RÉSUMÉ
Phytophthora species are oomycetes that have evolved a broad spectrum of biological processes and improved strategies to cope with host and environmental challenges. A growing body of evidence indicates that the high pathogen plasticity is based on epigenetic regulation of gene expression linked to Phytophthora's rapid adjustment to endogenous cues and various stresses. As 5mC DNA methylation has not yet been identified in Phytophthora, the reversible processes of acetylation/deacetylation of histone proteins seem to play a pivotal role in the epigenetic control of gene expression in oomycetes. To explore this issue, we review the structure, diversity, and phylogeny of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in six plant-damaging Phytophthora species: P. capsici, P. cinnamomi, P. infestans, P. parasitica, P. ramorum, and P. sojae. To further integrate and improve our understanding of the phylogenetic classification, evolutionary relationship, and functional characteristics, we supplement this review with a comprehensive view of HATs and HDACs using recent genome- and proteome-level databases. Finally, the potential functional role of transcriptional reprogramming mediated by epigenetic changes during Phytophthora species saprophytic and parasitic phases under nitro-oxidative stress is also briefly discussed.
Sujet(s)
Épigenèse génétique , Histone , Phytophthora , Phytophthora/génétique , Phytophthora/physiologie , Phytophthora/pathogénicité , Phytophthora/métabolisme , Histone/métabolisme , Acétylation , Histone deacetylases/métabolisme , Histone deacetylases/génétique , Histone acetyltransferases/métabolisme , Histone acetyltransferases/génétique , PhylogenèseRÉSUMÉ
Contamination of the soil with non-essential metals and metalloids is a serious problem in many regions of the world. These non-essential metals and metalloids are toxic to all organisms impacting crop yields and human health. Crop plants exposed to high concentrations of these metals leads to perturbed mineral homeostasis, decreased photosynthesis efficiency, inhibited cell division, oxidative stress, genotoxic effects and subsequently hampered growth. Plants can activate epigenetic and epitranscriptomic mechanisms to maintain cellular and organism homeostasis. Epigenetic modifications include changes in the patterns of cytosine and adenine DNA base modifications, changes in cellular non-coding RNAs, and remodeling histone variants and covalent histone tail modifications. Some of these epigenetic changes have been shown to be long-lasting and may therefore contribute to stress memory and modulated stress tolerance in the progeny. In the emerging field of epitranscriptomics, defined as chemical, covalent modifications of ribonucleotides in cellular transcripts, epitranscriptomic modifications are postulated as more rapid modulators of gene expression. Although significant progress has been made in understanding the plant's epigenetic changes in response to biotic and abiotic stresses, a comprehensive review of the plant's epigenetic responses to metals is lacking. While the role of epitranscriptomics during plant developmental processes and stress responses are emerging, epitranscriptomic modifications in response to metals has not been reviewed. This article describes the impact of non-essential metals and metalloids (Cd, Pb, Hg, Al and As) on global and site-specific DNA methylation, histone tail modifications and epitranscriptomic modifications in plants.
RÉSUMÉ
Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.
RÉSUMÉ
Nitroxyl (HNO), a one-electron reduced and protonated congener of nitric oxide (â¢NO), was recently discovered in Arabidopsis thaliana. Due to its distinct chemical properties, we believe HNO must be further studied to determine how many physiological processes it impacts.
Sujet(s)
Arabidopsis , Monoxyde d'azote , Oxydes d'azote/composition chimique , BiologieRÉSUMÉ
Nitrogen (N) is one of the most expensive nutrients to supply, therefore, improving the efficiency of N use is essential to reduce the cost of commercial fertilization in plant production. Since cells cannot store reduced N as NH3 or NH4 +, polyamines (PAs), the low molecular weight aliphatic nitrogenous bases, are important N storage compounds in plants. Manipulating polyamines may provide a method to increase nitrogen remobilization efficiency. Homeostasis of PAs is maintained by intricate multiple feedback mechanisms at the level of biosynthesis, catabolism, efflux, and uptake. The molecular characterization of the PA uptake transporter (PUT) in most crop plants remains largely unknown, and knowledge of polyamine exporters in plants is lacking. Bi-directional amino acid transporters (BATs) have been recently suggested as possible PAs exporters for Arabidopsis and rice, however, detailed characterization of these genes in crops is missing. This report describes the first systematic study to comprehensively analyze PA transporters in barley (Hordeum vulgare, Hv), specifically the PUT and BAT gene families. Here, seven PUTs (HvPUT1-7) and six BATs (HvBAT1-6) genes were identified as PA transporters in the barley genome and the detailed characterization of these HvPUT and HvBAT genes and proteins is provided. Homology modeling of all studied PA transporters provided 3D structures prediction of the proteins of interest with high accuracy. Moreover, molecular docking studies provided insights into the PA-binding pockets of HvPUTs and HvBATs facilitating improved understanding of the mechanisms and interactions involved in HvPUT/HvBAT-mediated transport of PAs. We also examined the physiochemical characteristics of PA transporters and discuss the function of PA transporters in barley development, and how they help barley respond to stress, with a particular emphasis on leaf senescence. Insights gained here could lead to improved barley production via modulation of polyamine homeostasis.
