Search

Frost tolerance is the main component of winter-hardiness. To express this trait, plants sense low temperature, and respond by activating the process of cold acclimation. The molecular mechanisms of this acclimation have not been fully understood in the agronomically important group of forage grasses, including Lolium-Festuca species. Herein, the introgression forms of L. multiflorum/F. arundinacea distinct with respect to their frost tolerance, were used as models for the comprehensive, proteomic and physiological, research to recognize the crucial components of cold acclimation in forage grasses. The obtained results stressed the importance of photosynthetic performance under acclimation to low temperature. The stable level of photochemical processes after three weeks of cold acclimation in the introgression form with a higher level of frost tolerance, combined simultaneously with only slightly (but not significantly) decreased level of CO2 assimilation after that period, despite significantly lower stomatal conductance, indicated the capacity for that form to acclimate its photosynthesis to low temperature. This phenomenon was driven by the Calvin cycle efficiency, associated with revealed here accumulation profiles and activities of chloroplastic aldolase. The capacity to acclimate the photosynthetic machinery to cold could be one of the most crucial components of forage grass metabolism to improve frost tolerance.

Giant miscanthus (Miscanthus × giganteus) is a valuable plant for bioenergy production and carbon sequestration in low-input agriculture. The newly establishing plantations of miscanthus are, however, at risk during the winter because of the small size of the clones and limited ability of reserves storage in the rhizomes. Increased freezing tolerance of both shoots and rhizomes of giant miscant has been observed after cold acclimation, when plants are exposed to low, non-freezing temperatures. In plants cold acclimation is under complex genetic control that is orchestrated by C-repeat binding factors (CBFs). The present paper aimed the identification and characterisation of CBF-family transcription factors in miscanthus and determined their expression profiles at a low temperature. Three miscanthus genes, homologous to sorghum and maize CBF-family genes were identified. However, only one – MgCBF6 was expressed in the cold-acclimated leaves of miscanthus with an expression profile typical of a CBF-family gene. The ability of miscanthus to cold acclimate was also confirmed using an electrolyte leakage test and survival count. An increase in freezing tolerance was observed both in leaves and rhizomes but only when an increase in the freezing tolerance of plasma membranes was considered. It may be concluded that CBF-induced gene expression pathway may be induced in M. × giganteus while this species use the dormancy strategy for overwintering.

Climate warming can change the winter weather patterns. Warmer temperatures during winter result in a lower risk of extreme freezing events. On the other hand the predicted warm gaps during winter will decrease their freezing tolerance. Both contradict effects will affect winter survival but their resultant effect is unclear. In this paper, we demonstrate that climate warming may result in a decrease in winter survival of plants. A field study of winterhardiness of common wheat and triticale was established at 11 locations and repeated during three subsequent winters. The freezing tolerance of the plants was studied after controlled cold acclimation and de-acclimation using both plant survival analysis and chlorophyll fluorescence measurements. Cold deacclimation resistance was shown to be independent from cold acclimation ability. Further, cold deacclimation resistance appeared to be crucial for overwintering when deacclimation conditions occurred in the field. The shortening of uninterrupted cold acclimation may increase cold deacclimation efficiency, which could threaten plant survival during warmer winters. Measurements of chlorophyll fluorescence transient showed some differences triggered by freezing before and after deacclimation. We conclude that cold deacclimation resistance should be considered in the breeding of winter cereals and in future models of winter damage risk.

