Cold Acclimation Process Research Articles

Transcriptome sequencing, de novo assembly and annotation of the freeze tolerant earthworm, Dendrobaena octaedra

The lumbricid earthworm Dendrobaena octaedra, widely distributed in the northern hemisphere, is a freeze tolerant worm species known for its ability to acclimate to the extreme cold condition. However, at the genome and transcriptome level, the species has not been explored to a great extent. The present study focuses on transcriptome analysis of Dendrobaena octaedra to identify and functionally annotate the potential transcripts and biological pathways associated with the freeze tolerance of the species. The Roche 454 pyrosequencing generated 181,856 reads for warm acclimated worms and 190,209 reads for cold acclimated worms which were de novo assembled to 6336 isotigs using the Newbler 2.3 assembler. In total, 2665 isotigs were annotated against the NCBI nr database and of them, 1753 isotigs were assigned to 9546 GO terms. The conserved domain analysis using InterProScan annotated 6309 isotigs into 1307 domains with EF-hand domain as the most highly represented domain. Further, the COG annotation classified 1426 isotigs into 25 functional categories and 895 isotigs were mapped to 90 KEGG pathways. Among them, 22 pathways were functionally enriched in the cold acclimated worms. The RNA seq analysis showed 96 differentially expressed genes of which 50 genes were upregulated in the cold acclimated worms. In addition, we identified 72 molecular chaperones which may have effect in regulating the cold tolerance of the species. The complete transcriptome resource of Dendrobaena octaedra can be effectively utilized further to interpret the key genetic, molecular and physiological mechanisms regulating the freeze tolerance and cold acclimation process of the species.

Increased leaf tocopherol and β‐carotene content is associated with the tolerance of winter wheat cultivars to frost

AbstractComparative studies in winter wheat cultivars have so far been focused on the content of tocopherols in the grains as tocopherols are an important source of vitamin E in the human diet. Our approach was to verify the following main hypothesis—whether a higher accumulation of nonenzymatic antioxidants (tocopherol, tocotrienols, β‐carotene) in the leaves of winter wheat during cold acclimation is accompanied by a higher frost tolerance of winter wheat cultivars. Seedlings of winter wheat cultivars—frost susceptible (Bystra) and frost tolerant (Nutka and Smuga)—were cold acclimated for a period of 3 weeks. To analyse the antioxidants (tocotrienols, tocopherols, β‐carotene), leaf samples were taken on the final day of the exposure to cold. Moreover, samples were also taken to estimate the content of abscisic acid, jasmonic acid and salicylic acid. After leaf chlorophyllafluorescence measurements, the plants were exposed to frost (−8 to −14°C) and left to regrow to estimate their survival. Cultivars of winter wheat with higher accumulation of tocopherols in leaves had higher frost tolerance than cultivar with fewer tocopherols in leaves. α‐tocopherol was accumulated in cv. Bystra in the amount of 137 μg gDW−1. The content of α‐tocopherol was about 26% and 90% higher in cv. Nutka and cv. Smuga, respectively. The β‐carotene content was also higher in cv. Nutka and cv. Smuga compared to cv. Bystra. This work indicates a need for further and more detailed studies on the role of tocopherols and carotenoids in the acclimation process of cereals (winter wheat) to low temperatures. The accumulation of nonenzymatic antioxidants in the cold acclimation process will be discussed against the background of the accumulation of the stress hormones and changes in the photosynthetic efficiency in the tested cultivars.

Janus-Faced Nature of Light in the Cold Acclimation Processes of Maize.

