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Heterologous expression of VaMYB44 gene in Arabidopsis and V. vinifera cv. 'Thompson Seedless' increases cold sensitivity, which is mediated by the interaction of VaMYC2 and VaTIFY5A with VaMYB44 MYB transcription factors play critical roles in plant stress response. However, the function of MYB44 under low temperature stress is largely unknown in grapes. Here, we isolated a VaMYB44 gene from Chinese wild Vitis amurensis acc. 'Shuangyou' (cold-resistant). The VaMYB44 is expressed in various organs and has lower expression levels in stems and young leaves. Exposure of the cold-sensitive V. vinifera cv. 'Thompson Seedless' and cold-resistant 'Shuangyou' grapevines to cold stress (-1 °C) resulted in differential expression of MYB44 in leaves with the former reaching 14 folds of the latter after 3 h of cold stress. Moreover, the expression of VaMYB44 was induced by exogenous ethylene, abscisic acid, and methyl jasmonate in the leaves of 'Shuangyou'. Notably, the subcellular localization assay identified VaMYB44 in the nucleus. Interestingly, heterologous expression of VaMYB44 in Arabidopsis and 'Thompson Seedless' grape increased freezing-induced damage compared to their wild-type counterparts. Accordingly, the transgenic lines had higher malondialdehyde content and electrolyte permeability, and lower activities of superoxide dismutase, peroxidase, and catalase. Moreover, the expression levels of some cold resistance-related genes decreased in transgenic lines. Protein interaction assays identified VaMYC2 and VaTIFY5A as VaMYB44 interacting proteins, and VaMYC2 could bind to the VaMYB44 promoter and promote its transcription. In conclusion, the study reveals VaMYB44 as the negative regulator of cold tolerance in transgenic Arabidopsis and transgenic grapes, and VaMYC2 and VaTIFY5A are involved in the cold sensitivity of plants by interacting with VaMYB44.

22 ABSTRACT 23 Study design Experimental study 24 Objectives To compare lipid metabolism in individuals with a cervical spinal cord injury (SCIC) 25 and able-bodied (AB) persons in response to mild cold stress. 26 Settings Laboratory of Wakayama Medical University, Japan. 27 Methods Nine males with SCIC and 11 AB wore a water-perfusion suit in a supine position. 28 Following 30-min rest thermoneutrality, the whole body was cooled by perfusing 25°C water 29 through the suit for 15-20 minutes (CS). Blood samples were collected before, immediately, and 60 30 (post-CS60) and 120 minutes after CS (post-CS120). Concentrations of serum free fatty acid 31 ([FFA]s), total ketone bodies ([tKB]s), insulin ([Ins]s) and plasma adrenaline ([Ad]p), noradrenaline 32 ([NA]p) and glucose ([Glc]p) were assessed. 33 Results [Ad]p in SCIC were lower than AB throughout the study (p=0.0002) and remained largely 34 unchanged in both groups. [NA]p increased after cold stress in AB only (p

Background Cold stress adversely influences rapeseeds (Brassica napus L.) growth and yield during winter and spring seasons. Hydrogen (H2) is a potential gasotransmitter that is used to enhance tolerance against abiotic stress, including cold stress. However, convenience and stability are two crucial limiting factors upon the application of H2 in field agriculture. To explore the application of H2 in field, here we evaluated the role of ammonia borane (AB), a new candidate for a H2 donor produced by industrial chemical production, in plant cold tolerance. Results The application with AB could obviously alleviate the inhibition of rapeseed seedling growth and reduce the oxidative damage caused by cold stress. The above physiological process was closely related to the increased antioxidant enzyme system and reestablished redox homeostasis. Importantly, cold stress-triggered endogenous H2S biosynthesis was further stimulated by AB addition. The removal or inhibition of H2S synthesis significantly abolished plant tolerance against cold stress elicited by AB. Further field experiments demonstrated that the phenotypic and physiological performances of rapeseed plants after challenged with cold stress in the winter and early spring seasons were significantly improved by administration with AB. Particularly, the most studied cold-stress response pathway, the ICE1-CBF-COR transcriptional cascade, was significantly up-regulated either. Conclusion Overall, this study clearly observed the evidence that AB-increased tolerance against cold stress could be suitable for using in field agriculture by stimulation of H2S signaling.

The importance of the temperature tolerance of fish is increasing due to climate change caused by global warming. This study examined the expression of the heat shock protein 70 (HSP70) gene, and plasma cortisol and glucose levels, as a stress response in red-spotted and hybrid groupers during exposure to heat and cold shock. Temperature in the tank where fishes acclimated at 20℃ was gradually increased or decreased, respectively, to examine the survival rate of fish. The result showed a higher survival rate of the hybrid than that of the red-spotted grouper upon exposure to a higher temperature. To further analyze the factors associated with temperature-associated stress, fishes were collected from different temperatures which changed from 20 to 30℃ or 10℃, respectively, and then back to 20℃. The expression levels of the gene encoding heat shock protein 70 (HSP70) were analyzed by qPCR using cDNA prepared from RNA extracted from the brain. A higher level of HSP70 transcript was detected in the hybrid during heat shock exposure. Analysis of cortisol and glucose from the blood of fish collected during the acclimation periods clearly indicated that the level of cortisol was increased upon temperature shift although a slight difference in the degrees of changes timing was slightly different between red-spotted grouper and hybrid. The results showed a correlation between the level of HSP70 and survival rate upon exposure to higher temperature shock. This study provides basic information regarding whether HSP70 expression increases the survival rate of fish subjected to rapid temperature changes.

Background: As an alpine plants，Rhododendron chrysanthum (R. chrysanthum) has evolved cold resistance mechanisms and become a valuable plant resource with the responsive mechanism of cold stress. Results: In my study, we adopt the phosphoproteomic and proteomic analysis combining with physiological measurement to illustrate the responsive mechanism of R. chrysanthum seedling under cold (4℃) stress. After chilling for 12 h, 350 significantly changed proteins and 274 significantly changed phosphoproteins were detected. COG analysis showed that significantly changed proteins and phosphoproteins were mainly involved in signal transduction and energy production and conversion under cold stress. The results indicated photosynthesis was inhibited under cold stress, but cold induced calcium-mediated signaling, reactive oxygen species (ROS) homeostasis and other transcription regulation factors could protect plants from destruction caused by cold stress. Conclusions: These data constitute a cold stress-responsive metabolic atlas in R. chrysanthum, which will springboard further investigations into the complex molecular mechanisms of plant cold adaptation.

Background Rice is one of the most important cereal crops in the world but is susceptible to cold stress (CS). In this study, we carried out parallel transcriptomic analysis at the reproductive stage on the anthers of two Japonica rice varieties with contrasting CS resistance: cold susceptible Longjing11 (LJ11) and cold resistant Longjing25 (LJ25). Results According to the obtained results, a total of 16,762 differentially expressed genes (DEGs) were identified under CS, including 7,050 and 14,531 DEGs in LJ25 and LJ11, respectively. Examining gene ontology (GO) enrichment identified 35 up- and 39 down-regulated biological process BP GO terms were significantly enriched in the two varieties, with ‘response to heat’ and ‘response to cold’ being the most enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 33 significantly enriched pathways. Only the carbon metabolism and amino acid biosynthesis pathways with down-regulated DEGs were enriched considerably in LJ11, while the plant hormone signal transduction pathway (containing 153 DEGs) was dramatically improved. Eight kinds of plant hormones were detected in the pathway, while auxin, abscisic acid (ABA), salicylic acid (SA), and ethylene (ETH) signaling pathways were found to be the top four pathways with the most DEGs. Furthermore, the protein-protein interaction (PPI) network analysis identified ten hub genes (co-expressed gene number ≥ 30), including six ABA-related genes. Various DEGs (such as OsDREB1A, OsICE1, OsMYB2, OsABF1, OsbZIP23, OsCATC, and so on) revealed distinct expression patterns among rice types when the DEGs between LJ11 and LJ25 were compared, indicating that they are likely responsible for CS resistance of rice in cold region. Conclusion Collectively, our findings provide comprehensive insights into complex molecular mechanisms of CS response and can aid in CS resistant molecular breeding of rice in cold regions.

