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Freezing tolerance of triticale is a major trait contributing to its winter hardiness. The identification of genomic regions – quantitative trait loci (QTLs) and molecular markers associated with freezing tolerance in winter hexaploid triticale was the aim of this study. For that purpose a new genetic linkage map was developed for the population of 92 doubled haploid lines derived from ‘Hewo’ × ‘Magnat’ F1 hybrid. Those lines, together with parents were subjected to freezing tolerance test three times during two winter seasons. Plants were grown and cold-hardened under natural fall/winter conditions and then subjected to freezing in controlled conditions. Freezing tolerance was assessed as the plants recovery (REC), the electrolyte leakage (EL) and chlorophyll fluorescence parameters (JIP) after freezing. Three consistent QTLs for several fluorescence parameters, electrolyte leakage and the percentage of the survived plants were identified with composite interval mapping (CIM) and single marker analysis (SMA). The first locus Qfr.hm-7A.1 explained 9 % of variation of both electrolyte leakage and plants recovery after freezing. Two QTLs explaining up to 12 % of variation in plants recovery and shared by selected chlorophyll fluorescence parameters were found on 4R and 5R chromosomes. Finally, main locus Qchl.hm-5A.1 was detected for chlorophyll fluorescence parameters that explained up to 19.6 % of phenotypic variation. The common QTLs located on chromosomes 7A.1, 4R and 5R, clearly indicated physiological and genetic relationship of the plant survival after freezing with the ability to maintain optimal photochemical activity of the photosystem II and preservation of the cell membranes integrity. The genes located in silico in the identified QTLs include those encoding transmembrane helix proteins like potassium channel and phosphoric ester hydrolase involved in response to osmotic stress as well as proteins involved in the regulation of the gene expression, chloroplast RNA processing and pyrimidine salvage pathway. Additionally, our results confirm that the JIP test is a valuable tool to evaluate freezing tolerance of triticale under unstable winter environments.

Combining to tolerance to biotic and abiotic stresses is important target for modern triticale breeding. Cultivation of varieties resistant to fungal pathogens is economically and environmentally important and may lead to reducing of the use of fungicides. Molecular markers are necessary for accumulation of advantageous alleles in the best genotypes by means of marker-assisted and genomic selection approaches. In present research, QTL regions associated with the powdery mildew resistance at adult plant stage were evaluated in order to provide the effective selection tools. Testing of DH population in multiple environments under natural infestation revealed 20 QTL on wheat (4A, 3B, 4B) and rye (2R, 4R, 5R, 6R) chromosomes. Regions explained 8.1% - 29.3% of phenotypic variation depending of the trait, localization and year of the experiment. Main QTL with effect exceeding 15% were found on chromosomes 3B, 4B, 2R, 5R and 6R. QTL and candidate genes located on chromosomes 4B, 2R, 5R and 6R are so far reported for the first time as regions associated with PM resistance in the adult triticale plants. Additionally, within all QTL, 21 candidate genes associated with the PM resistance were revealed. Predicted function of protein encoded by these genes include triggering a defense system which restricts the pathogen growth, enzyme activity, regulation of hormone-activated pathways, transcriptional corepressor complex and cell wall construction. Availability of QTL, molecular markers together with candidate genes linked with the powdery mildew resistance can be validated on triticale lines and varieties and then, used in MAS to improve modern breeding.

Plant overwintering may be affected in the future by climate change. Low-temperature waterlogging, associated with a predicted increase in rainfall during autumn and winter, can affect freezing tolerance, which is the main component of winter hardiness. The aim of this study was to elucidate the mechanism of change in freezing tolerance caused by low-temperature waterlogging in Lolium perenne, a cool-season grass that is well adapted to a cold climate. The work included: (i) a freezing tolerance test (plant regrowth after freezing), (ii) analysis of plant phytohormones production (abscisic acid [ABA] content and ethylene emission), (iii) measurement of leaf water content and stomatal conductance, (iv) carbohydrate analysis, and (v) analysis of Aco1, ABF2, and FT1 transcript accumulation. Freezing tolerance may be improved as a result of cold waterlogging. The mechanism of this change is reliant on multifaceted actions of phytohormones and carbohydrates, whereas ethylene may counteract ABA signaling. The regulation of senescence processes triggered by concerted action of phytohormones and glucose signaling may be an essential component of this mechanism.