Exposure of plants to low temperature in the light may induce photoinhibitory stress symptoms, including oxidative damage. However, it is also known that light is a critical factor for the development of frost hardiness in cold tolerant plants. In the present work the effects of light during the cold acclimation period were studied in chilling-sensitive maize plants. Before exposure to chilling temperature at 5°C, plants were cold acclimated at non-lethal temperature (15°C) under different light conditions. Although exposure to relatively high light intensities during cold acclimation caused various stress symptoms, it also enhanced the effectiveness of acclimation processes to a subsequent severe cold stress. It seems that the photoinhibition induced by low temperature is a necessary evil for cold acclimation processes in plants. Greater accumulations of soluble sugars were also detected during hardening at relatively high light intensity. Certain stress responses were light-dependent not only in the leaves, but also in the roots. The comparison of the gene expression profiles based on a microarray study demonstrated that the light intensity is at least as important a factor as the temperature during the cold acclimation period. Differentially expressed genes were mainly involved in most of assimilation and metabolic pathways, namely photosynthetic light capture via the modification of chlorophyll biosynthesis and the dark reactions, carboxylic acid metabolism, cellular amino acid, porphyrin or glutathione metabolic processes, ribosome biogenesis and translation. Results revealed complex regulation mechanisms and interactions between cold and light signalling processes.

Effect of Exogenous Salicylic Acid on the Physiological and Biochemical Processes of Ligustrum lucidum during Natural Cold Acclimation

The evergreen Ligustrum lucidum (glossy privet) suffers from freezing injury in northern China, where there are short growing seasons and early fall frost events. To investigate the influence of exogenous salicylic acid (SA) application on the natural cold acclimation of glossy privet, physiological and biochemical changes in glossy privet seedlings subjected to SA treatments at four concentrations (0, 150, 250, and 350 mg·L−1) were evaluated from Sept. to Dec. 2016. The optimum application concentrations were between 250 and 350 mg·L−1, which led to better freezing tolerance during natural cold acclimation. The improved freezing tolerance under exogenous SA application was associated with the accumulation of chlorophyll, proline, soluble protein, and soluble sugar, and the regulations of gibberellic acid (GA) and abscisic acid (ABA). Salicylic acid treatments started a cascade of steps for advancing the cold acclimation process of glossy privet. We suggest that exogenous SA application may be used on glossy privet grown in northern China.

Insight into cellular proteome of Lolium multiflorum/Festuca arundinacea introgression forms to decipher crucial mechanisms of cold acclimation in forage grasses

Frost tolerance is the main component of winter-hardiness. To express this trait, plants sense low temperature, and respond by activating the process of cold acclimation. The molecular mechanisms of this acclimation have not been fully understood in the agronomically important group of forage grasses, including Lolium-Festuca species. Herein, the introgression forms of L. multiflorum/F. arundinacea distinct with respect to their frost tolerance, were used as models for the comprehensive, proteomic and physiological, research to recognize the crucial components of cold acclimation in forage grasses. The obtained results stressed the importance of photosynthetic performance under acclimation to low temperature. The stable level of photochemical processes after three weeks of cold acclimation in the introgression form with a higher level of frost tolerance, combined simultaneously with only slightly (but not significantly) decreased level of CO2 assimilation after that period, despite significantly lower stomatal conductance, indicated the capacity for that form to acclimate its photosynthesis to low temperature. This phenomenon was driven by the Calvin cycle efficiency, associated with revealed here accumulation profiles and activities of chloroplastic aldolase. The capacity to acclimate the photosynthetic machinery to cold could be one of the most crucial components of forage grass metabolism to improve frost tolerance.

An Overview of Signaling Regulons During Cold Stress Tolerance in Plants.

Plants, being sessile organisms, constantly withstand environmental fluctuations, including low-temperature, also referred as cold stress. Whereas cold poses serious challenges at both physiological and developmental levels to plants growing in tropical or sub-tropical regions, plants from temperate climatic regions can withstand chilling or freezing temperatures. Several cold inducible genes have already been isolated and used in transgenic approach to generate cold tolerant plants. The conventional breeding methods and marker assisted selection have helped in developing plant with improved cold tolerance, however, the development of freezing tolerant plants through cold acclimation remains an unaccomplished task. Therefore, it is essential to have a clear understanding of how low temperature sensing strategies and corresponding signal transduction act during cold acclimation process. Herein, we synthesize the available information on the molecular mechanisms underlying cold sensing and signaling with an aim that the summarized literature will help develop efficient strategies to obtain cold tolerant plants.

Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley.

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light—including its spectra—also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 °C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeum vulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance.

Differential Mechanisms of Photosynthetic Acclimation to Light and Low Temperature in Arabidopsis and the Extremophile Eutrema salsugineum.

