Winter survival in red clover: experimental evidence for interactions among stresses.

BACKGROUND: There is a lack of knowledge on the combined effects of different stresses on plants, in particular different stresses that occur during winter in temperate climates. Perennial herbaceous plants in temperate regions are exposed to many different stresses during winter, but except for the fact that cold temperatures induce resistance to a number of them, very little is known about their interaction effects. Knowledge about stress interactions is needed in order to predict effects of climate change on both agricultural production and natural ecosystems, and to develop adaptation strategies, e.g., through plant breeding. Here, we conducted a series of experiments under controlled conditions to study the interactions between cold (low positive temperature), clover rot infection (caused by Sclerotinia trifoliorum) and freezing, in red clover (Trifolium pratense) accessions. We also compared our results with winter survival in field experiments and studied associations between stress and shoot growth. RESULTS: Exposure to low positive temperatures (cold acclimation) induced resistance to clover rot. There was a clear negative interaction effect between freezing stress and clover rot infection, resulting in up to 37% lower survival rate compared to what would have been expected from the additive effect of freezing and infection alone. Freezing tolerance could continue to improve during incubation under artificial snow cover at 3 °C in spite of darkness, and we observed compensatory shoot growth following freezing after prolonged incubation. At the accession level, resistance to clover rot was negatively correlated with growth in the field during the previous year at a Norwegian location. It was also negatively correlated with the shoot regrowth of control plants after incubation. Clover rot resistance tests under controlled conditions showed limited correlation with clover rot resistance observed in the field, suggesting that they may reveal variation in more specific resistance mechanisms. CONCLUSIONS: We here demonstrate, for the first time, a strong negative interaction between freezing and infection with a winter pathogen. We also characterize the effects of cold acclimation and incubation in darkness at different temperatures on winter stress tolerance, and present data that support the notion that annual cycles of growth and stress resistance are associated at the genetic level.

Biochemical changes after cold acclimation in Nordic red clover (Trifolium pratense L.) accessions with contrasting levels of freezing tolerance.

The ability to tolerate low freezing temperatures is an important component of winter survival and persistence of red clover. Cold acclimation (CA) allows plants to acquire higher levels of freezing tolerance. However, the biochemical responses to cold and the importance of such changes for the plant to acquire adequate freezing tolerance have not been investigated in red clover of Nordic origin, which has a distinct genetic background. To shed light on this, we selected five freezing tolerant (FT) and five freezing susceptible (FS) accessions and studied the effect of CA on the contents of carbohydrates, amino acids, and phenolic compounds in the crowns. Among those compounds which increased during CA, FT accessions had higher contents of raffinose, pinitol, arginine, serine, alanine, valine, phenylalanine, and one phenolic compound (a pinocembrin hexoside derivative) than FS accessions, suggesting a role for these compounds in the freezing tolerance in the selected accessions. These findings, together with a description of the phenolic profile of red clover crowns, significantly add to the current knowledge of the biochemical changes during CA and their role in freezing tolerance in Nordic red clover.

A genome-wide association study of freezing tolerance in red clover (Trifolium pratense L.) germplasm of European origin.

Improvement of persistency is an important breeding goal in red clover (Trifolium pratense L.). In areas with cold winters, lack of persistency is often due to poor winter survival, of which low freezing tolerance (FT) is an important component. We conducted a genome wide association study (GWAS) to identify loci associated with freezing tolerance in a collection of 393 red clover accessions, mostly of European origin, and performed analyses of linkage disequilibrium and inbreeding. Accessions were genotyped as pools of individuals using genotyping-by-sequencing (pool-GBS), generating both single nucleotide polymorphism (SNP) and haplotype allele frequency data at accession level. Linkage disequilibrium was determined as a squared partial correlation between the allele frequencies of pairs of SNPs and found to decay at extremely short distances (< 1 kb). The level of inbreeding, inferred from the diagonal elements of a genomic relationship matrix, varied considerably between different groups of accessions, with the strongest inbreeding found among ecotypes from Iberia and Great Britain, and the least found among landraces. Considerable variation in FT was found, with LT50-values (temperature at which 50% of the plants are killed) ranging from -6.0°C to -11.5°C. SNP and haplotype-based GWAS identified eight and six loci significantly associated with FT (of which only one was shared), explaining 30% and 26% of the phenotypic variation, respectively. Ten of the loci were found within or at a short distance (<0.5 kb) from genes possibly involved in mechanisms affecting FT. These include a caffeoyl shikimate esterase, an inositol transporter, and other genes involved in signaling, transport, lignin synthesis and amino acid or carbohydrate metabolism. This study paves the way for a better understanding of the genetic control of FT and for the development of molecular tools for the improvement of this trait in red clover through genomics assisted breeding.

