Freezing Tolerance of Lolium multiflorum/Festuca arundinacea Introgression Forms is Associated with the High Activity of Antioxidant System and Adjustment of Photosynthetic Activity under Cold Acclimation.
ABSTRACT: Though winter-hardiness is a complex trait, freezing tolerance was proved to be its main component. Species from temperate regions acquire tolerance to freezing in a process of cold acclimation, which is associated with the exposure of plants to low but non-freezing temperatures. However, mechanisms of cold acclimation in Lolium-Festuca grasses, important for forage production in Europe, have not been fully recognized. Thus, two L. multiflorum/F. arundinacea introgression forms with distinct freezing tolerance were used herein as models in the comprehensive research to dissect these mechanisms in that group of plants. The work was focused on: (i) analysis of cellular membranes' integrity; (ii) analysis of plant photosynthetic capacity (chlorophyll fluorescence; gas exchange; gene expression, protein accumulation, and activity of selected enzymes of the Calvin cycle); (iii) analysis of plant antioxidant capacity (reactive oxygen species generation; gene expression, protein accumulation, and activity of selected enzymes); and (iv) analysis of Cor14b accumulation, under cold acclimation. The more freezing tolerant introgression form revealed a higher integrity of membranes, an ability to cold acclimate its photosynthetic apparatus and higher water use efficiency after three weeks of cold acclimation, as well as a higher capacity of the antioxidant system and a lower content of reactive oxygen species in low temperature.
Project description: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 photo-oxidative 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 degrees C. 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 real-time 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.
Project description:Frost tolerance is the main component of winter-hardiness. To express this trait, plants have to sense low temperature, and respond by activating the process of cold acclimation. The molecular mechanisms of this acclimation have not been fully understood in the agronomically important group of forage grasses, including Lolium-Festuca species. Herein, the introgression forms of L. multiflorum/F. arundinacea distinct with respect to their frost tolerance, were used as models for the comprehensive, proteomic and physiological, research to recognize the crucial components of cold acclimation in forage grasses. The obtained results stressed the importance of photosynthetic performance under acclimation to low temperature. The stable level of photochemical processes after three weeks of cold acclimation in the introgression form with a higher level of frost tolerance, combined simultaneously with the stable level of CO2 assimilation after that period, despite decreased stomatal conductance, indicated the capacity for that form to acclimate its photosynthetic apparatus to low temperature. This phenomenon was driven by the Calvin cycle efficiency, associated with revealed here accumulation profiles and activities of chloroplastic aldolase. The capacity to acclimate the photosynthetic machinery to cold could be one of the most crucial components of forage grass metabolism to improve frost tolerance.
Project description:Plants adapted to cold winters go through annual cycles of gain followed by loss of freezing tolerance (cold acclimation and deacclimation). Warm spells during winter and early spring can cause deacclimation, and if temperatures drop, freezing damage may occur. Many plants are vernalized during winter, a process making them competent to flower in the following summer. In winter cereals, a coincidence in the timing of vernalization saturation, deacclimation, downregulation of cold-induced genes, and reduced ability to reacclimate, occurs under long photoperiods and is under control of the main regulator of vernalization requirement in cereals, VRN1, and/or closely linked gene(s). Thus, the probability of freezing damage after a warm spell may depend on both vernalization saturation and photoperiod. We investigated the role of vernalization and the VRN1-region on freezing tolerance of meadow fescue (Festuca pratensis Huds.), a perennial grass species. Two F2 populations, divergently selected for high and low vernalization requirement, were studied. Each genotype was characterized for the copy number of one of the four parental haplotypes of the VRN1-region. Clonal plants were cold acclimated for 2 weeks or vernalized/cold acclimated for a total of 9 weeks, after which the F2 populations reached different levels of vernalization saturation. Vernalized and cold acclimated plants were deacclimated for 1 week and then reacclimated for 2 weeks. All treatments were given at 8 h photoperiod. Flowering response, freezing tolerance and expression of the cold-induced genes VRN1, MADS3, CBF6, COR14B, CR7 (BLT14), LOS2, and IRI1 was measured. We found that some genotypes can lose some freezing tolerance after vernalization and a deacclimation-reacclimation cycle. The relationship between vernalization and freezing tolerance was complex. We found effects of the VRN1-region on freezing tolerance in plants cold acclimated for 2 weeks, timing of heading after 9 weeks of vernalization, expression of COR14B, CBF6, and LOS2 in vernalized and/or deacclimated treatments, and restoration of freezing tolerance during reacclimation. While expression of VRN1, COR14B, CBF6, LOS2, and IRI1 was correlated, CR7 was associated with vernalization requirement by other mechanisms, and appeared to play a role in freezing tolerance in reacclimated plants.
Project description:When grown under cool temperature, winter annuals upregulate photosynthetic capacity as well as freezing tolerance. Here, the role of three cold-induced C-repeat-binding factor (CBF1-3) transcription factors in photosynthetic upregulation and freezing tolerance was examined in two Arabidopsis thaliana ecotypes originating from Italy (IT) or Sweden (SW), and their corresponding CBF1-3-deficient mutant lines it:cbf123 and sw:cbf123. Photosynthetic, morphological and freezing-tolerance phenotypes, as well as gene expression profiles, were characterized in plants grown from the seedling stage under different combinations of light level and temperature. Under high light and cool (HLC) growth temperature, a greater role of CBF1-3 in IT versus SW was evident from both phenotypic and transcriptomic data, especially with respect to photosynthetic upregulation and freezing tolerance of whole plants. Overall, features of SW were consistent with a different approach to HLC acclimation than seen in IT, and an ability of SW to reach the new homeostasis through the involvement of transcriptional controls other than CBF1-3. These results provide tools and direction for further mechanistic analysis of the transcriptional control of approaches to cold acclimation suitable for either persistence through brief cold spells or for maximisation of productivity in environments with continuous low temperatures.
