Evolution of drought and frost responses in cool season grasses (Pooideae): was drought tolerance a precursor to frost tolerance?

Frost tolerance has evolved many times independently across flowering plants. However, conservation of several frost tolerance mechanisms among distant relatives suggests that apparently independent entries into freezing climates may have been facilitated by repeated modification of existing traits ('precursor traits'). One possible precursor trait for freezing tolerance is drought tolerance, because palaeoclimatic data suggest plants were exposed to drought before frost and several studies have demonstrated shared physiological and genetic responses to drought and frost stress. Here, we combine ecophysiological experiments and comparative analyses to test the hypothesis that drought tolerance acted as a precursor to frost tolerance in cool-season grasses (Pooideae). Contrary to our predictions, we measured the highest levels of frost tolerance in species with the lowest ancestral drought tolerance, suggesting that the two stress responses evolved independently in different lineages. We further show that drought tolerance is more evolutionarily labile than frost tolerance. This could limit our ability to reconstruct the order in which drought and frost responses evolved relative to each other. Further research is needed to determine whether our results are unique to Pooideae or general for flowering plants.

Testing the chilling- before drought-tolerance hypothesis in Pooideae grasses.

Temperate Pooideae are a large clade of economically important grasses distributed in some of the Earth's coldest and driest terrestrial environments. Previous studies have inferred that Pooideae diversified from their tropical ancestors in a cold montane habitat, suggesting that above-freezing cold (chilling) tolerance evolved early in the subfamily. By contrast, drought tolerance is hypothesized to have evolved multiple times independently in response to global aridification that occurred after the split of Pooideae tribes. To independently test predictions of the chilling-before-drought hypothesis in Pooideae, we assessed conservation of whole plant and gene expression traits in response to chilling vs. drought. We demonstrated that both trait responses are more similar across tribes in cold as compared to drought, suggesting that chilling responses evolved before, and drought responses after, tribe diversification. Moreover, we found significantly more overlap between drought and chilling responsive genes within a species than between drought responsive genes across species, providing evidence that chilling tolerance genes acted as precursors for the novel acquisition of increased drought tolerance multiple times independently, partially through the cooption of chilling responsive genes.

Flowering time runs hot and cold.

Evidence suggests that anthropogenically-mediated global warming results in accelerated flowering for many plant populations. However, the fact that some plants are late flowering or unaffected by warming, underscores the complex relationship between phase change, temperature, and phylogeny. In this review, we present an emerging picture of how plants sense temperature changes, and then discuss the independent recruitment of ancient flowering pathway genes for the evolution of ambient, low, and high temperature-regulated reproductive development. As well as revealing areas of research required for a better understanding of how past thermal climates have shaped global patterns of plasticity in plant phase change, we consider the implications for these phenological thermal responses in light of climate change.

Responsiveness to long days for flowering is reduced in Arabidopsis by yearly variation in growing season temperatures.

Conservative flowering behaviours, such as flowering during long days in summer or late flowering at a high leaf number, are often proposed to protect against variable winter and spring temperatures which lead to frost damage if premature flowering occurs. Yet, due the many factors in natural environments relative to the number of individuals compared, assessing which climate characteristics drive these flowering traits has been difficult. We applied a multidisciplinary approach to 10 winter-annual Arabidopsis thaliana populations from a wide climactic gradient in Norway. We used a variable reduction strategy to assess which of 100 climate descriptors from their home sites correlated most to their flowering behaviours when tested for responsiveness to photoperiod after saturation of vernalization; then, assessed sequence variation of 19 known environmental-response flowering genes. Photoperiod responsiveness inversely correlated with interannual variation in timing of growing season onset. Time to flowering appeared driven by growing season length, curtailed by cold fall temperatures. The distribution of FLM, TFL2 and HOS1 haplotypes, genes involved in ambient temperature response, correlated with growing-season climate. We show that long-day responsiveness and late flowering may be driven not by risk of spring frosts, but by growing season temperature and length, perhaps to opportunistically maximize growth.

