Z. Zwierzykowski

Identification of leaf proteins differentially accumulated during cold acclimation between Festuca pratensis plants with distinct levels of frost tolerance

Festuca pratensis (meadow fescue) as the most frost-tolerant species within the Lolium-Festuca complex was used as a model for research aimed at identifying the cellular components involved in the cold acclimation (CA) of forage grasses. The work presented here also comprises the first comprehensive proteomic research on CA in a group of monocotyledonous species which are able to withstand winter conditions. Individual F. pratensis plants with contrasting levels of frost tolerance, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on the analysis of leaf protein accumulation before and after 2, 8, and 26 h, and 3, 5, 7, 14, and 21 d of CA, using high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry. The analyses of approximately 800 protein profiles revealed a total of 41 (5.1%) proteins that showed a minimum of a 1.5-fold difference in abundance, at a minimum of one time point of CA for HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analysed plants appeared relatively early during cold acclimation, most often after 26 h (on the 2nd day) of CA and one-half of the differentially accumulated proteins were all parts of the photosynthetic apparatus. Several proteins identified here have been reported to be differentially accumulated during cold conditions for the first time in this paper. The functions of the selected proteins in plant cells and their probable influence on the level of frost tolerance in F. pratensis, are discussed.

GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum

The first backcross breeding programme for the transfer of freezing-tolerance genes from winter hardy Festuca pratensis to winter-sensitive Lolium multiflorum is described. A partly fertile, triploid F1 hybrid F. pratensis (2n=2x=14) × L. multiflorum (2n=4x=28) was employed initially, and after two backcrosses to L. multiflorum (2x) a total of 242 backcross two (BC2) plants were generated. Genomic in situ hybridisation (GISH) was performed on 61 BC2 plants selected for their good growth and winter survival characters in the spring following one Polish winter (2000–2001). Among the winter survivors, diploid chromosome numbers were present in 80% of plants. An appropriate single Festuca introgression in an otherwise undisturbed Lolium genome could provide increased freezing tolerance without compromise to the good growth and plant vigour found in Lolium. Among all the diploids, a total of 20 individuals were identified, each with a single F. pratensis chromosome segment. Another diploid plant contained 13 Lolium chromosomes and a large metacentric F. pratensis chromosome, identified as chromosome 4, with two large distal Lolium introgressions on each chromosome arm. Three of the diploid BC2, including the genotype with Festuca chromosome 4 DNA sequences, were found to have freezing tolerance in excess of that of L. multiflorum, and in one case in excess of the F. pratensis used as control. A detailed cytological analysis combining GISH and fluorescence in situ hybridisation analyses with rDNA probes revealed that the other two freezing-tolerant genotypes carried a Festuca chromosome segment at the same terminal location on the non-satellite arm of Lolium chromosome 2.

Genome constitution and evolution in Lolium × Festuca hybrid cultivars (Festulolium)

Festulolium hybrids are being increasingly used worldwide as forage grasses. This is due to their superior agronomic characteristics, which combine yield performance of ryegrasses (Lolium multiflorum and L. perenne) and tolerance against abiotic stress of fescues (Festuca pratensis, F. arundinacea and F. arundinacea var. glaucescens). Despite the widespread use, only fragmentary information exists on their genomic constitution. We used genomic in situ hybridization (GISH) to analyze genomic constitution of over 600 plants from almost all commercially available cultivars of Festulolium. Our results revealed a surprisingly large range of variation in the proportions of parental genomes and in the extent of intergenomic recombination. Using fluorescence in situ hybridization (FISH) with probes for ribosomal DNA, we assessed the frequency of recombination and elimination of particular chromosomes and chromosome groups in three contrasting Festulolium cultivars. This study provides novel information that will aid in understanding the relationship between a genetic make-up and the phenotype of Festulolium hybrids. Our results indicate that GISH might be a useful tool to aid in Festulolium breeding and provide data for a more detailed description of registered cultivars.

