Role In Cold Acclimation Research Articles

MYB Transcription Factor CDC5 Activates CBF3 Expression to Positively Regulates Freezing Tolerance via Cooperating With ICE1 and Histone Modification in Arabidopsis.

The freezing temperature greatly limits the growth, development and productivity of plants. The C-repeat/DRE binding factor (CBF) plays a major role in cold acclimation, enabling plants to increase their freezing tolerance. Notably, the INDUCER OF CBF EXPRESSION1 (ICE1) protein has garnered attention for its pivotal role in bolstering plants' resilience against freezing through transcriptional upregulation of DREB1A/CBF3. However, the research on the interaction between ICE1 and other transcription factors and its function in regulating cold stress tolerance is largely inadequate. In this study, we found that a R2R3 MYB transcription factor CDC5 interacts with ICE1 and regulates the expression of CBF3 by recruiting RNA polymerase II, overexpression of ICE1 can complements the freezing deficient phenotype of cdc5 mutant. CDC5 increases the expression of CBF3 in response to freezing. Furthermore, CDC5 influences the expression of CBF3 by altering the chromatin status through H3K4me3 and H3K27me3 modifications. Our work identified a novel component that regulates CBF3 transcription in both ICE1-dependent and ICE1-independent manner, improving the understanding of the freezing signal transduction in plants.

Low frequency of the wild-type freezing-tolerance LsCBF7 allele among lettuce population suggests a negative selection during domestication and breeding

Key messageSustainable winter production in lettuce requires freezing tolerant varieties. This study identified a wild-type allele of LsCBF7 that could contribute to freezing tolerance improvement in lettuce.Lettuce is one of the most consumed vegetables globally. While ideally grown in 13–21 °C, its cultivation extends into winter in milder climates. However, occasional freezing temperatures can significantly reduce yields. Therefore, the development of freezing-tolerant lettuce varieties has become a long-term goal of lettuce breeding programs. Despite its significance, our understanding of freezing tolerance in lettuce remains limited. Plants have evolved a coping mechanism against freezing, known as cold acclimation, whereby they can increase freezing tolerance when pre-exposed to low nonfreezing temperatures. The CBF pathway is well-known for its central role in cold acclimation. Previously, we identified 14 CBF genes in lettuce and discovered that one of them, LsCBF7, had a loss-of-function mutation. In this study, we uncovered that accessions from colder regions carried the wild-type allele of LsCBF7 and this allele likely contributed to increased freezing tolerance, with 14% of the lettuce population carrying this allele. Interestingly, in wild lettuce (L. serriola) that is considered a progenitor of cultivated lettuce, this wild-type allele was much more common, with a frequency of 90%. This finding suggests that this wild-type allele may have undergone negative selection during the domestication or breeding of lettuce. Our data strongly indicate that this allele could be linked to early bolting, an undesirable trait in lettuce, which may have driven the negative selection. While this wild-type allele shows promise for improving freezing tolerance in lettuce, it is crucial to decouple it from the early bolting trait to fully harness its potential in lettuce breeding.

Jasmonate enhances cold acclimation in jojoba by promoting flavonol synthesis.

Jojoba is an industrial oil crop planted in tropical arid areas, and its low-temperature sensitivity prevents its introduction into temperate areas. Studying the molecular mechanisms associated with cold acclimation in jojoba is advantageous for developing breeds with enhanced cold tolerance. In this study, metabolomic analysis revealed that various flavonols accumulate in jojoba during cold acclimation. Time-course transcriptomic analysis and weighted correlation network analysis (WGCNA) demonstrated that flavonol biosynthesis and jasmonates (JAs) signaling pathways played crucial roles in cold acclimation. Combining the biochemical and genetic analyses showed that ScMYB12 directly activated flavonol synthase gene (ScFLS). The interaction between ScMYB12 and transparent testa 8 (ScTT8) promoted the expression of ScFLS, but the negative regulator ScJAZ13 in the JA signaling pathway interacted with ScTT8 to attenuate the transcriptional activity of the ScTT8 and ScMYB12 complex, leading to the downregulation of ScFLS. Cold acclimation stimulated the production of JA in jojoba leaves, promoted the degradation of ScJAZ13, and activated the transcriptional activity of ScTT8 and ScMYB12 complexes, leading to the accumulation of flavonols. Our findings reveal the molecular mechanism of JA-mediated flavonol biosynthesis during cold acclimation in jojoba and highlight the JA pathway as a promising means for enhancing cold tolerance in breeding efforts.

