Habituation of the cold shock response: A systematic review and meta-analysis.

Cold water immersion (CWI) evokes the life-threatening reflex cold shock response (CSR), inducing hyperventilation, increasing cardiac arrhythmias, and increasing drowning risk by impairing safety behaviour. Repeated CWI induces CSR habituation (i.e., diminishing response with same stimulus magnitude) after ∼4 immersions, with variation between studies. We quantified the magnitude and coefficient of variation (CoV) in the CSR in a systematic review and meta-analysis with search terms entered to Medline, SportDiscus, PsychINFO, Pubmed, and Cochrane Central Register. Random effects meta-analyses, including effect sizes (Cohen's d) from 17 eligible groups (k), were conducted for heart rate (HR, n = 145, k = 17), respiratory frequency (fR, n = 73, k = 12), minute ventilation (Ve, n = 106, k = 10) and tidal volume (Vt, n = 46, k=6). All CSR variables habituated (p < 0.001) with large or moderate pooled effect sizes: ΔHR -14 (10) bt. min-1 (d: -1.19); ΔfR -8 (7) br. min-1 (d: -0.78); ΔVe, -21.3 (9.8) L. min-1 (d: -1.64); ΔVt -0.4 (0.3) L -1. Variation was greatest in Ve (control vs comparator immersion: 32.5&24.7%) compared to Vt (11.8&12.1%). Repeated CWI induces CSR habituation potentially reducing drowning risk. We consider the neurophysiological and behavioural consequences.

A C2H2-type zinc finger protein ZAT12 of Poncirus trifoliata acts downstream of CBF1 to regulate cold tolerance.

The Cys2/His2 (C2H2)-type zinc finger family has been reported to regulate multiple aspects of plant development and abiotic stress response. However, the role of C2H2-type zinc finger proteins in cold tolerance remains largely unclear. Through RNA-sequence analysis, a cold-responsive zinc finger protein, named as PtrZAT12, was identified and isolated from trifoliate orange (Poncirus trifoliata L. Raf.), a cold-hardy plant closely related to citrus. Furthermore, we found that PtrZAT12 was markedly induced by various abiotic stresses, especially cold stress. PtrZAT12 is a nuclear protein, and physiological analysis suggests that overexpression of PtrZAT12 conferred enhanced cold tolerance in transgenic tobacco (Nicotiana tabacum) plants, while knockdown of PtrZAT12 by virus-induced gene silencing (VIGS) increased the cold sensitivity of trifoliate orange and repressed expression of genes involved in stress tolerance. The promoter of PtrZAT12 harbors a DRE/CRT cis-acting element, which was verified to be specifically bound by PtrCBF1 (Poncirus trifoliata C-repeat BINDING FACTOR1). VIGS-mediated silencing of PtrCBF1 reduced the relative expression levels of PtrZAT12 and decreased the cold resistance of trifoliate orange. Based on these results, we propose that PtrZAT12 is a direct target of CBF1 and plays a positive role in modulation of cold stress tolerance. The knowledge gains new insight into a regulatory module composed of CBF1-ZAT12 in response to cold stress and advances our understanding of cold stress response in plants.

Molecular Mechanisms of Lipid-Based Metabolic Adaptation Strategies in Response to Cold.

Temperature changes and periods of detrimental cold occur frequently for many organisms in their natural habitats. Homeothermic animals have evolved metabolic adaptation strategies to increase mitochondrial-based energy expenditure and heat production, largely relying on fat as a fuel source. Alternatively, certain species are able to repress their metabolism during cold periods and enter a state of decreased physiological activity known as torpor. By contrast, poikilotherms, which are unable to maintain their internal temperature, predominantly increase membrane fluidity to diminish cold-related damage from low-temperature stress. However, alterations of molecular pathways and the regulation of lipid-metabolic reprogramming during cold exposure are poorly understood. Here, we review organismal responses that adjust fat metabolism during detrimental cold stress. Cold-related changes in membranes are detected by membrane-bound sensors, which signal to downstream transcriptional effectors, including nuclear hormone receptors of the PPAR (peroxisome proliferator-activated receptor) subfamily. PPARs control lipid metabolic processes, such as fatty acid desaturation, lipid catabolism and mitochondrial-based thermogenesis. Elucidating the underlying molecular mechanisms of cold adaptation may improve beneficial therapeutic cold treatments and could have important implications for medical applications of hypothermia in humans. This includes treatment strategies for hemorrhagic shock, stroke, obesity and cancer.