Photosynthetic organisms are able to sense energy imbalances brought about by the overexcitation of photosystem II (PSII) through the redox state of the photosynthetic electron transport chain, estimated as the chlorophyll fluorescence parameter 1-qL, also known as PSII excitation pressure. Plants employ a wide array of photoprotective processes that modulate photosynthesis to correct these energy imbalances. Low temperature and light are well established in their ability to modulate PSII excitation pressure. The acquisition of freezing tolerance requires growth and development a low temperature (cold acclimation) which predisposes the plant to photoinhibition. Thus, photosynthetic acclimation is essential for proper energy balancing during the cold acclimation process. Eutrema salsugineum (Thellungiella salsuginea) is an extremophile, a close relative of Arabidopsis thaliana, but possessing much higher constitutive levels of tolerance to abiotic stress. This comparative study aimed to characterize the photosynthetic properties of Arabidopsis (Columbia accession) and two accessions of Eutrema (Yukon and Shandong) isolated from contrasting geographical locations at cold acclimating and non-acclimating conditions. In addition, three different growth regimes were utilized that varied in temperature, photoperiod and irradiance which resulted in different levels of PSII excitation pressure. This study has shown that these accessions interact differentially to instantaneous (measuring) and long-term (acclimation) changes in PSII excitation pressure with regard to their photosynthetic behaviour. Eutrema accessions contained a higher amount of photosynthetic pigments, showed higher oxidation of P700 and possessed more resilient photoprotective mechanisms than that of Arabidopsis, perhaps through the prevention of PSI acceptor-limitation. Upon comparison of the two Eutrema accessions, Shandong demonstrated the greatest PSII operating efficiency (ΦPSII) and P700 oxidizing capacity, while Yukon showed greater growth plasticity to irradiance. Both of these Eutrema accessions are able to photosynthetically acclimate but do so by different mechanisms. The Shandong accessions demonstrate a stable response, favouring energy partitioning to photochemistry while the Yukon accession shows a more rapid response with partitioning to other (non-photochemical) strategies.

Characterization of BdCBF genes and genome-wide transcriptome profiling of BdCBF3-dependent and -independent cold stress responses in Brachypodium distachyon

Freezing stress substantially reduces crop yields and limits plant distribution. The identification of genes critical for cold acclimation is thus of great importance. C-repeat binding factors (CBFs) are transcription factors that play key regulatory roles in the cold acclimation process, which dramatically increases freezing tolerance in plants. We report here that B. distachyon can successfully cold acclimate and we identified a CBF gene family consisting of eight genes in a tandem array and are designated as BdCBF1-8. Expression analysis indicated that all the eight BdCBF genes are induced by cold. Freezing tolerance experiments showed that the knockdown of BdCBF3 gene in RNAi cbf3 mutant plants results in a significant reduction in survival after an exposure to freezing temperatures. RNA-seq transcriptomic analysis was conducted using the wild type and cbf3 mutant plants under both normal and cold conditions. We identified 460, 3213, 2839 and 1871 differentially expressed genes exhibiting different expression levels by pairwise comparisons of cbf3 (23°C) vs. WT (23°C), WT (23°C) vs. WT (4°C), cbf3 (23°C) vs. cbf3 (4°C), and cbf3 (4°C) vs. WT (4°C), respectively. These differentially expressed genes were enriched in several biological pathways. Combined analyses of differentially expressed genes in some of the enriched pathways provide insights into mechanisms of plant response to cold in the BdCBF3-dependent, –independent or –compensatory categories.

Prefoldins Negatively Regulate Cold Acclimation in Arabidopsis thaliana by Promoting Nuclear Proteasome-Mediated HY5 Degradation

The process of cold acclimation is an important adaptive response whereby many plants from temperate regions increase their freezing tolerance after being exposed to low non-freezing temperatures. The correct development of this response relies on proper accumulation of a number of transcription factors that regulate expression patterns of cold-responsive genes. Multiple studies have revealed a variety of molecular mechanisms involved in promoting the accumulation of these transcription factors. Interestingly, however, the mechanisms implicated in controlling such accumulation to ensure their adequate levels remain largely unknown. In this work, we demonstrate that prefoldins (PFDs) control the levels of HY5, an Arabidopsis transcription factor with a key role in cold acclimation by activating anthocyanin biosynthesis, in response to low temperature. Our results show that, under cold conditions, PFDs accumulate into the nucleus through a DELLA-dependent mechanism, where they interact with HY5, triggering its ubiquitination and subsequent degradation. The degradation of HY5 would result, in turn, in anthocyanin biosynthesis attenuation, ensuring the accurate development of cold acclimation. These findings uncover an unanticipated nuclear function for PFDs in plant responses to abiotic stresses.

