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Background Rice plants show yellowing, stunting, withering, reduced tillering and utimately low productivity in susceptible varieties under low temperature stress. Comparative transcriptome analysis was performed to identify novel transcripts, gain new insights into different gene expression and pathways involved in cold tolerance in rice. Results Comparative transcriptome analyses of 5 treatments based on chilling stress exposure revealed more down regulated genes in susceptible and higher up regulated genes in tolerant genotypes. A total of 13930 and 10599 differentially expressed genes (DEGs) were detected in cold susceptible variety (CSV) and cold tolerant variety (CTV), respectively. A continuous increase in DEGs at 6, 12, 24 and 48 h exposure of cold stress was detected in both the genotypes. Gene ontology (GO) analysis revealed 18 CSV and 28 CTV term significantly involved in molecular function, cellular component and biological process. GO classification showed a significant role of transcription regulation, oxygen, lipid binding, catalytic and hydrolase activity for tolerance response. Absence of photosynthesis related genes, storage products like starch and synthesis of other classes of molecules like fatty acids and terpenes during the stress were noticed in susceptible genotype. However, biological regulations, generation of precursor metabolites, signal transduction, photosynthesis, regulation of cellular process, energy and carbohydrate metabolism were seen in tolerant genotype during the stress. KEGG pathway annotation revealed more number of genes regulating different pathways resulting in more tolerant. During early response phase, 24 and 11 DEGs were enriched in CTV and CSV, respectively in energy metabolism pathways. Among the 1583 DEG transcription factors (TF) genes, 69 WRKY, 46 bZIP, 41 NAC, 40 ERF, 31/14 MYB/MYB-related, 22 bHLH, 17 Nin-like 7 HSF and 4C3H were involved during early response phase. Late response phase showed 30 bHLH, 65 NAC, 30 ERF, 26/20 MYB/MYB-related, 11 C3H, 12 HSF, 86 Nin-like, 41 AP2/ERF, 55 bZIP and 98 WRKY members TF genes. The recovery phase included 18 bHLH, 50 NAC, 31 ERF, 24/13 MYB/MYB-related, 4 C3H, 4 HSF, 14 Nin-like, 31 bZIP and 114 WRKY TF genes. Conclusions Transcriptome analysis of contrasting genotypes for cold tolerance detected the genes, pathways and transcription factors involved in the stress tolerance.

Summary In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH‐overexpressing apple plants possessed improved cold and salt tolerance compared to wild‐type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation–reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2O2 content, but a low O2·− production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt‐ and cold‐tolerant trees.

The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. Here we show that the capability ( capa ) neuropeptide gene is a desiccation- and cold stress-responsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, we demonstrate that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that capa accumulates in the capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. Our results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments our understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance. Significance Insects are among the most robust organisms on the planet, surviving in virtually all environments and capable of surmounting a range of environmental stresses including desiccation and cold. Although desiccation and cold tolerance share many common traits, potential mechanisms for such linked responses remain unclear. Here we show that an insect neuropeptide gene is associated with tolerance of both desiccation and cold in Drosophila melanogaster , suggesting a novel mechanism in renal tubule epithelia that enhances survival of both desiccation and cold. Also, we can reverse RNAi-induced stress tolerance phenotypes in intact flies using rationally designed peptide mimetic analogs. We thus demonstrate the power of intervention in physiological processes controlled by neuropeptides, with potential for insect pest control.

Although ethylene and strigolactone (SL) are key regulators of cold tolerance in plants, the molecular crosstalk between their signalling pathways is poorly understood. Here, we identified the transcription factor GOLDEN2‐LIKE1 (MdGLK1) as a central integrator of ethylene and SL signalling during the apple (Malus × domestica) cold stress response. MdGLK1 enhanced cold tolerance by recruiting BRASSINAZOLE‐RESISTANT1 (MdBZR1), a core component of brassinosteroid signalling, thereby promoting MdBZR1‐mediated transcriptional activation of the cold‐responsive genes C‐REPEAT BINDING FACTOR1 (MdCBF1) and MdCBF2. Ethylene signalling modulated this process through ETHYLENE INSENSITIVE3‐BINDING F‐BOX PROTEIN1 (MdEBF1), which targets MdGLK1 for proteasomal degradation, thereby attenuating MdGLK1‐enhanced cold tolerance. In addition, SUPPRESSOR OF MORE AXILLARY GROWTH2‐LIKE8 (MdSMXL8), a repressor of SL signalling, interfered with the formation of the MdGLK1‐MdBZR1 complex, further suppressing MdGLK1‐mediated cold tolerance. Collectively, the MdGLK1‐MdBZR1‐MdCBF1/2 regulatory module operates as a signalling hub that integrates the ethylene and SL pathways during cold stress adaptation in apple. These findings elucidate the molecular mechanism by which MdGLK1 coordinates ethylene and SL signals to enhance cold tolerance, thereby offering potential targets for optimising the trade‐off between growth and stress tolerance in plants.