Ten doubled haploid (DH) lines of winter barley with an increased range of freezing/drought tolerance were used to identify phytohormones involved in plant stress acclimation. Cold hardening and drought stress were applied at the most critical stages of plant development on young seedlings and heading plants, respectively. The level of the phytohormones was significantly higher at heading, more than 5-fold in respect of salicylic acid (SA) and total brassinosteroids (BRs) and 1.7-fold in the case of abscisic acid (ABA). Moreover, the spectrum of detectable BRs increased from one—homocastasterone (HCS)—found in seedlings to four BRs identified in heading plants [HCS, castasterone (CS), teasterone and dolicholide], with the last one detected for the first time in cereal species. To some extent freezing tolerance seems to be determined by native hormonal status as control seedlings of tolerant DH lines contained 1.4- and 2.3-fold lower amount of ABA and HCS and 2.3-fold higher amount of SA in comparison to freezing-sensitive ones. Such dependency was not observed in heading plants as significant variation in CS content was the only detected difference. Under stress treatments, tolerant DH lines accumulated significantly lower (75–81%) amount of ABA, which probably reflected lower stress intensity resulting from another defence strategy. In contrast, stress-induced significant almost 2-fold increase in HCS/CS and 2–3-fold decrease in SA content specific for tolerant DH lines of barley suggest the involvement of these molecules in freezing/drought defence. Detected correlations suggest their interaction with nonspecific peroxidase and low molecular weight antioxidants.

The increasing economic importance of triticale (×Triticosecale Wittm.) makes this synthetic hybrid cereal an interesting object of genetic studies. Genomic regions (QTL) of morphological winter triticale traits were determined using the mapping population of 89 doubled haploids lines (DHs) developed from F1 hybrid of cv. ‘Hewo’ and cv. ‘Magnat’ accompanied with the genetic map consisting of 20 linkage groups assigned to the A (7), B (7), and R (6) genomes (total of 3539 DArT, SNP-DArT and SSR markers, length of 4997.4 cM). Five independent experiments were performed in the field and greenhouse controlled conditions. A total of 12 major QTLs located on 2B, 5A, 5R, and 6B chromosomes connected to the stem length, the plant height, the spike length, the number of the productive spikelets per spike, the number of grains per spike, and the thousand kernel weight were identified by a composite interval mapping (CIM).

Mechanisms involved in the de-acclimation of herbaceous plants caused by warm periods during winter are poorly understood. This study identifies the genes associated with this mechanism in winter barley. Seedlings of eight accessions (four tolerant and four susceptible to de-acclimation cultivars and advanced breeding lines) were cold acclimated for three weeks and de-acclimated at 12 °C/5 °C (day/night) for one week. We performed differential expression analysis using RNA sequencing. In addition, reverse-transcription quantitative real-time PCR and enzyme activity analyses were used to investigate changes in the expression of selected genes. The number of transcripts with accumulation level changed in opposite directions during acclimation and de-acclimation was much lower than the number of transcripts with level changed exclusively during one of these processes. The de-acclimation-susceptible accessions showed changes in the expression of a higher number of functionally diverse genes during de-acclimation. Transcripts associated with stress response, especially oxidoreductases, were the most abundant in this group. The results provide novel evidence for the distinct molecular regulation of cold acclimation and de-acclimation. Upregulation of genes controlling developmental changes, typical for spring de-acclimation, was not observed during mid-winter de-acclimation. Mid-winter de-acclimation seems to be perceived as an opportunity to regenerate after stress. Unfortunately, it is competitive to remain in the cold-acclimated state. This study shows that the response to mid-winter de-acclimation is far more expansive in de-acclimation-susceptible cultivars, suggesting that a reduced response to the rising temperature is crucial for de-acclimation tolerance.