Associated analysis of GWAS with RNA-seq had detected candidate genes responsible for cold stress and chilling acclimation in rice. Haplotypes of two candidate genes and geographic distribution were analyzed. To explore new candidate genes and genetic resources for cold tolerance improvement in rice, genome-wide association study (GWAS) mapping experiments with 351 rice core germplasms was performed for three traits (survival rate, shoot length and chlorophyll content) under three temperature conditions (normal temperature, cold stress and chilling acclimation), yielding a total of 134 QTLs, of which 54, 59 and 21 QTLs were responsible for normal temperature, cold stress and chilling acclimation conditions, respectively. Integrated analysis of significant SNPs in 134 QTLs further identified 116 QTLs for three temperature treatments, 53, 43 and 18 QTLs responsible for normal temperature, cold stress and chilling acclimation, respectively, and 2 QTLs were responsible for both cold stress and chilling acclimation. Matching differentially expressed genes from RNA-seq to 43 and 18 QTLs for cold stress and chilling acclimation, we identified 69 and 44 trait-associated candidate genes, respectively, to be classified into six and five groups, particularly involved in metabolisms, reactive oxygen species scavenging and hormone signaling. Interestingly, two candidate genes LOC_Os01g04814, encoding a vacuolar protein sorting-associating protein 4B, and LOC_Os01g48440, encoding glycosyltransferase family 43 protein, showed the highest expression levels under chilling acclimation. Haplotype analysis revealed that both genes had a distinctive differentiation with subpopulation. Haplotypes of both genes with more japonica accessions have higher latitude distribution and higher chilling tolerance than the chilling sensitive indica accessions. These findings reveal the new insight into the molecular mechanism and candidate genes for cold stress and chilling acclimation in rice.

MsCML46 enhances tolerance to abiotic stresses through alleviating osmotic stress and oxidative damage by regulating the expression of stress-related genes to optimize osmolytes levels and antioxidant enzyme activity in transgenic tobacco. Abiotic stresses are major environmental factors that constraint crop productivity worldwide. Various stimuli regulate intracellular calcium levels and calcium-mediated signal transduction, and cellular responses. Ca2+ signals are perceived by different Ca2+ receptors. Calmodulin-like protein (CML) is one of the best-characterized Ca2+ sensors which shares sequence similarity with highly conserved calmodulin (CaM) ubiquitously expressed in plants. Currently, the molecular and physiological functions of CMLs are largely unknown. In this study, the MsCML46 was characterized in alfalfa (Medicago sativa cv. Zhaodong) under freezing stress. Results showed that MsCML46 was localized to the cytoplasm of Arabidopsis, and its expression was strongly elevated by cold, drought, salt, saline-alkali, and ABA treatments. Overexpressing MsCML46 in tobacco enhanced tolerance to freezing, drought, and salt stresses as evidenced by improved contents of osmotic regulatory solutes and antioxidant enzyme activity but decreased reactive oxygen species (ROS) accumulation. Furthermore, cold, drought, and salt stresses increased the expression of stress-related genes in transgenic tobacco. MsCML46 binds free Ca2+ to promote signal transduction and maintain higher K+/Na+ ratio. In this way, it protects intracellular homeostasis under sodium ion toxicity. These results suggest that MsCML46 plays a crucial role in resisting abiotic stresses and can be exploited in genetic engineering for crops.

NAC-type transcription factors are crucial players in the abiotic stress responses of plants. Soybean NAC-type transcription factor GmNAC20 was transformed into rice genome via Agrobacterium method of transformation to improve abiotic stress tolerance. Integration and expression of GmNAC20 were verified by the DNA blot hybridization, immunoblotting, RT-PCR, and quantitative RT-PCR in T3 generation of transgenic rice plants. Significant expression of GmNAC20 was found in transgenic plants under salinity, cold, and IAA treatments. The transgenic rice plants expressing GmNAC20 displayed enhanced salinity and cold stress tolerance via upregulating the abiotic stress-responsive genes. Furthermore, T3 transgenic plants retained relative water content, chlorophyll content with enhanced accumulation of proline content than wild-type plants under salinity, and cold stress environments. The decrease in MDA content and electrolyte leakage with a significant increase in antioxidant enzyme activities were noticed in transgenic rice plants under either salinity or cold stress conditions, compared to wild-type plants. Overexpression of GmNAC20 in rice plants also induced the lateral root formation, associated with upregulation of auxin signaling-related genes. Taken together, our results indicated that GmNAC20 acts as a positive regulator for conferring salinity and cold tolerance in rice plants and appropriate candidate for improving salinity and cold stress in other important food crops.

Background As maize originated in tropical or subtropical zones, most maize germplasm is extremely sensitive to low temperatures during the seedling stage. Clarifying the molecular mechanism of cold acclimation would facilitate the breeding of cold tolerant maize varieties, which is one of the major sustainability factors for crop production. To meet this goal, we investigated two maize inbred lines with contrasting levels of cold tolerance at the seedling stage (IL85, a cold tolerant line; B73, a cold sensitive line), and performed full-length transcriptome sequencing on the root tips of seedlings before and after 24 h of cold treatment. Results We identified 152,263 transcripts, including 20,993 novel transcripts, and determined per-transcript expression levels. A total of 1,475 transcripts were specifically up-regulated in the cold tolerant line IL85 under cold stress. GO enrichment analysis revealed that 25 transcripts were involved in reactive oxygen species (ROS) metabolic processes and 15 transcripts were related to the response to heat. Eight genes showed specific differential alternative splicing (DAS) in IL85 under cold stress, and were mainly involved in amine metabolism. A total of 1,111 lncRNAs were further identified, 62 of which were up-regulated in IL85 or B73 under cold stress, and their corresponding target genes were enriched in protein phosphorylation. Conclusions These results provide new insights into the molecular mechanism of cold acclimation during the seedling stage in maize, and will facilitate the development of cultivars with improved cold stress tolerance.

Background Cold is one of the main abiotic stresses that severely affect plant growth and development, and crop productivity as well. Transcriptional changes during cold stress have already been intensively studied in various plant species. However, the gene networks involved in the regulation of differential cold tolerance between tobacco varieties with contrasting cold resistance are quite limited. Results Here, we conducted multiple time-point transcriptomic analyses using Tai tobacco (TT, cold susceptibility) and Yan tobacco (YT, cold resistance) with contrasting cold responses. We identified similar DEGs in both cultivars after comparing with the corresponding control (without cold treatment), which were mainly involved in response to abiotic stimuli, metabolic processes, kinase activities. Through comparison of the two cultivars at each time point, in contrast to TT, YT had higher expression levels of the genes responsible for environmental stresses. By applying Weighted Gene Co-Expression Network Analysis (WGCNA), we identified two main modules: the pink module was similar while the brown module was distinct between the two cultivars. Moreover, we obtained 100 hub genes, including 11 important transcription factors (TFs) potentially involved in cold stress, 3 key TFs in the brown module and 8 key TFs in the pink module. More importantly, according to the genetic regulatory networks (GRNs) between TFs and other genes or TFs by using GENIE3, we identified 3 TFs (ABI3/VP1, ARR-B and WRKY) mainly functioning in differential cold responses between two cultivars, and 3 key TFs (GRAS, AP2-EREBP and C2H2) primarily involved in cold responses. Conclusion Collectively, our study provides valuable resources for transcriptome- based gene network studies of cold responses in tobacco. It helps to reveal how key cold responsive TFs or other genes are regulated through network. It also helps to identify the potential key cold responsive genes for the genetic manipulation of tobacco cultivars with enhanced cold tolerance in the future.

Background Plant species from Rosaceae family are economically important. One of the major environmental factors impacting those species is cold stress. Although several Rosaceae plant genomes have recently been sequenced, there have been very few research conducted on cold upregulated genes and their promoter binding sites. In this study, we used computational approaches to identify and analyse potential cold stress response genes across ten Rosaceae family members. Results Cold stress upregulated gene data from apple and strawberry were used to identify syntelogs in other Rosaceae species. Gene duplication analysis was carried out to better understand the distribution of these syntelog genes in different Rosaceae members. A total of 11,145 popular abiotic stress transcription factor-binding sites were identified in the upstream region of these potential cold-responsive genes, which were subsequently categorised into distinct transcription factor (TF) classes. MYB classes of transcription factor binding site (TFBS) were abundant, followed by bHLH, WRKY, and AP2/ERF. TFBS patterns in the promoter regions were compared among these species and gene families, found to be quite different even amongst functionally related syntelogs. A case study on important cold stress responsive transcription factor family, AP2/ERF showed less conservation in TFBS patterns in the promoter regions. This indicates that syntelogs from the same group may be comparable at the gene level but not at the level of cis-regulatory elements. Therefore, for such genes from the same family, different repertoire of TFs could be recruited for regulation and expression. Duplication events must have played a significant role in the similarity of TFBS patterns amongst few syntelogs of closely related species. Conclusions Our study overall suggests that, despite being from the same gene family, different combinations of TFs may play a role in their regulation and expression. The findings of this study will provide information about potential genes involved in the cold stress response, which will aid future functional research of these gene families involved in many important biological processes.