Transcriptome profiling of Eucalyptus nitens reveals deeper insight into the molecular mechanism of cold acclimation and deacclimation process

Eucalyptus nitens (H. Deane & Maiden) is a fast-growing species used principally for pulpwood and solid-wood production. Due to its cold tolerance, it is preferred over other Eucalyptus species at high elevations. To get a deeper insight in the molecular mechanisms of cold acclimation, the transcriptome profiling by RNA-Seq in plants of E. nitens under cold acclimation and deacclimation process was compared in order to identify differentially expressed genes (DEGs). Transcriptomes from control, cold acclimated to chilling temperature, cold acclimated at freezing temperature, and deacclimation condition were compared using Eucalyptus grandis as reference genome. The differential expression analysis allowed the identification of a total of 1600 DEGs out of which 1088 and 1071 were identified in response to cold acclimation and deacclimation, respectively. The gene ontology analysis revealed that DEGs were significantly enriched in response to stimulus, response to abiotic stimulus, membrane, catalytic activity, and cell periphery. Furthermore, the biochemical pathways analysis revealed a large number of DEGs represented in the biosynthesis of phenylpropanoids, specifically flavonoid biosynthesis likely to support ROS scarvening, genes related to photosynthesis, genes that take part in glycolysis/gluconeogenesis related to starch biosynthesis pathway, and genes represented in carotenoid biosynthesis pathway suggesting a role in the regulation of ABA synthesis, which has been previously involved in stress tolerance. A total of 115 DEGs corresponding to transcription factors were identified, being the most represented families AP2, MYB, and WRKY. Expression of six DEGs was validated using qRT-PCR that further supported the in silico results. The present study provides a comprehensive view of global gene expression and revealed valuable information about the dynamic and complex nature of gene expression occurring during cold acclimation and deacclimation process in E. nitens.

Physiological Analysis of Arabidopsis Ecotype to Investigate the Freezing Tolerance after Cold Acclimation Process

Physiological Analysis of Arabidopsis Ecotype to Investigate the Freezing Tolerance after Cold Acclimation Process

Variation in waterlogging-triggered stomatal behavior contributes to changes in the cold acclimation process in prehardened Lolium perenne and Festuca pratensis

According to predicted changes in climate, waterlogging events may occur more frequently in the future during autumn and winter at high latitudes of the Northern Hemisphere. If excess soil water coincides with the process of cold acclimation for plants, winter survival may potentially be affected. The effects of waterlogging during cold acclimation on stomatal aperture, relative water content, photochemical activity of photosystem II, freezing tolerance and plant regrowth after freezing were compared for two prehardened overwintering forage grasses, Lolium perenne and Festuca pratensis. The experiment was performed to test the hypothesis that changes in photochemical activity initiated by waterlogging-triggered modifications in the stomatal aperture contribute to changes in freezing tolerance. Principal component analysis showed that waterlogging activated different adaptive strategies in the two species studied. The increased freezing tolerance of F. pratensis was associated with increased photochemical activity connected with stomatal opening, whereas freezing tolerance of L. perenne was associated with a decrease in stomatal aperture. In conclusion, waterlogging-triggered stomatal behavior contributed to the efficiency of the cold acclimation process in L. perenne and F. pratensis.

