Role In Cold Stress Tolerance Research Articles

PhDREB1-PhZFP1-PhGolS1-1 regulator cascade contribute to cold tolerance by mediating galactinol and raffinose accumulation in petunia

The C-repeat/DRE Binding Factor (CBF/DREB1) gene, which plays a critical role in plant responses to cold stress, could positively regulate the accumulation of galactinol and raffinose, but the underlying molecular mechanisms remain elusive. We have previously confirmed that the C2H2-type zinc finger protein PhZFP1 plays a positive role in cold stress tolerance by modulating galactinol accumulation by directly regulating PhGolS1-1 in Petunia hybrida. However, whether and how CBF/DREB1 regulates PhZFP1 to mediate galactinol accumulation under cold stress remains largely unclear. Here, we report the biological and molecular characterization of PhDREB1F and PhDREB1I in petunia. We demonstrate that PhDREB1F and PhDREB1I function as nuclear transcription factors and activate PhZFP1 expression. Silencing of PhDREB1F or PhDREB1I mediated by virus-induced gene silencing (VIGS) increased the sensitivity to freezing stress in petunia plants. Besides, knocking-down of PhDREB1F or PhDREB1I decreased the expression of PhZFP1 and PhGolS1-1. Similarly, silencing of PhGolS1-1 increased cold sensitivity in petunia. And the contents of galactinol and raffinose in PhGolS1-1-VIGS plants were significantly decreased compared with those in TRV plants under chilling stress. What's more, PhDREB1F/1I, PhZFP1, and PhGolS1-1 all showed cold-induced expression patterns with different peaks. Collectively, our data indicate that PhDREB1F and PhDREB1I positively modulate cold stress tolerance through the PhDREB1s-PhZFP1-PhGolS1–1 pathway in petunia. This study provides new insights into the molecular mechanism of plant responsiveness to cold stress through CBF/DREB1 transcription factors.

Genome-wide analysis of fatty acid desaturase genes in chia (Salvia hispanica) reveals their crucial roles in cold response and seed oil formation

Chia (Salvia hispanica) is a functional food crop with high α-linolenic acid (ALA), the omega-3 essential fatty acid, but its worldwide plantation is limited by cold-intolerance and strict short-photoperiod flowering feature. Fatty acid desaturases (FADs) are responsible for seed oil accumulation, and play important roles in cold stress tolerance of plants. To date, there is no report on systemically genome-wide analysis of FAD genes in chia (ShiFADs). In this study, 31 ShiFAD genes were identified, 3 of which contained 2 alternative splicing transcripts, and they were located in 6 chromosomes of chia. Phylogenetic analysis classified the ShiFAD proteins into 7 groups, with conserved gene structure and MEME motifs within each group. Tandem and segmental duplications coursed the expansion of ShiFAD genes. Numerous cis-regulatory elements, including hormone response elements, growth and development elements, biotic/abiotic stress response elements, and transcription factor binding sites, were predicted in ShiFAD promoters. 24 miRNAs targeting ShiFAD genes were identified at whole-genome level. In total, 15 SSR loci were predicted in ShiFAD genes/promoters. RNA-seq data showed that ShiFAD genes were expressed in various organs with different levels. qRT-PCR detection revealed the inducibility of ShiSAD2 and ShiSAD7 in response to cold stress, and validated the seed-specific expression of ShiSAD11a. Yeast expression of ShiSAD11a confirmed the catalytic activity of its encoded protein, and its heterologous expression in Arabidopsis thaliana significantly increased seed oleic acid content. This work lays a foundation for molecular dissection of chia high-ALA trait and functional study of ShiFAD genes in cold tolerance.

Transcription factor CabHLH035 promotes cold resistance and homeostasis of reactive oxygen species in pepper

Plant basic helix-loop-helix (bHLH) transcription factors (TFs) play central roles in various abiotic stresses. However, its role in plant cold resistance is largely unknown. Previously, we characterised CaNAC035 in pepper, which positively regulates tolerance to cold, salt and drought stresses tolerance. Here, we identified CabHLH035, a CaNAC035-interacting protein in pepper. To explore its functions in cold stress tolerance, we silenced the gene in pepper via virus-induced gene silencing (VIGS) and overexpressed the gene in Arabidopsis. The results showed that CabHLH035 expression was induced by cold treatment, and silencing of CabHLH035 decreased cold stress tolerance. Conversely, overexpression of CabHLH035 in Arabidopsis increased cold stress tolerance. To investigate homologs genes of C-repeat binding factor (CBF) pathway proteins and reactive oxygen species (ROS) marker gene expression blocking by CabHLH035, we performed yeast one-hybrid (Y1H), dual luciferase and electrophoretic mobility shift assay experiments. The results showed that CabHLH035 bound to the region upstream of the CaCBF1A and CaAPX promoters. Additionally, CaCBF1A bound to the CaDHN4 promoter. Taken together, our results showed that CabHLH035 plays a crucial role in cold stress tolerance and its potential as a target for breeding cold-resistant crops. The findings provide a basis for studying the functions and regulatory network of cold stress tolerance in pepper.