Background Cold stress is one of the main abiotic stresses limiting cucumber (Cucumis sativus L.) growth and production. C-repeat binding factor/Dehydration responsive element-binding 1 protein (CBF/DREB1), containing conserved APETALA2 (AP2) DNA binding domains and two characteristic sequences, are key signaling genes that can be rapidly induced and play vital roles in plant response to low temperature. However, the CBF family has not been systematically elucidated in cucumber, and the expression pattern of this family genes under cold stress remains unclear. Results In this study, three CsCBF family genes were identified in cucumber genome and their protein conserved domain, protein physicochemical properties, gene structure and phylogenetic analysis were further comprehensively analyzed. Subcellular localization showed that all three CsCBFs were localized in the nucleus. Cis-element analysis of the promoters indicated that CsCBFs might be involved in plant hormone response and abiotic stress response. Expression analysis showed that the three CsCBFs could be significantly induced by cold stress, salt and ABA. The overexpression of CsCBFs in cucumber seedlings enhanced the tolerance to cold stress, and importantly, the transcript levels of CsCOR genes were significantly upregulated in 35S:CsCBFs transgenic plants after cold stress treatment. Biochemical analyses ascertained that CsCBFs directly activated CsCOR genes expression by binding to its promoter, thereby enhancing plant resistance to cold stress. Conclusion This study provided a foundation for further research on the function of CsCBF genes in cold stress resistance and elucidating its mechanism.

NAC (NAM, ATAF1/2, CUC2) transcription factors play important roles in plant growth, development, and responses to abiotic stress. In this study, we cloned an NAC2 subfamily transcription factor gene (SlNAC7) from the halophyte Suaeda liaotungensis K., and conducted a series of studies to determine the characteristics and functions of this gene. The SlNAC7 coding region contains 1719 base pairs that encode a 573 amino acid long protein. SlNAC7 is expressed in the roots, stems, and leaves of S. liaotungensis, with the highest expression in the leaves. We found that SlNAC7 expression can be induced by drought, salt, cold, and abscisic acid. Transient expression in onion epidermal cells revealed that SlNAC7 is located in both the nucleus and cytoplasm. A transcriptional activation experiment in yeast showed that the transcriptional activation domain of SlNAC7 is located at the C terminus. When SlNAC7 was transformed into Arabidopsis under the control of a CaMV 35S promoter its overexpression was found to enhance the ability of transgenic plants to resist drought, salt, and cold stress. Moreover, these plants showed multiple changes in growth characteristics and physiological and biochemical indices in response to different stresses, as well as the upregulation of numerous stress-related genes. We have thus characterized a new halophyte-derived NAC transcription factor, SlNAC7, which can regulate plant growth and physiological and biochemical changes under adverse conditions by regulating the expression of stress-related genes, thereby enhancing plant stress resistance. SlNAC7 is a promising candidate for breeding new varieties of stress-tolerant crops.

This study aimed to analyze the cold stress effects (in terms of hematology, energy reserves, and oxidative stress) in Piaractus mesopotamicus (pacú) and their mitigation by a Pyropia columbina red seaweed-supplemented diet. For this purpose, juvenile fish were fed with a control (CD) or a red seaweed-supplemented diet (RD) for 60 days, and then, the animals were exposed to a low temperature (14 °C) and a control temperature (24 °C) for 24 h. The cold shock generated an increase of hemoglobin levels in fish fed with both diets. In CD-fed fish, plasmatic triglycerides, cholesterol, and hepatic glycogen decreased after the thermal shock; meanwhile, the animals fed with RD showed decreased hepatic proteins, but increased cholesterol and hepatic glycogen. Regarding oxidative stress, antioxidant enzymes augmented their activity in the liver, intestine, and gills; meanwhile, lipid oxidative damage was observed in the liver and intestine of fish exposed to 14 °C and fed with both diets. Pacú was sensitive to cold shock, but no mitigation effects were observed in fish fed with the supplemented diet. Further research should target higher concentrations of P. columbina in supplemented diets to take advantage of this valuable resource.

Egg hatching in Meloidogyne incognita is a highly regulated developmental event and is strictly correlated with temperature. It has been demonstrated that exposure of M. incognita eggs to low temperature seriously affects their embryonic development. On the other hand, clear evidence has shown that M. incognita is able to overwinter at subzero soil temperatures in certain open fields. Therefore, subtle physiological and genetic adaptations may occur in M. incognita to minimize freezing injuries. A growing body of evidence indicates that cold acclimation plays a large role in an individual organism’s ability to cope with freezing-induced cellular damage. Given the decreasing temperatures in late autumn or early winter, we hypothesize that natural cold acclimation occurring during these periods may assist M. incognita in overwintering. Transcriptomic analysis and functional enrichment analyses were used to identify and annotate differentially expressed genes (DEGs) in acclimated eggs. The expression of DEGs involved in signal transduction and protein assembly was subsequently validated by reverse transcription quantitative PCR (RT-qPCR). Relatively long-term preacclimation at 4 °C significantly accelerated the hatching of M. incognita eggs that were subjected to freezing at − 1 °C. Using a transcriptomic approach, we further identified 686 and 460 up- and downregulated transcripts, respectively, in pre-cold-acclimated eggs. Additionally, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology annotations for functional enrichment analyses of the differentially expressed genes (DEGs). The phenomenon in which M. incognita safely overwinters at subzero soil temperatures in certain areas may be attributed to the natural cold acclimation occurring in late autumn. Here, the identification of DEGs between acclimated and nonacclimated eggs will provide us with promising directions for future studies on the mechanisms of M. incognita freezing tolerance.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

Significant changes in climate variables in the last decades resulted in changes of perceived climate conditions. However, there are only few studies discussing long-lasting changes in bioclimatic conditions. Thus, the purpose of this paper is to present the temporal and spatial distribution of hazardous heat and cold stress conditions in different regions of Poland. Its focus is on long-lasting changes in such conditions in the period 1951–2018. To assess changes in hazardous thermal stress conditions, the Universal Thermal Climate Index (UTCI) was used. UTCI values at 12 UTC hour (respectively 1 pm winter time, 2 pm summer time) were calculated daily based on air temperature, relative humidity, total cloud cover and wind speed at 24 stations representing the whole area of Poland. We found that the greatest changes were observed in minimum (1.33 °C/10 years) and average (0.52 °C/10 years) UTCI values as well as in cold stress frequency (− 4.00 days per 10 years). The changes vary seasonally and regionally. The greatest increase in UTCImin and decrease in cold stress days were noted from November to March and had the highest values in north-east and east Poland, and also in the foothills of the Carpathian Mountains. The trends in maximum UTCI are much smaller and not always positive. The spatially averaged trend in UTCImax for Poland as a whole was 0.35 °C/10 years and the increase in heat stress days was 0.80 days/10 years. The highest increases in UTCImax and heat stress days were noted in eastern and south-eastern Poland. Supplementary Information The online version contains supplementary material available at 10.1007/s00484-020-02069-7.

Background Red algae Porphyra sensu lato grow naturally in the unfavorable intertidal environment, in which they are exposed to substantial temperature fluctuations. The strategies of Porphyra to tolerate cold stress are poorly understood. Results Herein, investigations revealed that chilling and freezing induced alterations in the physiological properties, gene transcriptional profiles and metabolite levels in the economically important red algae species, Neoporphyra haitanensis. Control samples (kept at 20 °C) were compared to chilled thalli (10 and 4 °C) and to thalli under − 4 °C conditions. Chilling stress did not affect the health or photosynthetic efficiency of gametophytes, but freezing conditions resulted in the arrest of growth, death of some cells and a decrease in photosynthetic activity as calculated by Fv/Fm. Transcriptome sequencing analysis revealed that the photosynthetic system was down-regulated along with genes associated with carbon fixation and primary metabolic biosynthesis. Adaptive mechanisms included an increase in unsaturated fatty acids levels to improve membrane fluidity, an increase in floridoside and isofloridoside content to enhance osmotic resistance, and an elevation in levels of some resistance-associated phytohormones (abscisic acid, salicylic acid, and methyl jasmonic acid). These physiochemical alterations occurred together with the upregulation of ribosome biogenesis. Conclusions N. haitanensis adopts multiple protective mechanisms to maintain homeostasis of cellular physiology in tolerance to cold stress.