Brassinosteroids increase winter survival of winter rye (Secale cereale L.) by affecting photosynthetic capacity and carbohydrate metabolism during the cold acclimation process

The aim of the study was to verify the hypothesis that brassinosteroids would modify the cold acclimation process through the stimulation of photosynthesis and carbohydrate metabolism, and would consequently cause an increase in the plant’s capacity for winter survival. Seedlings of two winter rye cultivars (winter-resistant and moderately winter-resistant types) were sprayed with 24-epibrassinolide (EBR) and then subjected to cold acclimation at 4 °C. The foliar hormone application was studied for its effects on two components of winter resistance: tolerance to frost; and resistance to snow mould. These were examined after medium (3 weeks) and long (6 weeks) terms of cold acclimation. Additionally, as a control group, the level of endogenous brassinosteroids was measured in non-treated plants. The maximum acquired snow mould resistance was observed in the EBR-treated plants after 3 weeks of cold acclimation, while the maximum frost tolerance was recorded after 6 weeks of cold acclimation, as compared with control. In the winter-resistant cultivars, the EBR-induced snow mould resistance coincided with a lower photosynthetic efficiency and a lack of change in their Rubisco activity and level of total soluble carbohydrates. The elevated EBR-induced frost tolerance in the winter-resistant cultivars was associated with a carbohydrate metabolism that promoted a decrease in the monosaccharides of the leaves that stimulate photosynthetic efficiency and Rubisco activity. For both cultivars, the control plants developed frost tolerance between the third and sixth week of cold acclimation. However, the winter-resistant cultivar was characterised by a constant castasterone (CS) level, while the moderately winter-resistant one experienced a sharp increase in its endogenous CS after 3 weeks, and then after 6 weeks of cold acclimation.

Integrative "omic" analysis reveals distinctive cold responses in leaves and roots of strawberry, Fragaria × ananassa 'Korona'.

To assess underlying metabolic processes and regulatory mechanisms during cold exposure of strawberry, integrative “omic” approaches were applied to Fragaria × ananassa Duch. ‘Korona.’ Both root and leaf tissues were examined for responses to the cold acclimation processes. Levels of metabolites, proteins, and transcripts in tissues from plants grown at 18°C were compared to those following 1–10 days of cold (2°C) exposure. When leaves and roots were subjected to GC/TOF-MS-based metabolite profiling, about 160 compounds comprising mostly structurally annotated primary and secondary metabolites, were found. Overall, ‘Korona’ showed a modest increase of protective metabolites such as amino acids (aspartic acid, leucine, isoleucine, and valine), pentoses, phosphorylated and non-phosphorylated hexoses, and distinct compounds of the raffinose pathway (galactinol and raffinose). Distinctive responses were observed in roots and leaves. By 2DE proteomics a total of 845 spots were observed in leaves; 4.6% changed significantly in response to cold. Twenty-one proteins were identified, many of which were associated with general metabolism or photosynthesis. Transcript levels in leaves were determined by microarray, where dozens of cold associated transcripts were quantitatively characterized, and levels of several potential key contributors (e.g., the dehydrin COR47 and GADb) to cold tolerance were confirmed by qRT-PCR. Cold responses are placed within the existing knowledge base of low temperature-induced changes in plants, allowing an evaluation of the uniqueness or generality of Fragaria responses in photosynthetic tissues. Overall, the cold response characteristics of ‘Korona’ are consistent with a moderately cold tolerant plant.

Changes in carbohydrates triggered by low temperature waterlogging modify photosynthetic acclimation to cold in Festuca pratensis

Increased precipitation expected during autumn and winter at higher latitudes in the northern hemisphere may lead to low temperature short-term waterlogging. The temperature of waterlogging was shown to be an important factor controlling plant reaction to this stress. Photosynthetic apparatus response to water excess in the soil at low temperature was examined in Festuca pratensis genotypes with contrasting freezing tolerance. The study was aimed to test the hypothesis whether changes in leaf water-soluble carbohydrate concentration brought about alterations in Rubisco activity may affect the photoacclimation to cold under water excess in the soil. The study investigated the effects of waterlogging during cold acclimation process on a set of chlorophyll a fluorescence parameters, water-soluble carbohydrates, expression of Rubisco activase gene, and Rubisco activity. It was shown that carbohydrate status affected by Rubisco activity might be crucial for the activation of non-photochemical mechanism of photoacclimation to cold under waterlogging. Altered Rubisco activity was only partially attributed to the expression of Rubisco activase gene. Additionally, low carbohydrate concentration in the leaves of waterlogged plants was the condition preventing sugar repression of photosynthesis, including RcaA expression. This indicates that sugar de-repression of photosynthetic genes may be considered a component of photosynthetic acclimation to cold under waterlogging.

