BdRCN4, a Brachypodium distachyon TFL1 homologue, is involved in regulation of apical meristem fate.

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.

The Brachypodium distachyon DREB transcription factor BdDREB-39 confers oxidative stress tolerance in transgenic tobacco.

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.

Variation in TaSPL6-D confers salinity tolerance in bread wheat by activating TaHKT1;5-D while preserving yield-related traits.

Na+ exclusion from above-ground tissues via the Na+-selective transporter HKT1;5 is a major salt-tolerance mechanism in crops. Using the expression genome-wide association study and yeast-one-hybrid screening, we identified TaSPL6-D, a transcriptional suppressor of TaHKT1;5-D in bread wheat. SPL6 also targeted HKT1;5 in rice and Brachypodium. A 47-bp insertion in the first exon of TaSPL6-D resulted in a truncated peptide, TaSPL6-DIn, disrupting TaHKT1;5-D repression exhibited by TaSPL6-DDel. Overexpressing TaSPL6-DDel, but not TaSPL6-DIn, led to inhibited TaHKT1;5-D expression and increased salt sensitivity. Knockout of TaSPL6-DDel in two wheat genotypes enhanced salinity tolerance, which was attenuated by a further TaHKT1;5-D knockdown. Spike development was preserved in Taspl6-dd mutants but not in Taspl6-aabbdd mutants. TaSPL6-DIn was mainly present in landraces, and molecular-assisted introduction of TaSPL6-DIn from a landrace into a leading wheat cultivar successfully improved yield on saline soils. The SPL6-HKT1;5 module offers a target for the molecular breeding of salt-tolerant crops.

The H3K4 demethylase JMJ1 is required for proper timing of flowering in Brachypodium distachyon.

Flowering is a key developmental transition in the plant life cycle. In temperate climates, flowering often occurs in response to the perception of seasonal cues such as changes in day-length and temperature. However, the mechanisms that have evolved to control the timing of flowering in temperate grasses are not fully understood. We identified a Brachypodium distachyon mutant whose flowering is delayed under inductive long-day conditions due to a mutation in the JMJ1 gene, which encodes a Jumonji domain-containing protein. JMJ1 is a histone demethylase that mainly demethylates H3K4me2 and H3K4me3 in vitro and in vivo. Analysis of the genome-wide distribution of H3K4me1, H3K4me2, and H3K4me3 in wild-type plants by chromatin immunoprecipitation and sequencing combined with RNA sequencing revealed that H3K4m1 and H3K4me3 are positively associated with gene transcript levels, whereas H3K4me2 is negatively correlated with transcript levels. Furthermore, JMJ1 directly binds to the chromatin of the flowering regulator genes VRN1 and ID1 and affects their transcription by modifying their H3K4me2 and H3K4me3 levels. Genetic analyses indicated that JMJ1 promotes flowering by activating VRN1 expression. Our study reveals a role for JMJ1-mediated chromatin modification in the proper timing of flowering in B. distachyon.

The evolution of transposable elements in Brachypodium distachyon is governed by purifying selection, while neutral and adaptive processes play a minor role.

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 (<10%), while the vast majority of them are under moderate purifying selection regardless of their distance to genes. TE polymorphisms should not be ignored when conducting evolutionary studies, as they can be linked to adaptation. However, our study clearly shows that while they have a large potential to cause phenotypic variation in B. distachyon, they are not favored during evolution and adaptation over other types of mutations (such as point mutations) in this species.

Three near-complete genome assemblies reveal substantial centromere dynamics from diploid to tetraploid in Brachypodium genus.

