Cold Deacclimation Research Articles

Potential processes leading to winter reddening of young Douglas-fir Pseudotsuga menziesii Mirb. Franco. in Europe

Key messageWinter reddening of young Douglas-fir (Pseudotsuga menziesii Mirb. Franco), triggered by large thermal fluctuations in late winter, is a critical problem for European forestry. A literature review identified certain climatic conditions that are characteristic of ‘reddening’ years, including warm daily temperatures, high daily temperature amplitude, low relative humidity, moderate wind speeds, as well as the occurrence of freeze-thaw cycles with cold night temperatures.By describing the triggering environmental and stand factors, we propose three hypotheses for the physiological processes leading to winter reddening, namely (i) hydraulic failure due to winter drought stress, (ii) photo-oxidative stress in shade-acclimated trees, and (iii) early cold deacclimation during warm periods.i) Low soil temperature, by reducing root water uptake, combined with anticyclonic conditions, by increasing water losses, can induce hydraulic failure in the xylem. Hydraulic failure may be further accelerated by night frosts.ii) Winter reddening can occur when low temperature and high irradiance coincide, disrupting photostasis. Overwhelming of winter photo-protection may lead to photodamage and subsequent reddening.iii) Warm periods, by inducing cold deacclimation, make trees susceptible to frost damage.Finally, the three processes may interact under atypical anticyclonic conditions in late winter (e.g. cold or dry soils, warm days, high irradiance and/or freezing nights). Indeed, trees under water stress would develop a higher sensitivity to freezing night and photooxidative stress. We therefore proposed mitigation actions to avoid exposing trees to stressful conditions based on e.g. stand characteristics, understorey vegetation and planting.

The potential for an increasing threat of unseasonal temperature cycles to dormant plants.

Two functional responses largely guide woody plants' survival to winter conditions: cold hardiness and dormancy. Dormancy affects budbreak timing based on chill accumulation. Effects of warming on dormancy may appear time-shifted: fall and winter warming events decrease chill accumulation, delaying budbreak observed in spring. The same warming events also affect cold hardiness dynamics, having immediate implications. As cold deacclimation rates increase with dormancy progression, the same amount of warming has greater damage risk the later it occurs in the season, depending on return of low temperatures. Should frequency of erratic weather increase with climate change, more instances of risk are expected. However, understanding how plants fare through seasons now and in future climates still requires better knowledge of winter physiology.

Small Stomates and Xylem Vessels Associated With Freeze Tolerance in Winter Barley

ABSTRACTFreeze tolerance is a complex agronomic trait that is difficult to evaluate in the field because of year‐to‐year variation in weather. Discovering plant characteristics closely related to freeze tolerance would enable more effective selection for this important trait. To explore possible physiological mechanisms and search for useful characteristics related to freeze tolerance in winter barley, we conducted field and growth chamber experiments with seven freeze‐tolerant and seven freeze‐susceptible genotypes that exhibited contrasting winter survival in preliminary field screenings. In a 2‐year field experiment, malondialdehyde, proline and water‐soluble carbohydrate concentrations were measured during cold acclimation and deacclimation to investigate differences in osmoregulation and membrane stability between freeze‐tolerant and freeze‐sensitive genotypes. All parameters varied by sampling year, and significant differences between freeze tolerance groups were found mainly during cold deacclimation in the spring. In growth chamber experiments, the size of xylem vessels and stomates was measured with and without cold acclimation. Freeze‐tolerant genotypes had smaller xylem vessels and stomates than freeze‐sensitive genotypes with and without cold acclimation, and small stomatal length was associated with a small xylem vessel area. Thus, it may be possible to improve freeze tolerance in winter barley by selecting smaller xylem and stomate cells. This study also validated germplasms of winter barley with differential freeze tolerance for future projects on breeding for improving winter hardiness and on plant physiology and genetics in response to freezing stress.