Alternative splicing of the Rubisco activase gene was shown to be a point for optimization of photosynthetic carbon assimilation. It can be expected to be a stress-regulated event that depends on plant freezing tolerance. The aim of the study was to examine the relationships among Rubisco activity, the expression of two Rubisco activase splicing variants and photoacclimation to low temperature. The experiment was performed on two Lolium perenne genotypes with contrasting levels of freezing tolerance. The study investigated the effect of pre-hardening (15°C) and cold acclimation (4°C) on net photosynthesis, photosystem II photochemical activity, Rubisco activity and the expression of two splicing variants of the Rubisco activase gene. The results showed an induction of Rubisco activity at both 15°C and 4°C only in a highly freezing-tolerant genotype. The enhanced Rubisco activity after pre-hardening corresponded to increased expression of the splicing variant representing the large isoform, while the increase in Rubisco activity during cold acclimation was due to the activation of both transcript variants. These boosts in Rubisco activity also corresponded to an activation of non-photochemical mechanism of photoacclimation induced at low temperature exclusively in the highly freezing-tolerant genotype. In conclusion, enhanced expression of Rubisco activase splicing variants caused an increase in Rubisco activity during pre-hardening and cold acclimation in the more freezing-tolerant Lolium perenne genotype. The induction of the transcript variant representing the large isoform may be an important element of increasing the carbon assimilation rate supporting the photochemical mechanism of photosynthetic acclimation to cold.

Increased precipitation expected during autumn and winter at higher latitudes in the northern hemisphere may lead to low temperature short-term waterlogging. The temperature of waterlogging was shown to be an important factor controlling plant reaction to this stress. Photosynthetic apparatus response to water excess in the soil at low temperature was examined in Festuca pratensis genotypes with contrasting freezing tolerance. The study was aimed to test the hypothesis whether changes in leaf water-soluble carbohydrate concentration brought about alterations in Rubisco activity may affect the photoacclimation to cold under water excess in the soil. The study investigated the effects of waterlogging during cold acclimation process on a set of chlorophyll a fluorescence parameters, water-soluble carbohydrates, expression of Rubisco activase gene, and Rubisco activity. It was shown that carbohydrate status affected by Rubisco activity might be crucial for the activation of non-photochemical mechanism of photoacclimation to cold under waterlogging. Altered Rubisco activity was only partially attributed to the expression of Rubisco activase gene. Additionally, low carbohydrate concentration in the leaves of waterlogged plants was the condition preventing sugar repression of photosynthesis, including RcaA expression. This indicates that sugar de-repression of photosynthetic genes may be considered a component of photosynthetic acclimation to cold under waterlogging.

Diversity arrays technology (DArT) marker sequences for barley were used for identifying new potential candidate genes for freezing tolerance (FT). We used quantitative trait loci (QTL) genetic linkage maps for FT and photosynthetic acclimation to cold for six- and two-row barley populations, and a set of 20 DArT markers obtained using the association mapping of parameters for photosynthetic acclimation to low temperatures in barley for the bioinformatics analyses. Several nucleotide and amino acid sequence, annotation databases and associated algorithms were used to identify the similarities of six of the marker sequences to potential genes involved in plant low temperature response. Gene ontology (GO) annotations based on similarities to database sequences were assigned to these marker sequences, and indicated potential involvement in signal transduction pathways in response to stress factors and epigenetic processes, as well as auxin transport mechanisms. Furthermore, relative gene expressions for three of six of new identified genes (Hv.ATPase, Hv.DDM1, and Hv.BIG) were assessed within four barley genotypes of different FT. A physiological assessment of FT was conducted based on plant survival rates in two field-laboratory and one laboratory experiments. The results suggested that plant survival rate after freezing but not the degree of freezing-induced leaf damage between the tested accessions can be correlated with the degree of low-temperature downregulation of the studied candidate genes, which encoded proteins involved in the control of plant growth and development. Additionally, candidate genes for qRT-PCR suitable for the analysis of cold acclimation response in barley were suggested after validation.