Cold stress induces malformed tomato fruits by breaking the feedback loops of stem cell regulation in floral meristem.

Fruit malformation is a major constrain in fruit production worldwide resulting in substantial economic losses. The farmers for decades noticed that the chilling temperature before blooming often caused malformed fruits. However, the molecular mechanism underlying this phenomenon is unclear. Here we examined the fruit development in response to cold stress in tomato, and demonstrated that short-term cold stress increased the callose accumulation in both shoot apical and floral meristems, resulting in the symplastic isolation and altered intercellular movement of WUS. In contrast to the rapidly restored SlWUS transcription during the recovery from cold stress, the callose removal was delayed due to obstructed plasmodesmata. The delayed reinstatement of cell-to-cell transport of SlWUS prevented the activation of SlCLV3 and TAG1, causing the interrupted feedback inhibition of SlWUS expression, leading to the expanded stem cell population and malformed fruits. We further showed that the callose dynamics in response to short-term cold stress presumably exploits the mechanism of bud dormancy during the seasonal growth, involving two antagonistic hormones, abscisic acid and gibberellin. Our results provide a novel insight into the cold stress regulated malformation of fruit.

Two AT-Hook proteins regulate A/NINV7 expression to modulate sucrose catabolism for cold tolerance in Poncirus trifoliata.

Invertase (INV)-mediated sucrose (Suc) hydrolysis, leading to the irreversible production of glucose (Glc) and fructose (Frc), plays an essential role in abiotic stress tolerance of plants. However, the regulatory network associated with the Suc catabolism in response to cold environment remains largely elusive. Herein, the cold-induced alkaline/neutral INV gene PtrA/NINV7 of trifoliate orange (Poncirus trifoliata (L.) Raf.) was shown to function in cold tolerance via mediating the Suc hydrolysis. Meanwhile, a nuclear matrix-associated region containing A/T-rich sequences within its promoter was indispensable for the cold induction of PtrA/NINV7. Two AT-Hook Motif Containing Nuclear Localized (AHL) proteins, PtrAHL14 and PtrAHL17, were identified as upstream transcriptional activators of PtrA/NINV7 by interacting with the A/T-rich motifs. PtrAHL14 and PtrAHL17 function positively in the cold tolerance by modulating PtrA/NINV7-mediated Suc catabolism. Furthermore, both PtrAHL14 and PtrAHL17 could form homo- and heterodimers between each other, and interacted with two histone acetyltransferases (HATs), GCN5 and TAF1, leading to elevated histone3 acetylation level under the cold stress. Taken together, our findings unraveled a new cold-responsive signaling module (AHL14/17-HATs-A/NINV7) for orchestration of Suc catabolism and cold tolerance, which shed light on the molecular mechanisms underlying Suc catabolism catalyzed by A/NINVs under cold stress.

MnSOD functions as a thermoreceptor activated by low temperature.

A conservative characteristic of manganese superoxide dismutase is the rapid formation of product inhibition at high temperatures. At lower temperatures, the enzyme is less inhibited and undergoes more catalytic fast cycles before being product-inhibited. The temperature-dependent kinetics could be rationalized by the temperature-dependent coordination in the conserved center of manganese superoxide dismutase. As temperature decreases, a water molecule (WAT2) approaches or even coordinates Mn as the sixth ligand to interfere with O2•--Mn coordination and reduce product inhibition, so the dismutation should mainly proceed in the fast outer-sphere pathway at low temperatures. Cold-activation is an adaptive response to low temperature rather than a passive adaptation to excess superoxide levels since the cold-activated dismutase activity significantly exceeds the amount of superoxide in the cell or mitochondria. Physiologically speaking, cold activation of manganese superoxide dismutase mediates cold stress signaling and transduces temperature (physical signal) degree into H2O2 fluxes (chemical signal), which in turn may act as a second messenger to induce a series of physiological responses such as cold shock.

Aquaphotomics Research of Cold Stress in Soybean Cultivars with Different Stress Tolerance Ability: Early Detection of Cold Stress Response.

