Increased Freezing Tolerance Research Articles

Metabolomic analyses reveal substances that contribute to the increased freezing tolerance of alfalfa (Medicago sativa L.) after continuous water deficit

BackgroundAlfalfa is a high-quality forage cultivated widely in northern China. Recently, the failure of alfalfa plants to survive the winter has caused substantial economic losses. Water management has attracted considerable attention as a method for the potential improvement of winter survival. The aim of this study was to determine whether and how changes in the water regime affect the freezing tolerance of alfalfa.ResultsThe alfalfa variety WL353LH was cultivated under water regimes of 80 and 25% of water-holding capacity, and all the plants were subjected to low temperatures at 4/0 °C (light/dark) and then − 2/− 6 °C (light/dark). The semi-lethal temperatures were lower for water-stressed than well-watered alfalfa. The pool sizes of total soluble sugars, total amino acids, and proline changed substantially under water-deficit and low-temperature conditions. Metabolomics analyses revealed 72 subclasses of differential metabolites, among which lipid and lipid-like molecules (e.g., fatty acids, unsaturated fatty acids, and glycerophospholipids) and amino acids, peptides, and analogues (e.g., proline betaine) were upregulated under water-deficit conditions. Some carbohydrates (e.g., D-maltose and raffinose) and flavonoids were also upregulated at low temperatures. Finally, Kyoto Encyclopedia of Genes and Genomes analyses revealed 18 significantly enriched pathways involved in the biosynthesis and metabolism of carbohydrates, unsaturated fatty acids, amino acids, and glycerophospholipids.ConclusionsWater deficit significantly enhanced the alfalfa’ freezing tolerance, and this was correlated with increased soluble sugar, amino acid, and lipid and lipid-like molecule contents. These substances are involved in osmotic regulation, cryoprotection, and the synthesis, fluidity, and stability of the cellular membrane. Our study provides a reference for improving alfalfa’ winter survival through water management.

Drivers of plasticity in freeze tolerance in the intertidal mussel Mytilus trossulus.

Freezing is an extreme stress to living cells, and so freeze-tolerant animals often accumulate protective molecules (termed cryoprotectants) to prevent the cellular damage caused by freezing. The bay mussel, Mytilus trossulus, is an ecologically important intertidal invertebrate that can survive freezing. Although much is known about the biochemical correlates of freeze tolerance in insects and vertebrates, the cryoprotectants that are used by intertidal invertebrates are not well characterized. Previous work has proposed two possible groups of low-molecular weight cryoprotectants in intertidal invertebrates: osmolytes and anaerobic byproducts. In our study, we examined which group of candidate cryoprotectants correlate with plasticity in freeze tolerance in mussels using 1H NMR metabolomics. We found that the freeze tolerance of M. trossulus varies on a seasonal basis, along an intertidal shore-level gradient, and with changing salinity. Acclimation to increased salinity (30ppt compared with 15ppt) increased freeze tolerance, and mussels were significantly more freeze tolerant during the winter. Mussel freeze tolerance also increased with increasing shore level. There was limited evidence that anaerobic byproduct accumulation was associated with increased freeze tolerance. However, osmolyte accumulation was correlated with increased freeze tolerance after high salinity acclimation and in the winter. The concentration of most low molecular weight metabolites did not vary with shore level, indicating that another mechanism is likely responsible for this pattern of variation in freeze tolerance. By identifying osmolytes as a group of molecules that assist in freezing tolerance, we have expanded the known biochemical repertoire of the mechanisms of freeze tolerance.

Dynamics of Plant Metabolism during Cold Acclimation.

Plants have evolved strategies to tightly regulate metabolism during acclimation to a changing environment. Low temperature significantly constrains distribution, growth and yield of many temperate plant species. Exposing plants to low but non-freezing temperature induces a multigenic processes termed cold acclimation, which eventually results in an increased freezing tolerance. Cold acclimation comprises reprogramming of the transcriptome, proteome and metabolome and affects communication and signaling between subcellular organelles. Carbohydrates play a central role in this metabolic reprogramming. This review summarizes current knowledge about the role of carbohydrate metabolism in plant cold acclimation with a focus on subcellular metabolic reprogramming, its thermodynamic constraints under low temperature and mathematical modelling of metabolism.

Type II Ice-Binding Proteins Isolated from an Arctic Microalga Are Similar to Adhesin-Like Proteins and Increase Freezing Tolerance in Transgenic Plants.