Evaluation of Fall and Winter Trinexapac-ethyl Applications on Ultradwarf Bermudagrass Putting Green Color, Quality, and Green Cover

Ultradwarf bermudagrass (UDB) putting greens grown in subtropical and temperate climates can face elevated risk of winter injury from cold temperatures. Trinexapac-ethyl (TE) inhibits UDB growth potentially reducing spring green-up and overexertion of carbohydrate reserves for UDB during the cold de-acclimation period. A field study was conducted to determine the effect of fall and winter TE applications on the visual quality and color of UDB putting greens in Virginia from the cold acclimation phase through the cold de-acclimation phase. A second controlled-environment study was conducted to determine how TE applications to UDB during cold acclimation affected UDB cold tolerance. In the first study, plots were treated with 0.026 kg⋅ha−1 a.i. every 14 days, 0.013 kg⋅ha−1 a.i. every 14 days, or 0.013 kg⋅ha−1 a.i. every 7 days either in the fall only or in the fall and winter. A nontreated control was included for comparison. For the second study, cup-cutter plugs (10.8-cm diameter) of UDB were treated with 0.026 kg⋅ha−1 a.i. every 14 days from the time growth resumed after green-up through cold acclimation or not treated with TE. Plugs were then exposed to −9.4 °C for 4, 6, 8, or 10 hours and placed into a greenhouse to green up. The GC50 values (exposure time to reduce bermudagrass green cover by 50% 6 days after exposure to −9.4 °C) for the treatments were then calculated based on exposure time and percent green-up. In the first study, TE applications improved UDB quality >3.8%. However, TE applications reduced UDB color, and trends exhibited this reduction in color particularly during the late cold acclimation, winter dormancy, and early cold de-acclimation phases. In the second study, TE applications reduced GC50 values by >10.9% compared with nontreated plugs, suggesting TE reduces UDB cold tolerance during the cold acclimation phase.

Transcriptome Analysis of Diurnal and Nocturnal-Warmed Plants, the Molecular Mechanism Underlying Cold Deacclimation Response in Deschampsia antarctica.

Warming in the Antarctic Peninsula is one of the fastest on earth, and is predicted to become more asymmetric in the near future. Warming has already favored the growth and reproduction of Antarctic plant species, leading to a decrease in their freezing tolerance (deacclimation). Evidence regarding the effects of diurnal and nocturnal warming on freezing tolerance-related gene expression in D. antarctica is negligible. We hypothesized that freezing tolerance-related gene (such as CBF-regulon) expression is reduced mainly by nocturnal warming rather than diurnal temperature changes in D. antarctica. The present work aimed to determine the effects of diurnal and nocturnal warming on cold deacclimation and its associated gene expression in D. antarctica, under laboratory conditions. Fully cold-acclimated plants (8 °C/0 °C), with 16h/8h thermoperiod and photoperiod duration, were assigned to four treatments for 14 days: one control (8 °C/0 °C) and three with different warming conditions (diurnal (14 °C/0 °C), nocturnal (8 °C/6 °C), and diurnal-nocturnal (14 °C/6 °C). RNA-seq was performed and differential gene expression was analyzed. Nocturnal warming significantly down-regulated the CBF transcription factors expression and associated cold stress response genes and up-regulated photosynthetic and growth promotion genes. Consequently, nocturnal warming has a greater effect than diurnal warming on the cold deacclimation process in D. antarctica. The eco-physiological implications are discussed.

Physiological and molecular mechanisms of cold deacclimation for timely balancing stress release with growth resumption

Physiological and molecular mechanisms of cold deacclimation for timely balancing stress release with growth resumption

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

How Does Diurnal and Nocturnal Warming Affect the Freezing Resistance of Antarctic Vascular Plants?

The Antarctic Peninsula has rapidly warmed up in past decades, and global warming has exhibited an asymmetric trend; therefore, it is interesting to understand whether nocturnal or diurnal warming is the most relevant for plant cold deacclimation. This study aimed to evaluate the effect of diurnal and nocturnal warming on Antarctic vascular plant's freezing resistance under laboratory conditions. This was studied by measuring the lethal temperature for 50% of tissue (LT50), ice nucleation temperature (INT), and freezing point (FP) on Deschampsia antarctica and Colobanthus quitensis plants. Additionally, soluble carbohydrates content and dehydrin levels were analyzed during nocturnal and diurnal temperatures increase. Nocturnal warming led to a 7 °C increase in the LT50 of D. antarctica and reduced dehydrin-like peptide expression. Meanwhile, C. quitensis warmed plants reduce their LT50 to about 3.6 °C. Both species reduce their sucrose content by more than 28% in warming treatments. Therefore, nocturnal warming leads to cold deacclimation in both plant species, while C. quitensis plants are also cold-deacclimated upon warm days. This suggests that even when the remaining freezing resistance of both species allows them to tolerate summer freezing events, C. quitensis can reach its boundaries of freezing vulnerability in the near future if warming in the Antarctic Peninsula progress.

