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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.

Strawberry is one of the plants sensitive to salt and alkalinity stress. Light quality affects plant growth and metabolic activities. However, there is no clear answer in the literature on how light can improve the performance of the photosynthetic apparatus of this species under salt and alkalinity stress. The aim of this work was to investigate the effects of different spectra of supplemental light on strawberry (cv. Camarosa) under salt and alkalinity stress conditions. Light spectra of blue (with peak 460 nm), red (with peak 660 nm), blue/red (1:3), white/yellow (1:1) (400–700 nm) and ambient light were used as control. There were three stress treatments: control (no stress), alkalinity (40 mM NaHCO3), and salinity (80 mM NaCl). Under stress conditions, red and red/blue light had a positive effect on CO2 assimilation. In addition, blue/red light increased intrinsic water use efficiency (WUEi) under both stress conditions. Salinity and alkalinity stress decreased OJIP curves compared to the control treatment. Blue light caused an increase in its in plants under salinity stress, and red and blue/red light caused an increase in its in plants under alkalinity. Both salt and alkalinity stress caused a significant reduction in photosystem II (PSII) performance indices and quantum yield parameters. Adjustment of light spectra, especially red light, increased these parameters. It can be concluded that the adverse effects of salt and alkalinity stress on photosynthesis can be partially alleviated by changing the light spectra.

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.

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.

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.

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.

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.

The aim of the study was to assess the ef fects of low-temperature hardening (2 ◦ C) on the biochemical compounds and processes that can increase resistance of winter rape to inoculation with Leptosphaeria maculans spores. The study involved an evaluation of the entire pool of phenolic compounds, L-phenylalanine ammonia lyase (PAL) activity, excitation intensity for blue and green fluorescence, catalase (CAT) activity, respiration intensity and heat emission from leaf tissues. All the measurements were performed 24 and 72 hours after the inoculation. Low-temperature hardening, which preceded the inoculation of rape seedlings with spores of L. maculans, caused a significant increase in CAT activity and the level of phenolic compounds. The observed changes in PAL activity reflected the changes in phenolics content. The hardened plants showed a significantly higher intensity of blue fluorescence excitation at 24 and 72 hours after the inoculation, as compared to the non-hardened seedlings. Increased content of phenolic compounds and PAL and catalase activity triggered by the temperature of 2 ◦ C and maintained for 24 hours after the inoculation, may confirm the stimulating effect of the hardening temperature. Intensified emission of blue fluorescence indicating saturation of a cell wall with phenolic compounds makes the cell wall structure less stretchy, more tight and leakproof, and thereby hinders fungal growth through plant tissue.

Acclimation of photosynthetic apparatus to variable environmental conditions is an important component of tolerance to dehydration stresses, including salinity. The present study deals with the research on alterations in chloroplast proteome of the forage grasses. Based on chlorophyll fluorescence parameters, two genotypes of a model grass species—Festuca arundinacea with distinct levels of salinity tolerance: low salt tolerant (LST) and high salt tolerant (HST), were selected. Next, two-dimensional electrophoresis and mass spectrometry were applied under both control and salt stress conditions to identify proteins accumulated differentially between these two genotypes. The physiological analysis revealed that under NaCl treatment the studied plants differed in photosystem II activity, water content, and ion accumulation. The differentially accumulated proteins included ATPase B, ATP synthase, ribulose-1,5-bisphosphate carboxylase large and small subunits, cytochrome b6-f complex iron-sulfur subunit, oxygen-evolving enhancer proteins (OEE), OEE1 and OEE2, plastidic fructose-bisphosphate aldolase (pFBA), and lipocalin. A higher level of lipocalin, potentially involved in prevention of lipid peroxidation under stress, was also observed in the HST genotype. Our physiological and proteomic results performed for the first time on the species of forage grasses clearly showed that chloroplast metabolism adjustment could be a crucial factor in developing salinity tolerance.