Independent recruitment of FRUITFULL-like transcription factors in the convergent origins of vernalization-responsive grass flowering.

Flowering in response to low temperatures (vernalization) has evolved multiple times independently across angiosperms as an adaptation to match reproductive development with the short growing season of temperate habitats. Despite the context of a generally conserved flowering time network, evidence suggests that the genes underlying vernalization responsiveness are distinct across major plant clades. Whether different or similar mechanisms underlie vernalization-induced flowering at narrower (e.g., family-level) phylogenetic scales is not well understood. To test the hypothesis that vernalization responsiveness has evolved convergently in temperate species of the grass family (Poaceae), we carried out flowering time experiments with and without vernalization in several representative species from different subfamilies. We then determined the likelihood that vernalization responsiveness evolved through parallel mechanisms by quantifying the response of Pooideae vernalization pathway FRUITFULL (FUL)-like genes to extended periods of cold. Our results demonstrate that vernalization-induced flowering has evolved multiple times independently in at least five grass subfamilies, and that different combinations of FUL-like genes have been recruited to this pathway on several occasions.

Major niche transitions in Pooideae correlate with variation in photoperiodic flowering and evolution of CCT domain genes.

The external cues that trigger timely flowering vary greatly across tropical and temperate plant taxa, the latter relying on predictable seasonal fluctuations in temperature and photoperiod. In the grass family (Poaceae) for example, species of the subfamily Pooideae have become specialists of the northern temperate hemisphere, generating the hypothesis that their progenitor evolved a flowering response to long days from a short-day or day-neutral ancestor. Sampling across the Pooideae, we found support for this hypothesis, and identified several secondary shifts to day-neutral flowering and one to short-day flowering in a tropical highland clade. To explain the proximate mechanisms for the secondary transition back to short-day-regulated flowering, we investigated the expression of CCT domain genes, some of which are known to repress flowering in cereal grasses under specific photoperiods. We found a shift in CONSTANS 1 and CONSTANS 9 expression that coincides with the derived short-day photoperiodism of our exemplar species Nassella pubiflora. This sets up the testable hypothesis that trans- or cis-regulatory elements of these CCT domain genes were the targets of selection for major niche shifts in Pooideae grasses.

Understanding Past, and Predicting Future, Niche Transitions based on Grass Flowering Time Variation.

Since their origin in the early Cretaceous, grasses have diversified across every continent on Earth, with a handful of species (rice [Oryza sativa], maize [Zea mays], and wheat [Triticum aestivum]) providing most of the caloric intake of contemporary humans and their livestock. The ecological dominance of grasses can be attributed to a number of physiological innovations, many of which contributed to shifts from closed to open habitats that incur daily (e.g. tropical mountains) and/or seasonal extremes in temperature (e.g. temperate/continental regions) and precipitation (e.g. tropical savannas). In addition to strategies that allow them to tolerate or resist periodically stressful environments, plants can adopt escape behaviors by modifying the relative timing of distinct development phases. Flowering time is one of these behaviors that can also act as a postzygotic barrier to reproduction and allow temporal partitioning of resources to promote coexistence. In this review, we explore what is known about the phylogenetic pattern of flowering control in grasses, and how this relates to broad- and fine-scale niche transitions within the family. We then synthesize recent findings on the genetic basis of flowering time evolution as a way to begin deciphering why certain aspects of flowering are seemingly so conserved, and what the implications of this are for future adaptation under climate change.

Increased above-ground resource allocation is a likely precursor for independent evolutionary origins of annuality in the Pooideae grass subfamily.

