Daniel P. Woods

The Molecular Basis of Vernalization in Different Plant Groups

Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization, the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring, is an example of the influence of temperature on the timing of flowering. In different groups of plants, there are distinct genes involved in vernalization, indicating that vernalization systems evolved independently in different plant groups. The convergent evolution of vernalization systems is not surprising given that angiosperm families had begun to diverge in warmer paleoclimates in which a vernalization response was not advantageous. Here, we review what is known of the vernalization response in three different plant groups: crucifers (Arabidopsis), Amaranthaceae (sugar beet), and Pooideae (wheat, barley, and Brachypodium distachyon). We also discuss the advantages of using Brachypodium as a model system to study flowering and vernalization in the Pooids. Finally, we discuss the evolution and function of the Ghd7/VRN2 gene family in grasses.

Interaction of Photoperiod and Vernalization Determines Flowering Time of Brachypodium distachyon

The temperate grass, Brachypodium distachyon, is a useful model for studying gene networks controlling flowering. Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization is an example of temperature influencing the timing of flowering and is defined as the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring. In cereals, three genes (VERNALIZATION1 [VRN1], VRN2, and FLOWERING LOCUS T [FT]) have been identified that influence the vernalization requirement and are thought to form a regulatory loop to control the timing of flowering. Here, we characterize natural variation in the vernalization and photoperiod responses in Brachypodium distachyon, a small temperate grass related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Brachypodium spp. accessions display a wide range of flowering responses to different photoperiods and lengths of vernalization. In addition, we characterize the expression patterns of the closest homologs of VRN1, VRN2 (VRN2-like [BdVRN2L]), and FT before, during, and after cold exposure as well as in different photoperiods. FT messenger RNA levels generally correlate with flowering time among accessions grown in different photoperiods, and FT is more highly expressed in vernalized plants after cold. VRN1 is induced by cold in leaves and remains high following vernalization. Plants overexpressing VRN1 or FT flower rapidly in the absence of vernalization, and plants overexpressing VRN1 exhibit lower BdVRN2L levels. Interestingly, BdVRN2L is induced during cold, which is a difference in the behavior of BdVRN2L compared with wheat VRN2 during cold.

Evolution of VRN2/Ghd7-Like Genes in Vernalization-Mediated Repression of Grass Flowering

Despite widespread vernalization responsiveness in the grass subfamily Pooideae, the flowering repressor VERNALIZATION2 evolved more recently in core members of this subfamily. Flowering of many plant species is coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization provides competence to flower after prolonged cold exposure, and a vernalization requirement prevents flowering from occurring prior to winter. In winter wheat (Triticum aestivum) and barley (Hordeum vulgare), three genes VRN1, VRN2, and FT form a regulatory loop that regulates the initiation of flowering. Prior to cold exposure, VRN2 represses FT. During cold, VRN1 expression increases, resulting in the repression of VRN2, which in turn allows activation of FT during long days to induce flowering. Here, we test whether the circuitry of this regulatory loop is conserved across Pooideae, consistent with their niche transition from the tropics to the temperate zone. Our phylogenetic analyses of VRN2-like genes reveal a duplication event occurred before the diversification of the grasses that gave rise to a CO9 and VRN2/Ghd7 clade and support orthology between wheat/barley VRN2 and rice (Oryza sativa) Ghd7. Our Brachypodium distachyon VRN1 and VRN2 knockdown and overexpression experiments demonstrate functional conservation of grass VRN1 and VRN2 in the promotion and repression of flowering, respectively. However, expression analyses in a range of pooids demonstrate that the cold repression of VRN2 is unique to core Pooideae such as wheat and barley. Furthermore, VRN1 knockdown in B. distachyon demonstrates that the VRN1-mediated suppression of VRN2 is not conserved. Thus, the VRN1-VRN2 feature of the regulatory loop appears to have evolved late in the diversification of temperate grasses.

Winter Memory throughout the Plant Kingdom: Different Paths to Flowering

Molecular mechanisms contribute to the memory of winter in different plant groups.

PHYTOCHROME C Is an Essential Light Receptor for Photoperiodic Flowering in the Temperate Grass, Brachypodium distachyon

We show that in the temperate grass, Brachypodium distachyon, PHYTOCHROME C (PHYC), is necessary for photoperiodic flowering. In loss-of-function phyC mutants, flowering is extremely delayed in inductive photoperiods. PHYC was identified as the causative locus by utilizing a mapping by sequencing pipeline (Cloudmap) optimized for identification of induced mutations in Brachypodium. In phyC mutants the expression of Brachypodium homologs of key flowering time genes in the photoperiod pathway such as GIGANTEA (GI), PHOTOPERIOD 1 (PPD1/PRR37), CONSTANS (CO), and florigen/FT are greatly attenuated. PHYC also controls the day-length dependence of leaf size as the effect of day length on leaf size is abolished in phyC mutants. The control of genes upstream of florigen production by PHYC was likely to have been a key feature of the evolution of a long-day flowering response in temperate pooid grasses.

Memory of the vernalized state in plants including the model grass Brachypodium distachyon

Plant species that have a vernalization requirement exhibit variation in the ability to “remember” winter – i.e., variation in the stability of the vernalized state. Studies in Arabidopsis have demonstrated that molecular memory involves changes in the chromatin state and expression of the flowering repressor FLOWERING LOCUS C, and have revealed that single-gene differences can have large effects on the stability of the vernalized state. In the perennial Arabidopsis relative Arabis alpina, the lack of memory of winter is critical for its perennial life history. Our studies of flowering behavior in the model grass Brachypodium distachyon reveal extensive variation in the vernalization requirement, and studies of a particular Brachypodium accession that has a qualitative requirement for both cold exposure and inductive day length to flower reveal that Brachypodium can exhibit a highly stable vernalized state.