Redox regulation of the Calvin–Benson–Bassham cycle during cold acclimation

NADPH-dependent thioredoxin reductase C (NTRC) redox interaction with protein CP12 plays a role in cold acclimation. A recent study by Teh et al. describes the underlying molecular mechanisms that leads to dissociation of the autoinhibitory PRK/CP12/GAPDH (phosphoribulokinase/CP12/glyceraldehyde-3-phosphate dehydrogenase) supracomplex. We propose that chloroplast-to-nucleus retrograde signaling precedes the described mechanism.

SVALKA-POLYCOMB REPRESSIVE COMPLEX2 module controls C-REPEAT BINDING FACTOR3 induction during cold acclimation.

C-REPEAT BINDING FACTORS (CBFs) are highly conserved plant transcription factors that promote cold tolerance. In Arabidopsis (Arabidopsis thaliana), three CBFs (CBF1 to CBF3) play a critical role in cold acclimation, and the expression of their corresponding genes is rapidly and transiently induced during this adaptive response. Cold induction of CBFs has been extensively studied and shown to be tightly controlled, yet the molecular mechanisms that restrict the expression of each CBF after their induction during cold acclimation are poorly understood. Here, we present genetic and molecular evidence that the decline in the induction of CBF3 during cold acclimation is epigenetically regulated through the Polycomb Repressive Complex (PRC) 2. We show that this complex promotes the deposition of the repressive mark H3K27me3 at the coding region of CBF3, silencing its expression. Our results indicate that the cold-inducible long noncoding RNA SVALKA is essential for this regulation by recruiting PRC2 to CBF3. These findings unveil a SVALKA-PRC2 regulatory module that ensures the precise timing of CBF3 induction during cold acclimation and the correct development of this adaptive response.

Machine learning extracts marks of thiamine's role in cold acclimation in the transcriptome of Vitis vinifera.

The escalating challenge of climate change has underscored the critical need to understand cold defense mechanisms in cultivated grapevine Vitis vinifera. Temperature variations can affect the growth and overall health of vine. We used Self Organizing Maps machine learning method to analyze gene expression data from leaves of five Vitis vinifera cultivars each treated by four different temperature conditions. The algorithm generated sample-specific "portraits" of the normalized gene expression data, revealing distinct patterns related to the temperature conditions applied. Our analysis unveiled a connection with vitamin B1 (thiamine) biosynthesis, suggesting a link between temperature regulation and thiamine metabolism, in agreement with thiamine related stress response established in Arabidopsis before. Furthermore, we found that epigenetic mechanisms play a crucial role in regulating the expression of stress-responsive genes at low temperatures in grapevines. Application of Self Organizing Maps portrayal to vine transcriptomics identified modules of coregulated genes triggered under cold stress. Our machine learning approach provides a promising option for transcriptomics studies in plants.

Metabolic and genetic responses to simulated overwintering conditions of alfalfa-rhizobia associations contrasted in their freezing tolerance

AbstractThe study of winter stress tolerance in perennial legumes needs to consider the complete symbiotic system including both plants and bacteria since these two partners are differentially affected by stress conditions. Here, we compared the regrowth after a freezing stress of four different associations of two alfalfa populations differing in freezing tolerance (A-TF0 and A-TF7) inoculated with two Sinorhizobium (Ensifer) meliloti strains (B399 and NRG34) of contrasted adaptation to cold. To understand the contribution of each partner to a better regrowth performance of an association after freezing, we identified molecular traits having major roles in cold acclimation, freezing tolerance, and those involved in the crosstalk between alfalfa and its symbiotic partner. Regrowth after exposure to a freezing stress was 35% larger in the A-TF7 × NRG34 than in the A-TF0 × B399 association. The metabolomic study of roots, crowns and, more specifically, nodules, revealed profound changes in these organs, switching from a sink to support cold acclimation to a source of reserves enabling regrowth after deacclimation. Marked increases in concentrations of stachyose and raffinose, two sugars of the raffinose-family oligosaccharides (RFO), and in the expression level of a gene of the RFO synthetic pathway were observed in response to cold acclimation supporting the importance of a protective role for RFO in alfalfa. Both cold-adapted partners of the symbiotic association contributed to increases in arginine concentration in nodules in response to cold acclimation and deacclimation underscoring the importance of N storage and remobilization for a successful overwintering in alfalfa.