Microalgal ice-binding proteins (IBPs) in the polar region are poorly understood at the genome-wide level, although they are important for cold adaptation. Through the transcriptome study with the Arctic green alga Chloromonas sp. KNF0032, we identified six Chloromonas IBP genes (CmIBPs), homologous with the previously reported IBPs from Antarctic snow alga CCMP681 and Antarctic Chloromonas sp. They were organized with multiple exon/intron structures and low-temperature-responsive cis-elements in their promoters and abundantly expressed at low temperature. The biological functions of three representative CmIBPs (CmIBP1, CmIBP2 and CmIBP3) were tested using in vitro analysis and transgenic plant system. CmIBP1 had the most effective ice recrystallization inhibition (IRI) activities in both in vitro and transgenic plants, and CmIBP2 and CmIBP3 had followed. All transgenic plants grown under nonacclimated condition were freezing tolerant, and especially 35S::CmIBP1 plants were most effective. After cold acclimation, only 35S::CmIBP2 plants showed slightly increased freezing tolerance. Structurally, the CmIBPs were predicted to have β-solenoid forms with parallel β-sheets and repeated TXT motifs. The repeated TXT structure of CmIBPs appears similar to the AidA domain-containing adhesin-like proteins from methanogens. We have shown that the AidA domain has IRI activity as CmIBPs and phylogenetic analysis also supported that the AidA domains are monophyletic with ice-binding domain of CmIBPs, and these results suggest that CmIBPs are a type of modified adhesins.

Genetic dissection of cell wall defects and the strigolactone pathway in Arabidopsis.

Defects in the biosynthesis and/or deposition of secondary plant cell wall polymers result in the collapse of xylem vessels causing a dwarfed plant stature and an altered plant architecture termed irregular xylem (irx) syndrome. For example, reduced xylan O‐acetylation causes strong developmental defects and increased freezing tolerance. Recently, we demonstrated that the irx syndrome in the trichome birefringence‐like 29/eskimo1 (tbl29/esk1) mutant is dependent on MORE AXILLARY GROWTH 4 (MAX4), a key enzyme in the biosynthesis of the phytohormone strigolactone (SL). In this report, we show that other xylan‐ and cellulose‐deficient secondary wall mutants exhibit increased freezing tolerance correlated with the irx syndrome. In addition, these phenotypes are also dependent on MAX4, suggesting a more general interaction between secondary wall defects and SL biosynthesis. In contrast, MAX4 does not play a role in developmental defects triggered by primary wall deficiencies, suggesting that the interaction is restricted to vascular tissue. Through a reverse genetics approach, the requirement of different components of the SL pathway impacting the irx syndrome in tbl29 was evaluated. Our results show that the tbl29‐associated irx phenotypes are dependent on the MAX3 and MAX4 enzymes, involved in the early steps of SL biosynthesis. In contrast, this signaling is independent on downstream enzymes in the biosynthesis and perception of SL such as MAX1 and MAX2.

Fat body disintegration after freezing stress is a consequence rather than a cause of freezing injury in larvae of Drosophila melanogaster

Extracellular freezing of insect body water may cause lethal injury either by direct mechanical stress exerted by growing ice crystals on cells and tissues or, indirectly, by deleterious physico-chemical effects linked to freeze-induced cell dehydration. Here we present results showing that the macroscopic damage (cell ruptures, tissue disintegration) to fat body of Drosophila melanogaster is not directly caused by mechanical forces linked to growth of ice crystals but rather represents a secondary consequence of other primary freeze injuries occurring at subcellular or microscopic levels. Larvae of D. melanogaster were acclimated to produce variants ranging from freeze susceptible to freeze tolerant. Then, larvae were exposed to supercooling and freezing stresses at different subzero temperatures. The larval survival and macroscopic damage to fat body tissue was scored in 1632 larvae exposed to cold stress. In most cases, fat body damage was not evident immediately following cold stress but developed later. This suggests that the fat body disintegration is a consequence rather than a cause of cold injury. Analysis of fat body membrane phospholipids revealed that increased freeze tolerance was associated with increased relative proportion of phosphatidylethanolamines (PEs) at the expense of phosphatidylcholines (PCs). The PE/PC ratio increased from 1.08 in freeze-susceptible larvae to 2.10 in freeze-tolerant larvae. The potential effects of changing PE/PC ratio on phospholipid bilayer stability upon supercooling and freezing stress are discussed.