Lolium multiflorum/Festuca arundinacea introgression forms have been proved several times to be good models to identify key components of grass metabolism involved in the mechanisms of tolerance to water deficit. Here, for the first time, a relationship between photosynthetic and antioxidant capacities with respect to drought tolerance of these forms was analyzed in detail. Two closely related L. multiflorum/F. arundinacea introgression forms distinct in their ability to re-grow after cessation of prolonged water deficit in the field were selected and subjected to short-term drought in pots to dissect precisely mechanisms of drought tolerance in this group of plants. The studies revealed that the form with higher drought tolerance was characterized by earlier and higher accumulation of abscisic acid, more stable cellular membranes, and more balanced reactive oxygen species metabolism associated with a higher capacity of the antioxidant system under drought conditions. On the other hand, both introgression forms revealed the same levels of stomatal conductance, CO2 assimilation, and consequently, intrinsic water use efficiency under drought and recovery conditions. However, simultaneous higher adjustment of the Calvin cycle to water deficit and reduced CO2 availability, with respect to the accumulation and activity of plastid fructose-1,6-bisphosphate aldolase, were clearly visible in the form with higher drought tolerance.

Impact of photosynthetic and antioxidant capacities on drought tolerance of two closely related forage grasses, Festuca arundinacea and Festuca glaucescens, was deciphered. Within each species, two genotypes distinct in drought tolerance were subjected to a short-term drought, followed by a subsequent re-watering. The studies were focused on: (i) analysis of plant physiological performance, including: water uptake, abscisic acid (ABA) content, membrane integrity, gas exchange, and relative water content in leaf tissue, (ii) analysis of plant photosynthetic capacity (chlorophyll fluorescence, gene expression, protein accumulation, and activity of selected enzymes of the Calvin cycle), and (iii) analysis of plant antioxidant capacity (reactive oxygen species (ROS) generation, gene expression, protein accumulation and activity of selected enzymes). Though, F. arundinacea and F. glaucescens revealed different strategies in water uptake, and partially also in ABA signaling, their physiological reactions to drought and further re-watering, were similar. On the other hand, performance of the Calvin cycle and antioxidant system differed between the analyzed species under drought and re-watering periods. A stable efficiency of the Calvin cycle in F. arundinacea was crucial to maintain a balanced network of ROS/redox signaling, and consequently drought tolerance. The antioxidant capacity influenced mostly tolerance to stress in F. glaucescens.

Impact of photosynthetic and antioxidant capacities on drought tolerance of two closely related forage grasses, Festuca arundinacea and Festuca glaucescens, was deciphered. Within each species, two genotypes distinct in drought tolerance were subjected to a short-term drought, followed by a subsequent re-watering. The studies were focused on: (i) analysis of plant physiological performance, including: water uptake, abscisic acid (ABA) content, membrane integrity, gas exchange, and relative water content in leaf tissue; (ii) analysis of plant photosynthetic capacity (chlorophyll fluorescence; gene expression, protein accumulation, and activity of selected enzymes of the Calvin cycle); and (iii) analysis of plant antioxidant capacity (reactive oxygen species (ROS) generation; gene expression, protein accumulation and activity of selected enzymes). Though, F. arundinacea and F. glaucescens revealed different strategies in water uptake, and partially also in ABA signaling, their physiological reactions to drought and further re-watering, were similar. On the other hand, performance of the Calvin cycle and antioxidant system differed between the analyzed species under drought and re-watering periods. A stable efficiency of the Calvin cycle in F. arundinacea was crucial to maintain a balanced network of ROS/redox signaling, and consequently drought tolerance. The antioxidant capacity influenced mostly tolerance to stress in F. glaucescens.

Plant tolerance to environmental stress is determined by a very complicated network composed of many intra- and extracellular factors. The aim of this study was to select candidate genes involved in responses to freezing and drought in barley on the basis of previous proteomic studies and to analyze changes in their expression caused by application of both stress factors. Six candidate genes for freezing tolerance (namely the genes encoding elongation factor 1 alpha (EF1A), ferredoxin-NADP reductase, a 14-3-3a protein, &beta, fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (GroEs), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses. The expression levels of these genes were measured under control and stress conditions in six DH (doubled haploid) lines with differing freezing and drought tolerance. The results of gene expression analysis confirmed the roles of the candidate genes preselected in this study on the basis of previous proteome analysis in contributing to the differences in freezing and drought tolerance observed in the studied population of DH lines of winter barley.