Limitation of sucrose biosynthesis shapes carbon partitioning during plant cold acclimation.

Cold acclimation is a multigenic process by which many plant species increase their freezing tolerance. Stabilization of photosynthesis and carbohydrate metabolism plays a crucial role in cold acclimation. To study regulation of primary and secondary metabolism during cold acclimation of Arabidopsis thaliana, metabolic mutants with deficiencies in either starch or flavonoid metabolism were exposed to 4°C. Photosynthesis was determined together with amounts of carbohydrates, anthocyanins, organic acids and enzyme activities of the central carbohydrate metabolism. Starch deficiency was found to significantly delay soluble sugar accumulation during cold acclimation, while starch overaccumulation did not affect accumulation dynamics but resulted in lower total amounts of \sucrose and glucose. Anthocyanin amounts were lowered in both starch deficient and overaccumulating mutants. Vice versa, flavonoid deficiency did not result in a changed starch amount, which suggested a unidirectional signalling link between starch and flavonoid metabolism. Mathematical modelling of carbon metabolism indicated kinetics of sucrose biosynthesis to be limiting for carbon partitioning in leaf tissue during cold exposure. Together with cold-induced dynamics of citrate, fumarate and malate amounts, this provided evidence for a central role of sucrose phosphate synthase activity in carbon partitioning between biosynthetic and dissimilatory pathways which stabilizes photosynthesis and metabolism at low temperature.

Computational genomics insights into cold acclimation in wheat.

Development of cold acclimation in crops involves transcriptomic reprograming, metabolic shift, and physiological changes. Cold responses in transcriptome and lipid metabolism has been examined in separate studies for various crops. In this study, integrated computational approaches was employed to investigate the transcriptomics and lipidomics data associated with cold acclimation and vernalization in four wheat genotypes of distinct cold tolerance. Differential expression was investigated between cold treated and control samples and between the winter-habit and spring-habit wheat genotypes. Collectively, 12,676 differentially expressed genes (DEGs) were identified. Principal component analysis of these DEGs indicated that the first, second, and third principal components (PC1, PC2, and PC3) explained the variance in cold treatment, vernalization and cold hardiness, respectively. Differential expression feature extraction (DEFE) analysis revealed that the winter-habit wheat genotype Norstar had high number of unique DEGs (1884 up and 672 down) and 63 winter-habit genes, which were clearly distinctive from the 64 spring-habit genes based on PC1, PC2 and PC3. Correlation analysis revealed 64 cold hardy genes and 39 anti-hardy genes. Cold acclimation encompasses a wide spectrum of biological processes and the involved genes work cohesively as revealed through network propagation and collective association strength of local subnetworks. Integration of transcriptomics and lipidomics data revealed that the winter-habit genes, such as COR413-TM1, CIPKs and MYB20, together with the phosphatidylglycerol lipids, PG(34:3) and PG(36:6), played a pivotal role in cold acclimation and coordinated cohesively associated subnetworks to confer cold tolerance.

Cold-Induced Nuclear Import of CBF4 Regulates Freezing Tolerance.

C-repeat binding factors (CBFs) are crucial transcriptional activators in plant responses to low temperature. CBF4 differs in its slower, but more persistent regulation and its role in cold acclimation. Cold acclimation has accentuated relevance for tolerance to late spring frosts as they have become progressively more common, as a consequence of blurred seasonality in the context of global climate change. In the current study, we explore the functions of CBF4 from grapevine, VvCBF4. Overexpression of VvCBF4 fused to GFP in tobacco BY-2 cells confers cold tolerance. Furthermore, this protein shuttles from the cytoplasm to the nucleus in response to cold stress, associated with an accumulation of transcripts for other CBFs and the cold responsive gene, ERD10d. This response differs for chilling as compared to freezing and is regulated differently by upstream signalling involving oxidative burst, proteasome activity and jasmonate synthesis. The difference between chilling and freezing is also seen in the regulation of the CBF4 transcript in leaves from different grapevines differing in their cold tolerance. Therefore, we propose the quality of cold stress is transduced by different upstream signals regulating nuclear import and, thus, the transcriptional activation of grapevine CBF4.