The development of non-destructive methods for early detection of cold stress of plants and the identification of cold-tolerant cultivars is highly needed in crop breeding programs. Current methods are either destructive, time-consuming or imprecise. In this study, soybean leaves' spectra were acquired in the near infrared (NIR) range (588-1025 nm) from five cultivars genetically engineered to have different levels of cold stress tolerance. The spectra were acquired at the optimal growing temperature 27 °C and when the temperature was decreased to 22 °C. In this paper, we report the results of the aquaphotomics analysis performed with the objective of understanding the role of the water molecular system in the early cold stress response of all cultivars. The raw spectra and the results of Principal Component Analysis, Soft Independent Modeling of Class Analogies and aquagrams showed consistent evidence of huge differences in the NIR spectral profiles of all cultivars under normal and mild cold stress conditions. The SIMCA discrimination between the plants before and after stress was achieved with 100% accuracy. The interpretation of spectral patterns before and after cold stress revealed major changes in the water molecular structure of the soybean leaves, altered carbohydrate and oxidative metabolism. Specific water molecular structures in the leaves of soybean cultivars were found to be highly sensitive to the temperature, showing their crucial role in the cold stress response. The results also indicated the existence of differences in the cold stress response of different cultivars, which will be a topic of further research.

Transcriptome analysis provides new insights into plants responses under phosphate starvation in association with chilling stress.

BACKGROUND: Chilling temperature reduces the rate of photosynthesis in plants, which is more pronounced in association with phosphate (Pi) starvation. Previous studies showed that Pi resupply improves recovery of the rate of photosynthesis in plants much better under combination of dual stresses than in non-chilled samples. However, the underlying mechanism remains poorly understood. RESULTS: In this study, RNA-seq analysis showed the expression level of 41 photosynthetic genes in plant roots increased under phosphate starvation associated with 4 °C (-P 4 °C) compared to -P 23 °C. Moreover, iron uptake increased significantly in the stem cell niche (SCN) of wild type (WT) roots in -P 4 °C. In contrast, lower iron concentrations were found in SCN of aluminum activated malate transporter 1 (almt1) and its transcription factor, sensitive to protein rhizotoxicity 1 (stop1) mutants under -P 4 °C. The Fe content examined by ICP-MS analysis in -P 4 °C treated almt1 was 98.5 ng/µg, which was only 17% of that of seedlings grown under -P 23 °C. Average plastid number in almt1 root cells under -P 4 °C was less than -P 23 °C. Furthermore, stop1 and almt1 single mutants both exhibited increased primary root elongation than WT under combined stresses. In addition, dark treatment blocked the root elongation phenotype of stop1 and almt1. CONCLUSIONS: Induction of photosynthetic gene expression and increased iron accumulation in roots is required for plant adjustment to chilling in association with phosphate starvation.

How Cold Shock Affects Ploidy Level and Early Ontogenetic Development of the Sterlet, A. ruthenus L.

The objective of the present research was to study the effect of cold shock (3 °C and 6 °C) on fertilized eggs of the sterlet, Acipenser ruthenus L. Cold shock was applied for various durations (30, 60 and 90 min) and the ploidy levels, survival, and genotypes of the treated embryos/larvae were recorded. Analysis of ploidy levels confirmed the presence of diploid, triploid, and mosaic (1n/2n, 2n/3n, and 1n/2n/3n) genotypes in experimental groups, while it was strictly diploid in control groups. Microsatellite genotyping confirmed both the incidence of polyspermy and retention of the 2nd polar body in experimental groups. However, patterns of inheritance in all diploid offspring in experimental and control groups revealed classical Mendelian disomic inheritance. Interestingly, the observed mosaic sterlets had normal morphology and were alive. However, some larvae had abnormal morphology which may be due to haploid syndrome. In all treatment groups (treatments: 3 °C-30 min; 3 °C-60 min; 3 °C-90 min; 6 °C-60 min), where the percentage of polyploid/mosaic larvae were high, the mortality was also high. Whereas, in the control groups (where there were only diploid (2n) larvae), the mortality was relatively low.

Thermal stress affects proliferation and differentiation of turkey satellite cells through the mTOR/S6K pathway in a growth-dependent manner.

