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An attempt has been made to work out a simple and reliable method of fast frost resistance evaluation of winter oilseed rape using in vitro cultures. In winter rape cv. Gorczanski, there was investigated cold acclimation ability of hypocotyle sections from 5-days old seedlings and also of callus tissue formed on these sections after subsequent 4 weeks growth on induction medium. It has been found that hypocotyle sections are unable to cold-acclimate. Winter rape calli acclimated well and optimum conditions for acclimation is fortnight’s growth at +2°C. Exposure to light during hardening was not necessary for acquiring maximum resistance. On five winter rape cultivars freezing tests were performed using the best cold acclimation conditions. The differences in resistance between hypocotyle sections did not match the differences in field survival or frost resistance of whole plants. As distinct from hypocotyle sections callus tissue appeared to be suitable for evaluation of frost resistance. However, to ensure the objectiveness of assessment in this method is not so easy. The testing temperature must be chosen carefully, because the results can be reliable only at sufficiently low temperature. For correct estimation of the frost resistance level, it is possible to use both the decrease of triphenyltetrazolium chloride reduction rate during freezing and the increase of callus dry weight during 14 days after freezing. (Abbreviations: BAP — 6-Benzylaminopurine; 2,4-D — 2,4-dichlorophenoxyacetic acid; LT50 — temperature at which 50 % of the plants (or plant material) has been frost killed; MS(0.5;2) — Moorashige and Skoog Basic Medium with addition of 0.5 mg 2.4-D and 2 mg BAP; PPFD — photosynthetic photon flux density; TTC — 2,3,5 triphenyltetrazolium chloride)

The effect of prehardening (early stage of growth at moderate low temperatures (+ 12°C) during the day) on the reliability of frost resistance estimation in a controlled environment has been studied on three winter oilseed rape cultivars differing markedly in their field survival rate (Leo, Gorczanski and Idol). It has been also examined the relationship between the photosynthetic activity during the first stage of cold acclimation and the level of frost resistance observed in investigated cultivars. Presented results demonstrated that prehardening, which increases to a significant degree the effectiveness of the cold acclimation process, also increases the differences in cold hardiness between cultivars, and limits the magnitude of experimental errors made during frost resistance estimation under controlled conditions. In all studied cultivars, prehardening increases significantly the photosynthetic activity during cold acclimation. On the other hand, both in the prehardened and in the nonprehardened plants, no relation has been found between either gas exchange rates or chlorophyll 'a' fluorescence characteristics at low temperatures and cultivar ability for acclimation. Neither the existing differences in photosynthetic activity, nor the degree of photosynthetic apparatus acclimation to cold, which occurs during prehardening, are the factors responsible for the frost resistance variation observed between studied 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.

The objective of the research was to define the changes in photosynthetic activity induced by prehardening and to determine their involvement in frost tolerance of winter rape. Prehardening of winter rape, consisting of keeping the plants at + 12°C during the light periods from sprouting until the beginning of the 1st stage of cold hardening, contributed to increasing its effectiveness. After 42 days of hardening at + 2°C the resistance of the prehardened plants equalled that attained by winter rape in the most favourable seasons of vegetation in the field. Prehardening stimulated the efficiency of photosynthesis at chill temperatures (+ 2-5°C). Differences in photosynthetic efficiency, like those in frost resistance, increase with successive weeks of hardening. They also concern the leaves already developed at the hardening temperature. A prehardened photosynthetic apparatus is less susceptible to the progress of photoinactivation taking place when the seedlings are kept at + 2°C. It also demonstrated greater activity even during the first hour of hardening or in the newly expanding leaves, and also at higher temperatures, most probably because of the more efficient progress of the dark processes. The described changes in the functioning of the photosynthetic apparatus induced by prehardening were thus qualitatively very similar to those observed during long-term growth at + 5°C, already described in the literature.

Studies on the effects of temperature during the early stage of growth on frost resistance of winter rape seedlings under controlled conditions were performed. It was found that cold acclimation responses of plants were affected to a great extent by the conditions of the seedlings early growth. During this period, when the day temperatures were reduced to the range from +10 °C to +15 °C, a process termed “prehardening” was observed.

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.