Key message Association mapping of drought-related traits in barley was used to increase the density of existing QTL maps without recreating mapping populations. Abstract We used 109 spring barley genotypes exhibiting high or low drought tolerance to elucidate the associations between diversity array technology sequencing (DArTseq) and single nucleotide polymorphism (SNP) markers and various physiological parameters related to plant responses to drought conditions. The study was performed in controlled conditions (growth chambers), drought tolerance was phenotyped in the four-leaf seedlings. We identified 58 associations including 34 new markers (i.e., 16 DArTseq and 18 SNP markers). The results for three markers were consistent with the data obtained in an earlier traditional biparental QTL mapping study. The regions neighboring markers on linkage group 2H contained the highest number of significant marker–trait associations. Five markers related to the photosynthetic activity of photosystem II were detected on chromosome 4H. The lowest number of associations were observed for the sequences neighboring DArT markers on linkage group 6H. A chromosome 3H region related to water use efficiency and net photosynthesis rate in both biparental QTL, and association study, may be particularly valuable, as these parameters correspond to the ability of plants to remain highly productive under water deficit stress. Our findings confirm that association mapping can increase the density of existing QTL maps without recreating mapping populations. Electronic supplementary material The online version of this article (doi:10.1007/s00122-017-2994-1) contains supplementary material, which is available to authorized users.

Festuca arundinacea and F. pratensis are the models in forage grasses to recognize the molecular basis of drought, salt and frost tolerance, respectively. Transcription profiles of plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs) aquaporin genes were obtained for leaves of Festuca species treated with different abiotic stimuli. F. arundinacea plants were exposed to drought and salt stress, whereas F. pratensis plants were cold-hardened. Changes in genes expression measured with use of real time qRT-PCR method were compared between two genotypes characterized with a significantly different level of each stress tolerance. Under drought the transcript level of PIP1;2 and TIP1;1 aquaporin decreased in both analyzed F. arundinacea genotypes, whereas for PIP2;1 only in a high drought tolerant plant. A salt treatment caused a reduction of PIP1;2 transcript level in a high salt tolerant genotype and an increase of TIP1;1 transcript abundance in both F. arundinacea genotypes, but it did not influence the expression of PIP2;1 aquaporin. During cold-hardening a decrease of PIP1;2, PIP2;1, and TIP1;1 aquaporin transcripts was observed, both in high and low frost tolerant genotypes. The obtained results revealed that the selected genotypes responded in a different way to abiotic stresses application. A reduced level of PIP1;2 transcript in F. arundinacea low drought tolerant genotype corresponded with a faster water loss and a lowering of photosynthesis efficiency and gas exchange during drought conditions. In F. pratensis, cold acclimation was associated with a lower level of aquaporin transcripts in both high and low frost tolerant genotypes. This is the first report on aquaporin transcriptional profiling under abiotic stress condition in forage grasses. Electronic supplementary material The online version of this article (doi:10.1007/s13353-017-0403-8) contains supplementary material, which is available to authorized users.

Perennial ryegrass (Lolium perenne L.) belongs to the common cultivated grass species in Central and Western Europe. Despite being considered to be susceptible to drought, it is frequently used for forming the turf in urban green areas. In such areas, the water deficit in soil is recognized as one of the most important environmental factors, which can limit plant growth. The basic aim of this work was to explore the mechanisms standing behind the changes in the photosynthetic apparatus performance of two perennial ryegrass turf varieties grown under drought stress using comprehensive in vivo chlorophyll fluorescence signal analyses and plant gas exchange measurements. Drought was applied after eight weeks of sowing by controlling the humidity of the roots ground medium at the levels of 30, 50, and 70% of the field water capacity. Measurements were carried out at four times: 0, 120, and 240 h after drought application and after recovery (refilling water to 70%). We found that the difference between the two tested varieties’ response resulted from a particular re-reduction of P700+ (reaction certer of PSI) that was caused by slower electron donation from P680. The difference in the rate of electron flow from Photosystem II (PSII) to PSI was also detected. The application of the combined tools (plants’ photosynthetic efficiency analysis and plant gas exchange measurements) allowed exploring and explaining the specific variety response to drought stress.