Background Small interfering RNAs (siRNAs) target homologous genomic DNA sequences for cytosine methylation, known as RNA-directed DNA methylation (RdDM), plays an important role in transposon control and regulation of gene expression in plants. Repressor of silencing 1 (ROS1) can negatively regulate the RdDM pathway. Results In this paper, we investigated the molecular mechanisms by which an upstream regulator ACD6 in the salicylic acid (SA) defense pathway, an ABA pathway-related gene ACO3, and GSTF14, an endogenous gene of the glutathione S-transferase superfamily, were induced by various abiotic stresses. The results demonstrated that abiotic stresses, including water deficit, cold, and salt stresses, induced demethylation of the repeats in the promoters of ACD6, ACO3, and GSTF14 and transcriptionally activated their expression. Furthermore, our results revealed that ROS1-mediated DNA demethylation plays an important role in the process of transcriptional activation of ACD6 and GSTF14 when Arabidopsis plants are subjected to cold stress. Conclusions This study revealed that ROS1 plays an important role in the molecular mechanisms associated with genes involved in defense pathways in response to abiotic stresses.

Low temperature (LT) or cold stress is a major environmental stress that seriously affects plant growth and development, limiting crop productivity. Cold shock domain proteins (CSDPs), which are present in most living organism, are involved in RNA metabolisms influencing abiotic stress tolerance. The aims of this study are to identify target gene for LT-tolerance, like CSDPs, characterize genetics, and develop molecular marker distinguishing LT-tolerance in cabbage (Brassica oleracea var. capitata). Semi-quantitative RT-PCR or qRT-PCR was used in gene expression study. LT-tolerance was determined by electrolyte leakage and PCR with allelic specific primers. Allelic variation was found in BoCSDP5 coding sequence (CDs) between LT-tolerant (BN106 and BN553) and -susceptible inbred lines (BN107 and BN554). LT-tolerant inbred lines contained variant type of BoCSDP5 (named as BoCSDP5v) which encodes extra CCHC zinc finger domain at C-terminus. Association of LT-tolerance with BoCSDP5v was confirmed by electrolyte leakage and segregation using genetic population derived from BN553 and BN554 cross. Allelic variation in BoCSDP5 gene does not influence the rate of gene expression, but produces different proteins with different number of CCHC zinc finger domains. LT-tolerance marker designed on the basis of polymorphism between BoCSDP5 and BoCSDP5v was confirmed with samples used in previous B. oleracea CIRCADIAN CLOCK ASSOCIATED 1 (BoCCA1) marker validation. LT-tolerant allele (BoCSDP5v) is dominant and independent of CBF pathway, and sufficient to generate molecular markers to identify LT-tolerant cabbage when it is used in combination with another marker, like BoCCA1-derived one. Production and analysis of overexpressing plants of BoCSDP1, BoCSDP3, BoCSDP5 and BoCSDP5v will be required for elucidating the function of CCHC zinc finger domains in LT-tolerance.

The developmental stage of anther development is generally more sensitive to abiotic stress than other stages of growth. Specific ROS levels, plant hormones and carbohydrate metabolism are disturbed in anthers subjected to abiotic stresses. As sessile organisms, plants are often challenged to multiple extreme abiotic stresses, such as drought, heat, cold, salinity and metal stresses in the field, which reduce plant growth, productivity and yield. The development of reproductive stage is more susceptible to abiotic stresses than the vegetative stage. Anther, the male reproductive organ that generate pollen grains, is more sensitive to abiotic stresses than female organs. Abiotic stresses affect all the processes of anther development, including tapetum development and degradation, microsporogenesis and pollen development, anther dehiscence, and filament elongation. In addition, abiotic stresses significantly interrupt phytohormone, lipid and carbohydrate metabolism, alter reactive oxygen species (ROS) homeostasis in anthers, which are strongly responsible for the loss of pollen fertility. At present, the precise molecular mechanisms of anther development under adverse abiotic stresses are still not fully understood. Therefore, more emphasis should be given to understand molecular control of anther development during abiotic stresses to engineer crops with better crop yield.

Heat shock protein 60 (HSP60) is a well-recognized multifunctional protein, playing a substantial role in protecting organisms from environmental stress. The domestic pigeon (Columba livia) is a promising model organism, with important economic and ecological value, and its health is susceptible to temperature stress. To explore the molecular characteristics, tissue expression profile, and response to temperature stress for HSP60 of Columba livia (ClHSP60), we firstly cloned and characterized the complete cDNA sequence and investigated its expression profile under optimal conditions and acute temperature stress. The cDNA of ClHSP60 contained 2257 nucleotides, consisting of 12 exons with length ranging from 65 to 590 bp. The open reading frame (ORF) encoded 573 amino acids with calculated molecular weight of 60.97 kDa that contained a number of structurally prominent domains or motifs. Under optimal temperature conditions, levels of ClHSP60 expression differed between all the tested tissues (the highest was noted in liver and the lowest in pectoralis major muscle). Under acute temperature stress, five patterns of change were detected in the tested tissues, suggesting that different tissues in domestic pigeons differentially responded to various temperature stress conditions. Upregulation of ClHSP60 expression was highest in the lung and pectoralis major muscle, reflecting the crucial role of these two tissues in temperature regulation. However, the crop, cerebrum, and heart showed little change or decreased ClHSP60 expression. The results indicate that ClHSP60 may be sensitive to and play pivotal roles in responding to acute temperature stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-020-01160-7) contains supplementary material, which is available to authorized users.

The current study evaluated the effects of nano delivery of Spirulina platensis on growth performance, digestive enzymes, and biochemical, immunological, and antioxidative status, as well as resistance to Aeromonas veronii and some physical stressor challenges in Nile tilapia, Oreochromis niloticus. Three experimental fish groups (n = 270) with mean weights of 26 ± 0.30 g and mean lengths of 10 ± 0.5 cm were used; the first additive-free basal diet served as the control group, whereas the following two groups were supplemented with spirulina nanoparticles (SPNP) at 0 (control), 0.25, and 0.5%/kg diet for 4 weeks. Following the feeding trial, fish were challenged with hypoxia, cold stresses, and pathogenic bacteria (A. veronii) infection (9 × 108 CFU/ml). SPNP supplementation, especially 0.5%, (p 0.25% SPNP > 0% SPNP compared with the control. Protection in the incorporated fish groups exposed to A. veronii was 100% compared with the control group, which showed 50% cumulative mortalities. In conclusion, dietary SPNP supplementation improved growth performance, antioxidant activity, immune response, digestive enzymes, related gene expression, and resistance of Nile Tilapia to hypoxia, cold, and A. veronii infection. Thus, SPNP could be used as a natural therapy for controlling those stressors.

Prunus sibirica and Prunus mume are closely related plant species that differ in cold tolerance. Hybrids of P. sibirica and true mume, belonging to the apricot mei group, inherited strong cold resistance from P. sibirica. These materials are favourable for research on the molecular mechanisms of cold resistance. However, no suitable reference genes have been identified for analysing gene expression patterns between P. sibirica and P. mume. Ten candidate reference genes were assessed, namely, actins (ACT2-1, ACT2-2, ACT2-3, ACT2-4), protein phosphatase 2A-1 (PP2A-1), ubiquitins (UBQ2, UBQ3), ubiquitin extension protein (UBQ1) and tubulins (TUB1, TUB2), with four distinct algorithms (geNorm, NormFinder, BestKeeper and RefFinder). UBQ2 was recognized as the best reference gene in stems and buds across materials (P. sibirica; 'Xiaohong Zhusha', 'Beijing Yudie', and 'Xiao Lve' for true mume; and 'Dan Fenghou', 'Fenghou', and 'Yanxing' for apricot mei) under cold stress. In addition, the temporal and spatial expression patterns of PmCBF6 and PmLEA10 among seven varieties during winter periods were analysed using UBQ2 as a reference gene. The expression differed significantly among cultivars, which may contribute to their differences in cold tolerance. This paper confirmed the strong cold tolerance of apricot mei. And the best internal reference gene suitable for seven varieties was selected: UBQ2. Based on the above results, the expression of PmCBF6 and PmLEA10 genes during wintering in seven varieties was analysed. The molecular mechanisms of cold resistance were found to be possibly different in different varieties of P. sibirica and P. mume.