BACKGROUND: Centromeres are critical for maintaining genomic stability in eukaryotes, and their turnover shapes genome architectures and drives karyotype evolution. However, the co-evolution of centromeres from different species in allopolyploids over millions of years remains largely unknown. RESULTS: Here, we generate three near-complete genome assemblies, a tetraploid Brachypodium hybridum and its two diploid ancestors, Brachypodium distachyon and Brachypodium stacei. We detect high degrees of sequence, structural, and epigenetic variations of centromeres at base-pair resolution between closely related Brachypodium genomes, indicating the appearance and accumulation of species-specific centromere repeats from a common origin during evolution. We also find that centromere homogenization is accompanied by local satellite repeats bursting and retrotransposon purging, and the frequency of retrotransposon invasions drives the degree of interspecies centromere diversification. We further investigate the dynamics of centromeres during alloploidization process, and find that dramatic genetics and epigenetics architecture variations are associated with the turnover of centromeres between homologous chromosomal pairs from diploid to tetraploid. Additionally, our pangenomes analysis reveals the ongoing variations of satellite repeats and stable evolutionary homeostasis within centromeres among individuals of each Brachypodium genome with different polyploidy levels. CONCLUSIONS: Our results provide unprecedented information on the genomic, epigenomic, and functional diversity of highly repetitive DNA between closely related species and their allopolyploid genomes at both coarse and fine scale.

Polygenic architecture of flowering time and its relationship with local environments in the grass Brachypodium distachyon.

Synchronizing the timing of reproduction with the environment is crucial in the wild. Among the multiple mechanisms, annual plants evolved to sense their environment, the requirement of cold-mediated vernalization is a major process that prevents individuals from flowering during winter. In many annual plants including crops, both a long and short vernalization requirement can be observed within species, resulting in so-called early-(spring) and late-(winter) flowering genotypes. Here, using the grass model Brachypodium distachyon, we explored the link between flowering-time-related traits (vernalization requirement and flowering time), environmental variation, and diversity at flowering-time genes by combining measurements under greenhouse and outdoor conditions. These experiments confirmed that B. distachyon natural accessions display large differences regarding vernalization requirements and ultimately flowering time. We underline significant, albeit quantitative effects of current environmental conditions on flowering-time-related traits. While disentangling the confounding effects of population structure on flowering-time-related traits remains challenging, population genomics analyses indicate that well-characterized flowering-time genes may contribute significantly to flowering-time variation and display signs of polygenic selection. Flowering-time genes, however, do not colocalize with genome-wide association peaks obtained with outdoor measurements, suggesting that additional genetic factors contribute to flowering-time variation in the wild. Altogether, our study fosters our understanding of the polygenic architecture of flowering time in a natural grass system and opens new avenues of research to investigate the gene-by-environment interaction at play for this trait.

Comparative and functional analysis unveils the contribution of photoperiod to DNA methylation, sRNA accumulation, and gene expression variations in short-day and long-day grasses.

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.

Transposition of HOPPLA in siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility in Brachypodium distachyon.

Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual role in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grass Brachypodium distachyon to characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations in B. distachyon natural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transpositional activities of RLC_BdisC024 (HOPPLA) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility in B. distachyon and reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model.

Altering the substitution and cross-linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon.

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.

Reproducible growth of Brachypodium in EcoFAB 2.0 reveals that nitrogen form and starvation modulate root exudation.

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. We grew Brachypodium distachyon hydroponically in fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (nitrate, ammonium, and ammonium nitrate), followed by nitrogen starvation. Analyses of exudates with liquid chromatography-tandem mass spectrometry, biomass, medium pH, and nitrogen uptake showed EcoFAB 2.0's low intratreatment data variability. Furthermore, the three inorganic nitrogen forms caused differential exudation, generalized by abundant amino acids-peptides and alkaloids. Comparatively, nitrogen deficiency decreased nitrogen-containing compounds but increased shikimates-phenylpropanoids. Subsequent bioassays with two shikimates-phenylpropanoids (shikimic and p-coumaric acids) on soil bacteria or Brachypodium seedlings revealed their distinct capacity to regulate both bacterial and plant growth. Our results suggest that (i) Brachypodium alters exudation in response to nitrogen status, which can affect rhizobacterial growth, and (ii) EcoFAB 2.0 is a valuable standardized plant research tool.

Natural variation in Brachypodium distachyon responses to combined abiotic stresses.

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.

Systematic analysis of Prx genes in the Brachypodium genus and their expression pattern under abiotic constraints.