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately −7°C) and cold-acclimated (CA; leaf LT50 approximately −50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Evolution of Transient Helicity and Disorder in Late Embryogenesis Abundant Protein COR15A

Cold regulated protein 15A (COR15A) is a nuclear encoded, intrinsically disordered protein that is found in Arabidopsis thaliana. It belongs to the Late Embryogenesis Abundant (LEA) family of proteins and is responsible for increased freezing tolerance in plants. COR15A is intrinsically disordered in dilute solutions and adopts a helical structure upon dehydration or in the presence of co-solutes such as TFE and ethylene glycol. This helical structure is thought to be important for protecting plants from dehydration induced by freezing. Multiple protein sequence alignments revealed the presence of several conserved glycine residues that we hypothesize keeps COR15A from becoming helical in dilute solutions. Using AGADIR, the change in helical content of COR15A when these conserved glycine residues were mutated to alanine residues was predicted. Based on the predictions, glycine to alanine mutants were made at position 68, and 54,68,81, and 84. Labeled samples of wildtype COR15A and mutant proteins were purified and NMR experiments were performed to examine any structural changes induced by the mutations. To test the effects of dehydration on the structure of COR15A, trifluoroethanol, an alcohol based co solvent that is proposed to induce/stabilize helical structure in peptides was added to the NMR samples, and the results of the experiment showed an increase in helical content, compared to the samples without TFE. To test the functional differences between wild type and the mutants, liposome leakage assays were performed. The results from these assays suggest the more helical mutants may augment membrane stability.

A calcium sensor calcineurin B-like 9 negatively regulates cold tolerance via calcium signaling in Arabidopsis thaliana

ABSTRACTCalcineurin B-like protein 9 (CBL9) plays important roles in response to ABA, K+ deprivation in plants. However, whether CBL9 modulates plant adaptation to low-temperature stress is elusive. In this study, we demonstrated that the cbl9 mutants increased freezing tolerance under both cold-acclimating and nonacclimating conditions in Arabidopsis. Cold-induced changes of cytosolic free calcium concentration ([Ca2+]cyt) were then monitored by aequorin-expressed Arabidopsis plants. The results showed that the cold-triggered increases in [Ca2+]cyt levels in cbl9 mutants were clearly higher than those in wild type (WT) plants, while cold-affected changes in free calcium concentration within cytosolic microdomains adjacent to the vacuolar membrane ([Ca2+]md) in cbl9 mutants were similar to those in WT plants. In addition, treatments of seedlings with Ca2+ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and Ca2+ channel blocker lanthanum chloride markedly inhibit changes of [Ca2+]cyt in cbl9 mutants, while the inhibition of calcium release by lithium chloride from intracellular pools demonstrated consistent suppression of [Ca2+]cyt in cbl9 mutants and WT plants. Together, these results indicate that CBL9 negatively modulates cold tolerance through decreasing [Ca2+]cyt in Arabidopsis.

Molecular signatures associated with increased freezing tolerance due to low temperature memory in Arabidopsis.

Alternating temperatures require fast and coordinated adaptation responses of plants. Cold acclimation has been extensively investigated and results in increased freezing tolerance in Arabidopsis thaliana. Here, we show that the two Arabidopsis accessions, Col-0 and N14, which differ in their freezing tolerance, showed memory of cold acclimation, that is, cold priming. Freezing tolerance was higher in plants exposed to cold priming at 4°C, a lag phase at 20°C, and a second triggering cold stress (4°C) than in plants that were only cold primed. To our knowledge, this is the first report on cold memory improving plant freezing tolerance. The triggering response was distinguishable from the priming response at the levels of gene expression (RNA-Seq), lipid (ultraperformance liquid chromatography-mass spectrometry), and metabolite composition (gas chromatography-mass spectrometry). Transcriptomic responses pointed to induced lipid, secondary metabolism, and stress in Col-0 and growth-related functions in N14. Specific accumulation of lipids included arabidopsides with possible functions as signalling molecules or precursors of jasmonic acid. Whereas cold-induced metabolites such as raffinose and its precursors were maintained in N14 during the lag phase, they were strongly accumulated in Col-0 after the cold trigger. This indicates genetic differences in the transcriptomic and metabolic patterns during cold memory.