Satellite cells (SCs) are stem cells responsible for post-hatch muscle growth through hypertrophy and in birds are sensitive to thermal stress during the first week after hatch. The mechanistic target of rapamycin (mTOR) signaling pathway, which is highly responsive to thermal stress in differentiating turkey pectoralis major (p. major) muscle SCs, regulates protein synthesis and the activities of SCs through a downstream effector, S6 kinase (S6K). The objectives of this study were: 1) to determine the effect of heat (43°C) and cold (33°C) stress on activity of the mTOR/S6K pathway in SCs isolated from the p. major muscle of one-week-old faster-growing modern commercial (NC) turkeys compared to those from slower-growing Randombred Control Line 2 (RBC2) turkeys, and 2) to assess the effect of mTOR knockdown on the proliferation, differentiation, and expression of myogenic regulatory factors of the SCs. Heat stress increased phosphorylation of both mTOR and S6K in both turkey lines, with greater increases observed in the RBC2 line. With cold stress, greater reductions in mTOR and S6K phosphorylation were observed in the NC line. Early knockdown of mTOR decreased proliferation, differentiation, and expression of myoblast determination protein 1 and myogenin in both lines independent of temperature, with the RBC2 line showing greater reductions in proliferation and differentiation than the NC line at 38° and 43°C. Proliferating SCs are more dependent on mTOR/S6K-mediated regulation than differentiating SCs. Thus, thermal stress can affect breast muscle hypertrophic potential by changing satellite cell proliferation and differentiation, in part, through the mTOR/S6K pathway in a growth-dependent manner. These changes may result in irreversible effects on the development and growth of the turkey p. major muscle.

Transcriptional and physiological data revealed cold tolerance in a photo-thermo sensitive genic male sterile line Yu17S.

BACKGROUND: Rice is highly sensitive to chilling stress during the seedling stage. However, the adaptable photo-thermo sensitive genic male sterile (PTGMS) rice line, Yu17S, exhibits tolerance to low temperatures. Currently, the molecular characteristics of Yu17S are unclear. RESULTS: To evaluate the molecular mechanisms behind cold responses in rice seedlings, a comparative transcriptome analysis was performed in Yu17S during seedling development under normal temperature and low temperature conditions. In total, 9317 differentially expressed genes were detected. Gene ontology and pathway analyses revealed that these genes were involved mostly in photosynthesis, carotenoid biosynthesis, carbohydrate metabolism and plant hormone signal transduction. An integrated analysis of specific pathways combined with physiological data indicated that rice seedlings improved the performance of photosystem II when exposed to cold conditions. Genes involved in starch degradation and sucrose metabolism were activated in rice plants exposed to cold stress treatments, which was accompanied by the accumulation of soluble sugar, trehalose, raffinose and galactinol. Furthermore, chilling stress induced the expression of phytoene desaturase, 15-cis-ζ-carotene isomerase, ζ-carotene desaturase, carotenoid isomerase and ß-carotene hydroxylase; this was coupled with the activation of carotenoid synthase activity and increases in abscisic acid (ABA) levels in rice seedlings. CONCLUSIONS: Our results suggest that Yu17S exhibited better tolerance to cold stress with the activation of carotenoid synthase activity and increasing of ABA levels, and as well as the expression of photosynthesis-related genes under cold condition in rice seedlings.

Cold responses in rice: From physiology to molecular biology.

As rice originated in tropical or subtropical areas, it is generally sensitive to cold stress. Understanding the physiological and molecular mechanisms underlying rice responses to cold stress can provide new power for engineering cold-tolerant and high-yielding rice varieties.

F-box protein EBF1 and transcription factor ABI5-like regulate banana fruit chilling-induced ripening disorder.

Cold stress adversely affects plant production, both qualitatively and quantitatively. Banana (Musa acuminata) is sensitive to cold stress and suffers chilling injury (CI) when stored under 11°C, causing abnormal fruit softening. However, the mechanism underlying the abnormal fruit softening due to CI remains obscure. This study uncovered the coordinated transcriptional mechanism of ethylene F-box (EBF1) protein and abscisic acid-insensitive 5 (ABI5)-like protein in regulating chilling-induced softening disorders of Fenjiao banana. Cold stress severely inhibited the transcript and protein levels of EBF1, ABI5-like, and fruit softening-related genes. The ABI5-like protein bound to the promoters of key starch and cell wall degradation-related genes such as ß-amylase 8 (BAM8), pectate lyase 8 (PL8), and ß-D-xylosidase23-like (XYL23-like) and activated their activities. EBF1 physically interacted with ABI5-like and enhanced the transcriptional activity of the key starch and cell wall degradation-related genes but did not ubiquitinate or degrade ABI5-like protein. This promoted fruit ripening and ameliorated fruit CI in a manner similar to the effect of exogenous abscisic acid treatment. The ectopic and transient overexpression of EBF1 and ABI5-like genes in tomato (Solanum lycopersicum) and Fenjiao banana accelerated fruit ripening and softening by promoting ethylene production, starch and cell wall degradation, and decreasing fruit firmness. EBF1 interacted with EIL4 but did not ubiquitinate or degrade EIL4, which is inconsistent with the typical role of EBF1/2 in Arabidopsis (Arabidopsis thaliana). These results collectively highlight that the interaction of EBF1 and ABI5-like controls starch and cell wall metabolism in banana, which is strongly inhibited by chilling stress, leading to fruit softening and ripening disorder.