Semelparous annual plants flower a single time during their 1-yr life cycle, investing much of their energy into rapid reproduction. By contrast, iteroparous perennial plants flower multiple times over several years, and partition their resources between reproduction and persistence. To which extent evolutionary transitions between life-cycle strategies are internally constrained at the developmental, genetic and phylogenetic level is unknown. Here we study the evolution of life-cycle strategies in the grass subfamily Pooideae and test if transitions between them are facilitated by evolutionary precursors. We integrate ecological, life-cycle strategy and growth data in a phylogenetic framework. We investigate if growth traits are candidates for a precursor. Species in certain Pooideae clades are predisposed to evolve annuality from perenniality, potentially due to the shared inheritance of specific evolutionary precursors. Seasonal dry climates, which have been linked to annuality, were only able to select for transitions to annuality when the precursor was present. Allocation of more resources to above-ground rather than below-ground growth is a candidate for the precursor. Our findings support the hypothesis that only certain lineages can respond quickly to changing external conditions by switching their life-cycle strategy, likely due to the presence of evolutionary precursors.

Evolution of Cold Acclimation and Its Role in Niche Transition in the Temperate Grass Subfamily Pooideae.

The grass subfamily Pooideae dominates the grass floras in cold temperate regions and has evolved complex physiological adaptations to cope with extreme environmental conditions like frost, winter, and seasonality. One such adaptation is cold acclimation, wherein plants increase their frost tolerance in response to gradually falling temperatures and shorter days in the autumn. However, understanding how complex traits like cold acclimation evolve remains a major challenge in evolutionary biology. Here, we investigated the evolution of cold acclimation in Pooideae and found that a phylogenetically diverse set of Pooideae species displayed cold acclimation capacity. However, comparing differential gene expression after cold treatment in transcriptomes of five phylogenetically diverse species revealed widespread species-specific responses of genes with conserved sequences. Furthermore, we studied the correlation between gene family size and number of cold-responsive genes as well as between selection pressure on coding sequences of genes and their cold responsiveness. We saw evidence of protein-coding and regulatory sequence evolution as well as the origin of novel genes and functions contributing toward evolution of a cold response in Pooideae. Our results reflect that selection pressure resulting from global cooling must have acted on already diverged lineages. Nevertheless, conservation of cold-induced gene expression of certain genes indicates that the Pooideae ancestor may have possessed some molecular machinery to mitigate cold stress. Evolution of adaptations to seasonally cold climates is regarded as particularly difficult. How Pooideae evolved to transition from tropical to temperate biomes sheds light on how complex traits evolve in the light of climate changes.

Discrimination of grass pollen of different species by FTIR spectroscopy of individual pollen grains.

Fourier-transform infrared (FTIR) spectroscopy enables the chemical characterization and identification of pollen samples, leading to a wide range of applications, such as paleoecology and allergology. This is of particular interest in the identification of grass (Poaceae) species since they have pollen grains of very similar morphology. Unfortunately, the correct identification of FTIR microspectroscopy spectra of single pollen grains is hindered by strong spectral contributions from Mie scattering. Embedding of pollen samples in paraffin helps to retrieve infrared spectra without scattering artifacts. In this study, pollen samples from 10 different populations of five grass species (Anthoxanthum odoratum, Bromus inermis, Hordeum bulbosum, Lolium perenne, and Poa alpina) were embedded in paraffin, and their single grain spectra were obtained by FTIR microspectroscopy. Spectra were subjected to different preprocessing in order to suppress paraffin influence on spectral classification. It is shown that decomposition by non-negative matrix factorization (NMF) and extended multiplicative signal correction (EMSC) that utilizes a paraffin constituent spectrum, respectively, leads to good success rates for the classification of spectra with respect to species by a partial least square discriminant analysis (PLS-DA) model in full cross-validation for several species. PLS-DA, artificial neural network, and random forest classifiers were applied on the EMSC-corrected spectra using an independent validation to assign spectra from unknown populations to the species. Variation within and between species, together with the differences in classification results, is in agreement with the systematics within the Poaceae family. The results illustrate the great potential of FTIR microspectroscopy for automated classification and identification of grass pollen, possibly together with other, complementary methods for single pollen chemical characterization.

Importance of Individual Germination Receptor Subunits in the Cooperative Function between GerA and Ynd.