Winter conditions are subjected to rapid climate changes. Increased precipitation and snow melting during warmer winters may result in low-temperature flooding. These factors probably affect plant overwintering strategies. This study investigated the relationships between the photosynthetic adjustment and freezing tolerance following a short-term low-temperature flooding in two forage grasses (Lolium perenne and Festuca pratensis). The effect of flooding on the photosynthetic apparatus acclimation to cold was determined using chlorophyll a fluorescence measurements. Freezing tolerance was estimated using an electrolyte leakage test. It has been shown that genotypes activating non-photochemical mechanisms of photosynthetic acclimation to cold during low-temperature flooding may show increased freezing tolerance. Freezing tolerance reduction observed in Festuca pratensis was probably connected with decreased photochemical activity and photoinhibition of photosynthesis. It can be concluded that photosynthetic apparatus response to low-temperature flooding may contribute to changes in the freezing tolerance. The direction of the changes is associated with different photosynthetic apparatus performance.

The changes in protein abundance induced by cold hardening were analysed by 2 DE in ten doubled haploid (DH) lines of winter barley, highly differentiated with respect to freezing tolerance level. Among 45 differential proteins identified by MALDI-TOF/TOF, the majority was classified as related to photosynthesis, carbohydrate metabolism, oxidation-reduction reactions and stress response. Among the detected proteins, higher abundance of RuBisCO large and small subunits, RuBisCO activase, two Oxygen-evolving enhancer proteins, Ferredoxin-NADP reductase, Cytochrome P450-dependent fatty acid hydroxylase and 14-3-3 protein was associated with higher freezing tolerance level. Lower relative level of hypothetical ATP synthase beta subunit, uncharacterized mitochondrial protein AtMg00810 and ribosomal RNA small subunit methyltransferase G also seems to be important. The results of proteomic studies were complemented by the evaluation of photosynthetic apparatus acclimation, showing distinctive differences between the studied genotypes in the number of active PSII reaction centres (RC/CS m ). Additionally, the analysis of antioxidative enzyme activities suggests the importance of H 2 O 2 as a signalling molecule possibly involved in the initiation of cold-induced plant acclimation. However, in DH lines with high freezing tolerance, H 2 O 2 generation during cold hardening treatment was accompanied by more stable activity of catalase, H 2 O 2 -decomposing enzyme. Significance In the study, the changes in protein abundance induced by cold hardening treatment were analysed by two-dimensional gel electrophoresis in ten doubled haploid (DH) lines of winter barley. Harnessing DH technology resulted in distinctive widening of genetic variation with respect to freezing tolerance level. Both the cold-hardening effect on the protein pattern in an individual winter barley DH line as well as the variation among the selected DH lines were investigated, which resulted in the identification of 45 differentiated proteins classified as involved in 14 metabolic pathways and cellular processes. Among them, eight proteins: (1) the precursor of RuBisCO large subunit, (2) RuBisCO small subunit (partial), (3) RuBisCO activase small isoform, (4) the precursor of Oxygen-evolving enhancer protein 1-like (predicted protein), (5) Oxygen-evolving enhancer protein 2, (6) the leaf isozyme of Ferredoxin-NADP reductase, (7) hypothetical protein M569_12509 Cytochrome P450-dependent fatty acid hydroxylase-like and (8) hypothetical protein BRADI_1g11290 (14-3-3 protein A-like) were accumulated to a higher level in leaves of cold-hardened seedlings of freezing tolerant winter barley DH lines in comparison with susceptible ones. Three others: (9) hypothetical protein BRADI_5g05668 F1 ATP synthase beta subunit-like, (10) predicted protein uncharacterized mitochondrial protein AtMg00810-like and (11) BnaA02g08010D Ribosomal RNA small subunit methyltransferase G-like were detected at lower level in freezing tolerant seedlings in comparison with susceptible genotypes. The last two were for the first time linked to cold acclimation. The results of complementary analyses indicate that PSII activity and stability of antioxidative enzymes under low temperature are also very important for freezing tolerance acquisition.

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.

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.

Exogenously applied H2O2 (50, 100 and 200mM) to Mesembryanthemum crystallinum root medium induced a transition from C3 to Crassulacean Acid Metabolism (CAM), as evaluated by diurnal malate (Δmal) fluctuations. A very high concentration of H2O2 (400mM) reduced Δmal below the value measured in control plants. An increase of malate content during the night in 400mM H2O2-treated plants might suggest that malate decarboxylation is crucial for CAM functioning. We conclude that malate plays a dual role: i) a protective and signaling function before CAM expression, and ii) a storage form of CO2 in plants performing CAM. A slight stimulation of photosystem II (PSII) photochemistry and net photosynthesis observed during the C3-CAM shift indicated that neither photoinhibition nor reduction of the photosynthetic rate were prerequisites for CAM. Moreover, CAM induction corresponded to a decrease of catalase activity. In CAM-performing plants, α-tocopherol, polyamines (putrescine and spermidine) and proline showed daily alterations and the content of α-tocopherol and polyamines was lower at the end of the day. In contrast, the proline concentration correlated with the applied H2O2 concentration and was higher at the end of the day in treated plants. The dynamic changes of antioxidant and osmolyte levels suggest their active role in preventing oxidative damage, stress acclimation mechanisms and involvement in metabolic regulation and/or signal transduction cascades.