Increased precipitation and snowmelt during warmer winters may lead to low-temperature waterlogging of plants. Perennial ryegrass (Lolium perenne L.) is one of the most important cool-season grasses in agriculture. It is well adapted to cold climates, and may be considered as a model system for studying the mechanisms involved in cold acclimation. The aim of this study was to evaluate the effects of waterlogging on photosynthetic acclimation to cold in perennial ryegrass. Two L. perenne genotypes that differ in their responses to waterlogging in terms of freezing tolerance were compared. We evaluated the effects of waterlogging during cold acclimation on the water-soluble carbohydrate concentration, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, photochemical efficiency of PSII, and transcript levels of the Rubisco activase (RcaA) and sucrose-sucrose fructosyltransferase (1-SST) genes. The genotype that did not accumulate water-soluble carbohydrates in the leaf under waterlogging showed a lower degree of feedback inhibition of photosynthesis under low temperature, and activated a photochemical mechanism of photosynthetic acclimation to cold. The other genotype accumulated water-soluble carbohydrates in the leaf during waterlogging, and activated a non-photochemical mechanism under cold conditions. Different photosynthetic acclimation systems to cold under waterlogging may be activated in these two contrasting L. perenne genotypes.

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.

Lolium perenne is used extensively as a forage and turf grass due to its high nutritive values, rapid establishment and persistence. Festuca pratensis, a species closely related to L. perenne, has lower nutritive values but is one of the most winter-hardy species within the Lolium-Festuca complex. Frost tolerance is the main component of winter-hardiness and the plant species growing in temperate regions can acquire it through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation. Herein, we review our recent results of two proteomic projects, focused on F. pratensis and L. perenne. Each project involved the comparison of leaf protein accumulation profiles during cold acclimation between plants with different levels of frost tolerance by the use of two-dimensional electrophoresis and identification of differentially accumulated proteins by mass spectrometry. In the present paper similarities and differences in leaf proteome response to cold acclimation between F. pratensis and L. perenne are summarized.

Vernalization requirements and cold resistance of 13 lines of winter rape derived from doubled haploids obtained by androgenesis were investigated. The degree of vernalization requirements was examined in two parallel experiments. In the first experiment seedlings aged 2 weeks were vernalized during 63 and 35 days under controlled conditions, at 5 °C. In the second experiment the natural conditions of vernalization were differentiated by sowing the plants at four different dates in spring: March 15th, April 15th, May 15th, and at the latest date excluding vernalization, June 5th. The lines examined revealed differentiation of vernalization requirements that were similar in both experiments; all the lines required a period of exposure to cold before flowering. Cold resistance of lines was estimated in two successive series at −15 °C and −17 °C. Before testing the plants were kept in natural field conditions from the beginning of September until the middle of November. Testing of cold resistance was preceded by hardening under controlled conditions. Considerable differences in cold resistance was found in the forms investigated. The results obtained do not show any relation between frost resistance and the degree of vernalization requirements, as the line characterized by the strongest winter habit of growth showed low cold resistance, whereas the lines which were closer to spring growth habit showed the highest level of cold resistance; the lines with the least vernalization requirements revealed the highest level of cold resistance. This conclusion can be confirmed by the significance of the correlation coefficients between the various indices defining the frost resistance and the indices of vernalization requirements.

Strictly controlled and coordinated induction of CBF regulon (a set of genes regulated by CBF proteins) promotes plant freezing tolerance. CBFs regulate the expression of COR genes that confer freezing tolerance. COR14b in barley is one of the effector genes which seems to be important in resistance to combined freezing and photoinhibition of photosynthesis. LOS2 represses the transcription of STS/ZAT10 (a negative regulator of CBF-target genes) and thus acts as a positive regulator of COR genes. In Arabidopis, low temperature induction of CBFs was reported to be gated by the circadian clock. Moreover, light-quality signals have been shown to regulate some plants’ freezing tolerance genes. The aim of our study was to determine the effects of combined treatment with light and cold on the transcript levels of CBF6, FpCor14b and LOS2 genes in Festuca pratensis. We have demonstrated that the regulation of CBF6, FpCor14b and LOS2 induction kinetics in F. pratensis occurs through the interaction of temperature and light with time of day during low-temperature shift. The FpCOR14b transcript level was shown to be up-regulated by increasing light intensity. It was also proved that light quality strongly regulates CBF6, FpCor14b and LOS2 transcripts induction kinetics at low temperatures.

