Arkadiusz Kosmala

Molecular mechanisms underlying frost tolerance in perennial grasses adapted to cold climates

We review recent progress in understanding cold and freezing stress responses in forage grass species, notably Lolium and Festuca species. The chromosomal positions of important frost tolerance and winter survival QTLs on Festuca and Lolium chromosomes 4 and 5 are most likely orthologs of QTLs on Triticeae chromosome 5 which correspond to a cluster of CBF-genes and the major vernalization gene. Gene expression and protein accumulation analyses after cold acclimation shed light on general responses to cold stress. These responses involve modulation of transcription levels of genes encoding proteins involved in cell signalling, cellular transport and proteins associated with the cell membrane. Also, abundance levels of proteins directly involved in photosynthesis were found to be different between genotypes of differing frost tolerance levels, stressing the importance of the link between the function of the photosynthetic apparatus under cold stress and frost tolerance levels. The significance of the ability to undergo photosynthetic acclimation and avoid photoinhibition is also evident from numerous studies in forage grasses. Other interesting candidate mechanisms for freezing tolerance in forage grasses are molecular responses to cold stress which have evolved after the divergence of temperate grasses. This includes metabolic machinery for synthesis of fructans and novel ice-binding proteins.

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.

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.

Deciphering priming-induced improvement of rapeseed (Brassica napus L.) germination through an integrated transcriptomic and proteomic approach

Rape seeds primed with -1.2 MPa polyethylene glycol 6000 showed improved germination performance. To better understand the beneficial effect of osmopriming on seed germination, a global expression profiling method was used to compare, for the first time, transcriptomic and proteomic data for osmoprimed seeds at the crucial phases of priming procedure (soaking, drying), whole priming process and subsequent germination. Brassica napus was used here as a model to dissect the process of osmopriming into its essential components. A total number of 952 genes and 75 proteins were affected during the main phases of priming and post-priming germination. Transcription was not coordinately associated with translation resulting in a limited correspondence between mRNAs level and protein abundance. Soaking, drying and final germination of primed seeds triggered distinct specific pathways since only a minority of genes and proteins were involved in all phases of osmopriming while a vast majority was involved in only one single phase. A particular attention was paid to genes and proteins involved in the transcription, translation, reserve mobilization, water uptake, cell cycle and oxidative stress processes.

Changes in the chloroplast proteome following water deficit and subsequent watering in a high- and a low-drought-tolerant genotype of Festuca arundinacea

Festuca arundinacea is one of the most drought-tolerant species within the Lolium-Festuca complex and was used as a model for research aimed at identifying the chloroplast components involved in the proteomic response for drought stress in forage grasses. Individual F. arundinacea genotypes with contrasting levels of drought tolerance, the high-drought-tolerant (HDT) and the low-drought-tolerant (LDT) genotypes, were selected for comparative physiological and proteomic work. Measurements of water uptake, chlorophyll fluorescence, relative water content, electrolyte leakage, and gas exchange during drought and rewatering periods were followed by investigations on accumulation levels of chloroplast proteins before drought conditions, on d 3 and 11 of drought treatment, and after 10 d of subsequent watering, using two-dimensional gel electrophoresis. The proteins that were accumulated differentially between the selected plants were then identified by mass spectrometry. The LDT genotype revealed lower levels of water uptake and relative water content as drought progressed, and this was accompanied by lower levels of transpiration and net photosynthesis, and a higher level of electrolyte leakage observed in this genotype. Eighty-two protein accumulation profiles were compared between the HDT and LDT genotypes and ten proteins were shown to be differentially accumulated between them. The functions of the selected proteins in plant cells and their probable influence on the process of recovery after drought treatment in F. arundinacea 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.

Male gametogenesis and sterility in garlic (Allium sativum L.): barriers on the way to fertilization and seed production

Commercial cultivars of garlic (Allium sativum) do not produce flowers and seed; hence, information on microgametogenesis and genetic knowledge of this important crop is unavailable. Recently, physiological studies enabled flowering and fertility restoration in garlic bolting genotypes by environmental manipulations, thus broadening of the genetic variation and facilitating genetic studies. The present report provides first detailed description of the development of male gametophytes in 11 garlic genotypes varying in their fertility traits. Morphological and anatomical studies revealed completely fertile genotypes, as well as variation in anther and pollen development and disruption of the male organs and gametes at different developmental stages. Three types of plant sterility were observed, including complete sterility, male sterility and environmentally induced male sterility. The ITS1 and ITS2 regions of rRNA of the studied genotypes proved to be strongly conservative and thus did not correspond with the phenotypic expression of fertility or sterility in garlic. On the other hand, two-dimensional protein separation maps revealed significant differences between fertile and sterile genotypes, as well as between developmental stages of microsporogenesis. Further research is needed to investigate the internal mechanisms and environmental component of garlic sterility, as well as the possible molecular markers of these traits.

Are nitric oxide donors a valuable tool to study the functional role of nitric oxide in plant metabolism

In the present work, we tested known nitric oxide (NO) modulators generating the NO+ (sodium nitroprusside, SNP) and NO˙ forms (S-nitroso-N-acetyl-D-penicillamine, SNAP and nitrosoglutathione, GSNO). This allowed us to compare downstream NO-related physiological effects on proteins found in leaves of pelargonium (Pelargonium peltatum L.). Protein modification via NO donors generally affects plant metabolism in a distinct manner, manifested by a lower thiobarbituric acid reactive substance (TBARS) content and lipoxygenase (LOX) activity in response to SNAP and GSNO. This is in contrast to the response observed for SNP treatment. Most changes in enzyme activity (GR, glutathione reductase; GST, glutathione-S-transferase; GPX, glutathione peroxidase) are most spectacular and repeatable during the first 8 h of incubation, which is explained by the half-life of the applied donors. In particular, a close dependence was found between the time-course of NO emission from the applied donors and the temporary inhibition of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX). The observed changes were accompanied by time-dependent alterations in protein accumulation as analysed by two-dimensional gel electrophoresis (2-DE) in pelargonium leaves treated with NO donors (SNP, SNAP and GSNO). Using proteomics, different proteins were found to be down- and up-regulated. However, no new protein spots characteristic of all three donors were found. These results indicate that the form of NO emitted from the donor structure plays a key role in switching on appropriate metabolic modifications. It has been noted that several NO-affected metabolomic changes induced by the used donors were not comparable, which confirms the need to maintain caution when interpreting results obtained using the pharmacological approach with different NO modulator compounds.

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 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.