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.

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 ...

A number of physiological and molecular characteristics are proposed as selection criteria for drought tolerance. This study measured the associations between physiological and molecular characteristics of drought response in malting and fodder spring barleys. Plants of 13 malt- and 14 feed-type Polish genotypes were exposed to drought at the four-leaf stage for 7 days. Drought susceptibility indexes (DSI) were calculated for membrane integrity, water status, gas exchange and PSII photochemical activity. Accumulation of HVA1 and SRG6 transcripts in drought was measured with real-time PCR. A wide range of variation in the drought response was observed among studied genotypes. Malting barleys were less sensitive to drought than feed-barleys according to all the traits studied. In both groups, different patterns of relationships between traits were observed. In malting genotypes only, CO 2 assimilation rates in drought, as well as PSII efficiency were related to both water content and the accumulation of HVA1 transcript in leaves. On the other hand the SRG6 expression was highly correlated in both groups of barley with the photochemical efficiency of PSII. The results suggest that different physiological, biochemical and molecular characteristics should be applied in the selection towards drought resistance in the case of malting and fodder barleys.

The aim of the experiment was to verify whether changes in photosynthetic activity measured by means of chlorophyll fluorescence techniques and soluble carbohydrate level induced during pre-hardening and cold acclimation in androgenic forms of Festulolium are related to their winter hardiness and pathogen resistance. The study was performed on six androgenic genotypes of Festulolium, which differ in their degrees of winter hardiness. The chlorophyll fluorescence parameters and sugar content were measured in the control plants, pre-hardened at 12 °C and cold-acclimated at 2 °C. Cold acclimation at 2 °C, preceded by pre-hardening at 12 °C, induced a decline in maximum PSII quantum efficiency (F v /F m ) and in non-photochemical quenching (NPQ). Exposure to 2 °C also resulted in reduced efficiency of energy conversion in photochemical processes (qp). The soluble carbohydrate amount increased in the susceptible genotypes and was associated with a decline in NPQ and current quantum yield of PSII (φ PSII ). In resistant plants, a decrease in the sugar content was observed, which could be explained by their consumption in metabolic processes initiated during cold acclimation. The changes in sugar content were associated with a decline in NPQ and q P .

Associations of PCR markers with freezing tolerance and acclimation of photosynthetic apparatus to cold were tested on 28 winter barley cultivars and advanced breeding forms to select alleles for practical application in marker assisted selection (MAS). We found significant associations between freezing tolerance evaluated with field-laboratory method (FLM) and markers located on 5H chromosome in region of gene Fr-H2 (bin9-10: Xbmag812, Xmwg2230) and region of gene Fr-H1 (bin11: Xmwg514, HvBM5, Xmwg644). Additionally, significant associations with photochemical quenching of chlorophyll a fluorescence (qP) were found for PCR markers Xpsr115 and Xmwg2062. In our study variation in the promoter region of Vrn-H1 (HvBM5) was directly connected with freezing tolerance of plants partially de-acclimated in the field. The results obtained here showed that different loci of freezing tolerance may play role in variable selection pressure of winter conditions.

The analysis of fast chlorophyll fluorescence induction kinetics in dark-adapted leaves (JIP-test) is proposed as a rapid method for evaluating freezing tolerance in wheat. Wheat leaves were detached from the plants of 40 cultivars grown in an open-field in Smolice, during December and March of winters 2004/2005 and 2005/2006, transferred to laboratory in Krakow and frozen in −15°C. Next, measurements of chlorophyll fluorescence were taken. The correlation coefficients between the JIP-test parameters with freezing tolerance data obtained in multiple field-laboratory studies, varied between 0.58 and 0.70 depending on sampling time and parameter measured. The chlorophyll fluorescence parameter, which can always be used for the screening of freezing tolerance in the method described here, is the performance index (PI), which characterizes the overall energy flow efficiency in photosystem II (PSII). The results indicate, that the technique proposed here can be useful for freezing tolerance screening in plant breeding programmes. However, this method seems to be a more reliable tool in the selection of freezing tolerant germplasm, than for discarding freezing susceptible materials.