The effect of harvest time of forage on carbohydrate digestion in horses quantified by in vitro and mobile bag techniques.

Carbohydrates in forages constitute an important part of the feed ration for all horses. The aim of the present study was to investigate the effect of harvest time on carbohydrate composition and digestion of various grass species. The experiment was divided into three parts 1) characterization of the chemical composition of experimental feeds (6 grass species: meadow fescue [MF], cocksfoot [CF], perennial ryegrass [PR], smooth bromegrass [SB], tall fescue [TF], and timothy [TI], and 3 harvest times: early, medium, and late first cut), 2) measurements of the in vitro digestion of selected experimental feeds (the 6 grass species, and 2 harvest times [early and late]) measured by in vitro gas production, and 3) in vivo digestion of selected experimental feeds (2 grass species: CF and PR, 2 harvest times [early and late]) measured by the mobile bag technique using caecum cannulated horses. An experimental field was established with plots containing each of the grass species in three replicate blocks. Grass samples were cut between 1200 and 1400 h at 4th of June (early first cut), 17th of June (medium first cut), and 1st of July (late first cut) and analyzed for crude protein (CP), neutral detergent fiber with heat stable amylase and free of residual ash (aNDFom) and water-soluble carbohydrates (WSC). The in vitro fermentation was investigated using the ANKOM RF gas production technique, where feeds were incubated for 48 h using horse caecal fluid as an inoculum. Gas production was modeled, and maximum gas production (MGP) was used to evaluate the potential digestibility of the feeds. Based on the chemical analyses and the in vitro experiment, early and late harvested CF and PR were selected for the in vivo experiment, which was conducted as a randomized 4 × 4 Latin square design including four periods, four horses and four feeds. In general, the CP content decreased whereas the aNDFom content increased as the grasses matured. The content of WSC increased in SB and TI, but decreased in CF, and fructans increased in SB, TI, PR, and TF as they matured. The in vitro MGP showed a clearer difference between harvest times than between grass species. Harvest time had larger effect on digestibility than grass species, and a high precaecal disappearance of the WSC fraction was measured by the mobile bag technique. Cocksfoot was identified as a grass species with potentially low digestibility and low WSC content and could potentially be used more for horses.

Feedstuffs contain different carbohydrate fractions that are digested in different parts of the gastrointestinal tract of horses. Grass for grazing or harvesting contains variable amounts of structural carbohydrates such as cellulose and hemi-cellulose (named fibres) and nonstructural carbohydrates which in temperate grass species include sugars and fructans (named water soluble carbohydrates (WSC)). This study quantified carbohydrate composition and digestion of six grass species (perennial ryegrass, timothy, smooth bromegrass, tall fescue, cocksfoot, and meadow fescue) harvested at three different times (early, medium, and late) and preserved as hay. In general, fiber content increased as the grasses matured, whereas WSC content varied to a large extent. In vitro fermentation using horse caecal fluid was used to quantify digestion of early and late cut grass samples of all species. Harvest time (early vs. late) had a larger effect on in vitro fermentation compared to the effect of grass species. Early and late harvested perennial ryegrass and cocksfoot were further selected for detailed studies of precaecal digestion in vivo as these species had highest and lowest WSC content. In general, cocksfoot was identified as grass species with low digestibility and low WSC concentration compared to the other species investigated.

Identification of loci controlling timing of stem elongation in red clover using genotyping by sequencing of pooled phenotypic extremes.