In their role as molecular chaperones, heat shock proteins (Hsps) mediate protein folding thereby mitigating cellular damage caused by physiological and environmental stress. Nauplii of the crustacean Artemia franciscana respond to heat shock by producing Hsps; however, the effects of cold shock on Hsp levels in A. franciscana have not been investigated previously. The effect of cold shock at 1 °C followed by recovery at 27 °C on the amounts of ArHsp90, Hsp70, ArHsp40, and ArHsp40-2 mRNA and their respective proteins in A. franciscana nauplii was examined by quantitative PCR (qPCR) and immunoprobing of western blots. The same Hsp mRNAs and proteins were also quantified during incubation of nauplii at their optimal growth temperature of 27 °C. qPCR analyses indicated that the abundance of ArHsp90, Hsp70, and ArHsp40 mRNA remained relatively constant during both cold shock and recovery and was not significantly different compared with levels at optimal temperature. Western blotting revealed that ArHsp90, ArHsp40, and ArHsp40-2 were generally below baseline, but at detectable levels during the 6 h of cold shock, and persisted in early recovery stages before declining. Hsp70 was the only protein that remained constant in quantity throughout cold shock and recovery. By contrast, all Hsps declined rapidly during 6 h when nauplii were incubated continuously at 27 °C optimal temperature. Generally, the amounts of ArHsp90, ArHsp40, and ArHsp40-2 were higher during cold shock/recovery than those during continuous incubation at 27 °C. Our data support the conclusion that low temperature preserves Hsp levels, making them available to assist in protein repair and recovery after cold shock. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-020-01147-4) contains supplementary material, which is available to authorized users.

Heat shock protein 90 genes/proteins (Hsp90s) are related to the stress resistance found in various plant species. These proteins affect the growth and development of plants and have important effects on the plants under various stresses (cold, drought and salt) in the environment. In this study, we identified 334 Hsp90s from 43 plant species, and Hsp90s were found in all species. Phylogenetic tree and conserved domain database analysis of all Hsp90s showed three independent clades. The analysis of motifs, gene duplication events, and the expression data from PGSC website revealed the gene structures, evolution relationships, and expression patterns of the Hsp90s. In addition, analysis of the transcript levels of the 7 Hsp90s in potato (Solanum tuberosum) under low temperature and high temperature stresses showed that these genes were related to the temperature stresses. Especially StHsp90.2 and StHsp90.4, under high or low temperature conditions, the expression levels in leaves, stems, or roots were significantly up-regulated. Our findings revealed the evolution of the Hsp90s, which had guiding significance for further researching the precise functions of the Hsp90s.

Himalayan mountains are distinctly characterized for their unique climatic and topographic variations; therefore, unraveling the cold-adaptive mechanisms and processes of native life forms is always being a matter of concern for scientific community. In this perspective, the proteomic response of psychrophilic diazotroph Pseudomonas helmanticensis was studied towards low-temperature conditions. LC-MS-based analysis revealed that most of the differentially expressed proteins providing cold stress resistance were molecular chaperons and cold shock proteins. Enzymes involved in proline, polyamines, unsaturated fatty acid biosynthesis, ROS-neutralizing pathways, and arginine degradation were upregulated. However, proteins involved in the oxidative pathways of energy generation were severalfold downregulated. Besides these, the upregulation of uncharacterized proteins at low temperature suggests the expression of novel proteins by P. helmanticensis for cold adaptation. Protein interaction network of P. helmanticensis under cold revealed that Tif, Tig, DnaK, and Adk were crucial proteins involved in cold adaptation. Conclusively, this study documents the proteome and protein-protein interaction network of the Himalayan psychrophilic P. helmanticensis under cold stress.

Thirty MaFLAs vary in their molecular features. MaFLA14/18/27/29 are likely to be involved in banana chilling tolerance by facilitating the cold signaling pathway and enhancing the cell wall biosynthesis. Although several studies have identified the molecular functions of individual fasciclin-like arabinogalactan protein (FLA) genes in plant growth and development, little information is available on their involvement in plant tolerance to low-temperature (LT) stress, and the related underlying mechanism is far from clear. In this study, the different expression of FLAs of banana (Musa acuminata) (MaFLAs) in the chilling-sensitive (CS) and chilling-tolerant (CT) banana cultivars under natural LT was investigated. Based on the latest banana genome database, a genome-wide identification of this gene family was done and the molecular features were analyzed. Thirty MaFLAs were distributed in 10 out of 11 chromosomes and these clustered into four major phylogenetic groups based on shared gene structure. Twenty-four MaFLAs contained N-terminal signal, 19 possessed predicted glycosylphosphatidylinositol (GPI), while 16 had both. Most MaFLAs were downregulated by LT stress. However, MaFLA14/18/29 were upregulated by LT in both cultivars with higher expression level recorded in the CT cultivar. Interestingly, MaFLA27 was significantly upregulated in the CT cultivar, but the opposite occurred for the CS cultivar. MaFLA27 possessed only N-terminal signal, MaFLA18 contained only GPI anchor, MaFLA29 possessed both, while MaFLA14 had neither. Thus, it was suggested that the accumulation of these FLAs in banana under LT could improve banana chilling tolerance through facilitating cold signal pathway and thereafter enhancing biosynthesis of plant cell wall components. The results provide background information of MaFLAs, suggest their involvement in plant chilling tolerance and their potential as candidate genes to be targeted when breeding CT banana.

ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant-antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants.

DEAD-box protein family is the largest subfamily of RNA helicases and plays an important role in RNA metabolism and plant growth, development, and stress responses. Although DEAD-box genes have been characterized in various major crop plants, their identification and characterization in Convolvulaceae is still in infancy. Sweet potato (Ipomoea batatas, in Convolvulaceae) is the seventh most important crop in the world. Ipomoea trifida is one of the ancestors of sweet potato and is an effective resource for sweet potato cross-breeding. Identification and characterisation of DEAD-box transcription factors in sweet potato wild ancestor I. trifida genome. A systematic genome-wide analysis was conducted to identify the DEAD-box transcription factors in the I. trifida genome. We identified 17 ItfDEAD-box genes which distributed unevenly on the nine chromosomes of I. trifida and encoded 29 DEAD transcripts. The phylogenetic analysis classified the DEAD-box proteins into nine groups named I–IX. Homology model prediction of ItfDEAD-box proteins obtained 14 models which lay a preliminary foundation for the further functional exploration of the ItfDEAD-box proteins. The tissue-specific and abiotic stress-responsive expression profiles of ItfDEAD-box genes were analyzed in different tissues and under abiotic stress responses by RNA-seq data and confirmed by quantitative PCR analysis. Some genes were significantly up- or down-regulated by different abiotic stress, suggesting that ItfDEAD-box plays a crucial role in stress responses in I. trifida. The identification and gene expression of the ItfDEAD-box gene family might shed light on the function exploration of DEAD-box gene in I. trifida and promote the molecular breeding of sweet potato.

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 β-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.

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.

Background Liriodendron chinense (Lchi) is a tree species within the Magnoliaceae family and is considered a basal angiosperm. The too low or high temperature or soil drought will restrict its growth as the adverse environmental conditions, thus improving L. chinense abiotic tolerance was the key issues to study. WRKYs are a major family of plant transcription factors known to often be involved in biotic and abiotic stress responses. So far, it is still largely unknown if and how the LchiWRKY gene family is tied to regulating L. chinense stress responses. Therefore, studying the involvement of the WRKY gene family in abiotic stress regulation in L. chinense could be very informative in showing how this tree deals with such stressful conditions. Results In this research, we performed a genome-wide analysis of the Liriodendron chinense (Lchi) WRKY gene family, studying their classification relationships, gene structure, chromosomal locations, gene duplication, cis-element, and response to abiotic stress. The 44 members of the LchiWRKY gene family contain a significant amount of sequence diversity, with their lengths ranging from 525 bp to 40,981 bp. Using classification analysis, we divided the 44 LchiWRKY genes into three phylogenetic groups (I, II, II), with group II then being further divided into five subgroups (IIa, IIb, IIc, IId, IIe). Comparative phylogenetic analysis including the WRKY families from 17 plant species suggested that LchiWRKYs are closely related to the Magnolia Cinnamomum kanehirae WRKY family, and has fewer family members than higher plants. We found the LchiWRKYs to be evenly distributed across 15 chromosomes, with their duplication events suggesting that tandem duplication may have played a major role in LchiWRKY gene expansion model. A Ka/Ks analysis indicated that they mainly underwent purifying selection and distributed in the group IId. Motif analysis showed that LchiWRKYs contained 20 motifs, and different phylogenetic groups contained conserved motif. Gene ontology (GO) analysis showed that LchiWRKYs were mainly enriched in two categories, i.e., biological process and molecular function. Two group IIc members (LchiWRKY10 and LchiWRKY37) contain unique WRKY element sequence variants (WRKYGKK and WRKYGKS). Gene structure analysis showed that most LchiWRKYs possess 3 exons and two different types of introns: the R- and V-type which are both contained within the WRKY domain (WD). Additional promoter cis-element analysis indicated that 12 cis-elements that play different functions in environmental adaptability occur across all LchiWRKY groups. Heat, cold, and drought stress mainly induced the expression of group II and I LchiWRKYs, some of which had undergone gene duplication during evolution, and more than half of which had three exons. LchiWRKY33 mainly responded to cold stress and LchiWRKY25 mainly responded to heat stress, and LchiWRKY18 mainly responded to drought stress, which was almost 4-fold highly expressed, while 5 LchiWRKYs (LchiWRKY5, LchiWRKY23, LchiWRKY14, LchiWRKY27, and LchiWRKY36) responded equally three stresses with more than 6-fold expression. Subcellular localization analysis showed that all LchiWRKYs were localized in the nucleus, and subcellular localization experiments of LchiWRKY18 and 36 also showed that these two transcription factors were expressed in the nucleus. Conclusions This study shows that in Liriodendron chinense, several WRKY genes like LchiWRKY33, LchiWRKY25, and LchiWRKY18, respond to cold or heat or drought stress, suggesting that they may indeed play a role in regulating the tree’s response to such conditions. This information will prove a pivotal role in directing further studies on the function of the LchiWRKY gene family in abiotic stress response and provides a theoretical basis for popularizing afforestation in different regions of China.