AGC/AKT Protein Kinase SCH9 Is Critical to Pathogenic Development and Overwintering Survival in Magnaporthe oryzae

Primary inoculum that survives overwintering is one of the key factors that determine the outbreak of plant disease. Pathogenic resting structures, such as chlamydospores, are an ideal inoculum for plant disease. Puzzlingly, Magnaporthe oryzae, a devastating fungal pathogen responsible for blast disease in rice, hardly form any morphologically changed resting structures, and we hypothesize that M. oryzae mainly relies on its physiological alteration to survive overwintering or other harsh environments. However, little progress on research into regulatory genes that facilitate the overwintering of rice blast pathogens has been made so far. Serine threonine protein kinase AGC/AKT, MoSch9, plays an important role in the spore-mediated pathogenesis of M. oryzae. Building on this finding, we discovered that in genetic and biological terms, MoSch9 plays a critical role in conidiophore stalk formation, hyphal-mediated pathogenesis, cold stress tolerance, and overwintering survival of M. oryzae. We discovered that the formation of conidiophore stalks and disease propagation using spores was severely compromised in the mutant strains, whereas hyphal-mediated pathogenesis and the root infection capability of M. oryzae were completely eradicated due to MoSch9 deleted mutants’ inability to form an appressorium-like structure. Most importantly, the functional and transcriptomic study of wild-type and MoSch9 mutant strains showed that MoSch9 plays a regulatory role in cold stress tolerance of M. oryzae through the transcription regulation of secondary metabolite synthesis, ATP hydrolyzing, and cell wall integrity proteins during osmotic stress and cold temperatures. From these results, we conclude that MoSch9 is essential for fungal infection-related morphogenesis and overwintering of M. oryzae.

Functional Characterization of Cotton C-Repeat Binding Factor Genes Reveal Their Potential Role in Cold Stress Tolerance.

Low temperature is a common biological abiotic stress in major cotton-growing areas. Cold stress significantly affects the growth, yield, and yield quality of cotton. Therefore, it is important to develop more robust and cold stress-resilient cotton germplasms. In response to climate change and erratic weather conditions, plants have evolved various survival mechanisms, one of which involves the induction of various stress responsive transcript factors, of which the C-repeat-binding factors (CBFs) have a positive effect in enhancing plants response to cold stress. In this study, genomewide identification and functional characterization of the cotton CBFs were carried out. A total of 29, 28, 25, 21, 30, 26, and 15 proteins encoded by the CBF genes were identified in seven Gossypium species. A phylogenetic evaluation revealed seven clades, with Clades 1 and 6 being the largest. Moreover, the majority of the proteins encoded by the genes were predicted to be located within the nucleus, while some were distributed in other parts of the cell. Based on the transcriptome and RT-qPCR analysis, Gthu17439 (GthCBF4) was highly upregulated and was further validated through forward genetics. The Gthu17439 (GthCBF4) overexpressed plants exhibited significantly higher tolerance to cold stress, as evidenced by the higher germination rate, increased root growth, and high-induction levels of stress-responsive genes. Furthermore, the overexpressed plants under cold stress had significantly reduced oxidative damage due to a reduction in hydrogen peroxide (H2O2) production. Moreover, the overexpressed plants under cold stress had minimal cell damage compared to the wild types, as evidenced by the Trypan and 3,3′-Diaminobenzidine (DAB) staining effect. The results showed that the Gthu17439 (GthCBF4) could be playing a significant role in enhancing cold stress tolerance in cotton and can be further exploited in developing cotton germplasm with improved cold-stress tolerance.

RsmD, a Chloroplast rRNA m2G Methyltransferase, Plays a Role in Cold Stress Tolerance by Possibly Affecting Chloroplast Translation in Arabidopsis.