Variability of ribosomal DNA sites in Festuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH

This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploidFestuca pratensis andLolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploidF. pratensis ×L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) withL. perenne genomicDNAas a probe, andF. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. InF. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standardF. pratensis karyotypes. Losses of 45S rDNA loci were more frequent inL. perenne cultivars and intergeneric hybrids. Comparison of theF. pratensis andL. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location inL. perenne. A greater instability ofF. pratensis-genome-like andL. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 inF. pratensis and on chromosome 3 inL. perenne are useful markers for these chromosomes in intergenericFestuca ×Lolium hybrids.

Differences in leaf proteome response to cold acclimation between Lolium perenne plants with distinct levels of frost tolerance

Perennial ryegrass (Lolium perenne) is a high quality forage and turf grass mainly due to its excellent nutritive values and rapid establishment rate. However, this species has limited ability to perform in harsh winter climates. Though winter hardiness is a complex trait, it is commonly agreed that frost tolerance (FT) is its main component. Species growing in temperate regions can acquire FT through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation (CA). The research on molecular basis of FT has been performed on the model plants, but they are not well adapted to extreme winter climates. Thus, the mechanisms of cell response to low temperature in winter crops and agronomically important perennial grasses have yet to be revealed. Here, two L. perenne plants with contrasting levels of FT, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on analyses of leaf protein accumulation before and after 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA, using a high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry (MS). Analyses of 580 protein profiles revealed a total of 42 (7.2%) spots that showed at a minimum of 1.5-fold differences in protein abundance, at a minimum of at one time point of CA between HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analyzed plants appeared most often on the 5th (18 proteins) and the 7th (19 proteins) day of CA. The proteins derived from 35 (83.3%) spots were successfully identified by the use of MS and chloroplast proteins were shown to be the major group selected as differentially accumulated during CA. The functions of the identified proteins and their probable influence on the level of FT in L. perenne are discussed.

Chromosome pairing in allotetraploid hybrids of Festuca pratensis Lolium perenne revealed by genomic in situ hybridization (GISH)

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F1 hybrid plants of the allotetraploid Festuca pratensis × Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents – Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents – Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.

Influence of short-term drought conditions and subsequent re-watering on the physiology and proteome of Lolium multiflorum/Festuca arundinacea introgression forms, with contrasting levels of tolerance to long-term drought.

Festuca arundinacea is a drought tolerant species. Lolium multiflorum has better forage quality but lower tolerance to abiotic stresses. Their hybrids offer an opportunity to perform research on the molecular basis of tolerance to drought. The aim of this work was to recognise the mechanisms of response to short-term drought (11 days) in a glasshouse in two L. multiflorum/F. arundinacea introgression forms with distinct levels of tolerance to long-term drought (14 weeks) in the field. Measurements of physiological parameters, analyses of protein accumulation profiles using two-dimensional gel electrophoresis, and mass spectrometry identification of proteins, which were accumulated differentially between the selected genotypes during short-term drought, were performed. Genotype 7/6, with lower yield potential during 14 weeks of drought, and lower ability to re-grow after watering, had a higher capacity for photosynthesis during 11 days of drought. Genotype 4/10, more tolerant to long-term drought, was able to repair damaged cell membranes after watering and was also characterised by lower transpiration during short-term drought. A total of 455 proteins were analysed, and the 17 that were differentially accumulated between the two genotypes were identified. The results of physiological and proteomic research led to a hypothesis that the higher photosynthetic capacity of genotype 7/6 could be due to a more efficient Calvin cycle, supported by higher accumulation of crucial proteins involving chloroplast aldolase.

Chromosome pairing of individual genomes in tall fescue (Festuca arundinacea Schreb.), its progenitors, and hybrids with Italian ryegrass (Lolium multiflorum Lam.)