Methyl jasmonate enhances apple’ cold tolerance through the JAZ–MYC2 pathway

Improving the cold resistance of plants is important, because their growth and development are negatively affected by cold stress. In this study, exogenous applications of methyl jasmonate could enhance the cold resistance of ‘Orin’ apple (Malus × domestica) calli by increasing the expression levels of the cold-signal response genes MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3. In addition, yeast two-hybrid and pull-down assays demonstrated that MdCIbHLH1 interacts with MdJAZ1/4 and MdMYC2 in vitro and in vivo. Protein degradation experiments demonstrated that the stability of the MdJAZ1/4 proteins were affected by the application of exogenous methyl jasmonate, which was followed by their degradation by the 26S proteasome. MdJAZ1/4 act as repressors, binding MdMYC2 in the jasmonate-signaling pathway. The overexpression of MdMYC2 in ‘Orin’ calli increased the expression levels of MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3, resulting in an increased freeze tolerance. Furthermore, the overexpression of MdJAZ1 or MdJAZ4 in transgenic red-fleshed apple calli weakened the promotive effect of MdMYC2 on cold tolerance. Yeast one-hybrid and chromatin immunoprecipitation-PCR analyses showed that MdMYC2 could bind the G-box element found in the MdCBF1 promoter. Thus, jasmonate may function as a critical upstream signal in the ICE–CBF/DREB1 pathway to positively regulate apple freeze tolerance.

Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A

Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic α-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.

Transgenic creeping bentgrass plants expressing a Picea wilsonii dehydrin gene (PicW) demonstrate improved freezing tolerance.

Agrostis stolonifera L. 'Penn A-4' is a common creeping bentgrass species that is widely used in urban landscaping and golf courses. To prolong the green stage of this grass, a dehydrin gene PicW isolated from Wilson's spruce (Picea wilsonii) was transformed into plants of 'Penn A-4' cultivar via a straightforward stolon node infection system. A putative transgenic plant was obtained and its tolerance to low-temperature stress was evaluated. When the transgenic line was subjected to a freezing (- 5°C) treatment, it showed better viability and more robust physiology than wild type, as evidenced by higher soluble sugar and proline contents, and lower relative electrical conductivity and malondialdehyde content. The transgenic line also showed tolerance to a chilling treatment (5°C), although its performance was not significantly different from that of wild-type plants. Overall, the research here clearly revealed the explicit role of PicW in increasing freezing tolerance of grass at the whole-plant level, and demonstrated that the straightforward stolon node transformation method could be well used to genetically modify turfgrass. The obtained transgenic line might be as genetic resource for breeding program and practiced to grow in cold temperate zones.

Comprehensive transcriptome analysis reveals common and specific genes and pathways involved in cold acclimation and cold stress in tea plant leaves

Freezing temperatures during the winter and unusual temperature fluctuations during the winter and early spring are the most harmful ambient factors threatening tea plant winter survival and may cause marked economic losses in tea production during the spring. In this study, we simulate natural climate change, to established cold acclimation (CA) and rapid cold stress conditions to investigate the transcriptome changes involved in CA and cold stress. Results revealed transcriptional changes occurring during the initial period of CA and the cell wall changes that occur throughout the entire CA process; these changes play crucial roles in increasing freezing tolerance during this process. Comparing cold-acclimated plants without further treatment against cold-acclimated plants under cold-stress, different cold response mechanisms were rapidly activated under cold stress; however, the subsequent freezing-induced accumulation of reactive oxygen species could be the major signal and harmful factor stimulating stress-associated gene expression and impairing tea leaf physiology. Moreover, we investigated 60 differentially expressed genes shared by both processes and highlighted the importance of β-ketoacyl CoA synthases, HXXXD-type acyl-transferase family proteins, NAC domain containing protein 80, sugar SWEET transporters and enolases in the responses to various cold conditions. The results provide useful information for understanding the regulation mechanism in tea plant responding to complex low temperature conditions.

DIACYLGLYCEROL ACYLTRANSFERASE1 Contributes to Freezing Tolerance.