Potential roles for pattern molecule of PAMP-triggered immunity in improving crop cold tolerance.

KEY MESSAGE: The application of flagellin 22 (flg22), the most widely studied PAMP, enhance crop cold tolerance. ICE1-CBF pathway and SA signaling is involved in the alleviation of cold injury by flg22 treatment. Pathogen infection cross-activates cold response and increase cold tolerance of host plants. However, it is not possible to use the infection to increase cold tolerance of field plants. Here flagellin 22 (flg22), the most widely studied PAMP (pathogen-associated molecular patterns), was used to mimic the pathogen infection to cross-activate cold response. Flg22 treatment alleviated the injury caused by freezing in Arabidopsis, oilseed and tobacco. In Arabidopsis, flg22 activated the expression of immunity and cold-related genes. Moreover, the flg22 induced alleviation of cold injury was lost in NahG transgenic line (SA-deficient), sid2-2 and npr1-1 mutant plants, and flg22-induced expression of cold tolerance-related genes, which indicating that salicylic acid signaling pathway is required for the alleviation of cold injury by flg22 treatment. In short flg22 application can be used to enhance cold tolerance in field via a salicylic acid-depended pathway.

The role of humanin in natural stress tolerance: An underexplored therapeutic avenue.

BACKGROUND: The discovery of humanin (HN/MTRNR2) 20 years ago blazed a trail to identifying mitochondrial derived peptides with biological function. SCOPE: Humanin is associated with pro-survival, cytoprotective, anti-inflammatory, and anti-oxidative properties and may play a role in reducing neurodegenerative and metabolic disease progression. Although the role of humanin in vitro and in vivo laboratory models is well characterized, the regulation of humanin in natural models that encounter lethal cytotoxic and oxidative insults, as part of their natural history, require immediate research. In this review, we discuss the conservation of humanin-homologues across champion hibernators, anoxia and freeze-tolerant vertebrates and postulate on the putative roles of humanin in non-model species. SIGNIFICANCE: We hope characterization of humanin in animals that are naturally immune to cellular insults, that are otherwise lethal for non-tolerant species, will elucidate key biomarkers and cytoprotective pathways with therapeutic potential and help differentiate pro-survival mechanisms from cellular consequences of stress.

Cytosolic proline is required for basal freezing tolerance in Arabidopsis.

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze-thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2-1 (p5cs2-1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1-1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2-1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2-1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.

Function of Chloroplasts in Plant Stress Responses.

The chloroplast has a central position in oxygenic photosynthesis and primary metabolism. In addition to these functions, the chloroplast has recently emerged as a pivotal regulator of plant responses to abiotic and biotic stress conditions. Chloroplasts have their own independent genomes and gene-expression machinery and synthesize phytohormones and a diverse range of secondary metabolites, a significant portion of which contribute the plant response to adverse conditions. Furthermore, chloroplasts communicate with the nucleus through retrograde signaling, for instance, reactive oxygen signaling. All of the above facilitate the chloroplast's exquisite flexibility in responding to environmental stresses. In this review, we summarize recent findings on the involvement of chloroplasts in plant regulatory responses to various abiotic and biotic stresses including heat, chilling, salinity, drought, high light environmental stress conditions, and pathogen invasions. This review will enrich the better understanding of interactions between chloroplast and environmental stresses, and will lay the foundation for genetically enhancing plant-stress acclimatization.

A newly discovered behavior ('tail-belting') among wild rodents in sub zero conditions.