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.

† Background and Aims Cold acclimation modifies the balance of the energy absorbed and metabolized in the dark processes of photosynthesis, which may affect the expression of cold-regulated (COR) genes. At the same time, a gradual acclimation to the relatively high light conditions is observed, thereby minimizing the potential for photooxidative damage. As a result, the resistance to photoinhibition in the cold has often been identified as a trait closely related to freezing tolerance. Using four barley genotypes that differentially express both traits, the effect of cold acclimation on freezing tolerance and high-light tolerance was studied together with the expression of COR14b, one of the best-characterized barley COR genes. † Methods Plants were cold acclimated for 2 weeks at 2 8C. Freezing tolerance was studied by means of electrolyte leakage. Changes in photosynthetic apparatus and high-light tolerance were monitored by means of chlorophyll fluorescence. Accumulation of COR14b and some proteins important in photosynthetic acclimation to cold were studied with western analysis. COR14b transcript accumulation during cold acclimation was assessed with realtime PCR. † Key Results Cold acclimation increased both freezing tolerance and high-light tolerance, especially when plants were treated with high light after non-lethal freezing. In all plants, cold acclimation triggered the increase in photosynthetic capacity during high-light treatment. In two plants that were characterized by higher high-light tolerance but lower freezing tolerance, higher accumulation of COR14b transcript and protein was observed after 7 d and 14 d of cold acclimation, while a higher transient induction of COR14b expression was observed in freezing-tolerant plants during the first day of cold acclimation. High-light tolerant plants were also characterized with a higher level of PsbS accumulation and more efficient dissipation of excess light energy. † Conclusions Accumulation of COR14b in barley seems to be important for resistance to combined freezing and high-light tolerance, but not for freezing tolerance per se.

Subunits of high molecular weight glutenins strongly influence wheat bread making quality and can be associated with important agronomic traits. Polish winter wheats show a significant quantitative dominance of the null allele over the coding alleles of the Glu-A1 locus. To identify the causes of such skewed distribution, 116 F5 lines obtained from six cross combinations were analyzed for their HMW glutenin subunits and 11 agronomic characteristics, such as plant height and uniformity, leaf blotch and leaf rust resistance, grain yield per plot, number of grains per ear, grain yield per ear, 1000 kernel weight, frost tolerance, total protein content and the SDS-sedimentation value. The SDS-sedimentation value, resistance to leaf blotch and frost tolerance showed statistically significant associations with the status of the Glu-A1 locus. It appears that chromosome 1A with the null allele at Glu-A1 carries a closely linked locus responsible for frost tolerance. With early strong selection for winter hardiness, the null allele of Glu-A1 becomes fixed in advanced breeding materials despite its strong negative impact on the end use quality.

Genes for winter hardiness and frost tolerance were introgressed from Festuca arundinacea into winter-sensitive Lolium multiflorum. Two partly fertile, pentaploid (2n = 5x = 35) F(1) hybrids F. arundinacea (2n = 6x = 42) x L. multiflorum (2n = 4x = 28) were generated and backcrossed twice onto L. multiflorum (2x). The backcross 1 (BC(1)) and backcross 2 (BC(2)) plants were preselected for high vigor and good fertility, and subsequently, a total of 83 BC(2) plants were selected for winter hardiness after 2 Polish winters and by simulated freezing tests. Genomic in situ hybridization (GISH) was performed on 6 winter-hardy plants selected after the first winter and shown to be significantly (P0.05) more frost tolerant than the L. multiflorum control. Among the analyzed BC(2) winter survivors, only diploid (2n = 2x = 14) plants were found. Five plants carried 13 intact L. multiflorum chromosomes and 1 L. multiflorum chromosome with a single introgressed F. arundinacea terminal chromosome segment. The sixth BC(2) winter survivor appeared to be Lolium without any Festuca introgression capable of detection by GISH. A combined GISH and fluorescence in situ hybridization analysis with rDNA probes of the most winter-hardy (after 2 winters) and frost-tolerant BC(2) plant revealed the location of an F. arundinacea introgression on the nonsatellite arm of L. multiflorum chromosome 2, the same chromosome location reported previously as a site for frost tolerance genes in the diploid and winter-hardy species Festuca pratensis.