Photosynthetic acclimation to cold conditions is an important factor influencing freezing tolerance of plants. Photosynthetic enzyme activities increase as part of a photochemical mechanism underlying photosynthetic acclimation to low temperatures. Additionally, a non-photochemical mechanism may be activated to minimize photooxidative damage. The aim of this study was to test the hypothesis that differences in stomatal conductance in Hordeum vulgare plants with contrasting freezing tolerances induce various strategies for photosynthetic acclimation to cold stress. Different stomatal behaviors during the prehardening step resulted in diverse plant reactions to low-temperature stress. Plants with a relatively low freezing tolerance exhibited decreased stomatal conductance, resulting in decreased photochemical activity, faster induction of the non-photochemical mechanism, and downregulated expression of two Rubisco activase (RcaA) splicing variants. In contrast, plants with a relatively high freezing tolerance that underwent a prehardening step maintained the stomatal conductance at control level and exhibited delayed photochemical activity and RcaA expression decrease, and increased Rubisco activity, which increased net photosynthetic rate. Thus, in barley, the induction of photoinhibition avoidance (i.e., non-photochemical photoacclimation mechanism) is insufficient for an effective cold acclimation. An increase in cold-induced net photosynthetic rate due to open stomata is also necessary.

Common buckwheat (Fagopyrum esculentum Moench), a pseudocereal crop, produces a large number of flowers, but this does not guarantee high seed yields. This species demonstrates strong abortion of flowers and embryos. High temperatures during the generative growth phase result in an increase in the degeneration of embryo sacs. The aim of this study was to investigate proteomic changes in flowers and leaves of two common buckwheat accessions with different degrees of heat tolerance, Panda and PA15. Two-dimensional gel electrophoresis and mass spectrometry techniques were used to analyze the proteome profiles. Analyses were conducted for flower buds, open flowers capable of fertilization, and wilted flowers, as well as donor leaves, i.e., those growing closest to the inflorescences. High temperature up-regulated the expression of 182 proteins. The proteomic response to heat stress differed between the accessions and among their organs. In the Panda accession, we observed a change in abundance of 17, 13, 28, and 11 proteins, in buds, open and wilted flowers, and leaves, respectively. However, in the PA15 accession there were 34, 21, 63, and 21 such proteins, respectively. Fifteen heat-affected proteins were common to both accessions. The indole-3-glycerol phosphate synthase chloroplastic-like isoform X2 accumulated in the open flowers of the heat-sensitive cultivar Panda in response to high temperature, and may be a candidate protein as a marker of heat sensitivity in buckwheat plants.

Different pigments often occur together and affect photosynthetic characteristics of the respective leaf portions. In this study, photosynthetic activity in variegated leaves of five cultivars of the ornamental and medicinal plant, Coleus × hybridus hort., was estimated by image analysis and point data measurements of major chlorophyll (Chl) fluorescence parameters and related to the amount of photosynthetic pigments measured with a Chl meter or spectrophotometrically in leaf extracts. Significant differences in Chl and carotenoid (Car) contents were noticed among differentially pigmented sectors of a leaf and among the cultivars. Although the higher Chl concentration was noticed in purple parts compared to green parts of the leaves, the values of minimal and maximal fluorescence yield at the dark- and light-adapted state (F0, Fm, F0', Fm', respectively) were a little lower than those in the green sectors, indicating photoprotective effects provided by anthocyanins and Car, more abundant in the red parts. The lowest Chl and Car content was detected in creamy-yellow and pink sectors and this contributed to low F0, Fm, and Fm', maximal quantum yield of PSII photochemistry, and nonphotochemical and photochemical quenching but high PSII maximum efficiency and effective quantum yield of PSII photochemistry. Both methods of Chl fluorescence analysis revealed heterogeneity in capture, transfer, and dissipation of excitation energy but Chl fluorescence imaging was more suitable in examining very narrow pigmented leaf areas.