Rodents are among the most successful mammals because they have the ability to adapt to a broad range of environmental conditions. Here, we present the first record of a previously unknown thermal adaptation to cold stress that repeatedly occurred in two species of non-commensal rodents (Apodemus flavicollis and Apodemus agrarius). The classic rodent literature implies that rodents prevent heat loss via a broad range of behavioral adaptations including sheltering, sitting on their tails, curling into a ball, or huddling with conspecifics. Here, we have repeatedly observed an undescribed behavior which we refer to as "tail-belting". This behavior was performed under cold stress, whereby animals lift and curl the tail medially, before resting it on the dorsal, medial rump while feeding or resting. We documented 115 instances of the tail-belting behavior; 38 in Apodemus agrarius, and 77 in Apodemus flavicollis. Thermal imaging data show the tails remained near ambient temperature even when temperatures were below 0 °C. Since the tail-belting occurred only when the temperature dropped below - 6.9 °C (for A. flavicollis) and - 9.5 °C (for A. agrarius), we surmise that frostbite prevention may be the primary reason for this adaptation. It is likely that tail-belting has not previously been documented because free-ranging mice are rarely-recorded in the wild under extreme cold conditions. Given that these animals are so closely-related to laboratory rodents, this knowledge could potentially be relevant to researchers in various disciplines. We conclude by setting several directions for future research in this area.

Physiological and transcriptome analysis of Magnolia denudata leaf buds during long-term cold acclimation.

BACKGROUND: Magonlia denudata is an important perennial tree species of the Magnoliaceae family, known for its ornamental value, resistance to smoke pollution and wind, role in air purification, and robust cold tolerance. In this study, a high-throughput transcriptome analysis of leaf buds was performed, and gene expression following artificial acclimation 22 °C, 4 °C and 0 °C, was compared by RNA sequencing. RESULTS: Over 426 million clean reads were produced from three libraries (22 °C, 4 °C and 0 °C). A total of 74,503 non-redundant unigenes were generated, with an average length of 1173.7 bp (N50 = 1548). Based on transcriptional results, 357 and 235 unigenes were identified as being upregulated and downregulated under cold stress conditions, respectively. Differentially expressed genes were annotated using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses. The transcriptomic analysis focused on carbon metabolism and plant hormone signal transduction associated with cold acclimation. Transcription factors such as those in the basic helix-loop-helix and AP2/ERF families were found to play an important role in M. denudata cold acclimation. CONCLUSION: M. denudata exhibits responses to non-freezing cold temperature (4 °C) to increase its cold tolerance. Cold resistance was further strengthened with cold acclimation under freezing conditions (0 °C). Cold tolerance genes, and cold signaling transcriptional pathways, and potential functional key components for the regulation of the cold response were identified in M. denudata. These results provide a basis for further studies, and the verification of key genes involved in cold acclimation responses in M. denudata lays a foundation for developing breeding programs for Magnoliaceae species.

A novel LRR-RLK (CTLK) confers cold tolerance through regulation on the C-repeat-binding factor pathway, antioxidants, and proline accumulation.

Leucine-rich repeat-receptor-like kinase (LRR-RLK) is a large subfamily of plant RLKs; however, its role in cold tolerance is still unknown. A novel cold tolerance LRR-RLK gene (MtCTLK1) in Medicago truncatula was identified using the transgenic lines overexpressing MtCTLK1 (MtCTLK1-OE) and mtctlk1 lines with Tnt1 retrotransposon insertion. Compared with the wild-type, MtCTLK1-OE lines had increased cold tolerance and mtctlk1 showed decreased cold tolerance. The impaired cold tolerance in mtctlk1 could be complemented by the transgenic expression of MtCTLK1 or its homolog MfCTLK1 from Medicago falcata. Antioxidant enzyme activities and proline accumulation as well as transcript levels of the associated genes were increased in response to cold, with higher levels in MtCTLK1-OE or lower levels in mtctlk1 lines as compared with wild type. C-Repeat-Binding Factors (CBFs) and CBF-dependent cold-responsive genes were also induced in response to cold, and higher transcript levels of CBFs and CBF-dependent cold-responsive genes were observed in MtCTLK1-OE lines whereas lower levels in mtctlk1 mutants. The results validate the role of MtCTLK1 or MfCTLK1 in the regulation of cold tolerance through the CBF pathway, antioxidant defense system and proline accumulation. It also provides a valuable gene for the molecular breeding program to improve cold tolerance in crops.