Winter-hardiness is a complex trait limiting cultivation of winter barley (Hordeum vulgare ssp. vulgare) with respect to the regions of temperate climate. In the present studies, we verified whether inexpensive and fast physiological markers characterizing photosynthetic acclimation to cold may provide robust characteristics of winter barley genotypes for improved frost resistance. Freezing tolerance of 28 winter barley varieties and advanced breeding lines were tested for three winters in field-laboratory experiment and under fully controlled conditions. To increase the environmental variability of freezing tolerance, a part of the plants were also de-acclimated under semi-controlled conditions and re-acclimated in laboratory before freezing tests. After controlled cold acclimation, apparent quantum yield of photosystem II (F y /F m ) as well as photochemical (qp) and non-photochemical (NPQ) coefficients of chlorophyll fluorescence quenching were studied. Field-laboratory method assessment of freezing tolerance gives distinct and even opposite results in subsequent years. Also de-acclimation interacted with growth conditions in the field, giving different rankings of genotypes each year. The results obtained suggest that high level of freezing tolerance measured in laboratory, which is connected with photosynthetic acclimation to cold may be not sufficient for the expression of field resistance, especially when winter conditions are not favourable for cold acclimation.

Chlorophyll (Chl) a fluorescence measurements as evaluators of plant freezing tolerance are frequently insufficiently sensitive to detect the early metabolic changes that are initiated following exposure to freezing temperatures. Using cold-acclimated winter wheat, I analysed the polyphasic transience (from 50 µs to 1 s) of Chl a fluorescence. This enabled detailed studies of the progressive energy flows and efficiencies within the photosystem 2 (PS2) complex that ensue following initial exposure to freezing temperatures right through to the plant recovery stage. The initial consequences of mild frosts that may cause primary damage involve a disturbance to the energy transfer subsequent to QA (the primary quinone electron acceptor of PS2). Lower freezing temperatures, on the other hand, may deter energy flow between the PS2 reaction centre (RC), Chl, and QA. All primary damage could only be repaired partially. Further freezing-triggered dysfunction of the electron transfer between the PS2 RCs and QA was connected with secondary damage that could lead to PS2 deactivation. Both primary and secondary freezing damages were reflected in decreased PIABS, the Performance Index based on equal absorption that characterizes all energy bifurcations in PS2. PIABS also differentiated cultivars with contrasting freezing-tolerance either subsequent to the onset of freezing or during the recovery stage. In contrast, the potential quantum yield of PS2 (Fv/Fm), which characterizes efficiency of energy trapping in the PS2 RCs, was only different in cultivars with contrasting freezing-tolerance during the recovery stage.

OJIP analysis, which explores changes in photosystem II (PSII) photochemical performance, has been used as a measure of plant susceptibility to stress. However, in the case of freezing tolerance and winter hardiness, which are highly environmentally variable, the use of this method can give ambiguous results depending on the species as well as the sampling year and time. To clarify this issue, we performed chlorophyll fluorescence measurements over three subsequent winters (2010/11, 2011/12 and 2012/13) on 220 accessions of common winter wheat and 139 accessions of winter triticale. After freezing, leaves were collected from cold-acclimated plants in the laboratory and field-grown plants. Observations of field survival in seven locations across Poland and measurements of freezing tolerance of the studied plants were also recorded. Our results confirm that the OJIP test is a reliable indicator of winter hardiness and freezing tolerance of common wheat and triticale under unstable winter environments. Regardless of species, the testing conditions giving the most reliable results were identical, and the reliability of the test could be easily checked by analysis of some relationships between OJIP-test parameters. We also found that triticale is more winter hardy and freezing tolerant than wheat. In addition, the two species were characterized by different patterns of photosynthetic apparatus acclimation to cold.