One hundred and nine accessions of spring barley seedlings were phenotyped under soil drought conditions. Chlorophyll fluorescence induction (OJIP) parameters, leaf water content, relative turgidity, net assimilation rate (P N), and water use efficiency (WUE) of plants were measured. All the tested lines were genotyped by means of DArT sequencing (DArTseq) technology. For association mapping a 11,780 polymorphic DArTseq and 4,725 DArTseq SNP markers were used. Our results revealed dissimilar patterns of the relationships between OJIP-parameters under control and drought conditions. A high level of correlation between parameters characterizing Photosystem's II (PSII) energy trapping efficiency (Fv/Fm) and photochemical events downstream of PSII reaction center (e.g., Performance Index-PICSo) was observed only in the case of drought-treated plants. Generally, OJIP parameters were correlated with leaf water content (less in control). This correlation was weaker with WUE, and absent with P N. Under drought stress, 6,252 genotype × phenotype associations, which passed false discovery rate (FDR) verification, were found between all the studied phenotypic characteristics (23, including 19 OJIP parameters) and 2,721 markers. On the other hand, only 282 associations passed FDR test in the control. They comprised 22 phenotypic parameters and 205 markers. Probing for gene annotations of sequences was performed for markers associated with Fv/Fm for both drought and control, markers were associated with studied traits in both control and drought, as well as for markers associated with both OJIP and other physiological parameters in drought. Our work allowed us to conclude that drought treatment differentiates the studied lines through the revealing of relationships between water content and the damages to PSII reaction centers or different components of PSII energy transfer chain. Moreover, the former was not connected with net photosynthesis rate.

Photosynthetic acclimation to cold conditions is an important factor influencing freezing tolerance of plants. Photosynthetic enzyme activities increase as part of a photochemical mechanism underlying photosynthetic acclimation to low temperatures. Additionally, a non-photochemical mechanism may be activated to minimize photooxidative damage. The aim of this study was to test the hypothesis that differences in stomatal conductance in Hordeum vulgare plants with contrasting freezing tolerances induce various strategies for photosynthetic acclimation to cold stress. Different stomatal behaviors during the prehardening step resulted in diverse plant reactions to low-temperature stress. Plants with a relatively low freezing tolerance exhibited decreased stomatal conductance, resulting in decreased photochemical activity, faster induction of the non-photochemical mechanism, and downregulated expression of two Rubisco activase (RcaA) splicing variants. In contrast, plants with a relatively high freezing tolerance that underwent a prehardening step maintained the stomatal conductance at control level and exhibited delayed photochemical activity and RcaA expression decrease, and increased Rubisco activity, which increased net photosynthetic rate. Thus, in barley, the induction of photoinhibition avoidance (i.e., non-photochemical photoacclimation mechanism) is insufficient for an effective cold acclimation. An increase in cold-induced net photosynthetic rate due to open stomata is also necessary.

In a ×Festulolium population (FuRs0357) of parental origin Lolium perenne × Festuca pratensis, selection of freezing tolerance by freezing tests on whole plants (FT) and chlorophyll a (Chl-a) fluorimetry on frozen detached leaves (CF) was assessed in high and low directions during two cycles of selection. The original population went through two cycles of random mating. All selections and non-selected intercrossed generations of the original population were established in field trials at a coastal site and a continental site in Norway. At the coastal site, analyses of Chl-a fluorimetry parameters and leaf growth on individual plants in autumn and winter hardiness observed in field plots in spring showed that the first-generation selections for high freezing tolerance were associated with winter hardiness and early growth cessation. The second-generation FT-selections for high freezing tolerance were also associated with winter hardiness, whereas the CF-high selections diverged toward high photosynthetic activity. Both low selections were correlated with high photosynthetic activity. There were smaller variations between generations in unselected generations of the original population. Low accumulated leaf growth and early growth cessation were observed in the second-generation FT-selection for high freezing tolerance, whereas high normalized difference vegetation index (NDVI) were seen in Chl-a selections. Both selection methods distinguished diverging selections with significantly different high and low freezing tolerance, but selection efficiency was comparable only for the first selection cycle. Moreover, due to mixed ploidy level in the original population, selection by FT and CF generated diploid and tetraploid plants, respectively, which intensified the response of selection, particularly in the diploid selections. Total dry matter yield (DMY) (mean of three annual cuts for 3 years) of the FT-high selections was lower than for the CF-selections. At coastal sites, selection intensity using freezing tests on whole plants should be adapted to actual climate conditions, to obtain genotypes that balance photosynthetic activity during autumn and good winter hardiness, making them persistent and high yielding.

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.

