Photosynthetic Acclimation to Cold as a Potential Physiological Marker of Winter Barley Freezing Tolerance Assessed under Variable Winter Environment

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_v/F_m) as well as photochemical (q_P) 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.     

Increasing pre‐acclimation temperature reduces the freezing tolerance of winter‐type faba bean (Vicia faba L.)

Autumn‐sown winter‐type faba bean (Vicia faba L.) has been shown to have a yield advantage over spring sowing. Still, adoption of this overwintered pulse crop remains limited in temperate locations, due to inadequate winter hardiness. This research sought to understand how the prevailing temperature during emergence and seedling development, that is pre‐acclimation, influences freezing tolerance. Seedlings grown under a controlled “warm” 17/12°C (day/night) pre‐acclimation environment were initially less freezing tolerant than those grown under a “cold” 12/5°C temperature treatment. Stem and particularly root tissues were primarily responsible for slower cold acclimation, and there was a genotype specific response of above‐ground tissues to pre‐acclimation treatment. Both above and below‐ground tissues should be tested across a range of pre‐acclimation temperatures when screening faba bean germplasm for freezing tolerance.     

Associations of PCR markers with freezing tolerance and photosynthetic acclimation to cold in winter barley

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 (q_P) 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.     

Temperature Before Cold Acclimation Affects Cold Tolerance and Photoacclimation in Timothy (Phleum pratense L.), Perennial Ryegrass (Lolium perenne L.) and Red Clover (Trifolium pratense L.)

The expected temperature rise in late summer/early autumn can change the conditions for acclimation and affect the winter survival of perennial crops. This study examined the effect of the temperature just before the onset of cold acclimation (pre‐acclimation) on freezing tolerance of timothy (Phleum pratense L.), perennial ryegrass (Lolium perenne L.) and red clover (Trifolium pratense L.) populations (both cultivars and breeding populations) adapted to either northern or southern parts of Norway. Using phytotron experiments, we studied whether increasing pre‐acclimation temperature delays growth cessation, affects photoacclimation and reduces freezing tolerance. Furthermore, we assessed whether these effects were related to the latitudinal adaptation of the plant material. The results showed that a rise in pre‐acclimation temperature decreased both cold acclimation capacity and photoacclimation in these species. This affected the freezing tolerance, which was reduced significantly more in northern‐adapted population of timothy and perennial ryegrass compared with southern‐adapted populations. Red clover was less affected by temperature changes than the grasses.     

Chlorophyll Fluorescence‐Based Studies of Frost Damage and the Tolerance for Cold‐Induced Photoinhibition in Freezing Tolerance Analysis of Triticale (×Triticosecale Wittmack)

Freezing tolerance of 60 breeding lines of winter hexaploid triticale (×Triticosecale Wittmack) was studied in the field‐laboratory experiment. The experiment was repeated over three winters. The survey was also carried out in plants grown and cold‐acclimated in the laboratory. In both the experiments, plant survival analysis and chlorophyll fluorescence‐based studies on energy flows in photosystem II (PSII) (JIP‐test) after freezing of detached leaves were performed. In the laboratory experiment, the temperature of 50 % electrolyte leakage from leaves and the resistance against cold‐induced photoinhibition were additionally investigated. In the case of plants’ cold‐acclimated in the field, determination of the freezing tolerance of PSII gave similar results as the determination of freezing tolerance of whole plants. Both traits were strongly affected by genotype–environmental interactions, but these effects were less visible for PSII characteristics. In the laboratory experiments, a strong correlation between freezing tolerance and the tolerance for cold‐induced photoinhibition of photosynthesis was observed. The possibility of the use of chlorophyll fluorescence‐based techniques for the determination of freezing tolerance in triticale is discussed.     

Relationship between Polyamines and Other Cold‐induced Response Mechanisms in Different Cereal Species

To find a connection between polyamines and various protective effectors involved in the development of cold tolerance, eight different cereal genotypes, including wheat, barley and oat species, were investigated during the acclimation phase to low temperature. Exposure to low temperature induced different changes in the levels of polyamines, and other signalling molecules, such as salicylic acid and abscisic acid, and of other protective compounds, namely flavonols, sugars and antioxidant enzyme activity, and in the lipid composition of certain membrane factions. The most remarkable differences were observed in the oat varieties compared to the other cereal genotypes, which was manifested in the lack of spermidine accumulation and of decrease in trans‐Δ³‐hexadecanoic acid content, in lower initial and not cold‐inducible abscisic acid content and guaiacol peroxidase activity after cold treatment. Correlation analysis revealed that spermidine shows strong positive relationship with flavonols, abscisic acid and ascorbate peroxidase, while was in negative relationship with trans‐Δ³‐hexadecanoic acid. These results suggest that spermidine may have a crucial role in the cold acclimation signalling processes in cereals.     