Peroxiredoxins (Prx) are ubiquitous peroxidases required for the removal of excess free radicals produced under stress conditions. Peroxiredoxin genes (Prx) in the Brachypodium genus were identified using bioinformatics tools and their expression profiles were determined under abiotic stress using RT-qPCR. The promoter regions of Prx genes contain several cis-acting elements related to stress response. In silico expression analysis showed that B. distachyon Prx genes (BdPrx) are tissue specific. RT-qPCR analysis revealed their differential expression when exposed to salt or PEG-induced dehydration stress. In addition, the upregulation of BdPrx genes was accompanied by accumulation of H2 O2 . Exogenous application of H2 O2 induced expression of almost all BdPrx genes. The identified molecular interaction network indicated that Prx proteins may contribute to abiotic stress tolerance by regulating key enzymes involved in lignin biosynthesis. Overall, our findings suggest the potential role of Prx genes in abiotic stress tolerance and lay the foundation for future functional analyses aiming to engineer genetically improved cereal lines.

Group VII ethylene response factors forming distinct regulatory loops mediate submergence responses.

Group VII ethylene response factors (ERFVIIs), whose stability is oxygen concentration-dependent, play key roles in regulating hypoxia response genes in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) during submergence. To understand the evolution of flooding tolerance in cereal crops, we evaluated whether Brachypodium distachyon ERFVII genes (BdERFVIIs) are related to submergence tolerance. We found that three BdERFVIIs, BdERF108, BdERF018, and BdERF961, form a feedback regulatory loop to mediate downstream responses. BdERF108 and BdERF018 activated the expression of BdERF961 and PHYTOGLOBIN 1 (PGB1), which promoted nitric oxide turnover and preserved ERFVII protein stability. The activation of PGB1 was subsequently counteracted by increased BdERF961 accumulation through negative feedback regulation. Interestingly, we found that OsERF67, the orthologue of BdERF961 in rice, activated PHYTOGLOBIN (OsHB2) expression and formed distinct regulatory loops during submergence. Overall, the divergent regulatory mechanisms exhibited by orthologs collectively offer perspectives for the development of submergence-tolerant crops.

Genome-wide expansion and reorganization during grass evolution: from 30 Mb chromosomes in rice and Brachypodium to 550 Mb in Avena.

BACKGROUND: The BOP (Bambusoideae, Oryzoideae, and Pooideae) clade of the Poaceae has a common ancestor, with similarities to the genomes of rice, Oryza sativa (2n = 24; genome size 389 Mb) and Brachypodium, Brachypodium distachyon (2n = 10; 271 Mb). We exploit chromosome-scale genome assemblies to show the nature of genomic expansion, structural variation, and chromosomal rearrangements from rice and Brachypodium, to diploids in the tribe Aveneae (e.g., Avena longiglumis, 2n = 2x = 14; 3,961 Mb assembled to 3,850 Mb in chromosomes). RESULTS: Most of the Avena chromosome arms show relatively uniform expansion over the 10-fold to 15-fold genome-size increase. Apart from non-coding sequence diversification and accumulation around the centromeres, blocks of genes are not interspersed with blocks of repeats, even in subterminal regions. As in the tribe Triticeae, blocks of conserved synteny are seen between the analyzed species with chromosome fusion, fission, and nesting (insertion) events showing deep evolutionary conservation of chromosome structure during genomic expansion. Unexpectedly, the terminal gene-rich chromosomal segments (representing about 50 Mb) show translocations between chromosomes during speciation, with homogenization of genome-specific repetitive elements within the tribe Aveneae. Newly-formed intergenomic translocations of similar extent are found in the hexaploid A. sativa. CONCLUSIONS: The study provides insight into evolutionary mechanisms and speciation in the BOP clade, which is valuable for measurement of biodiversity, development of a clade-wide pangenome, and exploitation of genomic diversity through breeding programs in Poaceae.

Phytochromes transmit photoperiod information via the evening complex in Brachypodium.