Germination of Bacillus spores is triggered by the binding of specific nutrients to germinant receptors (GRs) located in the spore's inner membrane. The GRs typically consist of A, B, and C subunits, encoded by tricistronic ger operons. The Bacillus licheniformis genome contains the gerA family operons gerA, ynd, and gerK In contrast to the ABC(D) organization that characterizes gerA operons of many Bacillus species, B. licheniformis genomes contain a pentacistronic ynd operon comprising the yndD, yndE3 , yndE2 , yndF1 , and yndE1 genes encoding A, B, B, C, and B GR subunits, respectively (subscripts indicate paralogs). Here we show that B. licheniformis spores can germinate in the absence of the Ynd and GerK GRs, although cooperation between all three GRs is required for optimal germination with amino acids. Spores carrying an incomplete set of Ynd B subunits demonstrated reduced germination efficiencies, while depletion of all three Ynd B subunits restored germination of the spore population to levels only slightly lower than those of wild-type spores at high germinant concentrations. This suggests that the presence of an incomplete set of Ynd B subunits exhibits a dominant negative effect on germination and that the A and C subunits of the Ynd GR are sufficient for the cooperative functionality between Ynd and GerA. In contrast to the B subunits of Ynd, the B subunit of GerA was essential for amino acid-induced germination. This study provides novel insights into the role of individual GR subunits in the cooperative interaction between GRs in triggering spore germination.IMPORTANCE Spore-forming bacteria are problematic for the food industry, as spores can survive decontamination procedures and subsequently revive in food products, with the risk of food spoilage and foodborne disease. The Ynd and GerA germination receptors (GRs) cooperate in triggering efficient germination of Bacillus licheniformis spores when nutrients are present in the surrounding environment. This study shows that the single B subunit of GerA is essential for the cooperative function between Ynd and GerA, while the three B subunits of the Ynd GR are dispensable. The ability of GRs lacking individual subunits to stimulate germination together with other GRs could explain why ger operons lacking GR subunit genes are maintained in genomes of spore-forming species.

Evidence for an Early Origin of Vernalization Responsiveness in Temperate Pooideae Grasses.

The ability of plants to match their reproductive output with favorable environmental conditions has major consequences both for lifetime fitness and geographic patterns of diversity. In temperate ecosystems, some plant species have evolved the ability to use winter nonfreezing cold (vernalization) as a cue to ready them for spring flowering. However, it is unknown how important the evolution of vernalization responsiveness has been for the colonization and subsequent diversification of taxa within the northern and southern temperate zones. Grasses of subfamily Pooideae, including several important crops, such as wheat (Triticum aestivum), barley (Hordeum vulgare), and oats (Avena sativa), predominate in the northern temperate zone, and it is hypothesized that their radiation was facilitated by the early evolution of vernalization responsiveness. Predictions of this early origin hypothesis are that a response to vernalization is widespread within the subfamily and that the genetic basis of this trait is conserved. To test these predictions, we determined and reconstructed vernalization responsiveness across Pooideae and compared expression of wheat vernalization gene orthologs VERNALIZATION1 (VRN1) and VRN3 in phylogenetically representative taxa under cold and control conditions. Our results demonstrate that vernalization responsive Pooideae species are widespread, suggesting that this trait evolved early in the lineage and that at least part of the vernalization gene network is conserved throughout the subfamily. These results are consistent with the hypothesis that the evolution of vernalization responsiveness was important for the initial transition of Pooideae out of the tropics and into the temperate zone.

Evolution of the miR5200-FLOWERING LOCUS T flowering time regulon in the temperate grass subfamily Pooideae.