During cold acclimation by higher plants, temperature perception via changes in redox state of Photosystem II (PSII) and subsequent acclimation of the photosynthetic apparatus to cold is very important for achieving freezing tolerance. These properties were studied in two groups (A and B) of the same backcross 3 (BC3) progeny derived from a triploid hybrid of Festuca pratensis (2×) × Lolium multiflorum (4×) backcrossed three times onto diploid L. multiflorum cultivars. Leaves of Group A plants formed at 20°C at medium-low light were unable to acclimate their photosynthetic apparatus to cold. Compared to Group B, the Group A plants were also more frost sensitive. This acclimation ability correlated with the freezing tolerance of the plants. However, leaves of the same Group A plants developed at 20°C, but under higher-light conditions had increased ability to acclimate their photosynthetic apparatus to cold. It was concluded that Group A plants may have impaired PSII temperature perception, and this then resulted in their poor capability to cold acclimate.

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 F(1) hybrid F. pratensis (2n=2x=14) x L. multiflorum (2n=4x=28) was employed initially, and after two backcrosses to L. multiflorum (2x) a total of 242 backcross two (BC(2)) plants were generated. Genomic in situ hybridisation (GISH) was performed on 61 BC(2) 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 BC(2), 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.

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.

The aim of the present investigation was to determine if the loss of frost resistance observed in spring-type oilseed rape during winter may be the effect of the tendency to start elongation growth during the prolonged low-temperature stay. Interactions between elongation growth rate, properties of photosynthetic apparatus and frost resistance were studied under these conditions in spring and winter cultivars of oilseed rape. Both spring and winter cultivars of oilseed rape reached the maximal frost resistance after 6 weeks at +5 degrees C. Photosynthetic apparatus of both cultivars acclimated to functioning in cold. The resistance of winter type plants remained unchanged at the end of the experiment (10 weeks) whereas spring-type plants lost the maximal resistance in subsequent weeks. It was preceded in the 7th week of low-temperature stay by acceleration of elongation growth without an increase in dry matter accumulation. A gradual loss of photosynthetic activity was also observed during this period. It was manifested as a decrease in antenna trapping efficiency, photochemical and non-photochemical fluorescence quenching and actual quantum yield of PSII without affecting apparent quantum yield of PSII. At the 70th day of the experiment, a decrease in CO(2) exchange and dry matter accumulation were even observed. The possible relationships between growth rate and functioning of photosynthetic apparatus are discussed.

The chlorophyll fluorescence imaging technique is a valuable tool to study the impact of heavy metal stress in plants. The aim of this paper was to investigate the influence of Cd on photosynthetic apparatus of white cabbage ( Brassica oleracea subsp. capitata f. alba) plants. Two cabbage cultivars ‘Ditmarska Najwcześniejsza’ (‘DN’; early) and ‘Amager Polana’ (‘AP’; late) were used. Cd was applied before planting seedlings (10 mg Cd kg −1 DM of soil).. Measurements were performed at the 3rd leaf after 2 weeks of planting. The level of Cd-induced stress to plants was estimated by chlorophyll (Chl) content (photometrically) and analyses of images and numeric values of the major fluorescence parameters of Chl (Chl fluorescence imaging system FluorCam). Cd negatively affected the chlorophyll content and Fv/Fm, Fv’/Fm’, Φ PSII and qP in leaves of early cultivar of white cabbage. However, in the case of late cv. we did not observe such distinct changes. It suggests that late cultivars. are more resistant to Cd than the early ones. Considering methodological aspect of the study, Chl fluorescence imaging can better reveal some alterations within the leaf, because numeric values of specific parameters, which are the averaged data collected from the whole leaf, cannot reflect the tissue specificity.Abbreviations: HM – heavy metal, Cd – cadmium, Chl – chlorophyll, Fv/Fm – photochemical efficiency of PSII in the dark-adapted state, F‘v’/F‘m’ – PSII maximum efficiency, Φ PSII – quantum efficiency of PSII electron transport, NPQ – nonphotochemical quenching of maximal Chl fluorescence, qP – photochemical quenching coefficient.