Background Rice (Oryza sativa L.) is a food crop for humans worldwide. However, temperature has an effect during the vegetative and reproductive stages. In high-latitude regions where rice is cultivated, cold stress is a major cause of yield loss and plant death. Research has identified a group of plant-specific transcription factors, DNA binding with one zinc fingers (DOFs), with a diverse range of functions, including stress signaling and stress response during plant growth. The aim of this study was to identify Dof genes in two rice subspecies, indica and japonica, and screen for Dof genes that may be involved in cold tolerance during plant growth. Results A total of 30 rice Dofs (OsDofs) were identified using bioinformatics and genome-wide analyses and phylogenetically analyzed. The 30 OsDOFs were classified into six subfamilies, and 24 motifs were identified based on protein sequence alignment. The chromosome locations of OsDofs were determined and nine gene duplication events were identified. A joint phylogenetic analysis was performed on DOF protein sequences obtained from four monocotyledon species to examine the evolutionary relationship of DOF proteins. Expression profiling of OsDofs from two japonica cultivars (Longdao5, which is cold-tolerant, and Longjing11, which is cold-sensitive) revealed that OsDof1 and OsDof19 are cold-inducible genes. We examined the seed setting rates in OsDof1- and OsDof19-overexpression and RNAi lines and found that OsDof1 showed a response to cold stress. Conclusions Our investigation identified OsDof1 as a potential target for genetic breeding of rice with enhanced cold tolerance.

Faba bean (Vicia faba L.), a globally important grain legume providing a stable source of dietary protein, was one of the earliest plant cytogenetic models. However, the lack of draft genome annotations and unclear structural information on mRNA transcripts have impeded its genetic improvement. To address this, we sequenced faba bean leaf transcriptome using the PacBio single-molecule long-read isoform sequencing platform. We identified 28,569 nonredundant unigenes, ranging from 108 to 9669 bp, with a total length of 94.5 Mb. Many unigenes (3597, 12.5%) had 2–20 isoforms, indicating a highly complex transcriptome. Approximately 96.5% of the unigenes matched sequences in public databases. The predicted proteins and transcription factors included NB-ARC, Myb_domain, C3H, bHLH, and heat shock proteins, implying that this genome has an abundance of stress resistance genes. To validate our results, we selected WCOR413-15785, DHN2-12403, DHN2-14197, DHN2-14797, COR15-14478, and HVA22-15 unigenes from the ICE-CBF-COR pathway to analyze their expression patterns in cold-treated samples via qRT-PCR. The expression of dehydrin-related genes was induced by cold stress. The assembled data provide the first insights into the deep sequencing of full-length RNA from faba bean at the single-molecule level. This study provides an important foundation to improve gene modeling and protein prediction.

Background Chlorophyll (Chl) is a vital photosynthetic pigment involved in capturing light energy and energy conversion. In this study, the color conversion of inner-leaves from green to yellow in the new wucai (Brassica campestris L.) cultivar W7–2 was detected under low temperature. The W7–2 displayed a normal green leaf phenotype at the seedling stage, but the inner leaves gradually turned yellow when the temperature was decreased to 10 °C/2 °C (day/night), This study facilitates us to understand the physiological and molecular mechanisms underlying leaf color changes in response to low temperature. Results A comparative leaf transcriptome analysis of W7–2 under low temperature treatment was performed on three stages (before, during and after leaf color change) with leaves that did not change color under normal temperature at the same period as a control. A total of 67,826 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis revealed that the DEGs were mainly enriched in porphyrin and Chl metabolism, carotenoids metabolism, photosynthesis, and circadian rhythm. In the porphyrin and chlorophyll metabolic pathways, the expression of several genes was reduced [i.e. magnesium chelatase subunit H (CHLH)] under low temperature. Almost all genes [i.e. phytoene synthase (PSY)] in the carotenoids (Car) biosynthesis pathway were downregulated under low temperature. The genes associated with photosynthesis [i.e. photosystem II oxygen-evolving enhancer protein 1 (PsbO)] were also downregulated under LT. Our study also showed that elongated hypocotyl5 (HY5), which participates in circadian rhythm, and the metabolism of Chl and Car, is responsible for the regulation of leaf color change and cold tolerance in W7–2. Conclusions The color of inner-leaves was changed from green to yellow under low temperature in temperature-sensitive mutant W7–2. Physiological, biochemical and transcriptomic studies showed that HY5 transcription factor and the downstream genes such as CHLH and PSY, which regulate the accumulation of different pigments, are required for the modulation of leaf color change in wucai under low temperature.

Genome-wide identification reveals 55 PvuGRAS genes belonging to 16 subfamilies and their gene structures and evolutionary relationships were characterized. Expression analyses highlight their prominence in plant growth, development and abiotic stress responses. GRAS proteins comprise a plant-specific transcription factor family involved in multiple growth regulatory pathways and environmental cues including abiotic/biotic stresses. Despite its crucial importance, characterization of this gene family is still elusive in common bean. A systematic genome-wide scan identified 55 PvuGRAS genes unevenly anchored to the 11 common bean chromosomes. Segmental duplication appeared to be the key driving force behind expansion of this gene family that underwent purifying selection during evolution. Computational investigation unraveled their intronless organization and identified similar motif composition within the same subfamily. Phylogenetic analyses clustered the PvuGRAS proteins into 16 phylogenetic clades and established extensive orthologous relationships with Arabidopsis and rice. Analysis of the upstream promoter region uncovered cis-elements responsive to growth, development, and abiotic stresses that may account for their differential expression. The identified SSRs could serve as putative molecular markers facilitating future breeding programs. 37 PvuGRAS transcripts were post-transcriptionally regulated by different miRNA families, miR171 being the major player preferentially targeting members of the HAM subfamily. Global expression profile based on RNA-seq data indicates a clade specific expression pattern in various tissues and developmental stages. Additionally, nine PvuGRAS genes were chosen for further qPCR analyses under drought, salt, and cold stress suggesting their involvement in acclimation to environmental stimuli. Combined, the present results significantly contribute to the current understanding of the complexity and biological function of the PvuGRAS gene family. The resources generated will provide a solid foundation in future endeavors for genetic improvement in common bean.

Breeding for cold tolerance in maize is an important objective in temperate areas. The objective was to carry out a highly efficient study of quantitative trait loci (QTLs) for cold tolerance in maize. We evaluated 406 recombinant inbred lines from a multi-parent advanced generation intercross (MAGIC) population in a growth chamber under cold and control conditions, and in the field at early and normal sowing. We recorded cold tolerance-related traits, including the number of days from sowing to emergence, chlorophyll content and maximum quantum efficiency of photosystem II (F/F). Association mapping was based on genotyping with near one million single nucleotide polymorphism (SNP) markers. We found 858 SNPs significantly associated with all traits, most of them under cold conditions and early sowing. Most QTLs were associated with chlorophyll and F/F. Many candidate genes coincided between the current research and previous reports. These results suggest that (1) the MAGIC population is an efficient tool for identifying QTLs for cold tolerance; (2) most QTLs for cold tolerance were associated with F/F; (3) most of these QTLs were located in specific genomic regions, particularly bin 10.04; (4) the current study allows genetically improving cold tolerance with genome-wide selection., This research was supported by the Spanish Plan for Research and Development (AGL2016-77628-R) and funded in part by the European Regional Development Fund (FEDER). The genotypic data were provided by the Biotechnological Institute of the Cornell University USA). Q Yi acknowledges a grant from the China Scholarship Council (CSC).