Flowering time is a carefully regulated trait controlled primarily through the action of the central genetic regulator, FLOWERING LOCUS T (FT). Recently it was demonstrated that a microRNA, miR5200, targets the end of the second exon of FT under short-day photoperiods in the grass subfamily Pooideae, thus preventing FT transcripts from reaching threshold levels under non-inductive conditions. Pooideae are an interesting group in that they rapidly diversified from the tropics into the northern temperate region during a major global cooling event spanning the Eocene-Oligocene transition. We hypothesize that miR5200 photoperiod-sensitive regulation of Pooideae flowering time networks assisted their transition into northern seasonal environments. Here, we test predictions derived from this hypothesis that miR5200, originally found in bread wheat and later identified in Brachypodium distachyon, (1) was present in the genome of the Pooideae common ancestor, (2) is transcriptionally regulated by photoperiod, and (3) is negatively correlated with FT transcript abundance, indicative of miR5200 regulating FT. Our results demonstrate that miR5200 did evolve at or around the base of Pooideae, but only acquired photoperiod-regulated transcription within the Brachypodium lineage. Based on expression profiles and previous data, we posit that the progenitor of miR5200 was co-regulated with FT by an unknown mechanism.

Introduced Scotch broom (Cytisus scoparius) invades the genome of native populations in vulnerable heathland habitats.

Cytisus scoparius is a global invasive species that affects local flora and fauna at the intercontinental level. Its natural distribution spans across Europe, but seeds have also been moved among countries, mixing plants of native and non-native genetic origins. Hybridization between the introduced and native gene pool is likely to threaten both the native gene pool and the local flora. In this study, we address the potential threat of invasive C. scoparius to local gene pools in vulnerable heathlands. We used nuclear single nucleotide polymorphic (SNP) and simple sequence repeat (SSR) markers together with plastid SSR and indel markers to investigate the level and direction of gene flow between invasive and native heathland C. scoparius. Analyses of population structures confirmed the presence of two gene pools: one native and the other invasive. The nuclear genome of the native types was highly introgressed with the invasive genome, and we observed advanced-generation hybrids, suggesting that hybridization has been occurring for several generations. There is asymmetrical gene flow from the invasive to the native gene pool, which can be attributed to higher fecundity in the invasive individuals, measured by the number of flowers and seed pods. Strong spatial genetic structure in plastid markers and weaker structure in nuclear markers suggest that seeds spread over relatively short distances and that gene flow over longer distances is mainly facilitated by pollen dispersal. We further show that the growth habits of heathland plants become more vigorous with increased introgression from the invaders. Implications of the findings are discussed in relation to future management of invading C. scoparius.

Evidence for adaptive evolution of low-temperature stress response genes in a Pooideae grass ancestor.

Adaptation to temperate environments is common in the grass subfamily Pooideae, suggesting an ancestral origin of cold climate adaptation. Here, we investigated substitution rates of genes involved in low-temperature-induced (LTI) stress responses to test the hypothesis that adaptive molecular evolution of LTI pathway genes was important for Pooideae evolution. Substitution rates and signatures of positive selection were analyzed using 4330 gene trees including three warm climate-adapted species (maize (Zea mays), sorghum (Sorghum bicolor), and rice (Oryza sativa)) and five temperate Pooideae species (Brachypodium distachyon, wheat (Triticum aestivum), barley (Hordeum vulgare), Lolium perenne and Festuca pratensis). Nonsynonymous substitution rate differences between Pooideae and warm habitat-adapted species were elevated in LTI trees compared with all trees. Furthermore, signatures of positive selection were significantly stronger in LTI trees after the rice and Pooideae split but before the Brachypodium divergence (P < 0.05). Genome-wide heterogeneity in substitution rates was also observed, reflecting divergent genome evolution processes within these grasses. Our results provide evidence for a link between adaptation to cold habitats and adaptive evolution of LTI stress responses in early Pooideae evolution and shed light on a poorly understood chapter in the evolutionary history of some of the world's most important temperate crops.

Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses.

BACKGROUND: Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. RESULTS: Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. CONCLUSIONS: We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.

Phenotypic responses to light, water, and nutrient conditions in the allopolyploid Arabidopsis suecica and its parent species A. thaliana and A. arenosa: Does the allopolyploid outrange its parents?