A diploid-like pairing system prevents meiotic irregularities and improves the efficiency of gamete production in allopolyploid species. While the nature of the system is known in some polyploid crops including wheat, little is known about the control of chromosome pairing in polyploid fescues (Festuca spp.). In this work we studied chromosome pairing in allohexaploid F. arundinacea, its progenitors F. pratensis and F. glaucescens, and two intergeneric hybrids Lolium multiflorum (2x) ×F. arundinacea (6x) and L. multiflorum (4x) ×F. glaucescens(4x). The use of genomic in situ hybridization (GISH) permitted the analysis of homoeologous chromosome pairing and recombination of different genomes involved. We detected a diploid-like pairing system in polyploid fescues F. arundinacea and F. glaucescens, the latter being one of the progenitors of F. arundinacea. The pairing control system was absent in the second progenitor F. pratensis. Detailed analysis of intergeneric hybrids confirmed the presumed haploinsufficiency of the fescue system, which resulted in homoeologous pairing between all component genomes. This indicates that introgression of any specific chromosome segment from one genome to another is possible in all genome combinations. Our results not only contribute to the quest to discover the nature of the system controlling chromosome pairing in polyploid fescues, but may also have serious implications for design of hybrid breeding schemes in forage grasses.

Chromosome pairing in triploid intergeneric hybrids ofFestuca pratensis withLolium multiflorum, revealed by GISH

Genomicin situ hybridisation (GISH) was used to reveal chromosome pairing in two partly fertile, triploid (2n = 3x = 21) hybrids obtained by crossing the diploid (2n = 2x = 14)Festuca pratensis Huds. (designated FpFp), used as a female parent, with the autotetraploid (2n = 4x = 28)Lolium multiflorum Lam. (designated LmLmLmLm), used as a male parent. The pattern of chromosome pairing calculated on the basis of the mean values of chromosome configurations identified in all 100 PMCs analysed, was: 0.71I Lm + 2.24I Fp + 2.18II Lm/Lm + 0.54II Lm/Fp + 4.18III Lm/Lm/Fp. A relatively high number of Lm/Lm bivalents and Fp univalents, and a low number of Lm/Fp bivalents and Lm univalents indicated that the pairing was preferential betweenL. multiflorum chromosomes. Other observations regarding chromosome pairing within the Lm/Lm/Fp trivalents also confirmed this preferential pairing in the analysed triploids, as the Fp chromosome was not randomly located in the chain- and frying-pan-shaped trivalents. The similarities and differences in chromosome pairing at metaphase I and the level of preferential pairing betweenLolium chromosomes in the different triploidLolium-Festuca hybrids are discussed.

Molecular Breeding and Functional Genomics for Tolerance to Abiotic Stress

Sustainability is a measure of our ability to produce food with the maximium of efficiency combined with the minimum of damage to the environment. Grasslands represent over 40% of all agricultural land in the European Union, and over 70% in the United Kingdom. Whilst Lolium in Europe is considered to be the ideal source of profitable and safe high quality animal forage, its general poor persistency limits its use to favourable growing areas. Fortunately, genes for abiotic stress resistance are transferred readily from closely related Festuca species by conventional breeding technologies. Introgression mapping allows the assembly of desirable gene combinations and molecular markers to assist with their selection in breeding programmes. Additional new androgenesis techniques have led to novel genotypes rarely observed as outcomes of breeding programmes. Lolium × Festuca hybrids display promiscuous chromosome recombination enabling genes from one species to be transferred readily to homoeologous chromosome regions where they both function normally and remain stable. Despite the close homology between Lolium and Festuca species, repetitive DNA sequences differ sufficiently for their genomes to be distinguished, by genomic in situ hybridisation (GISH). This enables the physical mapping of genes for abiotic stress resistance transferred from Festuca to Lolium. Further chromosome recombination between homoeologous Lolium and Festuca sequences enables Festuca introgressions to be “dissected”, and recombination series created. Knowledge of synteny and gene sequences within model species amongst the Poaceae, combined with the development of sequenced molecular markers, and bacterial artificial chromosomes is enabling the isolation of genes for abiotic stress resistance.