RÉSUMÉ
Metal-tolerance proteins (MTPs) are divalent cation transporters that play critical roles in metal tolerance and ion homeostasis in plants. However, a comprehensive study of MTPs is still lacking in crop plants. The current study aimed to comprehensively identify and characterize the MTP gene family in barley (Hordeum vulgare, Hv), an important crop. In total, 12 HvMTPs were identified in the barley genome in this study. They were divided into three phylogenetic groups (Zn-cation diffusion facilitator proteins [CDFs], Fe/Zn-CDFs, and Mn-CDFs) and further subdivided into seven groups (G1, G5, G6, G7, G8, G9, and G12). The majority of MTPs were hydrophobic proteins found in the vacuolar membrane. Gene duplication analysis of HvMTPs revealed one pair of segmental-like duplications in the barley genome. Evolutionary analysis suggested that barley MTPs underwent purifying natural selection. Additionally, the HvMTPs were analyzed in the pan-genome sequences of barley (20 accessions), which suggests that HvMTPs are highly conserved in barley evolution. Cis-acting regulatory elements, microRNA target sites, and protein-protein interaction analysis indicated the role of HvMTPs in a variety of biological processes. Expression profiling suggests that HvMTPs play an active role in maintaining barley nutrient homeostasis throughout its life cycle, and their expression levels were not significantly altered by abiotic stresses like cold, drought, or heat. The expression of barley HvMTP genes in the presence of heavy metals such as Zn2+, Cu2+, As3+, and Cd2+ revealed that these MTPs were induced by at least one metal ion, implying their involvement in metal tolerance or transportation. The identification and comprehensive investigation of MTP gene family members will provide important gene resources for the genetic improvement of crops for metal tolerance, bioremediation, or biofortification of staple crops.
Sujet(s)
Hordeum , Hordeum/génétique , Phylogenèse , Séquence d'acides aminés , Protéines végétales/génétique , Stress physiologique/génétiqueRÉSUMÉ
Flooding entails different stressful conditions leading to low oxygen availability for respiration and as a result plants experience hypoxia. Stress imposed by hypoxia affects cellular metabolism, including the formation of toxic metabolites that dramatically reduce crop productivity. Aldehyde dehydrogenases (ALDHs) are a group of enzymes participating in various aspects of plant growth, development and stress responses. Although we have knowledge concerning the multiple functionalities of ALDHs in tolerance to various stresses, the engagement of ALDH in plant metabolism adjustment to hypoxia is poorly recognized. Therefore, we explored the ALDH gene superfamily in the model plant Arabidopsis thaliana. Genome-wide analyses revealed that 16 AtALDH genes are organized into ten families and distributed irregularly across Arabidopsis 5 chromosomes. According to evolutionary relationship studies from different plant species, the ALDH gene superfamily is highly conserved. AtALDH2 and ALDH3 are the most numerous families in plants, while ALDH18 was found to be the most distantly related. The analysis of cis-acting elements in promoters of AtALDHs indicated that AtALDHs participate in responses to light, phytohormones and abiotic stresses. Expression profile analysis derived from qRT-PCR showed the AtALDH2B7, AtALDH3H1 and AtALDH5F1 genes as the most responsive to hypoxia stress. In addition, the expression of AtALDH18B1, AtALDH18B2, AtALDH2B4, and AtALDH10A8 was highly altered during the post-hypoxia-reoxygenation phase. Taken together, we provide comprehensive functional information on the ALDH gene superfamily in Arabidopsis during hypoxia stress and highlight ALDHs as a functional element of hypoxic systemic responses. These findings might help develop a framework for application in the genetic improvement of crop plants.