Abiotic stresses, including low temperature, can significantly reduce plant yielding. The knowledge on the molecular basis of stress tolerance could help to improve its level in species of relatively high importance to agriculture. Unfortunately, the complex research performed so far mainly on model species and also, to some extent, on cereals does not fully cover the demands of other agricultural plants of temperate climate, including forage grasses. Two Lolium perenne (perennial ryegrass) genotypes with contrasting levels of frost tolerance, the high frost tolerant (HFT) and the low frost tolerant (LFT) genotypes, were selected for comparative metabolomic research. The work focused on the analysis of leaf metabolite accumulation before and after seven separate time points of cold acclimation. Gas chromatography–mass spectrometry (GC/MS) was used to identify amino acids (alanine, proline, glycine, glutamic and aspartic acid, serine, lysine and asparagine), carbohydrates (fructose, glucose, sucrose, raffinose and trehalose) and their derivatives (mannitol, sorbitol and inositol) accumulated in leaves in low temperature. The observed differences in the level of frost tolerance between the analysed genotypes could be partially due to the time point of cold acclimation at which the accumulation level of crucial metabolite started to increase. In the HFT genotype, earlier accumulation was observed for proline and asparagine. The increased amounts of alanine, glutamic and aspartic acids, and asparagine during cold acclimation could be involved in the regulation of photosynthesis intensity in L. perenne. Among the analysed carbohydrates, only raffinose revealed a significant association with the acclimation process in this species.

The fungus Leptosphaeria maculans that causes blackleg can attack winter rape throughout the entire growing season. Therefore, appropriate treatments were designed to simulate environmental factors that may occur under field conditions. The callus tissue of winter rape was treated with abiotic stress in the form of cold (2 °C) and temperature change (2/20 °C and 20/2 °C), and biotic stress in the form of secondary metabolites of L. maculans. The following parameters were analyzed: total phenolics, the activity of l-phenylalanine ammonia lyase (PAL), catalase, respiration intensity, heat emission, and metabolic activity determined by means of 2,3,5-triphenyl-tetrazolium chloride (TTC) assay. Additionally, the changes in the electrical potential, known as a sensitive indicator of biochemical changes in the callus tissue, were measured. The obtained results seem to indicate rather increased stress, caused by temperature (2 or 20 °C) or its changes (2/20 °C, 20/2 °C) and the secondary metabolites of L. maculans than a cross reaction. The factor that most strongly affected the metabolism of winter rape callus tissue was the temperature change, which masked the changes caused by the fungal secondary metabolites or intensified their adverse effects. The only parameters that could be regarded as reliable indicators of the callus tissue response to the elicitation with secondary metabolites of L. maculans were catalase activity and respiration intensity. Additionally, a protective function of low temperature involved a reduction in catalase activity, resulting in increased level of reactive oxygen species (ROS), key molecules inducing the defense mechanisms during pathogenesis. Only the changes in l-phenylalanine ammonia lyase (PAL) activity and the accompanying changes in the content of phenolic compounds can be considered as beneficial effects of low temperature (2 °C) on a more efficient callus acclimation to the biotic stress.

Previously published results showed that high relative reduction state of PSII (PSII excitation pressure) during both early seedling growth (prehardening) as well as cold deacclimation caused significant changes in growth pattern. The differences in elongation growth rate were related to the cold acclimation of photosynthetic apparatus and to frost resistance. To study changes in the hormonal balance connected with alterations in elongation growth rate observed during prehardening and deacclimation under different PSII excitation pressure (modulated by day-temperatures), endogenous concentration of ABA, GA3 and GA-like substances (GAs) were analysed. Analyses were also performed during cold acclimation and reacclimation of plants characterized by different elongation growth rate triggered by prehardening or deacclimation under different day-temperatures. Growth under high PSII excitation pressure (prehardening) resulted in a significant increase in ABA and a considerable decrease in GAs contents. On the other hand, different ABA content played almost no role in controlling growth rate during cold deacclimation and subsequent reacclimation, when the induction of elongation growth was connected with the changes in concentration of GAs including GA3. The possible role of ABA and GAs in controlling prehardening, cold acclimation and deacclimation is discussed.