冬前低温条件下冬小麦品种间叶绿素荧光参数的比较

为比较抗寒性不同的冬麦品种低温和不同光强作用下叶绿素荧光参数的变化,以3个抗寒性不同的冬麦品种为材料,分别在低温驯化期、低温驯化结束期和封冻期取样,室内暗处理后给予6个不同的光强,测定不同光强下的光合电子传递速率(ETR)、光化学猝灭系数(qP)和非光化学猝灭系数(qN)。结果表明,低温、高光强下抗寒品种东农冬麦1号的ETR、qP和qN显著高于其他2个品种,济麦22的这几个叶绿素荧光参数最低。封冻前抗寒中间型东农705品系的热耗散保持较高水平,但进入封冻期后其热耗散能力迅速降低。     

Temperature‐Dependent Growth Adaptation of Festuca pratensis from the Biophysical Viewpoint

Meadow fescue (Festuca pratensis), Italian ryegrass (Lolium multiflorum), their hybrid Festulolium braunii and perennial ryegrass (Lolium perenne) were grown hydroponically under vegetative (20 °C) and hardening (8 °C, 0 °C) regimes. The relative shoot/root growth ratio K, linear root growth rate and other parameters were estimated. When the temperature was lowered from 20 °C to 8 °C, the relative shoot/root growth ratio K of F. pratensis decreased to 0.6, i.e. much more than that of Lolium, suggesting that at 8 °C F. pratensis shoot growth blockage occurs. Further, by dropping the temperature from 8 °C down to 0 °C, the K ratio of F. pratensis increased significantly, while the linear root growth rate decreased much more markedly than in the other species – it suggests also a blockage of root growth. This growth slowdown of stress‐tolerant F. pratensis shoots and at a lower temperature also of its roots is inadequate to the direct impact of temperature and, thus, indicates a two‐step qualitative reorganization, i.e. transition into a qualitatively new state of stress. This phenomenon does not manifest itself in the less stress‐tolerant Lolium and Festulolium species but is expressed in stress‐tolerators, allowing them to achieve their strategic goal – to survive under extreme conditions.     

Improved Cold and Drought Tolerance of Doubled Haploid Maize Plants Selected for Resistance to Prooxidant tert‐Butyl Hydroperoxide

To improve the abiotic stress tolerance of maize (Zea mays L.), doubled haploid (DH) plants were produced by in vitro selection of microspores exposed to tert‐butyl hydroperoxide (t‐BuOOH) as a powerful prooxidant This study investigated the tolerance of the progenies of t‐BuOOH‐selected DH lines to oxidative stress, cold and drought in controlled environment pot experiments by analyses of photosynthetic electron transport and CO₂ assimilation processes, chlorophyll bleaching and lipid peroxidation of leaves. Our results demonstrated that the t‐BuOOH‐selected DH plants exhibited enhanced tolerance not only to oxidative stress‐induced by t‐BuOOH but also to cold and drought stresses. In addition, they showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase and glutathione S‐transferase when compared with the DH lines derived from microspores that were not exposed to t‐BuOOH and to the original hybrid plants. The results suggest that the simultaneous up‐regulation of several antioxidant enzymes may contribute to the oxidative and cold stress tolerance of the t‐BuOOH‐selected DH lines, and that the in vitro microspore selection represents a potential way to improve abiotic stress tolerance in maize.     