Temperature modulation of CAMTA3 gene induction activity is mediated through the DNA binding domain.

The calmodulin-binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C-REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3334 , which includes the N-terminal CG-1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN-LIKE A1 (EXPL1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold-treated plants. Rather, domain-swapping experiments indicated that the CAMTA3 CG-1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG-1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C-terminal calmodulin-binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature.

ICE‐SCREAM comes in different flavors: Angiosperms encode two types of ICE proteins predicted to differ in their regulation by OST1 and MPK3/6

AbstractINDUCER OF CBF EXPRESSION (ICE) proteins reportedly have a role in cold acclimation and stomata development, but the former has recently been questioned. Our aim was to analyze ICE, OPEN STOMATA 1 (OST1) and MITOGEN ACTIVATED PROTEIN KINASE 3/6 (MPK3/6) protein data and place the results in context with reports in the literature. Orthologous protein sequences from species representative of different plant lineages were retrieved via BLAST searches and assembled into multiple sequence alignments for a comprehensive comparison of key features. We identified one type of ICE protein in bryophytes, lycophytes, ferns and angiosperms, and a second type of ICE protein in angiosperms only, each with characteristic interaction domains. Due to the higher observed similarity of the identified orthologs to proteins from Vitis riparia than to those from Arabidopsis, they were referred to as VrICE2L and VrICE4L, respectively. Regulatory sequence motifs for the kinase OST1 were present in the VrICE2L proteins although less conserved in the non‐angiosperms, whereas OST1 orthologs could be identified in all plant species, including algae. Interestingly, MPK3/6 motifs were most clearly conserved in the VrICE2L and VrICE4L proteins of the more evolved angiosperms, at a time that the A subtype of MPK3/6 kinases and the CRT‐BINDING FACTOR (CBF)‐encoding genes first appeared in the plant lineages. These data are intended to stimulate further investigations of VrICE4L proteins in non‐model plants and provide some evolutionary context into the overlap between stomatal function and regulation, and the cold acclimation pathway.

Genome-Wide Study of Hsp90 Gene Family in Cabbage (Brassica oleracea var. capitata L.) and Their Imperative Roles in Response to Cold Stress.

Heat shock protein 90 (Hsp90) plays an important role in plant developmental regulation and defensive reactions. Several plant species have been examined for the Hsp90 family gene. However, the Hsp90 gene family in cabbage has not been well investigated to date. In this study, we have been discovered 12 BoHsp90 genes in cabbage (Brassica oleracea var. capitata L.). These B. oleracea Hsp90 genes were classified into five groups based on phylogenetic analysis. Among the five groups, group one contains five Hsp90 genes, including BoHsp90-1, BoHsp90-2, BoHsp90-6, BoHsp90-10, and BoHsp90-12. Group two contains three Hsp90 genes, including BoHsp90-3, BoHsp90-4, and BoHsp90. Group three only includes one Hsp90 gene, including BoHsp90-9. Group four were consisting of three Hsp90 genes including BoHsp90-5, BoHsp90-7, and BoHsp90-8, and there is no Hsp90 gene from B. oleracea in the fifth group. Synteny analysis showed that a total of 12 BoHsp90 genes have a collinearity relationship with 5 Arabidopsis genes and 10 Brassica rapa genes. The promoter evaluation revealed that the promoters of B. oleracea Hsp90 genes included environmental stress-related and hormone-responsive cis-elements. RNA-seq data analysis indicates that tissue-specific expression of BoHsp90-9 and BoHsp90-5 were highly expressed in stems, leaves, silique, and flowers. Furthermore, the expression pattern of B. oleracea BoHsp90 exhibited that BoHsp90-2, BoHsp90-3, BoHsp90-7, BoHsp90-9, BoHsp90-10, and BoHsp90-11 were induced under cold stress, which indicates these Hsp90 genes perform a vital role in cold acclimation and supports in the continual of normal growth and development process. The cabbage Hsp90 gene family was found to be differentially expressed in response to cold stress, suggesting that these genes play an important role in cabbage growth and development under cold conditions.

Genome-wide identification of CBF genes and their responses to cold acclimation in Taraxacum kok-saghyz.