Modeling wheat and triticale winter hardiness under current and predicted winter scenarios for Central Europe: A focus on deacclimation

Winter hardiness depends on the ability of plants to tolerate a wide spectrum of environmental stresses, which can be affected by climate change in complex ways. Here, empirical Partial Least Squares Regression (PLSS) models of winter survival (WS) of winter wheat (Triticum aestivum L.) and triticale (Triticosecale x Wittmack) were created using data from six years of field experiments at multiple locations throughout Poland. These included 553 winter wheat and 155 triticale accessions. Our aims were to: 1) predict WS on the basis of meteorological data; 2) identify the most critical weather events affecting WS of winter wheat and triticale under Polish conditions; and 3) predict WS for the simulated winters of 2040, 2060 and 2080 under climate change scenarios RCP2.6, RCP4.5, RCP6.0 and RCP8.5 for the experimental site with the lowest mean WS rate during the field experiments. The empirical models showed a high R2 for winter wheat (0.751), and a low R2 for winter triticale (0.160), because of the low winter damage to triticale observed during the experiments. The key climate factors affecting WS varied between species. Winter wheat was affected by winter severity, the number of freezing-thawing cycles, the thermal vegetation index and the freezing index in various winter months. Triticale was affected by late winter ice encasement and high numbers of freeze-thaw events. The predictions indicated that the WS of both winter wheat and triticale may decrease in the future, especially when more extreme climate change scenarios were considered. The main issue will be cold deacclimation connected with climate warming which will be more important for WS than the general increase in minimum winter temperatures. This finding indicates that deacclimation tolerance should be included in wheat and triticale breeding programs as a trait crucial for WS under future winters, at least in Central Europe.

Identification of novel microRNAs for cold deacclimation in barley

Cold acclimation is crucial for the overwintering process of plants. Cold deacclimation is also important for plant survival in winter, which results in loss of freezing tolerance and initiation of growth. MicroRNAs (miRNAs) play crucial roles in regulating various physiological activities including cold response in plants. However, there is no study on miRNAs and their target genes in response to cold deacclimation in a cold-tolerant crop – barley (Hordeum vulgare L.). Here, we performed high-throughput sequencing of miRNAs of leaves during the cold deacclimation process using two barley cultivars with contrasting cold tolerance (Nure, tolerant and Tremois, sensitive). We found a total of 36 known and 267 novel miRNAs, including 12 known and 112 novel ones that are differentially expressed during cold deacclimation. The number of detected differentially expressed miRNAs was larger in Nure than that in Tremois, and the expression profile of miRNAs was dramatically different between Nure and Tremois. Moreover, we identified 13 known and 97 novel miRNAs, which have putative target genes during cold deacclimation. The putative targets of the novel miRNAs included genes encoding C-repeat binding factor (CBF) transcription factors, phytohormones, antioxidant, osmopretectant and flower development. Our results suggest that barley miRNAs respond quickly to cold deacclimation, and the larger number of miRNAs differentially expressed in the cold tolerant cv. Nure indicating that miRNAs might play an important role in the process of deacclimation. It sets a solid foundation for future studies and breeding programs on low temperature tolerance in barley.

Tempo of gene regulation in wild and cultivated Vitis species shows coordination between cold deacclimation and budbreak

Dormancy release, loss of cold hardiness and budbreak are critical aspects of the annual cycle of deciduous perennial plants. Molecular control of these processes is not fully understood, and genotypic variation may be important for climate adaptation. To gain greater understanding of these processes, single-node cuttings from wild (Vitis amurensis, V. riparia) and cultivated Vitis genotypes (V. vinifera ‘Cabernet Sauvignon’, ‘Riesling’) were collected from the vineyard during winter and placed under forcing conditions. Cold hardiness was measured daily, and buds were collected for gene expression analysis until budbreak. Wild Vitis genotypes had faster deacclimation and budbreak than V. vinifera. Temperature-sensing related genes were quickly and synchronously differentially expressed in all genotypes. Significant changes in the pattern of expression changes for eight major metabolic and hormone related pathways were seen across all genotypes. Downregulation of ABA synthesis appears to play an important role in loss of cold hardiness and budbreak in all genotypes. This role was validated through an observed halt in cold hardiness loss of ‘Riesling’ buds treated with exogenous ABA. The gene expression cascade that occurs during deacclimation and budbreak phenology of fast (wild) and slow (cultivated) grapevines appears coordinated and temporally conserved within these phenotypes.

