Conclusions With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs.

Closely related to economically important crops, the grass Brachypodium distachyon has been originally established as a pivotal species for grass genomics but more recently flourished as a model for developmental biology. Grasses encompass more than 10,000 species and cover more than 40% of the world land area from tropical to temperate regions. Given that grasses also supply about a fifth of the world's dietary protein as cereal grains, unlocking the sources of phenotypic variation in B. distachyon is hence of prime interest in fundamental and applied research in agronomy, ecology and evolution. We present here the B. distachyon diversity panel, which encompasses 332 fully sequenced accessions covering the whole species distribution from Spain to Iraq. By combining population genetics, niche modeling and landscape genomics, we suggest that B. distachyon recolonized Europe and the Middle East following the last glacial maximum. Consequently, the species faced new environmental conditions which led to clear associations between bioclimatic variables and genetic factors as well as footprints of positive selection in the genome. Altogether, this genomic resource offers a powerful alternative to Arabidopsis thaliana to investigate the genetic bases of adaptation and phenotypic plasticity in plants and more specifically in monocots.

Losses due to disease and climate change are among the most important issues currently facing crop production. It is therefore important to establish the impact of climate change, and particularly of high carbon dioxide (hCO2), on plant immunity in cereals, which provide 60% of human calories. The aim of this study was to determine if hCO2 impacts Brachypodium distachyon immunity, a model plant for temperate cereals. Plants were grown in air (430 ppm CO2) and at two high CO2 conditions, one that is relevant to projections within the coming century (1000 ppm) and a concentration sufficient to saturate photosynthesis (3000 ppm). The following measurements were performed: phenotyping and growth, salicylic acid contents, pathogen resistance tests, and RNAseq analysis of the transcriptome. Improved shoot development was observed at both 1000 and 3000 ppm. A transcriptomic analysis pointed to an increase in primary metabolism capacity under hCO2. Alongside this effect, up-regulation of genes associated with secondary metabolism was also observed. This effect was especially evident for the terpenoid and phenylpropanoid pathways, and was accompanied by enhanced expression of immunity-related genes and accumulation of salicylic acid. Pathogen tests using the fungus Magnaporthe oryzae revealed that hCO2 had a complex effect, with enhanced susceptibility to infection but no increase in fungal development. The study reveals that immunity in B. distachyon is modulated by growth at hCO2 and allows identification of pathways that might play a role in this effect. [ABSTRACT FROM AUTHOR]

The Ultraviolet Resistance Locus 8 (UVR8) in plants recognizes ultraviolet‐B (UV‐B) light and plays a crucial role in regulating plant growth through a series of signal transduction events. However, the UVR8 in monocotyledon crops has not yet been systematically analysed.We identified BdUVR8 (BRADI_3g45740) from the genome of Brachypodium distachyon, a relative of wheat, by analysing the phylogenetic tree, the gene expression pattern, detecting accumulation of UV‐B response metabolites, and checking for phenotype recovery.The BdUVR8 protein sequence is similar to the known UVR8 of other species. The phylogenetic tree of UVR8 shows clear divergence between dicotyledons and monocotyledons. Expression analysis revealed that UV‐B downregulates BdUVR8 by 70% and upregulates the chalcone synthase (BdCHS) gene 3.4‐fold in B. distachyon. The pCAMBIA1300::BdUVR8‐mCherry construct introduced into Arabidopsis uvr8 mutants showed that the BdUVR8 protein is localized in the cytoplasm and translocates into the nucleus in response to UV‐B irradiation. The introduction of BdUVR8 into uvr8 rescued hypocotyl elongation caused by UV‐B and restored expression of HY5, Chalcone synthase, and Flavanone 3‐hydroxylase, as well as accumulation of total flavonoids.Together, our results show that BdUVR8 is a photoreceptor that perceives UV‐B in B. distachyon. [ABSTRACT FROM AUTHOR]