The most wide-spread "hostile" environmental factor for marine microorganisms is low temperature, which is usually accompanied by high hydrostatic pressure (HHP). Metabolic mechanisms of marine microorganisms adapting to prolonged low temperature under HHP remain to be clarified. To reveal the underlying metabolic mechanisms, we performed NMR-based metabolomic analysis of aqueous extracts derived from a psychrotolerant Microbacterium sediminis YLB-01, which was isolated from deep-sea sediment and possess great biotechnology potentials. The YLB-01 cells were firstly cultivated at the optimal condition (28 °C, 0.1 MPa) for either 18 h (logarithmic phase) or 24 h (stationary phase), then continually cultivated at either 28 °C or 4 °C under HHP (30 MPa) for 7 days. The cells cultivated at low temperature, which experienced cold stress, were distinctly distinguished from those at normal temperature. Cold stress primarily induced metabolic changes in amino acid metabolism and carbohydrate metabolism. Furthermore, the logarithmic and stationary phase cells cultivated at low temperature exhibited distinct metabolic discrimination, which was mostly reflected in the significantly disturbed carbohydrate metabolism. The logarithmic phase cells displayed suppressed TCA cycle, while the stationary phase cells showed decreased pyruvate and increased lactate. In addition, we performed transcriptome analysis for the stationary phase cells to support the metabolomic analysis. Our results suggest that the cold adaptation of the psychrotroph YLB-01 is closely associated with profoundly altered amino acid metabolism and carbohydrate metabolism. Our work provides a mechanistic understanding of the metabolic adaptation of marine psychrotrophs to prolonged low temperature under HHP.

Snap bean, Phaseolus vulgaris L., as a warm-season vegetable, low temperature stress seriously affect the yield and quality. At present, little is known about the genes and molecular regulation mechanism in cold response in snap bean exposed to low temperature. Our objectives were to identify the low temperature response genes in snap bean and to examine differences in the gene response between cold-tolerant and cold-sensitive genotypes. We used two highly inbred snap bean lines in this study, the cold-tolerant line ‘120’, and the cold-sensitive line ‘093’. The plants were grown to the three leaf and one heart stage and exposed to 4 °C low temperature. We used RNA sequencing (RNA-seq) to analyze the differences of gene expression. 988 and 874 cold-responsive genes were identified in ‘T120 vs CK120’ and ‘T093 vs CK093’ (‘T’ stands for low temperature treatment, and ‘CK’ stands for control at room temperature), respectively. Of these, 555 and 442 genes were unique to cold-stressed lines ‘120’ and ‘093’, respectively compared to the control. Our analysis of these differentially expressed genes indicates that Ca2+, ROS, and hormones act as signaling molecules that play important roles in low temperature response in P. vulgaris. Altering the expression of genes in these signaling pathways activates expression of downstream response genes which can interact with other signaling regulatory networks. This may maintained the balance of ROS and hormones, making line ‘120’ more cold-tolerant than line ‘093’. Our results provide a preliminarily understanding of the molecular basis of low temperature response in snap bean, and also establish a foundation for the future genetic improvement of cold sensitivity in snap bean by incorporating genes for cold tolerance.

Rice (Oryza sativa) growth is always threatened by heat as well as cold stress, when it is exposed to natural environment. Heat growing degree hours (HGDH) and cold growing degree hours (CGDH1 and CGDH2) were firstly proposed to quantify heat and cold stress occurred during different growing stages. Information diffusion method was effectively used to fit the distribution and estimate probability of single stress at each station, with an advantage of no limitation in data series. In terms of single stress, highest probability was seen in HGDH, followed by CGDH1 and CGDH2. Seven copula functions, i.e., normal and t, Gumbel-Hougaard, Clayton, Frank, Joe, and Ali-Mikhail-Haq, were applied to fit the distribution of multi-stress with significant dependence, and simple calculation based on single stress was used to quantify the probability for multi-stress with independence. In these copulas, t was the most chosen one in the fitting of HGDH-CGDH1, HGDH-CGDH2, CGDH1-CGDH2, and HGDH-CGDH1-CGDH2, selected by the statistic of Akaike information criterion. Regarding bi-stress, higher joint probability was in HGDH-CGDH1, relative to HGDH-CGDH2 and CGDH1-CGDH2. As expected, the co-occurrence probability of tri-stress was lower than that of bi-stress in the magnitude and spatial extent, while joint probability of tri-stress was larger. Given the condition of occurrence of HGDH or CGDH1, the joint probability of HGDH-CGDH1 was higher than other pairs of bi-stress and tri-stress. It was special that higher joint probability of CGDH1-CGDH2 was detected under the condition of CGDH2 relative to CGDH1. Joint probability of tri-stress was lower under the condition of HGDH-CGDH1, in comparison with HGDH-CGDH2 and CGDH1-CGDH2. Hazards of single stress and multi-stress were expressed by the integration of intensity of stress index and corresponding probability. Most consistent conclusions agreed that central Fujian, Zhejiang, and northeastern Jiangxi were exposed to higher hazard, derived from not only single stress but also multi-stress. These results can be helpful in provision of information regarding prevention and adaptation strategies for rice cultivation as a response to extreme temperature stress.

The AMP-activated protein kinase (AMPK) has important roles in the regulation of energy metabolism, and AMPK activity and its regulation have been the focus of relevant investigations. However, functional characterization of AMPK is still limited in insects. In this study, the full-length cDNA coding AMPKα (TcAMPKα) was isolated from the red flour beetle, Tribolium castaneum. The TcAMPKα gene contains an ORF of 1581 bp encoding a protein of 526 amino acid residues, which shared conserved domain structure with Drosophila melanogaster and mammalian orthologs. Exposure of female adults to oxidative, heat, and cold stresses caused an increase in TcAMPKα mRNA expression levels and phosphorylation of Thr-173 in the activation loop. The RNAi-mediated knockdown of TcAMPKα resulted in the increased sensitivity of T. castaneum to oxidative, heat, and cold stresses. These results suggest that stress signals regulate TcAMPKα activity, and TcAMPKα plays an important role in enabling protective mechanisms and processes that confer resistance to environmental stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-019-01030-x) contains supplementary material, which is available to authorized users.

Maize (Zea mays L.) is an important model plant with an important role in agriculture and national economies all over the world. The optimum growth temperature of maize is between 25 and 28 °C. At temperatures below 12 °C, maize is vulnerable to damage by chilling stress. MYB transcription factors play important roles in plants' response to low temperature stress. Maize ZmMYB31 encodes a R2R3-MYB transcription factor, ZmMYB31, which localized in the nucleus. ZmMYB31 expression was induced by chilling stress and the highest expression level was detected with the 24 h chilling treatment. ZmMYB31 expression also increased in overexpressing Arabidopsis lines. The minimal fluorescence (Fo) with all photosystem II reaction centers open increased in wild type (WT) and transgenic plants under chilling stress, with the highest increase in WT. The maximal photochemical efficiency of photosystem II (Fv/Fm) decreased more in WT than in transgenic plants during chilling stress. Furthermore, the ZmMYB31-overexpressing lines showed higher superoxide dismutase and ascorbate peroxidase activity and lower reactive oxygen species (ROS) content than the WT. The expression of genes related to chilling stress was higher in transgenic plants than in WT. These results suggest that ZmMYB31 plays a positive regulatory role in chilling and peroxide stress by regulating the expression of chilling stress-related genes to reduce ion extravasation, ROS content, and low-temperature photoinhibition, thereby improving low temperature resistance.