The Recovery of Plastid Function Is Required for Optimal Response to Low Temperatures in Arabidopsis.

Cold acclimation is an essential response in higher plants to survive freezing temperatures. Here, we report that two independent mutant alleles of the H-subunit of Mg-chelatase, CHLH, gun5-1 and cch in Arabidopsis are sensitive to low temperatures. Plants were grown in photoperiodic conditions and exposed to low temperatures for short- and long-term periods. Tetrapyrrole biosynthesis was initially significantly inhibited in response to low temperature but recovered in wild type (Col-0), although the tetrapyrrole levels were lower in cold compared to control conditions. The gun5-1 and cch alleles showed an inability to recover chlorophyll biosynthesis in addition to a significant decrease in freezing tolerance. We found that the impaired plastid function in the CHLH mutant plants resulted in compromised de novo protein synthesis at low temperatures. The expression of the transcription factors CBF1-3 was super-induced in gun5-1 and cch mutant alleles but expression levels of their target genes, COR15a, COR47 and COR78 were similar or even lower compared to Col-0. In addition, the protein levels of COR15a were lower in gun5-1 and cch and a general defect in protein synthesis could be seen in the gun5-1 mutant following a 35S labelling experiment performed at low temperature. Taken together, our results demonstrate the importance of a functional chloroplast for the cold acclimation process and further suggest that impaired plastid function could result in inhibition of protein synthesis at low temperature.

Physiological and Biochemical Processes of Magnolia wufengensis in Response to Foliar Abscisic Acid Application during Natural Cold Acclimation

The rare species Magnolia wufengensis frequently suffers from freezing injury in northern China. To investigate the influence of exogenous abscisic acid (ABA) application on the natural cold acclimation of M. wufengensis, physiological and biochemical changes in field-grown M. wufengensis seedlings subjected to foliar ABA treatments at four concentrations (0, 300, 600, and 900 mg·L−1) were evaluated from Sept. 2012 to Jan. 2013. The optimum foliar application concentrations of ABA for M. wufengensis were between 600 and 900 mg·L−1, which led to faster shoot growth cessation, leaf senescence, and development rates of bud endodormancy level and shoot freezing tolerance. The improved freezing tolerance under exogenous ABA application was associated with promoted dehydration and accumulation of proline, soluble protein, and certain soluble sugars such as glucose and fructose. Foliar ABA treatments initiated a cascade of steps for advancing the cold acclimation process of M. wufengensis. We suggest that exogenous ABA application may be used on M. wufengensis grown in northern China, where there are short growing seasons and early fall frost events.

Transcriptome Profile in Response to Frost Tolerance in Eucalyptus globulus

The genetic improvement of trees for freezing tolerance is one of the most important goals to extend the plantations to colder areas. RNA-Seq technology has become a key tool in transcriptome studies. It can quantify overall expression levels for each gene simultaneously with high efficiency and speed through in silico gene expression, where differentially expressed genes can be identified by measuring the reads mapped for each transcript. In this study, the results of ESTs libraries from two Eucalyptus globulus genotypes showing contrasting differences in frost tolerance after cold acclimation using mRNA-Seq and in silico gene expression are discussed. A total of 14,265 non-redundant transcripts were predicted, where 163 corresponded to upregulated and 537 to downregulated genes. Pathway analyses of upregulated transcripts indicated that differences in frost tolerance might be regulated by the tree response to chemical and osmotic stimulus and organic substances, principally by overexpressing proteins that respond to hormone stress. These results suggest that genes coding for dehydrins, outer envelope, and voltage-dependent anion channel proteins are likely to participate in the regulation of the cold acclimation process and may have an important role in frost tolerance. The transcription factor analysis allowed identifying that those most differentially expressed in a resistant genotype were participating in the regulation of transcription, hormone regulation, photosynthesis, and response to stress. Additionally, the screening of polymorphic EST-SSR in silico and the validation of these markers in a reference population lead to identify a polymorphic EST-SSR with potential use for plant breeding and genotype discrimination.

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.