Thioredoxins (Trxs) are thiol-disulfide oxidoreductase proteins that play an important role in plant development and abiotic stress tolerance through redox regulation of target proteins. In plants, the individual biological functions of H-type thioredoxins (Trxhs) are still poorly known. This work is the first to identify the Trxh gene family of the trio of Brachypodium genus (B. distachyon, B. stacei, and B. hybridium) and to characterize their evolutionary patterns and expression profiles under abiotic stress. In total, 28 Trxh proteins were identified in the trio of Brachypodium species that can be divided into three groups. Interestingly, a new unusual Trxh protein (Trxh7) harboring two Cysteine (Cys) residues in its N-terminal region was discovered. Gene structure and protein-conserved motif analyses further consolidated the classification of the phylogenetic tree and highlighted the functional divergence of the Trxh family. Tandem and whole-genome duplications were found as major forces driving Trxh gene expansion in Brachypodium species. The duplicated Trxh gene pairs underwent a purifying selection during the evolution. In silico expression analysis showed the differential expression of BdTrxhs during development. Moreover, based on RT-qPCR expression analysis, target BdTrxh members were found to be induced by salt and drought stresses concomitantly with an accumulation of hydrogen peroxide (H2O2) in root tissues. The exogenous application of H2O2 modulated almost the same BdTrxh genes in root and leaf tissues. These results suggest specific roles for Trxh genes in B. distachyon beside their potential implication in abiotic stress response via redox homoeostasis. [ABSTRACT FROM AUTHOR]

Root morphology and exudation define a plants' sphere of influence in soils. In turn, soil characteristics influence plant growth, morphology, root microbiome, and rhizosphere chemistry. Collectively, all these parameters have significant implications on the major biogeochemical cycles, crop yield, and ecosystem health. However, how plants are shaped by the physiochemistry of soil particles is still not well understood. We explored how particle size and chemistry of growth substrates affect root morphology and exudation of a model grass. We grew Brachypodium distachyon in glass beads with various sizes (0.5, 1, 2, 3 mm), as well as in sand (0.005, 0.25, 4 mm) and in clay (4 mm) particles and in particle-free hydroponic medium. Plant morphology, root weight, and shoot weight were measured. We found that particle size significantly influenced root fresh weight and root length, whereas root number and shoot weight remained constant. Next, plant exudation profiles were analyzed with mass spectrometry imaging and liquid chromatography-mass spectrometry. Mass spectrometry imaging suggested that both, root length and number shape root exudation. Exudate profiles were comparable for plants growing in glass beads or sand with various particles sizes, but distinct for plants growing in clay for in situ exudate collection. Clay particles were found to sorb 20% of compounds exuded by clay-grown plants, and 70% of compounds from a defined exudate medium. The sorbed compounds belonged to a range of chemical classes, among them nucleosides, organic acids, sugars, and amino acids. Some of the sorbed compounds could be desorbed by a rhizobacterium (Pseudomonas fluorescens WCS415), supporting its growth. This study demonstrates the effect of different characteristics of particles on root morphology, plant exudation and availability of nutrients to microorganisms. These findings further support the critical importance of the physiochemical properties of soils when investigating plant morphology, plant chemistry, and plant-microbe interactions.

Background: The adaptation of plants to cold stress involves changes in gene expression profiles that are associated with epigenetic regulation. Although the three-dimensional (3D) genome architecture is considered an important epigenetic regulator, the role of 3D genome organization in the cold stress response remains unclear. Results: In this study, we developed high-resolution 3D genomic maps using control and cold-treated leaf tissue of the model plant Brachypodium distachyon using Hi-C to determine how cold stress affects the 3D genome architecture. We generated ~ 1.5 kb resolution chromatin interaction maps and showed that cold stress disrupts different levels of chromosome organization, including A/B compartment transition, a reduction in chromatin compartmentalization and the size of topologically associating domains (TADs), and loss of long-range chromatin loops. Integrating RNA-seq information, we identified cold-response genes and revealed that transcription was largely unaffected by the A/B compartment transition. The cold-response genes were predominantly localized in compartment A. In contrast, transcriptional changes are required for TAD reorganization. We demonstrated that dynamic TAD events were associated with H3K27me3 and H3K27ac state alterations. Moreover, a loss of chromatin looping, rather than a gain of looping, coincides with alterations in gene expression, indicating that chromatin loop disruption may play a more important role than loop formation in the cold-stress response. Conclusions: Our study highlights the multiscale 3D genome reprogramming that occurs during cold stress and expands our knowledge of the mechanisms underlying transcriptional regulation in response to cold stress in plants. [ABSTRACT FROM AUTHOR]