Project description: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.
Project description:Understanding how plants respond to drought at different levels of cell metabolism is an important aspect of research on the mechanisms involved in stress tolerance. Furthermore, a dissection of drought tolerance into its crucial components by the use of plant introgression forms facilitates to analyze this trait more deeply. The important components of plant drought tolerance are the capacity for photosynthesis under drought conditions, and the ability of cellular membrane regeneration after stress cessation. Two closely related introgression forms of Lolium multiflorum/Festuca arundinacea, differing in the level of photosynthetic capacity during stress, and in the ability to regenerate their cellular membranes after stress cessation, were used as forage grass models in a primary metabolome profiling and in an evaluation of chloroplast 1,6-bisphosphate aldolase accumulation level and activity, during 11 days of water deficit, followed by 10 days of rehydration. It was revealed here that the introgression form, characterized by the ability to regenerate membranes after rehydration, contained higher amounts of proline, melibiose, galactaric acid, myo-inositol and myo-inositol-1-phosphate involved in osmoprotection and stress signaling under drought. Moreover, during the rehydration period, this form also maintained elevated accumulation levels of most the primary metabolites, analyzed here. The other introgression form, characterized by the higher capacity for photosynthesis, revealed a higher accumulation level and activity of chloroplast aldolase under drought conditions, and higher accumulation levels of most photosynthetic products during control and drought periods. The potential impact of the observed metabolic alterations on cellular membrane recovery after stress cessation, and on a photosynthetic capacity under drought conditions in grasses, are discussed.
Project description:<h4>Background and aims</h4>Cold is a major constraint for cereal cultivation under temperate climates. Winter-hardy plants interpret seasonal changes and can acquire the ability to resist sub-zero temperatures. This cold acclimation process is associated with physiological, biochemical and molecular alterations in cereals. Brachypodium distachyon is considered a powerful model system to study the response of temperate cereals to adverse environmental conditions. To date, little is known about the cold acclimation and freezing tolerance capacities of Brachypodium. The main objective of this study was to evaluate the cold hardiness of seven diploid Brachypodium accessions.<h4>Methods</h4>An integrated approach, involving monitoring of phenological indicators along with expression profiling of the major vernalization regulator VRN1 orthologue, was followed. In parallel, soluble sugars and proline contents were determined along with expression profiles of two COR genes in plants exposed to low temperatures. Finally, whole-plant freezing tests were performed to evaluate the freezing tolerance capacity of Brachypodium.<h4>Key results</h4>Cold treatment accelerated the transition from the vegetative to the reproductive phase in all diploid Brachypodium accessions tested. In addition, low temperature exposure triggered the gradual accumulation of BradiVRN1 transcripts in all accessions tested. These accessions exhibited a clear cold acclimation response by progressively accumulating proline, sugars and COR gene transcripts. However, whole-plant freezing tests revealed that these seven diploid accessions only have a limited capacity to develop freezing tolerance when compared with winter varieties of temperate cereals such as wheat and barley. Furthermore, little difference in terms of survival was observed among the accessions tested despite their previous classification as either spring or winter genotypes.<h4>Conclusions</h4>This study is the first to characterize the freezing tolerance capacities of B. distachyon and provides strong evidence that some diploid accessions such as Bd21 have a facultative growth habit.
Project description: <b>Synopsis</b> This work demonstrates that PpABI3 contributes to freezing tolerance regulation in Physcomitrella patens. Transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is known to play a major role in regulating seed dormancy, germination, seedling development as well as stress responses. ABI3 is conserved among land plants; however, its roles in non-seed plants under stress conditions have not been well characterized. In this study, we report that ABI3 is involved in freezing tolerance regulation during cold acclimation at least in part through ABA signaling pathway in moss <i>Physcomitrella patens</i> (<i>P. patens</i>). Deletion of <i>PpABI3</i> (?<i>abi3-1</i>) compromises the induction of genes related to cold response and antioxidative protection, resulting in reduced accumulation of cryoprotectants and antioxidants. In addition, photosystem II (PSII) activity is repressed in ?<i>abi3-1</i> during cold acclimation partially due to alternations of photosynthetic protein complexes compositions. The gametophyte of ?<i>abi3-1</i> displays severe growth inhibition and developmental deficiency under low temperature condition, while two independent complementary lines display phenotypes similar to that of wild-type <i>P. patens</i> (WT). Furthermore, the freezing tolerance of ?<i>abi3-1</i> was significantly affected by deletion of <i>PpABI3</i>. These data revealed that <i>PpABI3</i> plays an important role in low temperature response and freezing tolerance in <i>P. patens</i>.
Project description:Plant tolerance to freezing temperatures is governed by endogenous components and environmental factors. Exposure to low non-freezing temperatures is a key factor in the induction of freezing tolerance in the process called cold acclimation. The role of nitric oxide (NO) in cold acclimation was explored in Arabidopsis using triple nia1nia2noa1-2 mutants that are impaired in the nitrate-dependent and nitrate-independent pathways of NO production, and are thus NO deficient. Here, we demonstrate that cold-induced NO accumulation is required to promote the full cold acclimation response through C-repeat Binding Factor (CBF)-dependent gene expression, as well as the CBF-independent expression of other cold-responsive genes such as Oxidation-Related Zinc Finger 2 (ZF/OZF2). NO deficiency also altered abscisic acid perception and signaling and the cold-induced production of anthocyanins, which are additional factors involved in cold acclimation.
Project description: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.