The results of genetic analysis of 38 rye taxa (Secale cereale L.) represented by agricultural varieties originating from Central Europe and the Union of Soviet Socialist Republics (SUN) are presented. The genetic diversity of rye cultivars by 5 RAPD markers was evaluated. Five primers gave 42 polymorphic fragments (99.52 %) with an average of 8.4 bands per primer. The most polymorphic primer was RLZ12, where 10 polymorphic amplification products were detected. Overleaf the lowest polymorphic primer was RLZ5 with 7 polymorphic products. Genetic polymorphism was characterized based on diversity index (DI), probability of identity (PI) and polymorphic information content (PIC). The dendrogram of genetic similarity was constructed, based on the Jaccard´s coefficient. In dendrogram three clusters were differentiated. The first cluster contained genotypes from Czechoslovakia, Poland and Czech Republic. The second cluster contained cultivars coming from Union of Soviet Socialist Republics and Hungary. In the next cluster Poland, Czech Republic and Czechoslovakia genotypes were situated. Two genotypes Bosmo and Wibro have not been distinguished. For better distinction of the analysed rye genotypes, it is necessary to use a higher number of RAPD markers. In this experiment, RAPD proved to be a rapid, reliable and practicable method for revealing of polymorphism in the rye cultivars.

A chlorophyll fluorescence technique enabled early detection of disturbances in the activity of the photosynthet-ic apparatus under Phoma lingam infection. The photosynthetic apparatus of leaves and cotyledons exhibited a negative response to P. lingam inoculation. The effect disappeared more rapidly in leaves, which were not inoculated directly, than in cotyledons, which were inoculated directly. Photosynthetic apparatus disturbances were detected in cotyledons and leaves as early as 24 h after inoculation. Photosynthetic apparatus activity can be affected by the level of hydrogen peroxide, which in cotyledons probably was an element of the hypersensitive response and in leaves could induce an increase in pathogenesis-related proteins, chitinase and β-1,3-glucanase.

Perennial ryegrass (Lolium perenne L.) belongs to the common cultivated grass species in Central and Western Europe. Despite being considered to be susceptible to drought, it is frequently used for forming the turf in urban green areas. In such areas, the water deficit in soil is recognized as one of the most important environmental factors, which can limit plant growth. The basic aim of this work was to explore the mechanisms standing behind the changes in the photosynthetic apparatus performance of two perennial ryegrass turf varieties grown under drought stress using comprehensive in vivo chlorophyll fluorescence signal analyses and plant gas exchange measurements. Drought was applied after eight weeks of sowing by controlling the humidity of the roots ground medium at the levels of 30, 50, and 70% of the field water capacity. Measurements were carried out at four times: 0, 120, and 240 h after drought application and after recovery (refilling water to 70%). We found that the difference between the two tested varieties&rsquo, response resulted from a particular re-reduction of P700+ (reaction certer of PSI) that was caused by slower electron donation from P680. The difference in the rate of electron flow from Photosystem II (PSII) to PSI was also detected. The application of the combined tools (plants&rsquo, photosynthetic efficiency analysis and plant gas exchange measurements) allowed exploring and explaining the specific variety response to drought stress.

This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.

The objective of this study was to assess the effect of an elevated atmospheric CO2 molar ratio on freezing tolerance, photosynthetic apparatus performance and expression of CBF6, Cor14b and LOS2 in meadow fescue (Festuca pratensis Huds.). It was shown that cold acclimation under a CO2 molar ratio of 800 μmol mol(air)−1 decreased the freezing tolerance of meadow fescue when compared to the ambient CO2 level. This effect was not related either to changes observed in PSII redox state or to photosynthetic acclimation to cold, which was in fact more effective at an elevated CO2 level. The decrease in freezing tolerance was linked to changes in the expression of CBF6 and LOS2 genes, whereas the protective effect on photosynthetic apparatus was connected with the activation of a non-photochemical mechanism of photoprotection as well as upregulation of FpCOR14b expression.