RÉSUMÉ
Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid, which arises from redox-sensitive methionine metabolism. In plants, Hcy synthesis involves both cystathionine ß-lyase and S-adenosylhomocysteine hydrolase activities. Thus, Hcy itself is crucial for de novo methionine synthesis and S-adenosylmethionine recycling, influencing the formation of ethylene, polyamines, and nicotianamine. Research on mammalian cells has shown biotoxicity of this amino acid, as Hcy accumulation triggers oxidative stress and the associated lipid peroxidation process. In addition, the presence of highly reactive groups induces Hcy and Hcy derivatives to modify proteins by changing their structure and function. Currently, Hcy is recognized as a critical, independent hallmark of many degenerative metabolic diseases. Research results indicate that an enhanced Hcy level is also toxic to yeast and bacteria cells. In contrast, in the case of plants the metabolic status of Hcy remains poorly examined and understood. However, the presence of the toxic Hcy metabolites and Hcy over-accumulation during the development of an infectious disease seem to suggest harmful effects of this amino acid also in plant cells. The review highlights potential implications of Hcy metabolism in plant physiological disorders caused by environmental stresses. Moreover, recent research advances emphasize that recognizing the Hcy mode of action in various plant systems facilitates verification of the potential status of Hcy metabolites as bioindicators of metabolism disorders and thus may constitute an element of broadly understood biomonitoring.
RÉSUMÉ
We explored the polyamine (PA) metabolic pathway genes in barley (Hv) to understand plant development and stress adaptation in Gramineae crops with emphasis on leaf senescence. Bioinformatics and functional genomics tools were utilized for genome-wide identification, comprehensive gene features, evolution, development and stress effects on the expression of the polyamine metabolic pathway gene families (PMGs). Three S-adenosylmethionine decarboxylases (HvSAMDCs), two ornithine decarboxylases (HvODCs), one arginine decarboxylase (HvADC), one spermidine synthase (HvSPDS), two spermine synthases (HvSPMSs), five copper amine oxidases (HvCuAOs) and seven polyamine oxidases (HvPAOs) members of PMGs were identified and characterized in barley. All the HvPMG genes were found to be distributed on all chromosomes of barley. The phylogenetic and comparative assessment revealed that PA metabolic pathway is highly conserved in plants and the prediction of nine H. vulgare miRNAs (hvu-miR) target sites, 18 protein-protein interactions and 961 putative CREs in the promoter region were discerned. Gene expression of HvSAMDC3, HvCuAO7, HvPAO4 and HvSPMS1 was apparent at every developmental stage. SPDS/SPMS gene family was found to be the most responsive to induced leaf senescence. This study provides a reference for the functional investigation of the molecular mechanism(s) that regulate polyamine metabolism in plants as a tool for future breeding decision management systems.
Sujet(s)
Carboxy-lyases , Hordeum , Hordeum/génétique , Hordeum/métabolisme , Phylogenèse , Amélioration des plantes , Polyamines/métabolisme , Adenosylmethionine decarboxylase/génétique , Adenosylmethionine decarboxylase/métabolisme , Adenosylmethionine decarboxylase/pharmacologie , Carboxy-lyases/génétique , Carboxy-lyases/métabolisme , Plantes , Régulation de l'expression des gènes végétauxRÉSUMÉ
Nitric oxide (NO) is a critical molecule that links plant development with stress responses. Herein, new insights into the role of NO metabolism during leaf senescence in Arabidopsis are presented. A gradual decrease in NO emission accompanied dark-induced leaf senescence (DILS), and a transient wave of peroxynitrite (ONOO-) formation was detected by day 3 of DILS. The boosted ONOO- did not promote tryptophan (Trp) nitration, while the pool of 6-nitroTrp-containing proteins was depleted as senescence progressed. Immunoprecipitation combined with mass spectrometry was used to identify 63 and 4 characteristic 6-nitroTrp-containing proteins in control and individually darkened leaves, respectively. The potential in vivo targets of Trp nitration were mainly related to protein biosynthesis and carbohydrate metabolism. In contrast, nitration of tyrosine-containing proteins was intensified 2-fold on day 3 of DILS. Also, nitrative modification of RNA and DNA increased significantly on days 3 and 7 of DILS, respectively. Taken together, ONOO- can be considered a novel pro-senescence regulator that fine-tunes the redox environment for selective bio-target nitration. Thus, DILS-triggered nitrative changes at RNA and protein levels promote developmental shifts during the plant's lifespan and temporal adjustment in plant metabolism under suboptimal environmental conditions.