MAIN CONCLUSION: Through selective genotyping of pooled phenotypic extremes, we identified a number of loci and candidate genes putatively controlling timing of stem elongation in red clover. We have identified candidate genes controlling the timing of stem elongation prior to flowering in red clover (Trifolium pratense L.). This trait is of ecological and agronomic significance, as it affects fitness, competitivity, climate adaptation, forage and seed yield, and forage quality. We genotyped replicate pools of phenotypically extreme individuals (early and late-elongating) within cultivar Lea using genotyping-by-sequencing in pools (pool-GBS). After calling and filtering SNPs and GBS locus haplotype polymorphisms, we estimated allele frequencies and searched for markers with significantly different allele frequencies in the two phenotypic groups using BayeScan, an FST-based test utilizing replicate pools, and a test based on error variance of replicate pools. Of the three methods, BayeScan was the least stringent, and the error variance-based test the most stringent. Fifteen significant markers were identified in common by all three tests. The candidate genes flanking the markers include genes with potential roles in the vernalization, autonomous, and photoperiod regulation of floral transition, hormonal regulation of stem elongation, and cell growth. These results provide a first insight into the potential genes and mechanisms controlling transition to stem elongation in a perennial legume, which lays a foundation for further functional studies of the genetic determinants regulating this important trait.

Nuclear DNA Content Variation in Different Life Cycle Stages of Sugar Kelp, Saccharina latissima.

Ploidy variants can be utilized to increase yield, introduce sterility, and modify specific traits with an economic impact. Despite economic importance of Saccharina species, their nuclear DNA content in different cell types and life stages remain unclear. The present research was initiated to determine the nuclear DNA content and intraindividual variation at different life cycle stages of the Laminarialean kelp Saccharina latissima. Nuclear DNA content in embryonic and mature sporophytes, released and unreleased zoospores, female, and male gametophytes from Sør-Trøndelag county in Norway were estimated by image analysis using the DNA-localizing fluorochrome DAPI and chicken's red blood cells as a standard. DNA content of a total of 6905 DAPI-stained nuclei was estimated. This is the first study of nuclear DNA content which covered the life cycle of kelp. The lowest level of DNA content (1C) was observed in zoospores with an average of 0.76 pg. Male and female single spore gametophyte cultures presented higher average DNA content, more than double that of zoospores, suggesting the presence of polyteny. Female gametophyte nuclei were slightly larger and more variable in size than those of male gametophytes. The DNA content observed in embryonic sporophytes and in meristoderm cells from older sporophytes (1.51 pg) was 2C as expected and in the range of previously published studies of sporophytes of S. latissima. Mature sporophytes showed intra-plant variation with DNA content values ranging from 2-16C. The main difference was between meristoderm cells (mostly 2C) and cortical and medullary cells (2-16C).

Functional Characterization of Pseudoidium neolycopersici Photolyase Reveals Mechanisms Behind the Efficacy of Nighttime UV on Powdery Mildew Suppression.

Powdery mildews can be controlled by brief exposure to ultraviolet (UV) radiation with devastating effect on their developmental stages including conidia germination. The treatment effect can be impaired by subsequent exposure to UV-A/blue light. UV-A/blue light-activated photolyase may be responsible for this and therefore we tested the function of three cryptochrome/photolyase family (CPF)-like genes (OINE01015670_T110144, OINE01000912_T103440, and OINE01005061_T102555) identified in the obligate biotrophic fungus Pseudoidium neolycopersici, the cause of tomato powdery mildew. A photolyase-deficient mutant of Escherichia coli transformed with coding sequence of OINE01000912_T103440 and exposed to brief (UV)-C treatment (peak emission at 254 nm) showed photoreactivation and cell survival when exposed to subsequent blue light, indicating complementation of photolyase activity. In contrast, the same photolyase-deficient E. coli transformed with the coding sequences of other two CPF-like genes did not survive this treatment, even though their expression were confirmed at protein level. This confirmed that OINE01000912_T103440 is a gene encoding photolyase, here named PnPHR1, with functionality similar to the native photolyase in E. coli, and classified as a class I cyclobutane pyrimidine dimer (CPD) photolyase. Modeling of the 634-amino acid sequence of PnPHR1 suggested that it is capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). However, spectroscopic data of the protein produced in an E. coli expression system could only reveal the presence of a reduced form of FAD, i.e., FADH- as an intrinsic chromophore. Within the tested wavelength range of 365-525 nm, the survival of photolyase-deficient mutant E. coli transformed with PnPHR1 showed a broad action spectrum from 365 to 454 nm. This was very similar to the previously characterized action spectrum for survival of P. neolycopersici conidia that had been treated with UV-C. Quantitative RT-PCR revealed that the expression of PnPHR1 in P. neolycopersici conidia was induced by UV-C, and peak expression occurred 4 h after brief UV-C treatment. The expression of PnPHR1 was repressed when incubated in red light after the UV-C treatment, but not when incubated in UV-A/blue light. The results may explain why the disease-reducing effect of short wavelength UV is impaired by exposure to UV-A and blue light.