As sessile organisms, plants possess a complex system to cope with environmental changes. Ca2+ functions as a vital second messenger in the stress signaling of plants, and the CBL-interacting protein kinases (CIPKs) serve as essential elements in the plant Ca2+ signaling pathway. In this study, calcineurin B-like protein-interacting protein kinase 26 (BdCIPK26) from Brachypodium distachyon was characterized. Overexpression of BdCIPK26 enhanced tolerance to drought and salt stress of transgenic plants. Further investigations revealed that BdCIPK26 participated in abscisic acid (ABA) signaling, conferred hypersensitivity to exogenous ABA in transgenic plants, and promoted endogenous ABA biosynthesis. Moreover, BdCIPK26 was found to maintain ROS homeostasis in plants under stress conditions. Therefore, this study indicates that BdCIPK26 functions as a positive regulator in drought and salt stress response. [ABSTRACT FROM AUTHOR]

Flavonoids are a kind of plant-specific secondary metabolites, which play an important role in regulating plant growth and development, stress response, and also have medicinal value. Chalcone synthase is the key enzyme in the synthesis of flavonoids. The function of chalcone synthase in Arabidopsis thaliana has been well studied, but its homologous protein in Brachypodium distachyon has not been reported. In this study, we identified a homolog of AtCHS in B. distachyon, named BdCHS, and described its function. Phylogenetic tree analysis showed that BdCHS was most closely related to CHS in Triticum aestivum. Transgene analysis revealed that BdCHS protein was localized in the cytoplasm of Arabidopsis root cells. BdCHS protein can complement the phenotype of AtCHS mutants with lighter seed coat color and increased lateral root density. The content of superoxide anion in the cortical cells above the lateral root primordium in AtCHS mutants was higher than that in the wild-type, and BdCHS protein could restore the content of superoxide anion in AtCHS mutant to the level of that in the wild-type. The results showed that BdCHS was a functional homolog of AtCHS, which laid a foundation for the subsequent application of BdCHS in genetic breeding and crop improvement. [ABSTRACT FROM AUTHOR]

Background: Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. Results: We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). Conclusions: With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs. [ABSTRACT FROM AUTHOR]

Plants have been recognized as key components of bioregenerative life support systems for space exploration, and many experiments have been carried out to evaluate their adaptability to spaceflight. Unfortunately, few of these experiments have involved monocot plants, which constitute most of the crops used on Earth as sources of food, feed, and fiber. To better understand the ability of monocot plants to adapt to spaceflight, we germinated and grew Brachypodium distachyon seedlings of the Bd21, Bd21-3, and Gaz8 accessions in a customized growth unit on the International Space Station, along with 1-g ground controls. At the end of a 4-day growth period, seedling organ's growth and morphologies were quantified, and root and shoot transcriptomic profiles were investigated using RNA-seq. The roots of all three accessions grew more slowly and displayed longer root hairs under microgravity conditions relative to ground control. On the other hand, the shoots of Bd21-3 and Gaz-8 grew at similar rates between conditions, whereas those of Bd21 grew more slowly under microgravity. The three Brachypodium accessions displayed dramatically different transcriptomic responses to microgravity relative to ground controls, with the largest numbers of differentially expressed genes (DEGs) found in Gaz8 (4527), followed by Bd21 (1353) and Bd21-3 (570). Only 47 and six DEGs were shared between accessions for shoots and roots, respectively, including DEGs encoding wall-associated proteins and photosynthesis-related DEGs. Furthermore, DEGs associated with the "Oxidative Stress Response" GO group were up-regulated in the shoots and down-regulated in the roots of Bd21 and Gaz8, indicating that Brachypodium roots and shoots deploy distinct biological strategies to adapt to the microgravity environment. A comparative analysis of the Brachypodium oxidative-stress response DEGs with the Arabidopsis ROS wheel suggests a connection between retrograde signaling, light response, and decreased expression of photosynthesis-related genes in microgravity-exposed shoots. In Gaz8, DEGs were also found to preferentially associate with the "Plant Hormonal Signaling" and "MAP Kinase Signaling" KEGG pathways. Overall, these data indicate that Brachypodium distachyon seedlings exposed to the microgravity environment of ISS display accession- and organ-specific responses that involve oxidative stress response, wall remodeling, photosynthesis inhibition, expression regulation, ribosome biogenesis, and post-translational modifications. The general characteristics of these responses are similar to those displayed by microgravity-exposed Arabidopsis thaliana seedlings. However, organ- and accession-specific components of the response dramatically differ both within and between species. These results suggest a need to directly evaluate candidate-crop responses to microgravity to better understand their specific adaptability to this novel environment and develop cultivation strategies allowing them to strive during spaceflight. [ABSTRACT FROM AUTHOR]