Sujet(s)
Arabidopsis , Arabidopsis/génétique , Tryptophane/métabolisme , Sénescence des plantes , Monoxyde d'azote/métabolisme , Tyrosine/composition chimique , Tyrosine/métabolisme , Plantes/métabolisme , ARN/métabolisme , Acide peroxynitreux/métabolismeRÉSUMÉ
Transglutaminases (TGases) are calcium-dependent enzymes that catalyse an acyl-transfer reaction between primary amino groups and protein-bound Gln residues. They are widely distributed in nature, being found in vertebrates, invertebrates, microorganisms, and plants. TGases and their functionality have been less studied in plants than humans and animals. TGases are distributed in all plant organs, such as leaves, tubers, roots, flowers, buds, pollen, and various cell compartments, including chloroplasts, the cytoplasm, and the cell wall. Recent molecular, physiological, and biochemical evidence pointing to the role of TGases in plant biology and the mechanisms in which they are involved allows us to consider their role in processes such as photosynthesis, plant fertilisation, responses to biotic and abiotic stresses, and leaf senescence. In the present paper, an in-depth description of the biochemical characteristics and a bioinformatics comparison of plant TGases is provided. We also present the phylogenetic relationship, gene structure, and sequence alignment of TGase proteins in various plant species, not described elsewhere. Currently, our knowledge of these proteins in plants is still insufficient. Further research with the aim of identifying and describing the regulatory components of these enzymes and the processes regulated by them is needed.
Sujet(s)
Plantes , Transglutaminases , Animaux , Paroi cellulaire/métabolisme , Chloroplastes/génétique , Chloroplastes/métabolisme , Phylogenèse , Plantes/génétique , Plantes/métabolisme , Transglutaminases/métabolismeRÉSUMÉ
Nitric oxide (NO) has been proven to be involved in the regulation of many physiological processes in plants. Though the contribution of NO in plant response to drought has been demonstrated in numerous studies, this phenomenon remains still not fully recognized. The research presented here was performed to decipher the role of NO metabolism in drought tolerance and the ability to recover after stress cessation in two closely related species of forage grasses, important for agriculture in European temperate regions: Festuca arundinacea and F. glaucescens. In both species, two genotypes with distinct levels of drought tolerance were selected to compare their physiological reactions to simulated water deficit and further re-watering, combined with a simultaneous application of NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). The results clearly indicated a strong relationship between scavenging of NO in leaves and physiological response of both analyzed grass species to water deficit and re-watering. It was revealed that NO generated under drought was mainly located in mesophyll cells. In plants with reduced NO level a higher photosynthetic capacity and delay in stomatal closure under drought, were observed. Moreover, NO scavenging resulted also in the increased membrane permeability and higher accumulation of ROS in cells of analyzed plants both under drought and re-watering. This phenomena indicate that lower NO level might reduce drought tolerance and the ability of F. arundinacea and F. glaucescens to recover after stress cessation.
Sujet(s)
Festuca , Lolium , Sécheresses , Festuca/génétique , Lolium/génétique , Monoxyde d'azote/métabolisme , Photosynthèse/physiologie , Feuilles de plante/métabolisme , Poaceae/métabolisme , Stress physiologique/génétique , Eau/métabolismeRÉSUMÉ
Heavy metal (HM) contamination of the environment is a major problem worldwide. The rate of global deposition of HMs in soil has dramatically increased over the past two centuries and there of facilitated their rapid accumulation also in living systems. Although the effects of HMs on plants, animals and humans have been extensively studied, yet little is known about their effects on the (patho)biology of the microorganisms belonging to a unique group of filamentous eukaryotic pathogens, i.e., fungi and oomycetes. Much of the literature concerning mainly model species has revealed that HM stress affects their hyphal growth, morphology, and sporulation. Toxicity at cellular level leads to disturbance of redox homeostasis manifested by the formation of nitro-oxidative intermediates and to the induction of antioxidant machinery. Despite such adverse effects, published data is indicative of the fact that fungal and oomycete pathogens have a relatively high tolerance to HMs in comparison to other groups of microbes such as bacteria. Likely, these pathogens may harbor a network of detoxification mechanisms that ensure their survival in a highly HM-polluted (micro)habitat. Such a network may include extracellular HMs immobilization, biosorption to cell wall, and/or their intracellular sequestration to proteins or other ligands. HMs may also induce a hormesis-like phenomenon allowing the pathogens to maintain or even increase fitness against chemical challenges. Different scenarios linking HMs stress and modification of the microorganisms pathogenicity are disscused in this review.