Changes in abscisic acid, salicylic acid and phenylpropanoid concentrations during cold acclimation of androgenic forms of Festulolium (Festuca pratensis × Lolium multiflorum) in relation to resistance to pink snow mould (Microdochium nivale)

We investigated changes in concentrations of abscisic (ABA) and salicylic acid (SA), phenolic compounds and phenylalanine ammonia-lyase (PAL) activity in relation to cold-induced tolerance of four androgenic genotypes of Festulolium ( Festuca  ×  Lolium hybrids ) to frost and to the snow mould fungus Microdochium nivale . Cold acclimation increased frost tolerance and resistance to snow mould. Resistant genotypes were characterized by higher ABA concentrations during the first 54 h of cold acclimation and lower concentrations of SA than susceptible genotypes. After cold acclimation, the content of phenolics was significantly lower in genotypes tolerant to frost and M. nivale infection than in susceptible genotypes, while PAL activity was significantly higher. Signalling networks controlling cold acclimation to frost (abiotic) and mould infection (biotic) appears to involve increases in foliar concentrations of ABA and decreases in the SA level during successful cold acclimation. Higher PAL activity and lower concentrations of phenolic compounds also appear to be associated with enhanced tolerance to frost and fungal attack.     

Glycine max and Glycine soja are capable of cold acclimation

Soybean has been considered a cold intolerant species; based largely upon seed germination and soil emergent evaluations. This study reports a distinct acquisition of cold tolerance, in seedlings, following short acclimation periods. Diversity in cold responses was assessed in eight cultivars of Glycine max and six accessions of G. soja. All varieties of soybean significantly increased in freezing tolerance following acclimation. This study indicates soybean seedlings are indeed capable of sensing cold and acquiring cold tolerance. Germination rates after cold imbibition were negatively correlated with maturity group, but positively correlated with cold acclimation potential in G. soja. Seed fatty acid composition was varied between the species, with Glycine soja accessions containing about 2‐times more linolenic acid (18:3) than G. max. Furthermore, high levels of linoleic acid (18:2) in seeds were positively correlated with germination rates following cold imbibition in G. soja only. We suggest that domestication has not impacted the overall ability of soybean to cold acclimate at the seedling stage and that there is little variation within the domesticated species for ability to cold acclimate. Thus, this brief comparative study reduces the enthusiasm for the “wild” species as an additional source of genetic diversity for cold tolerance.     

Field‐grown Cotton Exhibits Seasonal Variation in Photosynthetic Heat Tolerance without Exposure to Heat‐stress or Water‐deficit Conditions

Thermotolerance acclimation of photosystem II to heat and drought is well documented, but studies demonstrating developmental impacts on heat tolerance in field‐grown plants are limited. Consequently, climatic variables, estimated canopy temperature, predawn leaf water potential (Ψ_PD), and the temperature responses of maximum quantum yield of photosystem II (F_v/F_m), variable fluorescence (F_v/F₀), quantum yield of electron transport (φ_Eο) and efficiency of PSI electron acceptor reduction (REο/ABS) were characterized for Gossypium hirsutum at three sample times during the growing season (21 June, 2 July and 18 July 2013) under well‐watered conditions. The temperature decreasing a given photosynthetic parameter 15% from the optimum is referred to as T₁₅ and served as a standardized measure of heat tolerance. Ambient and estimated canopy temperatures were well within the optimal range for cotton throughout the sample period, and leaves were verified well watered using Ψ_PD measurements. However, T₁₅ varied with sample date (highest on July 2 for all parameters), being 2 °C (F_v/F₀) to 5.5 °C (φ_Eο) higher on July 2 relative to June 21, despite optimal temperature conditions and predawn leaf water potential on all sample dates. These findings suggest that even under optimum temperature conditions and water availability, heat tolerance could be influenced by plant developmental stage.     

Evaluation and development of flood‐tolerant soybean cultivars

Soybean (Glycine max [L.] Merr.) cultivars are generally sensitive to flooding stress. The plant growth is severely affected and grain yield is largely reduced in the flooded field. It is important to develop flood‐tolerant soybean cultivars for grain production in regions of heavy rainfalls worldwide. In this study, a total of 722 soybean genotypes were evaluated for flooding tolerance at R1 stages (first flower at any node) in the 5‐year flooding screening tests. Differential soybean genotypes exhibited diverse responses to flooding stress with that plant foliar damage score (FDS) and plant survival rate (PSR) ranged from 1.9 to 8.8 and 3.4% to 81.7%, respectively (p < .0001). Based on our standard of flooding evaluation, most genotypes were sensitive to flooding with 6.0 of average FDS and 38.7% of PSR. Fifty‐two soybean genotypes showed flooding tolerance and 11 genotypes were with consistent flooding tolerance during 4‐ to 5‐year continual evaluations. In the meantime, six genotypes were identified with consistent high sensitivity to flooding. The group analysis showed that genotypes from different sources had distinguishable responses to flooding stress (p < .0001). The interacting analysis of year and flooding tolerance indicated that FDS and PSR means were significantly different among 5 years due to weather temperature and flooding treatment time influences of each year (p < .0001). Furthermore, five breeding lines with high‐yielding and flood‐tolerant traits were developed using selected consistent flood‐tolerant and high‐yielding genotypes through conventional breeding approach.     