SUMMARY: Xylan is the most abundant non‐cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of β‐1,4‐xylose (Xyl) residues that may be substituted with α‐1,2‐linked (4‐O‐methyl)‐glucuronic acid (GlcA), α‐1,3‐linked arabinofuranose (Araf), and sometimes acetylation at the O‐2 and/or O‐3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution‐dependent endo‐xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components. Significance Statement: Xylan substitutions and their arrangement can influence xylan interaction with cellulose fibrils or lignin; however, the importance in grass cell walls is poorly understood. A deeper understanding of the nature of the xylan–cellulose and xylan–lignin interactions will allow scientists to develop cell wall models, predict wall properties and identify molecular targets for bioengineering, enabling the development of better approaches to use plant biomass to produce sustainable fuels and biomaterials. [ABSTRACT FROM AUTHOR]

There is a dynamic reciprocity between plants and their environment: soil physiochemical properties influence plant morphology and metabolism, and root morphology and exudates shape the environment surrounding roots. Here, we investigate the reproducibility of plant trait changes in response to three growth environments. We utilized fabricated ecosystem (EcoFAB) devices to grow the model grass Brachypodium distachyon in three distinct media across four laboratories: phosphate-sufficient and -deficient mineral media allowed assessment of the effects of phosphate starvation, and a complex, sterile soil extract represented a more natural environment with yet uncharacterized effects on plant growth and metabolism. Tissue weight and phosphate content, total root length, and root tissue and exudate metabolic profiles were consistent across laboratories and distinct between experimental treatments. Plants grown in soil extract were morphologically and metabolically distinct, with root hairs four times longer than with other growth conditions. Further, plants depleted half of the metabolites investigated from the soil extract. To interact with their environment, plants not only adapt morphology and release complex metabolite mixtures, but also selectively deplete a range of soil-derived metabolites. The EcoFABs utilized here generated high interlaboratory reproducibility, demonstrating their value in standardized investigations of plant traits.

The development of model systems requires a detailed assessment of standing genetic variation across natural populations. The Brachypodium species complex has been promoted as a plant model for grass genomics with translation to small grain and biomass crops. To capture the genetic diversity within this species complex, thousands of Brachypodium accessions from around the globe were collected and genotyped by sequencing. Overall, 1897 samples were classified into two diploid or allopolyploid species, and then further grouped into distinct inbred genotypes. A core set of diverse B. distachyon diploid lines was selected for whole genome sequencing and high resolution phenotyping. Genome-wide association studies across simulated seasonal environments was used to identify candidate genes and pathways tied to key life history and agronomic traits under current and future climatic conditions. A total of 8, 22, and 47 QTL were identified for flowering time, early vigor, and energy traits, respectively. The results highlight the genomic structure of the Brachypodium species complex, and the diploid lines provided a resource that allows complex trait dissection within this grass model species.

FLOWERING LOCUS T2 (FT2) is the closest paralog of the FT1 flowering gene in the temperate grasses. Here we show that overexpression of FT2 in Brachypodium distachyon and barley results in precocious flowering and reduced spikelet number, while down-regulation by RNA interference results in delayed flowering and a reduced percentage of filled florets. Similarly, truncation mutations of FT2 homeologs in tetraploid wheat delayed flowering (2-4 d) and reduced fertility. The wheat ft2 mutants also showed a significant increase in the number of spikelets per spike, with a longer spike development period potentially contributing to the delayed heading time. In the wheat leaves, FT2 was expressed later than FT1, suggesting a relatively smaller role for FT2 in the initiation of the reproductive phase. FT2 transcripts were detected in the shoot apical meristem and increased during early spike development. Transversal sections of the developing spike showed the highest FT2 transcript levels in the distal part, where new spikelets are formed. Our results suggest that, in wheat, FT2 plays an important role in spike development and fertility and a limited role in the timing of the transition between the vegetative and reproductive shoot apical meristem.