RÉSUMÉ
Nitric oxide (NO) is an essential redox-signaling molecule operating in many physiological and pathophysiological processes. However, evidence on putative NO engagement in plant immunity by affecting defense gene expressions, including histone modifications, is poorly recognized. Exploring the effect of biphasic NO generation regulated by S-nitrosoglutathione reductase (GNSOR) activity after avr Phytophthora infestans inoculation, we showed that the phase of NO decline at 6 h post-inoculation (hpi) was correlated with the rise of defense gene expressions enriched in the TrxG-mediated H3K4me3 active mark in their promoter regions. Here, we report that arginine methyltransferase PRMT5 catalyzing histone H4R3 symmetric dimethylation (H4R3sme2) is necessary to ensure potato resistance to avr P. infestans. Both the pathogen and S-nitrosoglutathione (GSNO) altered the methylation status of H4R3sme2 by transient reduction in the repressive mark in the promoter of defense genes, R3a and HSR203J (a resistance marker), thereby elevating their transcription. In turn, the PRMT5-selective inhibitor repressed R3a expression and attenuated the hypersensitive response to the pathogen. In conclusion, we postulate that lowering the NO level (at 6 hpi) might be decisive for facilitating the pathogen-induced upregulation of stress genes via histone lysine methylation and PRMT5 controlling potato immunity to late blight.
Sujet(s)
Phytophthora infestans , Solanum tuberosum , Histone/métabolisme , Méthylation , Monoxyde d'azote/métabolisme , Phytophthora infestans/génétique , Maladies des plantes/génétique , Solanum tuberosum/métabolismeRÉSUMÉ
Crop losses caused by climate change and various (a)biotic stressors negatively affect agriculture and crop production. Therefore, it is vital to develop a proper understanding of the complex response(s) to (a)biotic stresses and delineate them for each crop plant as a means to enable translational research. In plants, the improvement of crop quality by m6A editing is believed to be a promising strategy. As a reaction to environmental changes, m6A modification showed a high degree of sensitivity and complexity. We investigated differences in gene medleys between dark-induced leaf senescence (DILS) and developmental leaf senescence in barley, including inter alia RNA modifications active in DILS. The identified upregulated genes in DILS include RNA methyltransferases of different RNA types, embracing enzymes modifying mRNA, tRNA, and rRNA. We have defined a decisive moment in the DILS model which determines the point of no return, but the mechanism of its control is yet to be uncovered. This indicates the possibility of an unknown additional switch between cell survival and cell death. Discoveries of m6A RNA modification changes in certain RNA species in different stages of leaf senescence may uncover the role of such modifications in metabolic reprogramming. Nonetheless, there is no such data about the process of leaf senescence in plants. In this scope, the prospect of finding connections between the process of senescence and m6A modification of RNA in plants seems to be compelling.
RÉSUMÉ
Our previous study concerning the pathogen-induced biphasic pattern of nitric oxide (NO) burst revealed that the decline phase and a low level of NO, due to S-nitrosoglutathione reductase (GSNOR) activity, might be decisive in the upregulation of stress-sensitive genes via histone H3/H4 methylation in potato leaves inoculated with avr P. infestans. The present study refers to the NO-related impact on genes regulating DNA (de)methylation, being in dialog with histone methylation. The excessive amounts of NO after the pathogen or GSNO treatment forced the transient upregulation of histone SUVH4 methylation and DNA hypermethylation. Then the diminished NO bioavailability reduced the SUVH4-mediated suppressive H3K9me2 mark on the R3a gene promoter and enhanced its transcription. However, we found that the R3a gene is likely to be controlled by the RdDM methylation pathway. The data revealed the time-dependent downregulation of the DCL3, AGO4, and miR482e genes, exerting upregulation of the targeted R3a gene correlated with ROS1 overexpression. Based on these results, we postulate that the biphasic waves of NO burst in response to the pathogen appear crucial in establishing potato resistance to late blight through the RdDM pathway controlling R gene expression.
RÉSUMÉ
Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms; however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment.
Sujet(s)
Cadmium/toxicité , Phytophthora infestans/effets des médicaments et des substances chimiques , Phytophthora infestans/métabolisme , Antioxydants/métabolisme , Homéostasie/effets des médicaments et des substances chimiques , Stress oxydatif/effets des médicaments et des substances chimiques , Phytophthora infestans/pathogénicité , Maladies des plantes/étiologie , Espèces réactives de l'azote/métabolisme , Espèces réactives de l'oxygène/métabolisme , Polluants du sol/toxicité , Solanum tuberosum/microbiologie , Stress physiologique , Virulence/effets des médicaments et des substances chimiquesRÉSUMÉ
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.