Allele Frequency Changes Provide Evidence for Selection and Identification of Candidate Loci for Survival in Red Clover (Trifolium pratense L.).

Survivor populations of red clover (Trifolium pratense L.) from plots in a field experiment in southern Norway were genetically characterized using genotyping by sequencing, and compared with the original population and each other. Genetic differentiation between populations was characterized on the basis of allele frequencies of single nucleotide polymorphisms (SNPs), using principal component analysis. SNPs that had been under selection, i.e., SNPs with significantly different allele frequencies in survivor populations relative to the original population, or between survivor populations that had received different treatments, were identified by analysis of F ST values, using BayeScan and a simple and stringent F ST-based test utilizing replicate populations from the field experiment. In addition, we tested the possibility of pooling DNA samples prior to sequencing, and pooling leaf samples prior to DNA extraction and sequencing, followed by allele frequency estimation on the basis of number of variant reads. Overall, survivor populations were more different from each other than from the original population, indicating random changes in allele frequency, selection in response to local variation in conditions between plots in the field experiment, or sampling error. However, some differentiation was observed between plots sown as pure stands or species mixtures, plots sown at different densities, and plots subjected to different harvesting regimes. Allele frequencies could be accurately estimated from pooled DNA, and SNPs under selection could be identified when leaf samples were pooled prior to DNA extraction. However, substantial sampling error required replicate populations and/or a high number of sampled individuals. We identified a number of chromosomal loci that had been under selection in pure stand plots relative to the original sown population, and loci that had been under differential selection in pure stands of red clover vs. red clover grown in species mixtures. These are all candidate loci for establishment success or persistence in red clover.

Freezing tolerance revisited-effects of variable temperatures on gene regulation in temperate grasses and legumes.

Climate change creates new patterns of seasonal climate variation with higher temperatures, longer growth seasons and more variable winter climates. This is challenging the winter survival of perennial herbaceous plants. In this review, we focus on the effects of variable temperatures during autumn/winter/spring, and its interactions with light, on the development and maintenance of freezing tolerance. Cold temperatures induce changes at several organizational levels in the plant (cold acclimation), leading to the development of freezing tolerance, which can be reduced/lost during warm spells (deacclimation) in winters, and attained again during cold spells (reacclimation). We summarize how temperature interacts with components of the light regime (photoperiod, PSII excitation pressure, irradiance, and light quality) in determining changes in the transcriptome, proteome and metabolome.

Global transcriptome changes in perennial ryegrass during early infection by pink snow mould.

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar 'Fagerlin' based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould.

Vernalization Requirement and the Chromosomal VRN1-Region can Affect Freezing Tolerance and Expression of Cold-Regulated Genes in Festuca pratensis.

Plants adapted to cold winters go through annual cycles of gain followed by loss of freezing tolerance (cold acclimation and deacclimation). Warm spells during winter and early spring can cause deacclimation, and if temperatures drop, freezing damage may occur. Many plants are vernalized during winter, a process making them competent to flower in the following summer. In winter cereals, a coincidence in the timing of vernalization saturation, deacclimation, downregulation of cold-induced genes, and reduced ability to reacclimate, occurs under long photoperiods and is under control of the main regulator of vernalization requirement in cereals, VRN1, and/or closely linked gene(s). Thus, the probability of freezing damage after a warm spell may depend on both vernalization saturation and photoperiod. We investigated the role of vernalization and the VRN1-region on freezing tolerance of meadow fescue (Festuca pratensis Huds.), a perennial grass species. Two F2 populations, divergently selected for high and low vernalization requirement, were studied. Each genotype was characterized for the copy number of one of the four parental haplotypes of the VRN1-region. Clonal plants were cold acclimated for 2 weeks or vernalized/cold acclimated for a total of 9 weeks, after which the F2 populations reached different levels of vernalization saturation. Vernalized and cold acclimated plants were deacclimated for 1 week and then reacclimated for 2 weeks. All treatments were given at 8 h photoperiod. Flowering response, freezing tolerance and expression of the cold-induced genes VRN1, MADS3, CBF6, COR14B, CR7 (BLT14), LOS2, and IRI1 was measured. We found that some genotypes can lose some freezing tolerance after vernalization and a deacclimation-reacclimation cycle. The relationship between vernalization and freezing tolerance was complex. We found effects of the VRN1-region on freezing tolerance in plants cold acclimated for 2 weeks, timing of heading after 9 weeks of vernalization, expression of COR14B, CBF6, and LOS2 in vernalized and/or deacclimated treatments, and restoration of freezing tolerance during reacclimation. While expression of VRN1, COR14B, CBF6, LOS2, and IRI1 was correlated, CR7 was associated with vernalization requirement by other mechanisms, and appeared to play a role in freezing tolerance in reacclimated plants.

Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement.

Red clover (Trifolium pratense L.) is a globally significant forage legume in pastoral livestock farming systems. It is an attractive component of grassland farming, because of its high yield and protein content, nutritional value and ability to fix atmospheric nitrogen. Enhancing its role further in sustainable agriculture requires genetic improvement of persistency, disease resistance, and tolerance to grazing. To help address these challenges, we have assembled a chromosome-scale reference genome for red clover. We observed large blocks of conserved synteny with Medicago truncatula and estimated that the two species diverged ~23 million years ago. Among the 40,868 annotated genes, we identified gene clusters involved in biochemical pathways of importance for forage quality and livestock nutrition. Genotyping by sequencing of a synthetic population of 86 genotypes show that the number of markers required for genomics-based breeding approaches is tractable, making red clover a suitable candidate for association studies and genomic selection.

Overwintering of herbaceous plants in a changing climate. Still more questions than answers.

The increase in surface temperature of the Earth indicates a lower risk of exposure for temperate grassland and crop to extremely low temperatures. However, the risk of low winter survival rate, especially in higher latitudes may not be smaller, due to complex interactions among different environmental factors. For example, the frequency, degree and length of extreme winter warming events, leading to snowmelt during winter increased, affecting the risks of anoxia, ice encasement and freezing of plants not covered with snow. Future climate projections suggest that cold acclimation will occur later in autumn, under shorter photoperiod and lower light intensity, which may affect the energy partitioning between the elongation growth, accumulation of organic reserves and cold acclimation. Rising CO2 levels may also disturb the cold acclimation process. Predicting problems with winter pathogens is also very complex, because climate change may greatly influence the pathogen population and because the plant resistance to these pathogens is increased by cold acclimation. All these factors, often with contradictory effects on winter survival, make plant overwintering viability under future climates an open question. Close cooperation between climatologists, ecologists, plant physiologists, geneticists and plant breeders is strongly required to predict and prevent possible problems.

QTL analyses and comparative genetic mapping of frost tolerance, winter survival and drought tolerance in meadow fescue (Festuca pratensis Huds.).

Quantitative trait loci (QTLs) for frost and drought tolerance, and winter survival in the field, were mapped in meadow fescue (Festuca pratensis Huds.) and compared with corresponding traits in Triticeae and rice to study co-location with putatively orthologous QTLs and known abiotic stress tolerance genes. The genomes of grass species are highly macrosyntenic; however, the Festuca/Lolium and Triticeae homoeologous chromosomes 4 and 5 show major structural differences that is especially interesting in comparative genomics of frost tolerance. The locations of two frost tolerance/winter survival QTLs on Festuca chromosome 5F correspond most likely to the Fr-A1 and Fr-A2 loci on wheat homoeologous group 5A chromosomes. A QTL for long-term drought tolerance on chromosome 3F (syntenic with rice 1) support evidence from introgression of Festuca genome segments onto homoeologous Lolium chromosomes (3L) that this genome region is an excellent source of tolerance towards drought stress. The coincident location of several stress tolerance QTL in Festuca with QTL and genes in Triticeae species, notably dehydrins, CBF transcription factors and vernalisation response genes indicate the action of structural or regulatory genes conserved across evolutionarily distant species.