Clinostats are instruments that continuously rotate biological specimens along an axis, thereby averaging their orientation relative to gravity over time. Our previous experiments indicated that low-speed clinorotation may itself trigger directional root tip curvature. In this project, we have investigated the root curvature response to low-speed clinorotation using Arabidopsis thaliana and Brachypodium distachyon seedlings as models. We show that low-speed clinorotation triggers root tip curvature in which direction is dictated by gravitropism during the first half-turn of clinorotation. We also show that the angle of root tip curvature is modulated by the speed of clinorotation. Arabidopsis mutations affecting gravity susception (pgm) or gravity signal transduction (arg1, toc132) are shown to affect the root tip curvature response to low-speed clinorotation. Furthermore, low-speed vertical clinorotation triggers relocalization of the PIN3 auxin efflux facilitator to the lateral membrane of Arabidopsis root cap statocytes, and creates a lateral gradient of auxin across the root tip. Together, these observations support a role for gravitropism in modulating root curvature responses to clinorotation. Interestingly, distinct Brachypodium distachyon accessions display different abilities to develop root tip curvature responses to low-speed vertical clinorotation, suggesting the possibility of using genome-wide association studies to further investigate this process. [ABSTRACT FROM AUTHOR]

The annual temperate grass Brachypodium distachyon has become a model system for monocot biomass crops and for understanding lignocellulosic recalcitrance to employ better saccharification and fermentation approaches. It is a monocot plant used to study the grass cell walls that differ from the cell walls of dicot plants such as the eudicot model Arabidopsis. The B. distachyon cell wall is predominantly composed of cellulose, arabinoxylans, and mixed-linkage glucans, and it resembles the cell walls of other field grasses. It has a vascular bundle anatomy similar to C3 grasses. These features make Brachypodium an ideal model to study cell walls. Cell walls are composed of polymers with complex structures that vary between cell types and at different developmental stages. Antibodies that recognize specific cell wall components are currently one of the most effective and specific molecular probes to determine the location and distribution of polymers in plant cell walls in situ. Here, we investigated the glycan distribution in the cell walls of the root and leaf tissues of Brachypodium by employing cell-wall-directed antibodies against diverse glycan epitopes. There are distinct differences in the presence of the epitopes between the root and leaf tissues as well as in the cell type level, which gives insights into monocot biomass. [ABSTRACT FROM AUTHOR]

Potassium (K) is one of the most essential macronutrients for plants. However, K+ is deficient in some cultivated soils. Hence, improving the efficiencies of K+ uptake and utilization is important for agricultural production. Ca2+ signaling pathways play an important role in regulation of K+ acquisition. In the present study, BdCIPK31, a Calcineurin B-like protein interacting protein kinase (CIPK) from Brachypodium distachyon , was found to be a potential positive regulator in plant response to low K+ stress. The expression of BdCIPK31 was responsive to K+-deficiency, and overexpression of BdCIPK31 conferred enhanced tolerance to low K+ stress in transgenic tobaccos. Furthermore, BdCIPK31 was demonstrated to promote the K+ uptake in root, and could maintain normal root growth under K+-deficiency conditions. Additionally, BdCIPK31 functioned in scavenging excess reactive oxygen species (ROS), reduced oxidative damage caused by low K+ stress. Collectively, our study indicates that BdCIPK31 is a vital regulatory component in K+-acquisition system in plants. • A new multi-function regulator in plant K+ utilization has been discovered. • BdCIPK31 enhances root K+ uptake, but doesn't affect root-shoot K+ transport. • BdCIPK31 promotes sugar transport under low K+ stress, contributes to root growth. • BdCIPK31 enhances ROS-scavenging, reduces oxidative damage caused by K-starving. [ABSTRACT FROM AUTHOR]