Drought tolerant plant genotypes are able to maintain stability and integrity of cellular membranes in unfavorable conditions, and to regenerate damaged membranes after stress cessation. The profiling of cellular glycerolipids during drought stress performed on model species such as Arabidopsis thaliana does not fully cover the picture of lipidome in monocots, including grasses. Herein, two closely related introgression genotypes of Lolium multiflorum (Italian ryegrass) × Festuca arundinacea (tall fescue) were used as a model for other grass species to describe lipid rearrangements during drought and re-hydration. The genotypes differed in their level of photosynthetic capacity during drought, and in their capacity for membrane regeneration after stress cessation. A total of 120 lipids, comprising the classes of monogalactosyldiacyloglycerol, digalactosyldiacyloglycerol, sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, diacylglicerol, and triacylglicerol, were analyzed. The results clearly showed that water deficit had a significant impact on lipid metabolism in studied forage grasses. It was revealed that structural and metabolic lipid species changed their abundance during drought and re-watering periods and some crucial genotype-dependent differences were also observed. The introgression genotype characterized by an ability to regenerate membranes after re-hydration demonstrated a higher accumulation level of most chloroplast and numerous extra-chloroplast membrane lipid species at the beginning of drought. Furthermore, this genotype also revealed a significant reduction in the accumulation of most chloroplast lipids after re-hydration, compared with the other introgression genotype without the capacity for membrane regeneration. The potential influence of observed lipidomic alterations on a cellular membrane stability and photosynthetic capacity, are discussed. HIGHLIGHTS A higher drought tolerance of grasses could be associated with an earlier lipidome response to a stress signal and with a membrane regeneration after stress cessation accompanied by a turnover of chloroplast lipids

PsbO, the manganese-stabilizing protein, plays a crucial role in oxygen-evolving complex functioning and stabilization, by maintaining optimal manganese, calcium and chloride concentrations at the active state of PSII. In this paper we present a study focused on recognizing the relationship between psbO gene activity and acclimation of the photosynthetic apparatus under abiotic stresses in the grasses Festuca arundinacea and F. pratensis. PsbO expression was compared between two distinct genotypes within each species which differed in their levels of stress tolerance (drought and frost, respectively) during drought treatment (F. arundinacea) and cold acclimation (F. pratensis). The research involved: (1) the analyses of psbO gene expression profiles using real-time PCR, and (2) the analyses of PsbO protein accumulation profiles using protein gel blot hybridization. The results indicate that PsbO plays a protective function with respect to the photosynthetic apparatus during abiotic stresses. In cold-treated F. pratensis plants the accumulation of PsbO seems to be responsible for differences in the PSII photochemical efficiency. Higher stability of PSII during drought, observed in the high-drought tolerant F. arundinacea genotype, is not associated with PsbO accumulation, although the degradation of this protein affects destabilization of the oxygen-evolving complex in drought.

Drought is the most significant abiotic stress in agriculture; thus, this area of studies seems to be one of the most important challenges in plant biology. Data about gene expression under drought are crucial to study drought response mechanisms and to select the genes for a transgenic approach. Quantitative RT-PCR is a powerful method for gene expression analysis; however, obtaining proper data normalization requires internal reference genes with stable level of expression. In the present paper ten potential reference genes were examined in two developmental stages of barley for their expression stability during leaf growth and increasing drought level. The results indicated that leaf growth per se affects the expression of studied genes to the similar extent as the drought and showed that different genes were most stably expressed in the seedling and the heading stage. As a result, different sets of reference genes were selected for different applications. For instance, ADP-ribozylation factor 1 and ubiquitin encoding genes were most suitable to study drought-induced changes in gene expression at the seedling stage, whereas actin and GAPDH genes were useful during heading, and ADP-ribozylation factor 1 and HSP90 allowed for the comparison between these two stages. Our data proved the necessity for validation of commonly used reference genes. The results indicate that expression of ADP seems to be the least affected by all the factors studied in the present experiment. However, when the effect of only one factor among those investigated in this work will be studied, different genes should be considered to be used as the references due to the higher stability of their expression.

Changes in non-structural carbohydrates before and during winter are one of the major plant responses to winter stress. However, the observed pattern of changes is variable, not only between grass species but also between different experiments. This study examined the effect of developmental stage on carbohydrate reserves during winter in two grass species differing in winter hardiness. Timothy (Phleum pratense L.) and perennial ryegrass (Lolium perenne L.), sown early or late in the growing season, were sampled for total non-structural carbohydrates (TNC) in late October (end of main growing season), and then in late January, early March and late April. Quantitative and qualitative patterns of carbohydrate accumulation in crown segments were influenced both by plant genotype and developmental stage of the plants prior to winter. TNC increased from October to March, demonstrating considerable cell activity during winter, and declined to their lowest level in April. Sucrose proved to be the major ...