Autumnal growth cessation in non-native grass species is inadequate for sufficient cold acclimation. Cold acclimation is influenced to various degrees by growth cessation and photoacclimation. The effects of both factors on winter survival were investigated by measuring photosynthetic activity with Handy Pea fluorimeter and leaf elongation growth rate (LER) and their effects on winter survival. Triplicate field trials were established at two locations in Norway: Fureneset (61 °N) and Vagones (67 °N). In total, ten entries of perennial ryegrass ( Lolium perenne ), Festulolium hybrids and introgression lines, and meadow fescue ( Festuca pratensis ) were investigated weekly by measuring selected leaves ( n = 3 × 10) of the regrowth after the third harvest taken in late August in two successive years. The results showed that the relationship between photosynthetic performance in autumn, LER and winter survival differed between the locations. In the south (Fureneset), there was a positive correlation between photosynthetic activity before winter and winter survival. In the north (Vagones), there was no correlation or even a negative correlation between photosynthetic activity before winter and winter survival. Low photosynthetic activity in autumn was associated with a higher level of winter survival in the north for F. pratensis cv. Norild and two northern-adapted cultivars of L. perenne . Northern-adapted forage grasses can be assumed to have an alternative mechanism for growth inhibition, since in the north the amount of light seems to be insufficient to trigger the changes in photosynthetic apparatus that are responsible for growth cessation in the south. Moreover, with progressing climate change, this adaptation pattern will increasingly be required in more southerly areas of the Nordic region because the light intensity decrease and temperature increase predicted for these areas will delay cold acclimation.

Rubisco activase is required to regulate the catalytic activity of Rubisco in plants, in an ATP-dependent manner. One or two Rubisco activase proteins have been identified in different plant species. In some species, the two isoforms are the products of alternative splicing of the Rubisco activase gene. The aim of this study was to confirm that Lolium perenne and Festuca pratensis plants have two isoforms of Rubisco activase and that they are the products of alternative splicing of common pre-mRNA. Protein gel blot analyses indicated that L. perenne and F. pratensis leaves contained two Rubisco activase proteins. Sequence analysis of cDNA and genomic DNA showed that differential splicing generated two mRNAs that differed in sequence only in the inclusion of 48 bp. The insertion contains a stop codon leading to the synthesis of a shorter polypeptide. Under the conditions of our experiment, the shorter splicing variant of L. perenne and F. pratensis Rubisco activase gene was preferentially produced. Any further studies concerning Rubisco activase genes in L. perenne and/or F. pratensis plants should take into consideration the mechanism of its expression.

Quantitative PCR studies need proper reference genes with expression stability exclusively validated under certain experimental conditions. The expression stability of several genes commonly used as references was tested under 24-epibrassinolide (EBR) and temperature treatment. Different statistical approaches (qBase PLUS , BestKeeper, NormFinder) were used to prepare rankings of expression stability in two species of an economic importance: common wheat ( Triticum aestivum ) and perennial ryegrass ( Lolium perenne ). Candidate reference genes were shown to be regulated differentially in these two plant species. The maximum stability values indicated that the expression stability was higher in T. aestivum . Taking into account of all ranks it seems that TBP-1 and UBI in ryegrass and ACT , ADP and EF1A in wheat should be used as reference genes in the brassinosteroids and temperature involving studies.

Increased precipitation and snow melt during warmer winters may lead to low temperature flooding and ice encasement formation. These conditions are stressful to plants and may affect their winter survival and spring regrowth. The aim of this study was to assess the effects of low temperature flooding on frost tolerance, photosynthetic performance, osmotic potential, water soluble carbohydrate content and expression of CBF6, Cor14b and LOS2 genes in four genotypes of Festuca pratensis with distinct levels of frost tolerance. It was shown that plants cold acclimated under flooding increase their frost tolerance faster and/or to a greater extent than in non-flooded controls. Changes in the induction kinetics of transcription factors encoding genes are connected with transient growth of frost tolerance in two out of the four genotypes, irrespective of their frost tolerance. A significant and stable increase in frost tolerance observed in the genotype with the lowest tolerance under control conditions was related to higher carbohydrate concentration in the flooded plants. In more frost tolerant genotypes, low temperature flooding also improved their resistance to low-temperature induced photoinhibition of photosynthesis. In conclusion, low-temperature flooding of the plant roots and crowns may boost cold acclimation efficiency in F. pratensis, but this effect is genotype-dependent and varies according to the background.

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.

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.