The plant proteins called ERECTA family play important role in inflorescence architecture, stomatal patterning and phloem-xylem organization. ERECTA proteins belong to the moonlighting proteins family containing the guanylyl cyclase (GC) catalytic center embedded within the intracellular kinase domain. This characteristic architecture of ERECTA proteins prompted us to experimentally confirm of enzymatic activity of one of these, BdERL1 (ERECTA-like1 from Brachypodium distachyon). We have shown that BdERL1 is dual-function protein with both kinase and GC activity. Moreover, our mutagenesis studies also revealed the catalytic roles of key conserved amino acid residues at the GC center and importantly, probing of the kinase and GC with Ca2+ and/or cGMP, shed light on the intramolecular regulations of BdERL1.

Grass stomata recruit lateral subsidiary cells (SCs), which are key to the unique stomatal morphology and the efficient plant-atmosphere gas exchange in grasses. Subsidiary mother cells (SMCs) strongly polarise before an asymmetric division forms a SC. Yet apart from a proximal polarity module that includes PANGLOSS1 (PAN1) and guides nuclear migration, little is known regarding the developmental processes that form SCs. Here, we used comparative transcriptomics of developing wild-type and SC-less bdmute leaves in the genetic model grass Brachypodium distachyon to identify novel factors involved in SC formation. This approach revealed BdPOLAR, which forms a novel, distal polarity domain in SMCs that is opposite to the proximal PAN1 domain. Both polarity domains are required for the formative SC division yet exhibit various roles in guiding pre-mitotic nuclear migration and SMC division plane orientation, respectively. Nonetheless, the domains are linked as the proximal domain controls polarisation of the distal domain. In summary, we identified two opposing polarity domains that coordinate the SC division, a process crucial for grass stomatal physiology.

B3 is a class of plant-specific transcription factors with important roles in plant development and growth. Here, we identified 69 B3 transcription factors in Brachypodium distachyon that were unevenly distributed across all five chromosomes. The ARF, REM, LAV, and RAV subfamilies were grouped based on sequence characteristics and phylogenetic relationships. The phylogenetically related members in the B3 family shared conserved domains and gene structures. Expression profiles showed that B3 genes were widely expressed in different tissues and varied in response to different abiotic stresses. BdB3-54 protein from the REM subfamily was located in the nucleus by subcellular localization and processed transcriptional activation activity. Overexpression of BdB3-54 in Arabidopsis increased primary root length. Our study provides a basis for further research on the functions of BdB3 genes. [ABSTRACT FROM AUTHOR]

Fusarium head blight (FHB) and Fusarium root rot (FRR) are important diseases of small-grain cereals caused by Fusarium species. While host response to FHB has been subject to extensive study, very little is known about response to FRR and the transcriptome responses of FHB and FRR have not been thoroughly compared. Brachypodium distachyon (Bd) is an effective model for investigating host responses to both FHB and FRR. In this study the transcriptome response of Bd to F. graminearum (Fg) infection of heads and roots was investigated. An RNA-seq analysis was performed on both Bd FHB and FRR during the early infection. Additionally, an RNA-seq analysis was performed on in vitro samples of Fg for comparison with Fg gene expression in planta. Differential gene expression and gene-list enrichment analyses were used to compare FHB and FRR transcriptome responses in both Bd and Fg. Differential expression of selected genes was confirmed using RT-qPCR. Most genes associated with receptor signalling, cell-wall modification, oxidative stress metabolism, and cytokinin and auxin biosynthesis and signalling genes were generally upregulated in FHB or were downregulated in FRR. In contrast, Bd genes involved in jasmonic acid and ethylene biosynthesis and signalling, and antimicrobial production were similarly differentially expressed in both tissues in response to infection. A transcriptome analysis of predicted Fg effectors with the same infected material revealed elevated expression of core tissue-independent genes including cell-wall degradation enzymes and the gene cluster for DON production but also several tissue-dependent genes including those for aurofusarin production and cutin degradation. This evidence suggests that Fg modulates its transcriptome to different tissues of the same host. [ABSTRACT FROM AUTHOR]