Overwintering rice can survive through the natural cold-winter field environment, sprout from rice tillering node in the following spring, tiller, flower, seed, and being harvested in the following autumn, which is a type of an extreme case of cold tolerance of rice. The successful utilization of cold tolerance rice is the most economical strategy for the cold tolerance rice cultivar breeding project. This work aims to identify the OW rice for the future development of cold tolerance cultivars. Altogether 1034 Chinese existing rice cultivars including 735 (71.08%) conventional Japonica rice cultivars and 299 (28.92%) conventional Indica rice cultivars were collected and evaluated for their responses to low temperatures under the natural field cold-winter environment. Among them, altogether 262 (25.34%) conventional Japonica rice cultivars could withstand cold tolerance to 4 °C of the daily minimum temperatures in December 2019 throughout the cold-winter season and distributed in 13 provinces of China, survive through the natural cold-winter field environment, and sprout from rice tillering node in March 2020. Only 24 (2.32%) conventional japonica rice cultivars could withstand cold tolerance to 0 °C of the daily minimum temperatures in January 2021 throughout the cold-winter season, which could also sprout from rice tillering node in March 2021 and distributed in seven provinces of China. The present cold tolerance rice cultivars will provide beneficial breeding germplasm for the future cold tolerance rice breeding project and new strategies involved in elucidating the molecular mechanism of the cold tolerance of rice.

Background Rice, being a thermophilic crop, exhibits high sensitivity to low-temperature stress throughout its growth and development. Consequently, enhancing cold tolerance (CT) has been a paramount objective in rice breeding programs. The budding and seedling stages are particularly susceptible to low-temperature damage, making it crucial to improve CT during these stages to ensure the stable establishment and development of the rice population. Results In this study, we exposed the parental lines Nipponbare (NIP) and Searice 86 (SR86), along with their derived 170 doubled-haploid (DH) population lines, to cold treatments during both the budding and seedling stages. Quantitative trait locus (QTL) mapping was performed using statistical indices such as the survival rate at the budding stage (SRBS), severity of damage at the budding stage (SDBS), survival rate at the seedling stage (SRSS), and wilting degree at the seedling stage (WDSS). This analysis identified four QTLs at the budding stage and eight QTLs at the seedling stage. Furthermore, by integrating differentially expressed genes (DEGs) from transcriptomic data with genes located within the QTL regions, we identified 10 candidate genes for the budding stage and 11 candidate genes for the seedling stage. Based on DNA sequence variations between the parental lines, changes in gene expression under cold treatment, and haplotype analyses, the key candidate genes were ultimately determined to be Os02g0250600 for the budding stage and Os06g0696600 for the seedling stage. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of transcriptomic data from both stages revealed significant differences in the regulatory pathways involved in CT between the budding and seedling stages. Conclusion The results indicate that Os02g0250600 is the pivotal gene responsible for CT at the budding stage, with haplotype 4 exhibiting the highest level of CT. Meanwhile, Os06g0696600 plays a crucial role in CT at the seedling stage, where haplotypes 2 and 4 have been identified as advantageous. A comprehensive analysis integrating QTL and transcriptome data from both stages revealed distinct differences in CT mechanisms, highlighting stage-specific variations. This study provides valuable theoretical insights and practical references for the cloning of CT genes and the development of cold-tolerant rice varieties during the budding and seedling stages.

Walnut ( Juglans regia L.) is prized worldwide for both its nutritional value and economic importance, yet it remains vulnerable to cold stress, with significant differences in tolerance among varieties. This study combined physiological analyses with whole-genome resequencing (WGS) to evaluate the cold stress responses of two varieties, ‘Qingxiang’ and ‘Liaoning No.8’. Under chilling stress (0 °C), we measured electrolyte leakage and antioxidant enzyme activity, applying both exogenous methyl jasmonate (MeJA) and the jasmonate inhibitor DIECA. Genomic variations were analyzed using WGS. Results showed that ‘Liaoning No.8’ exhibited superior cold tolerance. Application of MeJA reduced electrolyte leakage by 37% and MDA accumulation by 52% on average, whereas DIECA exacerbated stress-related damage. WGS achieved 16.24–16.26× coverage and identified 2.73–2.78 million SNPs, 378–382k InDels, 25–26k SVs, and 7.2–7.9k CNVs. Twenty genes containing sequence variants showed transcriptional responses under cold stress that were significantly correlated with mutation density ( r  = 0.62, P  < 0.01). One gene, XM_018985465.2, which lacked SNPs in ‘Liaoning No.8’, was expressed 4.2 times more in this variety, suggesting cis-regulatory influence. These findings highlight the role of jasmonic acid signaling in enhancing cold tolerance in walnut and offer genomic insights into its underlying adaptive mechanisms.