Cold deacclimation mechanisms and reacclimation potential in flower buds of blackcurrant (Ribes nigrum).

As a consequence of global climate change, cold acclimation and deacclimation cycles are becoming increasingly frequent during winter in temperate regions. However, little is known about plant deacclimation and in particular reacclimation mechanisms, although deacclimation resistance and the ability to reacclimate may have wide-ranging consequences regarding plant productivity in a changing climate. Here, we report time-dependent responses of freezing tolerance, respiration rates, metabolite contents (high-resolution magic angle spinning NMR) and fatty acid levels (gas chromatography) in flower buds of two ecodormant Ribes nigrum cultivars exposed to three different deacclimation temperatures followed by a reacclimation treatment at 4°C. The data reveal that despite differences in the progression of deacclimation, the capacity of blackcurrant flower buds to reharden in late winter is virtually non-existing, implying that increasingly irregular temperature patterns is critical for blackcurrant fruit yield. The early phase of deacclimation is associated with a transient increase in respiration and decreasing contents of amino acids, tricarboxylic acid (TCA) cycle intermediates and sugars, indicating an increased need for carbon sources and respiratory energy production for the activation of growth. Decreasing sugar levels may additionally cause loss of freezing tolerance. Deacclimation also involves desaturation of membrane lipids, which likely also contributes to decreased freezing tolerance but may also reflect biosynthesis of signaling molecules stimulating growth and floral organ differentiation. These data provide new insights into the under-researched deacclimation mechanisms and the ability of blackcurrant to reacclimate following different advancements of deacclimation and contribute to our understanding of plant responses to increasingly irregular temperature patterns.

Comparative proteomic analysis of QTL CTS-12 derived from wild rice (Oryza rufipogon Griff.), in the regulation of cold acclimation and de-acclimation of rice (Oryza sativa L.) in response to severe chilling stress

BackgroundRice (Oryza sativa L.) is a thermophilic crop vulnerable to chilling stress. However, common wild rice (Oryza rufipogon Griff.) in Guangxi (China) has the ability to tolerate chilling stress. To better understand the molecular mechanisms underlying chilling tolerance in wild rice, iTRAQ-based proteomic analysis was performed to examine CTS-12, a major chilling tolerance QTL derived from common wild rice, mediated chilling and recovery-induced differentially expressed proteins (DEPs) between the chilling-tolerant rice line DC90 and the chilling-sensitive 9311.ResultsComparative analysis identified 206 and 155 DEPs in 9311 and DC90, respectively, in response to the whole period of chilling and recovery. These DEPs were clustered into 6 functional groups in 9311 and 4 in DC90. The majority were enriched in the ‘structural constituent of ribosome’, ‘protein-chromophore linkage’, and ‘photosynthesis and light harvesting’ categories. Short Time-series Expression Miner (STEM) analysis revealed distinct dynamic responses of both chloroplast photosynthetic and ribosomal proteins between 9311 and DC90.ConclusionCTS-12 might mediate the dynamic response of chloroplast photosynthetic and ribosomal proteins in DC90 under chilling (cold acclimation) and recovery (de-acclimation) and thereby enhancing the chilling stress tolerance of this rice line. The identified DEPs and the involvement of CTS-12 in mediating the dynamic response of DC90 at the proteomic level illuminate and deepen the understanding of the mechanisms that underlie chilling stress tolerance in wild rice.

Bark cells and xylem cells in Japanese white birch twigs initiate deacclimation at different temperatures

Appropriate timing of cold deacclimation is an important component of winter survival of perennial plants, such as trees, in temperate and boreal zones. Recently, concerns about predicted global climate change disturbing deacclimation timing have been increasing. The relationship between ambient temperatures and the manner by which cells' freezing resistance changes is essential for forecasting the timing of deacclimation. In this study, Japanese white birch twigs that underwent deacclimation treatment at a constant temperature of −2, 0, 4, 10, or 20 °C were separated into bark in which cells adapted to subfreezing temperatures by extracellular freezing and xylem in which cells adapted to subfreezing temperatures by deep supercooling, and the freezing resistance of cells in each tissue type was investigated by measuring percentage electrolyte leakage. Birch cells deacclimated in a different manner according to tissue type. Within 7 days under deacclimation treatment, xylem cells decreased their freezing resistance significantly at a high subfreezing temperature (−2 °C). In contrast, bark cells required a temperature of 10 or 20 °C for a detectable decrease in freezing resistance to occur within the same period. At a temperature lower than 0 °C, bark cells did not decrease their freezing resistance, even after 28 days of treatment. The difference in freezing behavior of cells might involve the difference in how deacclimation occurred in bark and xylem cells.