The genetic mechanisms underlying the cattle resilience to severe cold temperatures are still unknown. In this study, we observed that four blood biochemical parameters were significantly altered, i.e., blood adrenocorticotropic hormone (ACTH), triiodothyronine (T3), thyroxine (T4), and potassium (K(+)) after expose to − 32 °C for 3 h. This was observed using 105 healthy Sanhe heifers with similar weight (398.17 ± 34.06 kg) and age (19.30 ± 4.91 months). A total of 20 single nucleotide polymorphisms (SNPs) were identified in 5′-flanking region of the hsp70 gene in Sanhe cattle, while only 10 SNPs were segregating when comparing genetic variations between Sanhe cattle and 285 Chinese Holstein samples. Statistically significant associations between the genomic markers SNP-42(−), SNP-105(+), SNP-181(+), and SNP-205(+) with blood T3 and between SNP-105(+) and blood T4 were observed by applying the general linear model procedure and Bonferroni t test. Furthermore, we demonstrated that the T alleles of SNP-42(−) and SNP-205(+) in the GC box and Kozak sequence of the hsp70 gene, respectively, significantly decreased the green fluorescent proteins activity in vitro GFP reporter assays. These findings suggest that these two SNPs are causative polymorphisms involved in the regulation of hsp70 promoter activity and might contribute to the observed association between the hsp70 gene and T3 and T4 levels in Sanhe cattle. Thus, hsp70 gene is a promising candidate gene to be validated in independent cattle populations and functional studies related to cold stress resilience in cattle. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-019-00973-5) contains supplementary material, which is available to authorized users.

Understanding the genetic basis of cold tolerance is a key step towards obtaining new and improved crop varieties. Current geographical distribution of durum wheat in Argentina exposes the plants to frost damage when spikes have already emerged. Biochemical pathways involved in cold tolerance are known to be early activated at above freezing temperatures. In this study we reported the transcriptome of CBW0101 spring durum wheat by merging data from untreated control and cold (5 °C) treated plant samples at reproductive stage. A total of 128,804 unigenes were predicted. Near 62% of the unigenes were annotated in at least one database. In total 876 unigenes were differentially expressed (DEGs), 562 were up-regulated and 314 down-regulated in treated samples. DEGs are involved in many critical processes including, photosynthetic activity, lipid and carbohydrate synthesis and accumulation of amino acids and seed proteins. Twenty-eight transcription factors (TFs) belonging to 14 families resulted differentially expressed from which eight families comprised of only TFs induced by cold. We also found 31 differentially expressed Long non-coding RNAs (lncRNAs), most of them up-regulated in treated plants. Two of these lncRNAs could operate via microRNAs (miRNAs) target mimic. Our results suggest a reprogramming of expression patterns in CBW0101 that affects a number of genes that is closer to the number reported in winter genotypes. These observations could partially explain its moderate tolerance (low proportion of frost-damaged spikes) when exposed to freezing days in the field. Fil: Díaz, Marina Lucía. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Soresi, Daniela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Basualdo, Jessica. Universidad Nacional del Sur. Departamento de Agronomía; Argentina Fil: Cuppari, Selva Yanet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Carrera, Alicia Delia. Universidad Nacional del Sur. Departamento de Agronomía; Argentina

The inner membrane protein lipopolysaccharide assembly protein B (LapB) is an adaptor protein that activates the proteolysis of LpxC by an essential inner membrane metalloprotease, FtsH, leading to a decrease in the level of lipopolysaccharide in the membrane. In this study, we revealed the mechanism by which the essential inner membrane protein YejM regulates LapB and analyzed the role of the transmembrane domain of LapB in Escherichia coli. The transmembrane domain of YejM genetically and physically interacted with LapB and inhibited its function, which led to the accumulation of LpxC. The transmembrane domain of LapB was indispensable for both its physical interaction with YejM and its regulation of LpxC proteolysis. Notably, we found that the lapB mutant exhibited strong cold sensitivity and this phenotype was not associated with increased accumulation of LpxC. The transmembrane domain of LapB was also required for its role in adaptation to cold stress. Taken together, these results showed that LapB plays an important role in both the regulation of LpxC level, which is controlled by its interaction with the transmembrane domain of YejM, and adaptation to cold stress, which is independent of LpxC.

Dof genes enhance cold tolerance in grapevine and VaDof17d is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides. DNA-binding with one finger (Dof) proteins comprise a large family that plays important roles in the regulation of abiotic stresses. No in-depth analysis of Dof genes has been performed in the grapevine. In this study, we analyzed a total of 25 putative Dof genes in grapevine at genomic and transcriptomic levels, compiled expression profiles of 11 selected VaDof genes under cold stress and studied the potential function of the VaDof17d gene in grapevine calli. The 25 Dof proteins can be classified into four phylogenetic groups. RNA-seq and qRT-PCR results demonstrated that a total of 11 VaDof genes responded to cold stress. Comparative mRNA sequencing of 35S::VaDof17d grape calli showed that VaDof17d was tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GolS) and raffinose synthase genes. We found that the Dof17d-ED (CRISPR/Cas9-mediated mutagenesis of Dof17d-ED) mutant had low cold tolerance with a decreased RFOs level during cold stress. These results formed the fundamental knowledge for further analysis of the biological roles of Dof genes in the grapevine's adaption to cold stresses.

Background Low temperature is a major abiotic stress that seriously limits mangrove productivity and distribution. Kandelia obovata is the most cold-resistance specie in mangrove plants, but little is known about the molecular mechanism underlying its resistance to cold. Osmotin is a key protein associated with abiotic and biotic stress response in plants but no information about this gene in K. obovata was reported. Results In this study, a cDNA sequence encoding osmotin, KoOsmotin (GenBank accession no. KP267758), was cloned from mangrove plant K. obovata. The KoOsmotin protein was composed of 221 amino acids and showed a calculated molecular mass of 24.11 kDa with pI 4.92. The KoOsmotin contained sixteen cysteine residues and an N-terminal signal peptide, which were common signatures to most osmotins and pathogenesis-related 5 proteins. The three-dimensional (3D) model of KoOsmotin, contained one α-helix and eleven β-strands, was formed by three characteristic domains. Database comparisons of the KoOsmotin showed the closest identity (55.75%) with the osmotin 34 from Theobroma cacao. The phylogenetic tree also revealed that the KoOsmotin was clustered in the branch of osmotin/OLP (osmotin-like protien). The KoOsmotin protein was proved to be localized to both the plasma membrane and cytoplasm by the subcellular localization analysis. Gene expression showed that the KoOsmotin was induced primarily and highly in the leaves of K. obovata, but less abundantly in stems and roots. The overexpressing of KoOsmotin conferred cold tolerance in Escherichia coli cells. Conclusion As we known, this is the first study to explore the osmotin of K. obovata. Our study provided valuable clues for further exploring the function of KoOsmotin response to stress.

Background Tropical stenothermal fish exhibit special tolerance and response to cold stress. However current knowledge of the molecular mechanisms response to cold stress in aquatic ectotherms is largely drawn from eurythermal or extreme stenothermal species. The tiger barb Puntius tetrazona is a tropical stenothermal fish, with great popularity in aquarium trade and research. Results To investigate the response mechanism of P. tetrazona to low temperature, fish were exposed to increasing levels of acute cold stress. Histopathological analysis showed that the brain, gill, liver and muscle tissues appeared serious damage after cold stress (13 °C). Brain, gill, liver and muscle tissues from control (CTRL) groups (27 °C) and COLD stress groups (13 °C) of eight-month fish (gender-neutral) were sampled and assessed for transcriptomic profiling by high-throughput sequencing. 83.0 Gb of raw data were generated, filtered and assembled for de novo transcriptome assembly. According to the transcriptome reference, we obtained 392,878 transcripts and 238,878 unigenes, of which 89.29% of the latter were annotated. There were 23,743 differently expressed genes (DEGs) been filtered from four pairs of tissues (brain, gill, liver and muscle) between these cold stress and control groups. These DEGs were mainly involved in circadian entrainment, circadian rhythm, biosynthesis of steroid and fatty acid. There were 64 shared DEGs between the four pairs of groups, and five were related to ubiquitylation/deubiquitylation. Our results suggested that ubiquitin-mediated protein degradation might be necessary for tropical stenothermal fish coping with acute cold stress. Also, the significant cold-induced expression of heat shock 70 kDa protein (HSP70) and cold-induced RNA-binding protein (CIRBP) was verified. These results suggested that the expression of the molecular chaperones HSP70 and CIRBP in P. tetrazona might play a critical role in coping with acute cold stress. Conclusions This is the first transcriptome analysis of P. tetrazona using RNA-Seq technology. Novel findings about tropical stenothermal fish under cold stress (such as HSP70 and CIRBP genes) are presented here. This study contributes new insights into the molecular mechanisms of tropical stenothermal species response to acute cold stress.