CsbHLH18, a bHLH transcription factor of Citrus sinensis, functions as a positive regulator of cold tolerance due to promoted ROS scavenging, and directly targets a POD gene. Abstract The basic helix–loop–helix (bHLH) transcription factors (TFs) comprise one of the largest gene families in plants, and participate in various physiological processes, but the physiological role and regulatory function of the majority of bHLHs remain poorly understood. Here, a total of 56 putative CsbHLH genes were identified in sweet orange (Citrus sinensis) based on a genome-wide analysis. The CsbHLH genes, except four members, were distributed throughout nine chromosomes and divided into 19 subgroups. Most of the CsbHLH genes were responsive to cold stress, with the greatest up-regulation being observed in CsbHLH18. CsbHLH18 is localized in the nuclei and has transcriptional activation activity. Overexpression of CsbHLH18 conferred enhanced cold tolerance in transgenic tobacco. The transgenic plants accumulated significantly less reactive oxygen species (ROS), concurrent with increased activities and transcript levels of antioxidant enzymes. In contrast, knockdown of bHLH18 by RNAi in trifoliate orange promoted cold susceptibility, accompanied by down-regulation of antioxidant genes and accumulation of more ROS. Protein–DNA interaction assays demonstrate that CsbHLH18 directly and specifically binds to and activates the promoter of CsPOD. Taken together, these findings indicate that CsbHLH18 plays a positive role in cold tolerance through, at least partly, modulation of ROS homeostasis by directly regulating the antioxidant gene.

Takifugu fasciatus is an aquaculture species with high economic value. In recent years, problems such as environmental pollution and inbreeding have caused a serious decline in T. fasciatus germplasm resources. In this study, a high-density genetic linkage map was constructed by whole-genome resequencing. The map consists of 4891 bin markers distributed across 22 linkage groups (LGs), with a total genetic coverage of 2381.353 cM and a mean density of 0.535 cM. Quantitative trait locus (QTL) localization analysis showed that a total of 19 QTLs associated with growth traits of T. fasciatus in the genome-wide significance threshold range, distributed on 11 LGs. In addition, 11 QTLs associated with cold tolerance traits were identified, each scattered on a different LG. Furthermore, we used QTL localization analysis to screen out three candidate genes ( IGF1 , IGF2 , ADGRB ) related to growth in T. fasciatus . Meanwhile, we screened three candidate genes ( HSP90 , HSP70 , and HMGB1 ) related to T. fasciatus cold tolerance. Our study can provide a theoretical basis for the selection and breeding of cold-tolerant or fast-growing T. fasciatus.

Background The basic helix-loop-helix (bHLH) transcription factors play important roles in many processes in plant growth, metabolism and responses to abiotic stresses. Although, the sequence of Chinese white pear genome (cv. ‘Dangshansuli’) has already been reported, there is still a lack of clarity regarding the bHLH family genes and their evolutionary history. Results In this work, a genome-wide identification of the bHLH genes in Chinese white pear was performed, and we characterized the functional roles of these PbrbHLH genes in response to abiotic stresses. Based on the phylogenetic analysis and structural characteristics, 197 identified bHLH genes could be well classified into 21 groups. Expansion of PbrbHLH gene family was mainly driven by WGD and dispersed duplication with the purifying selection from the recent WGD. The functional annotation enrichment showed that the majority of PbrbHLHs were enriched in the GO terms and KEGG pathways involved in responds to stress conditions as TFs. Transcriptomic profiles and qRT-PCR revealed that PbrbHLH7 , PbrbHLH8 , PbrbHLH128 , PbrbHLH160 , PbrbHLH161 and PbrbHLH195 were significantly up-regulated under cold and drought treatments. In addition, PbrbHLH195 -silenced pear seedlings display significant reduced cold tolerance, exhibiting reduced chlorophyll content, as well as increased electrolyte leakage and concentrations of malondialdehyde and H 2 O 2 . Conclusion For the first time, a comprehensive analysis identified the bHLH genes in Chinese white pear and demonstrated that PbrbHLH195 is involved in the production of ROS in response to cold stress, suggesting that members of the PbrbHLH family play an essential role in the stress tolerance of pear.