Deacclimation may be crucial for winter survival of cereals under warming climate

Climate warming can change the winter weather patterns. Warmer temperatures during winter result in a lower risk of extreme freezing events. On the other hand the predicted warm gaps during winter will decrease their freezing tolerance. Both contradict effects will affect winter survival but their resultant effect is unclear. In this paper, we demonstrate that climate warming may result in a decrease in winter survival of plants. A field study of winterhardiness of common wheat and triticale was established at 11 locations and repeated during three subsequent winters. The freezing tolerance of the plants was studied after controlled cold acclimation and de-acclimation using both plant survival analysis and chlorophyll fluorescence measurements.Cold deacclimation resistance was shown to be independent from cold acclimation ability. Further, cold deacclimation resistance appeared to be crucial for overwintering when deacclimation conditions occurred in the field. The shortening of uninterrupted cold acclimation may increase cold deacclimation efficiency, which could threaten plant survival during warmer winters. Measurements of chlorophyll fluorescence transient showed some differences triggered by freezing before and after deacclimation.We conclude that cold deacclimation resistance should be considered in the breeding of winter cereals and in future models of winter damage risk.

Natural Variation of Cold Deacclimation Correlates with Variation of Cold-Acclimation of the Plastid Antioxidant System in Arabidopsis thaliana Accessions.

Temperature variations impact on the balance between photosynthetic electron transport and electron-consuming assimilation reactions and transiently increase generation of reactive oxygen species (ROS). Previous studies demonstrated that the expression of C-repeat binding factors (CBFs), which activate cold acclimation reactions, respond to chloroplast ROS signals and that cold deacclimation is partly halted for days after the transfer of acclimated plants to optimal growth conditions in four Arabidopsis accessions from cold-continental habitats. We hypothesized that these accessions differ from others in the regulation of the plastid antioxidant system (PAS). In the present study, we compared the expression intensity of the 12 most prominent PAS genes for peroxidases, superoxide dismutase and low molecular weight antioxidant regenerating enzymes in 10 Arabidopsis accessions with regulation of CBF and COR (cold regulated genes) transcript levels and cold-regulated metabolite levels prior to cold, after 2 week long cold acclimation and during the first 3 days of deacclimation. In the accessions with prolonged activation of cold responses, by trend, weaker induction of various cold-inducible PAS genes and stronger decreases in the expression of negatively cold-regulated PAS genes were observed. Low PAS gene expression delayed the post-cold decrease in H2O2 levels after transfer of the plants from cold to optimal growth conditions. We conclude that weaker expression of various PAS genes in the cold is an adapted strategy of the Arabidopsis accessions N14, N13, Ms-0, and Kas-1 to avoid full inactivation of cold-responses in the first days after the end of the cold period.

COLD ACCLIMATION PATTERNS OF ACTINIDIA ARGUTE CULTIVARS IN A TEMPERATE CLIMATE ZONE

The kiwi berry, Actinidia arguta Planch is gaining importance in temperate Europe. Warm transient intervals during the winter season can initiate cold deacclimation and bud break of woody plants. Most A. arguta cultivars are characterised by an early bud burst which may result in horticultural limitations due to a high sensitivity to spring frost of these young shoots/inflorescences. The evolution of bud dormancy was observed in two Actinidia arguta 'Weiki' and 'Geneva' cultivars. From September through mid-October the final 30-day shooting percentage (FS) was low. Between mid-October and mid-December a steep rise in FS indicates that bud endodormancy was broken. 'Weiki' appears to have a lesser need for cold exposure in order to release it from dormancy. The seasonal pattern of cold acclimation and deacclimation was determined for both cultivars using the electrolyte leakage test. Maximal cold tolerance was achieved in midwinter. In early spring cold tolerance of the buds declined again. 'Geneva' was more cold tolerant than 'Weiki'.