The experiments were performed to check the effects of exogenous ABA and gibberellin on photosynthetic apparatus and leaf resistance to freezing. In the experiment, two cultivars (winter and spring) of oilseed rape were used in the experiment. Discs, cut out from leaves of cold acclimated plants grown at 12 and 20 °C at similar PPFD levels, were immersed for 72 hours in growth regulator solutions. Some of discs were additionally subjected to high radiation. Independently on cultivar studied, the effects of growth regulator treatments were significant only in leaves developed at 20 °C. ABA treatment increased frost resistance, promoted photosynthetic activity measured in cold and inhibited expansion of leaf-disc area, whereas GA3 evoked opposite effects. The treatment with growth regulators particularly affected the resistance of photosynthetic apparatus to high light. In this case ABA treatment decreased, whereas GA3 increased photoinhibition of PSII. The outcomes may suggest that in the ABA-treated plants PSII is better protected against photoinduced inactivation both by the increase in effectiveness of photosynthetic dark reactions at high light/low temperature conditions, increased energy dissipation in xantophyll cycle and enhanced accumulation of anthocyanins. GA3 treatment may affect the resistance to photoinhibition directly via decrease in anthocyanins contents and indirectly through increase of elongation growth rate in the tissue.

A possible role of photosynthetic apparatus during cold de-acclimation was studied in oilseed rape (Brassica napus var. oleifera). Plants of spring (Star) and winter (Górczañski) cultivars were cold acclimated at + 5 degrees C, and de-acclimated during 4 weeks at combinations of + 12 and + 20 degrees C operating in the light or/and dark, with a 12-h photoperiod. Evidence is presented that the photosynthetic apparatus may be involved in temperature perception during de-acclimation. De-acclimation was faster under a 20/12 degrees C (day/night) treatment than under the reverse 12/20 degrees C (day/night). De-acclimation rate was constant when the day temperature was constant, irrespective of the night temperature both under cold day temperature regimes (12/20, 12/12 degrees C (day/night) and warm-day treatments (20/12, 20/20 degrees C (day/night). The fast decrease in frost resistance observed under warm-day de-acclimation was always accompanied by an acceleration of elongation growth. In the spring cultivar, elongation growth increased starting from the second week of de-acclimation, regardless of temperature conditions. Once elongation growth had commenced during de-acclimation, it continued throughout the period necessary for re-acclimation to low temperature. Re-acclimation to the initial freezing tolerance level was only possible when plant elongation was reduced. In addition re-acclimation of the photosynthetic apparatus to low temperature was impossible in fast growing plants. A possible relationship between PSII, growth rate and frost resistance during cold acclimation and de-acclimation is discussed.

Seedlings of spring barley, meadow fescue, and winter rape were fumigated with 180 μg kg−1 of ozone for 12 d, and effect of O3 on photosynthesis and cell membrane permeability of fumigated plants was determined. Electrolyte leakage and chlorophyll fluorescence were measured after 6, 9, and 12 d of fumigation, while net photosynthetic rate (PN) and stomatal conductance (gs) were measured 9 d after the start of ozone exposure. O3 treatment did not change membrane permeability in fescue and barley leaves, while in rape a significant decrease in ion leakage was noted within the whole experiment. O3 did not change the photochemical efficiency of photosystem 2 (PS2), i.e., Fv/Fm, and the initial fluorescence (F0). The values of half-rise time (t1/2) from F0 to maximal fluorescence (Fm) decreased in fescue and barley after 6 and 9 d of fumigation. PN decreased significantly in ozonated plants, in the three species. The greatest decrease in PN was observed in ozonated barley plants (17 % of the control). The ozone-induced decrease in PN was due to the closure of stomata. Rape was more resistant to ozone than fescue or barley. Apparently, the rape plants show a large adaptation to ozone and prevent loss of membrane integrity leading to ion leakage.

Cultivars of European winter oilseed rape cultivated in the second half of the 1970s and in the mid-1990s were screened for their winter hardiness, frost resistance and vernalization requirement. A strong correlation between winter hardiness and frost resistance in both groups of rape has been noticed. Among oilseed rapes cultivated in the late 1970s, low erucic acid and particularly double zero cultivars were less winter hard than high erucic acid cultivars. Double zero cultivars were characterized by lower frost resistance and lower vernalization requirement. A significant correlation between vernalization requirement and both frost resistance and field survival has also been shown. Frost resistance of the 1990s (double zero) cultivars was higher than that of double low cultivars from the late 1970s. Their vernalization requirement was still small and did not correlate with either frost resistance or winter hardiness. It was concluded that reduction in the content of glucosinolates in the 1970s involved decrease in winter hardiness and vernalization requirement of cultivars. During the following 20 years winter hardiness of double low cultivars has been improved, but vernalization requirements have not changed. As a result no correlation between winter hardiness and vernalization requirement in contemporary canola cultivars has been observed.

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.

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.

† 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.

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.

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.