Cold acclimation of winter and spring peas: carbon partitioning as affected by light intensity

Like most plants, pea (Pisum sativum L.) becomes tolerant to frost if it is first exposed to low non-freezing temperatures, a process known as cold acclimation. Cold acclimation is a complex process involving many physiological and metabolic changes. Two spring dry peas, two winter dry peas and one winter forage line were exposed to cold temperature in a controlled environment in two experiments, one using low light intensity and the other regular light intensity. Plants were harvested throughout the experiment and dry matter accumulation, water content, soluble and insoluble sugar concentrations were determined from shoot and root samples. Cold acclimation did not occur when temperatures were low if light intensity was low, even in winter peas. In contrast, with regular light intensity, the winter peas acquired more freezing tolerance than spring peas and a close relationship was found between the soluble sugar concentration of leaves just before the frost and the degree of freezing tolerance obtained by the different genotypes. Relationships between freezing tolerance and carbon partitioning between shoot and roots are discussed.     

Androgenesis as a means of dissecting complex genetic and physiological controls: selecting useful gene combinations for breeding freezing tolerant grasses

The advantages of androgenesis from Lolium × Festuca hybrids as a means towards enhanced gene expression and capture of rare genetic variation are reviewed. New evidence is presented for the technique’s use in combination with introgression-mapping for targeting Festuca-derived genes for enhanced freezing-tolerance. As a starting point, a dihaploid genotype derived by androgenesis from a Lolium multiflorum × Festuca pratensis amphiploid (2n = 4x = 28) hybrid cultivar is used as female parent in a backcross breeding programme with L. multiflorum (2n = 2x = 14). A derivative of the backcross␣breeding programme was a genotype of L.␣multiflorum (2n = 2x = 14) incorporating a F.␣pratensis introgression on chromosome 4 that was more freezing-tolerant than Lolium. New evidence of the importance in Lolium and Festuca species of the adaptive capabilities of Photosystem II (PSII) in relation to subsequent freezing-tolerance, is presented. Non-photochemical quenching (NPQ) mechanisms for expulsion of excess light energy during cold acclimation are found in F. pratensis but not in L. multiflorum. Screens of a backcross population derived from an initial dihaploid genotype (n + n = 14) produced by androgenesis from a L. multiflorum × F.␣pratensis amphiploid, indicate a direct relationship between cold acclimation induced increases in NPQ and freezing-tolerance. Preliminary evidence of a role for genes found on chromosome 4 of F. pratensis for increased NPQ expression, is presented.     

Use of chlorophyll fluorescence to evaluate the cold acclimation and freezing tolerance of winter and spring oats

The efficiency of the excitation capture by open Photosystem II (PSII) reaction centres was measured by the F_v/F_m ratios in a collection of winter and spring oats in order to assess the effects of hardening and freezing on the functionality of PSII and also the suitability of a chlorophyll fluorescence‐based method to screen oat cultivars for frost tolerance. A significant reversible decrease in F_v/F_m was found in all genotypes during acclimation to low, non‐freezing temperatures. F_v/F_m analysis appears to be an attractive test for the evaluation of frost tolerance in oats, being rapid, non‐invasive and capable of monitoring a trait related to a crucial stage in the acquisition of frost tolerance. It is more sensitive and precise than other standard methods and highly correlated with field‐evaluated frost damage. The measurements made during recovery 1 or 2 days after stress when the visual symptoms are not yet expressed, were especially advantageous because of the large variability in genotype response. The r‐values (close to 0.8) were reduced due to the non‐standard behaviour of the winter cultivar ‘Aintree’. The cold acclimation response of this genotype has been analysed in detail and the limits of artificial freezing tests are discussed.     

