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.

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 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.

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.

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.

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.

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.

Scattered differentiation of unlinked loci across the genome underlines ecological divergence of the selfing grass Brachypodium stacei.

Ecological divergence without geographic isolation, as an early speciation process that may lead finally to reproductive isolation through natural selection, remains a captivating topic in evolutionary biology. However, the pattern of genetic divergence underlying this process across the genome may vary between species and mating systems. Here, we present evidence that Brachypodium stacei, an annual and highly selfing grass model species, has undergone sympatric ecological divergence without geographic isolation. Genomic, transcriptomic, and metabolomic analyses together with lab experiments mimicking the two opposite environmental conditions suggest that diploid B. stacei populations have diverged sympatrically in two slopes characterized by distinct biomes at Evolution Canyon I (ECI), Mount Carmel, Israel. Despite ongoing gene flow, primarily facilitated by seed dispersal, the level of gene flow has progressively decreased over time. This local adaptation involves the scattered divergence of many unlinked loci across the total genome that include both coding genes and noncoding regions. Additionally, we have identified significant differential expressions of genes related to the ABA signaling pathway and contrasting metabolome composition between the arid- vs. forest-adapted B. stacei populations in ECI. These results suggest that multiple small loci involved in environmental responses act additively to account for ecological adaptations by this selfing species in contrasting environments.

Characterization of diterpene synthase genes in Brachypodium distachyon, a monocotyledonous model plant, provides evolutionary insight into their multiple homologs in cereals.

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 gibberellin-biosynthetic 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.

Submergence Stress Alters the Expression of Clock Genes and Configures New Zeniths and Expression of Outputs in Brachypodium distachyon.

Plant networks of oscillating genes coordinate internal processes with external cues, contributing to increased fitness. We hypothesized that the response to submergence stress may dynamically change during different times of the day. In this work, we determined the transcriptome (RNA sequencing) of the model monocotyledonous plant, Brachypodium distachyon, during a day of submergence stress, low light, and normal growth. Two ecotypes of differential tolerance, Bd21 (sensitive) and Bd21-3 (tolerant), were included. We submerged 15-day-old plants under a long-day diurnal cycle (16 h light/8 h dark) and collected samples after 8 h of submergence at ZT0 (dawn), ZT8 (midday), ZT16 (dusk), ZT20 (midnight), and ZT24 (dawn). Rhythmic processes were enriched both with up- and down-regulated genes, and clustering highlighted that the morning and daytime oscillator components (PRRs) show peak expression in the night, and a decrease in the amplitude of the clock genes (GI, LHY, RVE) was observed. Outputs included photosynthesis-related genes losing their known rhythmic expression. Up-regulated genes included oscillating suppressors of growth, hormone-related genes with new late zeniths (e.g., JAZ1, ZEP), and mitochondrial and carbohydrate signaling genes with shifted zeniths. The results highlighted genes up-regulated in the tolerant ecotype such as METALLOTHIONEIN3 and ATPase INHIBITOR FACTOR. Finally, we show by luciferase assays that Arabidopsis thaliana clock genes are also altered by submergence changing their amplitude and phase. This study can guide the research of chronocultural strategies and diurnal-associated tolerance mechanisms.

PHYTOCHROME C regulation of photoperiodic flowering via PHOTOPERIOD1 is mediated by EARLY FLOWERING 3 in Brachypodium distachyon.

Daylength sensing in many plants is critical for coordinating the timing of flowering with the appropriate season. Temperate climate-adapted grasses such as Brachypodium distachyon flower during the spring when days are becoming longer. The photoreceptor PHYTOCHROME C is essential for long-day (LD) flowering in B. distachyon. PHYC is required for the LD activation of a suite of genes in the photoperiod pathway including PHOTOPERIOD1 (PPD1) that, in turn, result in the activation of FLOWERING LOCUS T (FT1)/FLORIGEN, which causes flowering. Thus, B. distachyon phyC mutants are extremely delayed in flowering. Here we show that PHYC-mediated activation of PPD1 occurs via EARLY FLOWERING 3 (ELF3), a component of the evening complex in the circadian clock. The extreme delay of flowering of the phyC mutant disappears when combined with an elf3 loss-of-function mutation. Moreover, the dampened PPD1 expression in phyC mutant plants is elevated in phyC/elf3 mutant plants consistent with the rapid flowering of the double mutant. We show that loss of PPD1 function also results in reduced FT1 expression and extremely delayed flowering consistent with results from wheat and barley. Additionally, elf3 mutant plants have elevated expression levels of PPD1, and we show that overexpression of ELF3 results in delayed flowering associated with a reduction of PPD1 and FT1 expression, indicating that ELF3 represses PPD1 transcription consistent with previous studies showing that ELF3 binds to the PPD1 promoter. Indeed, PPD1 is the main target of ELF3-mediated flowering as elf3/ppd1 double mutant plants are delayed flowering. Our results indicate that ELF3 operates downstream from PHYC and acts as a repressor of PPD1 in the photoperiod flowering pathway of B. distachyon.