Polyploid species possess more than two sets of chromosomes and may show high gene redundancy, hybrid vigor, and masking of deleterious alleles compared to their parent species. Following this, it is hypothesized that this makes them better at adapting to novel environments than their parent species, possibly due to phenotypic plasticity. The allopolyploid Arabidopsis suecica and its parent species A. arenosa and A. thaliana were chosen as a model system to investigate relationships between phenotypic plasticity, fitness, and genetic variation. Particularly, we test if A. suecica is more plastic, show higher genetic diversity, and/or have higher fitness than its parent species. Wild Norwegian populations of each species were analyzed for phenotypic responses to differences in availability of nutrient, water, and light, while genetic diversity was assessed through analysis of AFLP markers. Arabidopsis arenosa showed a higher level of phenotypic plasticity and higher levels of genetic diversity than the two other species, probably related to its outbreeding reproduction strategy. Furthermore, a general positive relationship between genetic diversity and phenotypic plasticity was found. Low genetic diversity was found in the inbreeding A. thaliana. Geographic spacing of populations might explain the clear genetic structure in A. arenosa, while the lack of structure in A. suecica could be due to coherent populations. Fitness measured as allocation of resources to reproduction, pointed toward A. arenosa having lower fitness under poor environmental conditions. Arabidopsis suecica, on the other hand, showed tendencies toward keeping up fitness under different environmental conditions.

Convergent evolution of the annual life history syndrome from perennial ancestors.

Despite most angiosperms being perennial, once-flowering annuals have evolved multiple times independently, making life history traits among the most labile trait syndromes in flowering plants. Much research has focused on discerning the adaptive forces driving the evolution of annual species, and in pinpointing traits that distinguish them from perennials. By contrast, little is known about how 'annual traits' evolve, and whether the same traits and genes have evolved in parallel to affect independent origins of the annual syndrome. Here, we review what is known about the distribution of annuals in both phylogenetic and environmental space and assess the evidence for parallel evolution of annuality through similar physiological, developmental, and/or genetic mechanisms. We then use temperate grasses as a case study for modeling the evolution of annuality and suggest future directions for understanding annual-perennial transitions in other groups of plants. Understanding how convergent life history traits evolve can help predict species responses to climate change and allows transfer of knowledge between model and agriculturally important species.

A Vernalization Response in a Winter Safflower (Carthamus tinctorius) Involves the Upregulation of Homologs of FT, FUL, and MAF.

Safflower (Carthamus tinctorius) is a member of the Asteraceae family that is grown in temperate climates as an oil seed crop. Most commercially grown safflower varieties can be sown in late winter or early spring and flower rapidly in the absence of overwintering. There are winter-hardy safflower accessions that can be sown in autumn and survive over-wintering. Here, we show that a winter-hardy safflower possesses a vernalization response, whereby flowering is accelerated by exposing germinating seeds to prolonged cold. The impact of vernalization was quantitative, such that increasing the duration of cold treatment accelerated flowering to a greater extent, until the response was saturated after 2 weeks exposure to low-temperatures. To investigate the molecular-basis of the vernalization-response in safflower, transcriptome activity was compared and contrasted between vernalized versus non-vernalized plants, in both 'winter hardy' and 'spring' cultivars. These genome-wide expression analyses identified a small set of transcripts that are both differentially expressed following vernalization and that also have different expression levels in the spring versus winter safflowers. Four of these transcripts were quantitatively induced by vernalization in a winter hardy safflower but show high basal levels in spring safflower. Phylogenetic analyses confidently assigned that the nucleotide sequences of the four differentially expressed transcripts are related to FLOWERING LOCUS T (FT), FRUITFUL (FUL), and two genes within the MADS-like clade genes. Gene models were built for each of these sequences by assembling an improved safflower reference genome using PacBio-based long-read sequencing, covering 85% of the genome, with N50 at 594,000 bp in 3000 contigs. Possible evolutionary relationships between the vernalization response of safflower and those of other plants are discussed.