Differential Physio‐Biochemical Responses to Cold Stress of Cold‐Tolerant and Non‐Tolerant Grapes (Vitis L.) from China

There are many grape species exhibiting differences in cold tolerance in China. ‘Zuoshan1’ (Vitis amurensis Rupr. cv. Zuoshan1), one kind of the most cold tolerant grapes, can endure ?40 to ?50 °C, whereas, ‘Maoputao’ (Vitis quinqanguoari Rehd. cv. Maoputao) is sensitive to the cold stress. To understand the physiological mechanism, we investigated responses of both species to a range of decreasing temperatures under the controlled condition. The cold‐tolerant ‘Zuoshan1’ showed higher accumulation of abscisic acid than non‐tolerant ‘Maoputao’, which was correlated with a more dramatic increase in solutes including sugars, proteins at early stage of our detection. At latter stages, these solutes showed more decline in ‘Zuoshan1’. Meanwhile, earlier leaf yellowing and necrosis of ‘Zuoshan1’ were detected with concomitant decrease in the chlorophyll (Chl) content along with remarkable increase in electrolyte leakage and malondialdehyde, which indicated that the earlier leaf senescence occurred to ‘Zuoshan1’. Based on the leaf morphology and parameter investigation, it was concluded that cold‐tolerant grape ‘Zuoshan1’ made quicker responses to the low temperature (LT), indicating it sensed the cold‐signal more strongly than the non‐tolerant grape ‘Maoputao’. Therefore the ability to take more active response to cold stress contributes to the higher tolerance in ‘Zuoshan1’.     

Relationship between Frost Tolerance and Cold-Induced Resistance of Spring Barley, Meadow Fescue and Winter Oilseed Rape to Fungal Pathogens

Plants exposed to one stress factor may become more tolerant to another. Cold is the most often documented factor inducing plant resistance to pathogens. The aim of this work was to investigate whether resistance of spring barley and meadow fescue to Bipolaris sorokiniana and resistance of winter oilseed rape to Phoma lingam induced at 5 °C for 2, 4 or 6 weeks are associated with frost tolerance, water potential and soluble carbohydrate content. Cold‐acclimated plants of each species showed increased resistance to the studied pathogens. Barley, fescue and rape plants demonstrated higher frost tolerance after hardening, but only in the case of fescue a correlation between resistance to frost and resistance to B. sorokiniana was found. A significant decrease in the water potential of leaf cells was observed in cold‐acclimated barley and fescue. In these two species, water potential greatly affected resistance to B. sorokiniana. However, only in barley did accumulation of fructose, glucose and sucrose correlate as well with changes in water potential as with cold‐induced resistance to the pathogen. In the case of hardened rape, no correlation between the studied parameters was found. The results obtained indicated that the temperature of 5 °C used during cold acclimation was not favourable for hardening of this plant species.     

Identification of an ice recrystallisation inhibition gene family in winter‐hardy wheat and its evolutionary relationship to other members of the Triticeae

Ice recrystallisation inhibition (IRI) proteins are important for plants that exist in cold environments. Six novel genes encoding IRI proteins were isolated from winter‐hardy wheat (Tricticum aestivum) cultivar (Mironovskaya808). Conserved domain analysis confirmed that all the predicted proteins contained the basic characteristics of IRI proteins, including leucine‐rich repeats (LRR) and IRI‐domains (NxVx_1‐2G). In addition, these proteins possessed an N‐terminal signal peptide and some conserved cysteines. Despite the high conservation of the amino acid sequences, the characteristic regions still exist among these IRI‐domains of IRI sequences. All IRI sequences in this study are highly homologous with phytosulfokine receptors (PSR). Quantitative real‐time PCR (qPCR) analysis indicated that each member of the IRI gene family was induced after exposure to low temperature. Heterologous expression of IRI proteins enhanced freezing tolerance in host cells. A total of 28 IRI genes were isolated from Triticeae, and the evolutionary relationship of the IRI gene family in Triticeae and related species showed high interspecific specificity. The IRI genes from Aegilops, which is one of the progenitors of allohexaploid wheat, shared the closest relationship with those from Triticum. With high interspecific specificity in Triticeae, however, there was no specificity among different species of Triticum. Our results suggest that IRI genes have potential value to improve cold resistance of crops.