BACKGROUND: Daylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood. RESULTS: We find that phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night. CONCLUSIONS: We propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.

INDETERMINATE1-mediated expression of FT family genes is required for proper timing of flowering in Brachypodium distachyon.

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.

Subgenomic Stability of Progenitor Genomes During Repeated Allotetraploid Origins of the Same Grass Brachypodium hybridum.

Both homeologous exchanges and homeologous expression bias are generally found in most allopolyploid species. Whether homeologous exchanges and homeologous expression bias differ between repeated allopolyploid speciation events from the same progenitor species remains unknown. Here, we detected a third independent and recent allotetraploid origin for the model grass Brachypodium hybridum. Our homeologous exchange with replacement analyses indicated the absence of significant homeologous exchanges in any of the three types of wild allotetraploids, supporting the integrity of their progenitor subgenomes and the immediate creation of the amphidiploids. Further homeologous expression bias tests did not uncover significant subgenomic dominance in different tissues and conditions of the allotetraploids. This suggests a balanced expression of homeologs under similar or dissimilar ecological conditions in their natural habitats. We observed that the density of transposons around genes was not associated with the initial establishment of subgenome dominance; rather, this feature is inherited from the progenitor genome. We found that drought response genes were highly induced in the two subgenomes, likely contributing to the local adaptation of this species to arid habitats in the third allotetraploid event. These findings provide evidence for the consistency of subgenomic stability of parental genomes across multiple allopolyploidization events that led to the same species at different periods. Our study emphasizes the importance of selecting closely related progenitor species genomes to accurately assess homeologous exchange with replacement in allopolyploids, thereby avoiding the detection of false homeologous exchanges when using less related progenitor species genomes.

REGULATOR OF FLOWERING AND STRESS manipulates stomatal density and size in Brachypodium.

Stomata are crucial for gas exchange and water evaporation, and environmental stimuli influence their density (SD) and size (SS). Although genes and mechanisms underlying stomatal development have been elucidated, stress-responsive regulators of SD and SS are less well-known. Previous studies have shown that the stress-inducible Brachypodium RFS (REGULATOR OF FLOWERING AND STRESS, BdRFS) gene affects heading time and enhances drought tolerance by reducing leaf water loss. Here, we report that overexpression lines (OXs) of BdRFS have reduced SD and increased SS, regardless of soil water status. Furthermore, biomass and plant water content of OXs were significantly increased compared to wild type. CRISPR/Cas9-mediated BdRFS knockout mutant (KO) exhibited the opposite stomatal characteristics and biomass changes. Reverse transcription-quantitative polymerase chain reaction analysis revealed that expression of BdICE1 was reversely altered in OXs and KO, pointing to a potential cause for the observed changes in stomatal phenotypes. Stomatal and transcriptional changes were not observed in the Arabidopsis rfs double mutant. Taken together, RFS is a novel regulator of SD and SS and is a promising candidate for genetic engineering of climate-resilient crops.

The reference genome and abiotic stress responses of the model perennial grass Brachypodium sylvaticum.

Perennial grasses are important forage crops and emerging biomass crops and have the potential to be more sustainable grain crops. However, most perennial grass crops are difficult experimental subjects due to their large size, difficult genetics, and/or their recalcitrance to transformation. Thus, a tractable model perennial grass could be used to rapidly make discoveries that can be translated to perennial grass crops. Brachypodium sylvaticum has the potential to serve as such a model because of its small size, rapid generation time, simple genetics, and transformability. Here, we provide a high-quality genome assembly and annotation for B. sylvaticum, an essential resource for a modern model system. In addition, we conducted transcriptomic studies under 4 abiotic stresses (water, heat, salt, and freezing). Our results indicate that crowns are more responsive to freezing than leaves which may help them overwinter. We observed extensive transcriptional responses with varying temporal dynamics to all abiotic stresses, including classic heat-responsive genes. These results can be used to form testable hypotheses about how perennial grasses respond to these stresses. Taken together, these results will allow B. sylvaticum to serve as a truly tractable perennial model system.