Background: Fusarium head blight (FHB) is a serious fungal disease affecting crop plants, causing substantial yield reductions and the production of mycotoxins in the infected grains. Achieving progress in the breeding of crops with increased resistance and maintaining a high yield is not possible without a thorough examination of the molecular basis of plant immunity responses. Methods: LC-MS-based metabolomics approaches powered by three-way ANOVA and the selec-tion of differentially accumulated metabolites (DAMs) were used for studying plant immunity. A correlation network and functional enrichment analysis were conducted on grains of barley and wheat genotypes that were resistant or susceptible to FHB, as well as on the model grass Brachypodium distachyon (Bd), as this is still poorly understood at the metabolomic level. Results: We selected common and genotype-specific DAMs in response to F. culmorum inoculation. The immunological reaction at the metabolomic level was strongly diversified between resistant and susceptible genotypes. DAMs that were common to all tested species from the porphyrin, flavonoid, and phenylpropanoid metabolic pathways were highly correlated, reflecting con-servativeness in the FHB response in the Poaceae family. Resistance-related DAMs belonged to different structural classes, including tryptophan-derived metabolites, pyrimidines, the amino acids proline and serine, as well as phenylpropanoids and flavonoids. The physiological re-sponse to F. culmorum of Bd was close to that of barley and wheat genotypes; however, metabo-lomic changes were strongly diversified. Conclusions: Combined targeted and untargeted metabolomics provides comprehensive knowledge about significant elements of plant immuni-ty that have the potential to be molecular biomarkers of enhanced resistance to FHB in the grass family. Thorough examination of the Bd metabolome in juxtaposition with diversified geno-types of barley and wheat facilitated its use as a model grass for plant–microbe interaction. [ABSTRACT FROM AUTHOR]

A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon. However, like many other ligninmodified plants, C3H/APX-KDs show unpredictable pleiotropic phenotypes, including stunted growth, delayed senescence, and reduced seed yield. A system-wide level understanding of altered biological processes in ligninmodified plants can help pinpoint the lignin-modification associated growth defects to benefit future studies aiming to negate the yield penalty. Here, a multi-omic approach was used to characterize molecular changes resulting from C3H/APX-KD associated lignin modification and negative growth phenotype in Brachypodium distachyon. Our findings demonstrate that C3H/APX knockdown in Brachypodium stems substantially alters the abundance of enzymes implicated in the phenylpropanoid biosynthetic pathway and disrupt cellular redox homeostasis. Moreover, it elicits plant defense responses associated with intracellular kinases and phytohormone-based signaling to facilitate growth-defense trade-offs. A deeper understanding along with potential targets to mitigate the pleiotropic phenotypes identified in this study could aid to increase the economic feasibility of lignocellulosic biofuel production. [ABSTRACT FROM AUTHOR]

Brachypodium distachyon, because of its fully sequenced genome, is frequently used as a model grass species. However, its metabolome, which constitutes an indispensable element of complex biological systems, remains poorly characterized. In this study, we conducted comprehensive, liquid chromatography-mass spectrometry (LC-MS)-based metabolomic examination of roots, leaves and spikes of Brachypodium Bd21 and Bd3-1 lines. Our pathway enrichment analysis emphasised the accumulation of specialized metabolites representing the flavonoid biosynthetic pathway in parallel with processes related to nucleotide, sugar and amino acid metabolism. Similarities in metabolite profiles between both lines were relatively high in roots and leaves while spikes showed higher metabolic variance within both accessions. In roots, differences between Bd21 and Bd3-1 lines were manifested primarily in diterpenoid metabolism, while differences within spikes and leaves concerned nucleotide metabolism and nitrogen management. Additionally, sulphate-containing metabolites differentiated Bd21 and Bd3-1 lines in spikes. Structural analysis based on MS fragmentation spectra enabled identification of 93 specialized metabolites. Among them phenylpropanoids and flavonoids derivatives were mainly determined. As compared with closely related barley and wheat species, metabolic profile of Brachypodium is characterized with presence of threonate derivatives of hydroxycinnamic acids. [ABSTRACT FROM AUTHOR]

Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction. [ABSTRACT FROM AUTHOR]