Relationship between cold hardiness and dehydrin gene expression in peach shoot tissues under field conditions

The relationship between cold hardiness and expression of dehydrin genes in the shoots of four peach cultivars (Prunus persica ‘Daewol’, ‘Aikawanakajima’, ‘Fukuyokabijin’, and ‘Kiraranokiwami’) was quantitatively evaluated during seasonal cold acclimation and deacclimation. Cold hardiness was determined by electrolyte leakage analysis, which increased gradually during fall, reached a maximum level in midwinter, and decreased again in spring. According to the temperatures at which 50% injury occurred (LT50), differences in cold hardiness of ‘Daewol’ and ‘Kiraranokiwami’ were the most obvious during deacclimation, with the exception of April 2012. The expression patterns of PpDhn1 and PpDhn3 showed parallel fluctuations in cold hardiness in the four cultivars, whereas PpDhn2 did not show any seasonal changes in any of the cultivars.

Cold De-acclimation Tolerance among Polish Accessions of Common Wheat and Triticale

Cold de-acclimation is a phenomenon the frequency of which is expected to increase due to unstable winter temperatures predicted in the future [1] The aim of the present work was to screen for cold-de-acclimation tolerance the breeding materials of common wheat and triticale, and to find out whether this trait may be, at least partially, responsible for their winter survival.The experiment was performed during winter 2013/14 with 44 accessions of triticale and 78 accessions of wheat. Winter hardiness was studied in seven locations. The studies of freezing tolerance/de-acclimation were conducted at two experimental points where the plants were tested for the freezing tolerance before and after a warm spell during winter. Freezing tolerance on the cellular level was additionally assessed by means of chlorophyll fluorescence measurements after freezing.Our results indicated that the studied accessions of both species were highly variable in their tolerance to de-acclimation. Although no correlation between the freezing tolerance and de-acclimation tolerance was observed, all de-acclimation tolerant accessions were also highly freezing-tolerant. The maximum level of de-acclimation tolerance observed in triticale was higher than in wheat. It was also found that de-acclimation tolerance was responsible for different winter survival of triticale plants in one out of seven field locations, while in five locations no differences in winter survival were observed. In the case of common wheat, de-acclimation tolerance was not related to winter survival at any of three locations in which it varied between the studied materials.Similar observations, together with the studies of other risk factors for winter survival during future winters, will be continued for the next four winters to create models of future winter hardiness risk for wheat and triticale and to select plants with higher resistance to these factors.Founding: the Ministry of Agriculture and Rural Development (Poland) grant HOR hn 801-10/14.

Analysis of Differential Expression Patterns of mRNA and Protein During Cold-acclimation and De-acclimation in Arabidopsis

Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through the process of cold acclimation (CA). In contrast to CA, the acquired freezing tolerance is rapidly reduced during cold de-acclimation (DA) and plants resume growth after sensing warm temperatures. In order to better understand plant growth and development, and to aid in the breeding of cold-tolerant plants, it is important to decipher the functional mechanisms of the DA process. In this study, we performed comparative transcriptomic and proteomic analyses during CA and DA. As revealed by shotgun proteomics, we identified 3987 peptides originating from 1569 unique proteins and the corresponding mRNAs were analyzed. Among the 1569 genes, 658 genes were specifically induced at the transcriptional level during the process of cold acclimation. In order to investigate the relationship between mRNA and the corresponding protein expression pattern, a Pearson correlation was analyzed. Interestingly, 199 genes showed a positive correlation of mRNA and protein expression pattern, indicating that both their transcription and translation occurred during CA. However, 226 genes showed a negative correlation of mRNA and protein expression pattern, indicating that their mRNAs were transcribed during CA and were stored for the subsequent DA step. Under this scenario, those proteins were specifically increased during DA without additional transcription of mRNA. In order to confirm the negative correlation of mRNA and protein expression patterns, qRT-PCR and western blot analyses were performed. Mitochondrial malate dehydrogenase 1 (mMDH1) exhibited a negative correlation of mRNA and protein levels, which was characterized by CA-specific mRNA induction and protein accumulation specifically during DA. These data indicate that the expression of specific mRNAs and subsequent accumulation of corresponding proteins are not always in accordance under low temperature stress conditions in plants.