Freezing limits plant growth and crop productivity, and plant species in temperate zones have the capacity to develop freezing tolerance through complex modulation of gene expression affecting various aspects of metabolism and physiology. While many components of freezing tolerance have been identified in model species under controlled laboratory conditions, little is known about the mechanisms that impart freezing tolerance in natural populations of wild species. Here, we performed a quantitative trait locus (QTL) study of acclimated freezing tolerance in seedlings of Boechera stricta, a highly adapted relative of Arabidopsis (Arabidopsis thaliana) native to the Rocky Mountains. A single QTL was identified that contained the gene encoding ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (BstDGAT1), whose expression is highly cold responsive. The primary metabolic enzyme DGAT1 catalyzes the final step in assembly of triacylglycerol (TAG) by acyl transfer from acyl-CoA to diacylglycerol. Freezing tolerant plants showed higher DGAT1 expression during cold acclimation than more sensitive plants, and this resulted in increased accumulation of TAG in response to subsequent freezing. Levels of oligogalactolipids that are produced by SFR2 (SENSITIVE TO FREEZING2), an indispensable element of freezing tolerance in Arabidopsis, were also higher in freezing-tolerant plants. Furthermore, overexpression of AtDGAT1 led to increased freezing tolerance. We propose that DGAT1 confers freezing tolerance in plants by supporting SFR2-mediated remodeling of chloroplast membranes.

Molecular Regulation of CBF Signaling in Cold Acclimation.

Cold stress restricts plant growth, development, and distribution. Understanding how plants transduce and respond to cold signals has long been a topic of interest. Traditional genetic and molecular analyses have identified C-repeat/DREB binding factors (CBFs) as key transcription factors that function in cold acclimation. Recent studies revealed the involvement of pivotal protein kinases and transcription factors in CBF-dependent signaling, expanding our knowledge of cold signal transduction from perception to downstream gene expression events. In this review, we summarize recent advances in our understanding of the molecular regulation of these core components of the CBF cold signaling pathway. Knowledge of the mechanism underlying the ability of plants to survive freezing temperatures will facilitate the development of crop plants with increased freezing tolerance.

Effects of drought stress on cold hardiness of non-acclimated viola ( Viola × wittrockiana ‘Iona Gold with Blotch’) in controlled conditions

Abstract Predicted increases in winter temperatures may negatively impact plant survival by preventing maximal cold acclimation prior to cold temperatures. Accordingly, research is needed to identify strategies that may help promote cold hardiness and increase freezing tolerance of cold season ornamental plants. Therefore, the objectives of this research were to study the effects of drought stress on freezing tolerance of Viola × wittrockiana Iona Gold with Blotch during cold season under non-cold acclimating (20 ± 1 °C) conditions and examine the physiological and biochemical changes in plants in response to freezing temperature. After being grown in the greenhouse, plants were first subjected to different levels of soil water availability including control (90%), 70% and 50% field capacity (FC). Then, some traits including proline, carbohydrate and chlorophyll were determined. Finally, plants were transferred to the Thermo Gradient Freezer with six freezing temperatures (0, −3, −6, −9, −12, and −15 °C), as well as 20 °C as the control treatment. Electrolyte leakage (EL) after temperature treatments, plant survival percentage (SU) and some traits related to regrowth after recovery period were determined. Result showed that drought stress increased some factors such as carbohydrate, proline and chlorophyll. Electrolyte leakage significantly increased by lowering temperature and increased by 46% at 15 °C compared to control. Plant survival was significantly affected by treatments so that lowering the temperature to −15 °C caused total mortality in all the plants of irrigation treatments. Plants under 70% FC at 0 °C had the highest increased reproductive component. Number of leaf and leaf area peaked under the moderate water deficit (70% FC) conditions at 0 °C. Plants under 70% FC at 0 °C had the highest increases (87, 134, 90 and 101%, respectively) in dry weights of vegetative, reproductive, root, and total dry weights compared to control. Drought stress can increase freezing tolerance of viola depending on temperature regime.