Spring forms of oilseed rape are perceived as having low frost resistance. This is consistent with some field observations and laboratory studies in which spring and winter forms showed different extents of metabolic changes during cold acclimation. This is somewhat surprising, since rapeseed cultivars are classified as winter or spring types based on their requirements for vernalization to flower, not on their levels of frost resistance. This discrepancy may be due to the limited abilities of spring-type rape, in commonly used growth conditions, to cease growth and, simultaneously, maintain a high photosynthetic activity during cold acclimation, which is considered necessary for proper hardening. This study was conducted to determine if spring oilseed rape could acclimate to the level observed in winter-type plants after modification of growth and photosynthetic characteristics induced by prehardening. After prehardening spring cultivars of oilseed rape showed frost resistance comparable with winter-type plants and their response to prehardening was even stronger than that in winter-type plants. It is caused by the exceptionally low cold acclimation abilities of non-prehardened spring forms. No differences were noted between the frost resistances of callus tissue derived from winter and spring-type cultivars.

Photosynthetic apparatus acclimation to low temperature is a prerequisite for proper hardening and acquired freezing tolerance. It has been shown that Lolium perenne plants have two isoforms of Rubisco activase, products of two splicing variants (RcaA1, RcaA2) of common pre-mRNA (Jurczyk et al., 2015). The aim of the study was to determine the relationship between the activation of mechanisms of photosynthetic acclimation to cold, the Rubisco activity and the expression of alternatively spliced Rubisco activase isoforms in Lolium perenne. Transcript levels of alternatively spliced mRNAs coding for two Rubisco activase isoforms were measured using real-time PCR. Rubisco activity has been determined spectrophotometrically. The measurements of chlorophyll-a fluorescence parameters, including photochemical and non-photochemical quenching, were made with a FMS2 fluorymeter. Gas exchange measurements were performed using an infrared gas analyser. All measurements were taken before cold acclimation, after 2 weeks of prehardening, and after 1 and 3 weeks of cold acclimation in two Lolium perenne genotypes with contrasting levels of freezing tolerance. In more freezing tolerant genotype the Rubisco activity was higher in treated plants relative to control. The increased Rubisco activity during cold acclimation was due to higher expression of both transcripts variants, representing two isoforms of Rubisco activase, but the shorter isoform seemed to be more active. The photochemical, but also non-photochemical mechanism of photosynthetic activation to cold has been activated in freezing tolerant genotype. It may be concluded that the increase in photochemical mechanism of photosynthetic acclimation to cold observed in Lolium perenne may be attributed to enhanced expression of Rubisco activase leading to increased Rubisco activity.The research was supported under a project DEC-2011/03/B/NZ2/00536 “The role of alternate mRNA splicing of Rubisco activase gene in acclimation of photosynthetic apparatus to cold” financed by the Polish National Research Centre.

Cold de-acclimation is a phenomenon the frequency of which is expected to increase due to unstable winter temperatures predicted in the future [1] The aim of the present work was to screen for cold-de-acclimation tolerance the breeding materials of common wheat and triticale, and to find out whether this trait may be, at least partially, responsible for their winter survival. The experiment was performed during winter 2013/14 with 44 accessions of triticale and 78 accessions of wheat. Winter hardiness was studied in seven locations. The studies of freezing tolerance/de-acclimation were conducted at two experimental points where the plants were tested for the freezing tolerance before and after a warm spell during winter. Freezing tolerance on the cellular level was additionally assessed by means of chlorophyll fluorescence measurements after freezing. Our results indicated that the studied accessions of both species were highly variable in their tolerance to de-acclimation. Although no correlation between the freezing tolerance and de-acclimation tolerance was observed, all de-acclimation tolerant accessions were also highly freezing-tolerant. The maximum level of de-acclimation tolerance observed in triticale was higher than in wheat. It was also found that de-acclimation tolerance was responsible for different winter survival of triticale plants in one out of seven field locations, while in five locations no differences in winter survival were observed. In the case of common wheat, de-acclimation tolerance was not related to winter survival at any of three locations in which it varied between the studied materials. Similar observations, together with the studies of other risk factors for winter survival during future winters, will be continued for the next four winters to create models of future winter hardiness risk for wheat and triticale and to select plants with higher resistance to these factors. Founding: the Ministry of Agriculture and Rural Development (Poland) grant HOR hn 801-10/14.