C-repeat binding factors (CBFs) are transcription factors that are known to play important roles in plant cold acclimation. They are highly conserved in most higher plants. Taraxacum kok-saghyz (TKS) is an herb native to China and Kazakhstan and is well-known for its production of rubber silk with industrial and economic value. To understand cold acclimation mechanisms, we conducted a genome-wide discovery of the CBF family genes in TKS and revealed ten CBF genes. A bioinformatic analysis of the CBF genes was carried out to analyze the phylogenetic relationship, protein conservative motifs, protein physicochemical properties, gene structure, promoter cis-acting elements, and the gene expression patterns under cold acclimation and control conditions. It was found that most of these genes were highly responsive at the late stage of cold acclimation, indicating that they play important roles in the cold acclimation processes of TKS. This study provides a theoretical basis for the study of the molecular functions of the CBF gene family in TKS, and a useful guidance for the genetic improvement of the cold tolerance traits of TKS and other plants, including crops.

Transcriptomic analysis of grapevine in response to ABA application reveals its diverse regulations during cold acclimation and deacclimation

Abscisic acid (ABA) plays crucial regulatory roles in cold acclimation and deacclimation of grapevine, making it a potential tool to be utilized in vineyards for the acquisition of preferred phenotypes in winter and spring. To understand the function of ABA, we conducted experiments during cold acclimation and deacclimation and evaluated the impact of exogenous ABA on the grapevine transcriptome. RNA-seq data were collected periodically hours or days after ABA treatment. Transcriptomic data were analyzed using principal component analysis (PCA), hierarchical clustering, unsupervised weighed gene co-expression network analysis (WGCNA), contrast-based differentially expressed genes (DEGs) identification and pre-ranked gene set enrichment analysis (GSEA). Our results suggest that ABA functions differently during cold acclimation and deacclimation by selectively regulating key pathways including auxin/indole acetic acid (IAA) metabolism, galactose metabolism and ribosome biogenesis. We also identified the activation of several apparent negative feedback systems that regulated ABA-induced transcriptomic changes, suggesting the existence of a balancing system in response to excessive ABA. This balancing systems potentially eliminates the long-term negative effect on grapevine growing from using ABA in the field. These findings advance our understanding about the regulation of grapevine physiology during dormancy and supports the potential of applying ABA as a cultural practice to mitigate cold injury in winter and spring.

Norway spruce deploys tissue-specific responses during acclimation to cold.

Climate change in the conifer-dominated boreal forest is expected to lead to warmer but more dynamic winter air temperatures, reducing the depth and duration of snow cover and lowering winter soil temperatures. To gain insight into the mechanisms that have enabled conifers to dominate extreme cold environments, we performed genome-wide RNA-Seq analysis from needles and roots of non-dormant two-year Norway spruce (Picea abies (L.) H. Karst), and contrasted these response to herbaceous model Arabidopsis We show that the main transcriptional response of Norway spruce needles exposed to cold was delayed relative to Arabidopsis, and this delay was associated with slower development of freezing tolerance. Despite this difference in timing, Norway spruce principally utilizes early response transcription factors (TFs) belonging to the same gene families as Arabidopsis, indicating broad evolutionary conservation of cold response networks. In keeping with their different metabolic and developmental states, needles and root of Norway spruce showed contrasting results. Regulatory network analysis identified both conserved TFs with known roles in cold acclimation (e.g. homologs of ICE1, AKS3, and of the NAC and AP2/ERF superfamilies), but also a root-specific bHLH101 homolog, providing functional insights into cold stress response strategies in Norway spruce.

Different G6PDH isoforms show specific roles in acclimation to cold stress at various growth stages of barley (Hordeum vulgare) and Arabidopsis thaliana

Low temperatures (0–10 °C) represent a major physiological stress for plants, negatively affecting both their growth rates and overall growth. Cold stress may induce a wide range of negative physiological effects, from oxidative stress to photosynthetic damage. We investigated the effects of low temperatures in two different model plants, Arabidopsis thaliana and Hordeum vulgare. We tested whether the oxidative pentose phosphate pathway (OPPP) is involved in the increase of reductants’ levels needed to counteract oxidative stress induced by cold. The expression, occurrence, and activity of different glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) isoforms during cold stress and plant recovery from low temperatures, were measured at different growth stages from early germinated to mature pot-grown plants. Our results showed plants exhibited changes in different stress markers; ascorbate peroxidase – APX, catalase - CAT, proline, malondialdehyde, H2O2, NADPH/NADP+. We found that a major role in cold acclimation for cytosolic- and peroxisome-located G6PDHs, and different roles for plastidial/chloroplastic isoforms. This suggests that G6PDH isoforms may regulate redox homeostasis in low temperatures, in order to support the increased and continued demand of reductants during both cold stress and recovery stages. Furthermore, we found a significant involvement of (6PGDH), strengthening the idea that the contribution of the whole oxidative pentose phosphate pathway (OPPP) is required to sustain reductant supply under cold stress.