C-repeat binding transcription factors from Miscanthus × giganteus and their expression at a low temperature

Giant miscanthus (Miscanthus × giganteus) is a valuable plant for bioenergy production and carbon sequestration in low-input agriculture. The newly establishing plantations of miscanthus are, however, at risk during the winter because of the small size of the clones and limited ability of reserves storage in the rhizomes. Increased freezing tolerance of both shoots and rhizomes of giant miscant has been observed after cold acclimation, when plants are exposed to low, non-freezing temperatures. In plants cold acclimation is under complex genetic control that is orchestrated by C-repeat binding factors (CBFs). The present paper aimed the identification and characterisation of CBF-family transcription factors in miscanthus and determined their expression profiles at a low temperature. Three miscanthus genes, homologous to sorghum and maize CBF-family genes were identified. However, only one – MgCBF6 was expressed in the cold-acclimated leaves of miscanthus with an expression profile typical of a CBF-family gene. The ability of miscanthus to cold acclimate was also confirmed using an electrolyte leakage test and survival count. An increase in freezing tolerance was observed both in leaves and rhizomes but only when an increase in the freezing tolerance of plasma membranes was considered. It may be concluded that CBF-induced gene expression pathway may be induced in M. × giganteus while this species use the dormancy strategy for overwintering.

Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves

Salicylic acid (SA)-treatment has been reported to improve plant tolerance to various abiotic stresses. However, its effect on freezing tolerance has not been well investigated. We investigated the effect of exogenous SA on freezing tolerance of spinach (Spinacia oleracea L.) leaves. We also explored if nitric oxide (NO) and/or hydrogen peroxide (H2O2)-mediation was involved in this response, since these are known as primary signaling molecules involved in many physiological processes. A micro-centrifuge tube-based system used to apply SA to petiolate spinach leaves (0.5 mM over 4-d) was effective, as evident by SA content of leaf tissues. SA-treatment did not hamper leaf growth (fresh and dry weight; equatorial and longitudinal length) and was also not significantly different from 25% Hoagland controls vis-à-vis growth. SA application significantly improved freezing tolerance as evidenced by reduced ion-leakage and alleviated oxidative stress (lower accumulation of O2·- and H2O2) following freeze-thaw stress treatments (−6.5, −7.5, and −8.5 °C). Improved freezing tolerance of SA-treated leaves was paralleled by increased proline and ascorbic acid (AsA) accumulation. A 9-d cold acclimation (CA) treatment also improved leaf freezing tolerance (compared to non-acclimated control) and was accompanied by accumulation of SA and proline. Our results indicate that increased freezing tolerance may be associated with accumulation of compatible solutes (proline) and antioxidants (AsA). Notably, the beneficial effect of SA on freezing tolerance was abolished when either H2O2- or NO-scavenger (1 μM N-acetylneuraminic acid, NANA or 100 μM hemoglobin, HB, respectively) was added to SA as pretreatment. Our data suggest that SA-induced freezing tolerance in spinach may be mediated by NO and H2O2 signaling.

Transcriptomic changes reveal gene networks responding to the overexpression of a blueberry DWARF AND DELAYED FLOWERING 1 gene in transgenic blueberry plants

BackgroundConstitutive expression of the CBF/DREB1 for increasing freezing tolerance in woody plants is often associated with other phenotypic changes including dwarf plant and delayed flowering. These phenotypic changes have been observed when Arabidopsis DWARF AND DELAYED FLOWERING 1 (DDF1) was overexpressed in A. thaliana plants. To date, the DDF1 orthologues have not been studied in woody plants. The aim of this study is to investigate transcriptomic responses to the overexpression of blueberry (Vaccinium corymbosum) DDF1 (herein, VcDDF1-OX).ResultsThe VcDDF1-OX resulted in enhanced freezing tolerance in tetraploid blueberry plants and did not result in significant changes in plant size, chilling requirement, and flowering time. Comparative transcriptome analysis of transgenic ‘Legacy-VcDDF1-OX’ plants containing an overexpressed VcDDF1 with non-transgenic highbush blueberry ‘Legacy’ plants revealed the VcDDF1-OX derived differentially expressed (DE) genes and transcripts in the pathways of cold-response, plant flowering, DELLA proteins, and plant phytohormones. The increase in freezing tolerance was associated to the expression of cold-regulated genes (CORs) and the ethylene pathway genes. The unchanged plant size, dormancy and flowering were due to the minimal effect of the VcDDF1-OX on the expression of DELLA proteins, flowering pathway genes, and the other phytohormone genes related to plant growth and development. The DE genes in auxin and cytokinin pathways suggest that the VcDDF1-OX has also altered plant tolerance to drought and high salinity.ConclusionA DDF1 orthologue in blueberry functioned differently from the DDF1 reported in Arabidopsis. The overexpression of VcDDF1 or its orthologues is a new approach to increase freezing tolerance of deciduous woody plant species with no obvious effect on plant size and plant flowering time.