Extensive gene content variation in the Brachypodium distachyon pan-genome correlates with population structure

While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of differentially present genes in eukaryotes remains poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yield a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g., defense and development), display faster evolutionary rates, lie closer to transposable elements and are less likely to be syntenic with orthologous genes in other grasses. Our data suggest that differentially present genes contribute substantially to phenotypic variation within a eukaryote species, these genes have a major influence in population genetics, and transposable elements play a key role in pan-genome evolution.The role of differential gene content in the evolution and function of eukaryotic genomes remains poorly explored. Here the authors assemble and annotate the Brachypodium distachyon pan-genome consisting of 54 diverse lines and reveal the differential present genes as a major driver of phenotypic variation.

Genetic Architecture of Flowering-Time Variation in Brachypodium distachyon

QTL associated with VERNALIZATION1/PHYC and VERNALIZATION2 account for much of the natural variation in flowering time between the Brachypodium distachyon lines Bd21 and Bd1-1. The transition to reproductive development is a crucial step in the plant life cycle, and the timing of this transition is an important factor in crop yields. Here, we report new insights into the genetic control of natural variation in flowering time in Brachypodium distachyon, a nondomesticated pooid grass closely related to cereals such as wheat (Triticum spp.) and barley (Hordeum vulgare L.). A recombinant inbred line population derived from a cross between the rapid-flowering accession Bd21 and the delayed-flowering accession Bd1-1 were grown in a variety of environmental conditions to enable exploration of the genetic architecture of flowering time. A genotyping-by-sequencing approach was used to develop SNP markers for genetic map construction, and quantitative trait loci (QTLs) that control differences in flowering time were identified. Many of the flowering-time QTLs are detected across a range of photoperiod and vernalization conditions, suggesting that the genetic control of flowering within this population is robust. The two major QTLs identified in undomesticated B. distachyon colocalize with VERNALIZATION1/PHYTOCHROME C and VERNALIZATION2, loci identified as flowering regulators in the domesticated crops wheat and barley. This suggests that variation in flowering time is controlled in part by a set of genes broadly conserved within pooid grasses.

Establishment of a vernalization requirement in Brachypodium distachyon requires REPRESSOR OF VERNALIZATION1

Significance A key feature in the evolution of all vernalization systems is a cold-regulated component. In pooid grasses, up-regulation of the flowering promoter VERNALIZATION1 (VRN1) by prolonged cold is a key feature of vernalization, although little is known about the genes that repress VRN1 prior to cold exposure or activate it afterward. Here, we report the identification of REPRESSOR OF VERNALIZATION1 (RVR1), a repressor of VRN1 that is involved in creating a vernalization requirement in Brachypodium distachyon. RVR1 is present in all sequenced flowering plant genomes but is not found outside the plant kingdom. This report describes a role for the RVR1 class of genes in plants and an upstream component of the VRN1 regulatory system. A requirement for vernalization, the process by which prolonged cold exposure provides competence to flower, is an important adaptation to temperate climates that ensures flowering does not occur before the onset of winter. In temperate grasses, vernalization results in the up-regulation of VERNALIZATION1 (VRN1) to establish competence to flower; however, little is known about the mechanism underlying repression of VRN1 in the fall season, which is necessary to establish a vernalization requirement. Here, we report that a plant-specific gene containing a bromo-adjacent homology and transcriptional elongation factor S-II domain, which we named REPRESSOR OF VERNALIZATION1 (RVR1), represses VRN1 before vernalization in Brachypodium distachyon. That RVR1 is upstream of VRN1 is supported by the observations that VRN1 is precociously elevated in an rvr1 mutant, resulting in rapid flowering without cold exposure, and the rapid-flowering rvr1 phenotype is dependent on VRN1. The precocious VRN1 expression in rvr1 is associated with reduced levels of the repressive chromatin modification H3K27me3 at VRN1, which is similar to the reduced VRN1 H3K27me3 in vernalized plants. Furthermore, the transcriptome of vernalized wild-type plants overlaps with that of nonvernalized rvr1 plants, indicating loss of rvr1 is similar to the vernalized state at a molecular level. However, loss of rvr1 results in more differentially expressed genes than does vernalization, indicating that RVR1 may be involved in processes other than vernalization despite a lack of any obvious pleiotropy in the rvr1 mutant. This study provides an example of a role for this class of plant-specific genes.

Dissecting the Control of Flowering Time in Grasses Using Brachypodium distachyon

The timing of flowering is a critical life history trait that has been shaped over evolutionary time to maximize the ability to flower at a time that optimizes reproductive success. Furthermore, timing of flowering is one of many traits that has been manipulated by humans for increased crop productivity. It can be difficult to determine the molecular underpinnings controlling flowering in cereals due to their large complex genomes and larger stature. However, many attributes of Brachypodium distachyon makes it a useful model grass system to accelerate understanding of the genetic basis of flowering time in grasses. Here we will first discuss what is currently known about flowering in temperate grasses, which largely comes from studies of natural variation for flowering in wheat and barley, followed by some of the progress made in B. distachyon. We will then discuss practical considerations of flowering behavior when growing different accessions of B. distachyon for studies of other traits of interest.