Key message An effective approach for the further evolution of QTL markers, may be to create mapping populations for locally adapted gene pools, and to phenotype the studied trait under local conditions. Abstract Mapping populations of Polish fodder and malting spring barleys (Hordeum vulgare L.) were used to analyze traits describing short-time drought response at the seedlings stage. High-throughput genotyping (Diversity Array Technology (DArT) markers) and phenotyping techniques were used. The results showed high genetic diversity of the studied populations which allowed the creation of high-density linkage maps. There was also high diversity in the physiological responses of the barleys. Quantitative trait locus (QTL) analysis revealed 18 QTLs for nine physiological traits on all chromosomes except 1H in malting barley and 15 QTLs for five physiological traits on chromosomes 2H, 4H, 5H and 6H in fodder barley. Chromosomes 4H and 5H contained QTLs which explained most of the observed phenotypic variations in both populations. There was a major QTL for net photosynthetic rate in the malting barley located on chromosome 5H and two major QTLs for overall photochemical performance (PI) located on 5H and 7H. One major QTL related to photochemical quenching of chlorophyll fluorescence was located on chromosome 4H in fodder barley. Three QTL regions were common to both mapping populations but the corresponding regions explained different drought-induced traits. One region was for QTLs related to PSII photosynthetic activity stress index in malting barley, and the corresponding region in fodder barley was related to the water content stress index. These results are in accordance with previous studies which showed that different traits were responsible for drought tolerance variations in fodder and malting barleys. Electronic supplementary material The online version of this article (doi:10.1007/s00122-013-2190-x) contains supplementary material, which is available to authorized users.

Prehardening of winter rape, i.e. its early growth in a reduced day temperature (+ 12°C) from emergence to the beginning of cold acclimation at chilling temperatures (the 1 st stage of cold acclimation), has a beneficial influence on frost resistance. In earlier studies it has been demonstrated that during prehardening, plants formed leaf rosettes and increased the photosynthetic efficiency at chilling temperatures. In the present study investigations were carried out on the effect of prehardening on the growth rate of the plants during the 1 st stage of cold acclimation, and the progress of selected physiological processes occurring during this stage and resulting in increased frost resistance. It has been demonstrated that a greater inflow of photoassimilates during the 1 st stage of cold acclimation results in greater increments of the dry mass of prehardened plants. These plants show also a distinct inhibition both of the elongation growth and the rate of expanding new leaves. The acquired energy is thus spent to a greater extent on the processes associated with increasing frost resistance, and not on growth. In prehardened seedlings, during the 1 st stage of cold acclimation, there have been observed in leaves a higher rate of both water content decrease and drop in the osmotic potential of the cell sap and water potential in the tissues. Also an increased accumulation of soluble sugars and free proline was noticed. However, the beginning of these processes was not observed during the prehardening period. Prehardening stimulated the effectiveness of the 1 st stage indirectly through changes leading to the increased amount of available energy and enabling the utilisation of the greater part of acquired energy in the cold acclimation process.

Drought tolerance in barley is highly correlated with the expression of two genes: Hordeum vulgare aleurone 1 (HVA1) and stress-responsive gene 6 (SRG6). Though their role in the mechanism of drought response in barley has been confirmed in transgenic plants, the regulation pathways of these genes’ expression have not been sufficiently studied, especially on the level of whole plants. We used four barley genotypes of different drought tolerance to establish and compare the expression profiles of SRG6 and HVA1 and to associate them with the possible physiological and biochemical signals of water deficit. Both genes studied were expressed to a greater extent in drought tolerant genotypes. The highest level of HVA1 transcript accumulation was observed under conditions where the leaf water potential decreased significantly. In tolerant genotype this signal was partially replaced with abscisic acid (ABA) signal of soil water deficit and the final transcript accumulation was about 14 times lower than in the case of leaf water deficit. In the case of SRG6 the main signal which can triggered the transcript accumulation was ABA but in the case of tolerant genotype the direct effect of leaf water deficit was also observed. Thus, it seems to be possible that in drought tolerant barley genotypes, HVA1 and SRG6, are not only more expressed during drought, but tolerant genotypes may be also more sensitive to various internal signals confirming environmental water deficit. The putative role of hydrogen peroxide as a signal of water deficit in the regulation of both genes expression was not confirmed. The drought-induced expression of both HVA1 and SRG6 was additionally reduced in the light. Because of this powerful complexity, a true understanding of plant response to drought requires further studies integrating gene expression and cell signaling analysis in single organs or tissues with whole plant physiology and long distance signaling.