THERMAL REGIME DURING COLD ACCLIMATION AND DORMANT SEASON OF GRAPEVINES IN CONTEXT OF CLIMATE CHANGES- HILLS OF CRAIOVA VINEYARD (ROMANIA)

The thermal regime during the autumn-winter seasons plays a decisive role in cold acclimation and survival grapevine in cold climates. This study aims at assessing the time variability of the thermal regime during dormant season, between 1961-2021, in the Hills of Craiova vineyard and its potential implications on the grapevine, especially regarding its acclimation to cold and its vulnerability to frost. The following observations and analytical data were used in order to assess parameters and indicators with negative thermal stress potential: minimum and maximum temperature (daily, monthly averages), absolute maximum and minimum monthly temperatures, the duration of frost (days), the frequency of several low-temperature thresholds and the temperatures preceding frost episodes.

Gene expression and metabolic changes of Momordica charantia L. seedlings in response to low temperature stress.

Low temperature is one of the abiotic factors limiting germination, growth and distribution of the plant in current plant-products industry, especially for the tropical vegetables in non-tropical area or other fields under cold temperature. Screening the plant with ability against cold temperature captured worldwide attention and exerted great importance. In our previous work, the anti-cold specie of Momordica Charantia L. seedlings was screened out. Yet, the molecular and physiological mechanisms underlying this adaptive process still remain unknown. This study was aimed to investigate adaption mechanism of anti-cold species of Momordica Charantia L. seedlings in genetical and metabolomics levels. Two species, cold-susceptible group (Y17) and cold-resistant group (Y54), were evaluated containing the indexes of malondialdehyde (MDA), hydrogen peroxide (H2O2), proline content, activities of antioxidant enzymes, metabolites changes and genes differentiation in plant tissues after cold treatment. It found that low temperature stress resulted in increased accumulation of MDA, H2O2 and proline content in two species, but less expressions in cold-resistant species Y54. As compared to Y17, cold-resistant species Y54 presented significantly enhanced antioxidant enzyme activities of POD (peroxidase), CAT (cataalase) and SOD (superoxide dismutase). Meanwhile, higher expressed genes encoded antioxidant enzymes and transcription factors when exposure to the low temperature were found in cold-resistant species Y54, and core genes were explored by Q-PCR validation, including McSOD1, McPDC1 and McCHS1. Moreover, plant metabolites containing amino acid, sugar, fatty acid and organic acid in Y54 were higher than Y17, indicating their important roles in cold acclimation. Meanwhile, initial metabolites, including amimo acids, polypeptides, sugars, organic acids and nucleobases, were apparently increased in cold resistant species Y54 than cold susceptible species Y17. Our results demonstrated that the Momordica Charantia L. seedlings achieved cold tolerance might be went through mobilization of antioxidant systems, adjustment of the transcription factors and accumulation of osmoregulation substance. This work presented meaning information for revealing the anti-cold mechanism of the Momordica Charantia L. seedlings and newsight for further screening of anti-cold species in other plant.

Identification of Chloroplast Envelope Proteins with Critical Importance for Cold Acclimation.

The ability of plants to withstand cold temperatures relies on their photosynthetic activity. Thus, the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of the chloroplast envelope, we analyzed this membrane system in Arabidopsis (Arabidopsis thaliana) using mass spectrometry-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across the plastid and the enriched membrane fraction under both normal and cold conditions. We used multivariable logistic regression to model the probabilities for the classification of an envelope localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carriers, such as the ATP/ADP antiporter nucleotide transporter2 (NTT2; substantially increased abundance) or the maltose exporter MEX1 (substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function, we propose scenarios explaining their possible roles in cold acclimation. Furthermore, spatial proteomics introduces an additional layer of complexity and enables the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.