Plants have developed different strategies to adapt to various stress conditions including drought. In the present study the drought-induced changes in the actin filament (AFs) network was studied, for the first time, in two barley cultivars of contrasting drought tolerance level. Detached leaves of drought-tolerant (cv. ‘CAM/B1/CI’) and drought-susceptible (cv. ‘Maresi’) cultivars were dried under controlled conditions. The water relations as well as the transcript accumulation of actin (ACT11), actin depolymerization factor (ADF1) and dehydrine (HVA1) encoding genes were studied using qRT-PCR. Quantitative (the relative fluorescence index; RFI) and qualitative drought-induced changes in AF cytoskeleton were observed following staining with phalloidin. It was noticed that tolerant cultivar was characterized with relative water content decreased during drought treatment which was accompanied by increase in HVA1 expression together with decrease in ACT11 and ADF1 transcripts accumulation induced by drought. In drought-susceptible cultivar the expressions of both ACT11 and ADF1 were slightly lower than those in the control. Drought triggered an extensive AF cytoskeleton reorganization within different types of leaf-blade cells. Remarkable changes in AF configuration and its increased amount (fluorescence intensity) were observed mainly in drought-tolerant cultivar. In addition, drought-induced changes in AFs were closely associated with chloroplasts. Those AFs probably controlled drought-induced intracellular chloroplast positioning in mesophyll. Based on the results obtained in the present study, the possible role of AF rearrangements in drought response is discussed.

Recent studies (Rapacz 1999) have shown that cultivars of spring-type oilseed rape are able to cold-acclimate to the level comparable with winter cultivars, but only after prehardening which results both in the increase of photosynthetic activity and in growth cessation. It is commonly known that under field conditions spring-type cultivars could not survive winter. Present studies were undertaken to explain the reasons for low winter hardiness of spring type rape plants. Six cultivars of spring and two of winter rape were sown in the open-air vegetation room at the end of August. The obtained results indicate that the degree of frost damage in spring-type plants increased in the course of winter and this increase was parallel to elongation of generative shoots observed after periods of warming. Each spring cultivar was completely killed by frost just after its generative shoot reached 15–20 cm, irrespective of its frost resistance level, determined previously under laboratory conditions. In the case of winter cultivars survival rate was consistent with laboratory-estimated frost resistance. It is suggested that spring rape could not survive winter because of its limited ability to prevent shoot elongation during winter at temperatures slightly above 0 °C. It was also found that less efficient photosynthetic electron transport in autumn was observed in these spring cultivars in which the elongation of generative shoots was observed already during the first warm break in winter.

Experiments performed under controlled conditions showed that level of PPFD (photosynthetic photon flux density) during early seedlings growth (preceding cold acclimation at +2 °C) was not the key factor for the development of frost resistance. It did not modify the beneficial effects of prehardening (Rapacz 1997, in this issue) at moderately low (+12 °C) day temperature.

The aim of the presented work was to study whether the efficiency of photosynthesis may influence resistance of hardened plants to disease. Seedlings of spring barley, meadow fescue and winter oilseed rape were chilled at 5 °C for 2, 4 or 6 weeks and at these deadlines the changes in cell membrane permeability (expressed as electrolyte leakage), chlorophyll fluorescence (initial fluorescence - F0, maximal fluorescence - Fm, quantum yield of PSII - Fv/Fm) and net photosynthesis rate (FN) were measured. Also, the influence of cold on the degree of plant resistance to economically important pathogens -Bipolaris sorokiniana or Phoma lingam was estimated. Two, four or six week-hardened plants were artificially infected: barley and fescue by B. sorokiniana, and oilseed rape by P. lingam.