Ribosomal RNA (rRNA) methylation is a pivotal process in the assembly and activity of ribosomes, which in turn play vital roles in the growth, development and stress responses of plants. Although few methyltransferases responsible for rRNA methylation have been identified in plant chloroplasts, the nature and function of these enzymes in chloroplasts remain largely unknown. In this study, we characterized ArabidopsisRsmD (At3g28460), an ortholog of the methyltransferase responsible for N2-methylguanosine (m2G) modification of 16S rRNA in Escherichia coli. Confocal microscopic analysis of an RsmD- green fluorescent protein fusion protein revealed that RsmD is localized to chloroplasts. Primer extension analysis indicated that RsmD is responsible for m2G methylation at position 915 in the 16S rRNA of Arabidopsis chloroplasts. Under cold stress, rsmd mutant plants exhibited retarded growth, i.e. had shorter roots, lower fresh weight and pale-green leaves, compared with wild-type (WT) plants. However, these phenotypes were not detected in response to drought or salt stress. Notably, the rsmd mutant was hypersensitive to erythromycin or lincomycin and accumulated fewer chloroplast proteins compared with the WT, suggesting that RsmD influences translation in chloroplasts. Complementation lines expressing RsmD in the rsmd mutant background recovered WT phenotypes. Importantly, RsmD harbored RNA methyltransferase activity. Collectively, the findings of this study indicate that RsmD is a chloroplast 16S rRNA methyltransferase responsible for m2G915 modification that plays a role in the adaptation of Arabidopsisto cold stress.

A systems biology approach identifies a regulator, <i>BplERF1</i>, of cold tolerance in <i>Betula platyphylla</i>

Cold is an abiotic stress that can greatly affect the growth and survival of plants. Here, we reported that an <i>AP2/ERF</i> family gene, <i>BplERF1</i>, isolated from <i>Betula platyphylla</i> played a contributing role in cold stress tolerance. Overexpression of <i>BplERF1</i> in <i>B. platyphylla</i> transgenic lines enhanced cold stress tolerance by increasing the scavenging capability and reducing H₂O₂ and malondialdehyde (MDA) content in transgenic plants. Construction of BplERF-mediated multilayered hierarchical gene regulatory network (ML-hGRN), using Top-down GGM algorithm and the transcriptomic data of <i>BplERF1</i> overexpression lines, led to the identification of five candidate target genes of BplERF1 which include <i>MPK20</i>, <i>ERF9</i>, <i>WRKY53</i>, <i>WRKY70</i>, and <i>GIA1</i>. All of them were then verified to be the true target genes of BplERF1 by chromatin-immunoprecipitation PCR (ChIP-PCR) assay. Our results indicate that BplERF1 is a positive regulator of cold tolerance and is capable of exerting regulation on the expression of cold signaling and regulatory genes, causing mitigation of reactive oxygen species.

Ectopic Expression of OsDREB1G, a Member of the OsDREB1 Subfamily, Confers Cold Stress Tolerance in Rice.

Plants adapt to adverse environmental conditions through physiological responses, such as induction of the abscisic acid signaling pathway, stomatal regulation, and root elongation. Altered gene expression is a major molecular response to adverse environmental conditions in plants. Several transcription factors function as master switches to induce the expression of stress-tolerance genes. To find out a master regulator for the cold stress tolerance in rice, we focused on functionally identifying DREB subfamily which plays important roles in cold stress tolerance of plants. Here, we characterized OsDREB1G (LOC_Os02g45450), a functionally unidentified member of the DREB1 subgroup. OsDREB1G is specifically induced under cold stress conditions among several abiotic stresses examined. This gene is dominantly expressed in leaf sheath, blade, node, and root. Transgenic rice overexpressing this gene exhibited strong cold tolerance and growth retardation, like transgenic rice overexpressing other OsDREB1 genes. However, unlike these rice lines, transgenic rice overexpressing OsDREB1G did not exhibit significant increases in drought or salt tolerance. Cold-responsive genes were highly induced in transgenic rice overexpressing DREB1G compared to wild type. In addition, OsDREB1G overexpression directly induced the expression of a reporter gene fused to the promoters of cold-induced genes in rice protoplasts. Therefore, OsDREB1G is a typical CBF/DREB1 transcription factor that specifically functions in the cold stress response. Therefore, OsDREB1G could be useful for developing transgenic rice with enhanced cold-stress tolerance.

Overexpression of two cold-responsive ATAF-like NAC transcription factors from fine-stem stylo (Stylosanthes guianensis var. intermedia) enhances cold tolerance in tobacco plants

Stylosanthes (stylo) species are economically important tropical and subtropical forage legumes. They are vulnerable to chilling and frost, and little is known about the genetic and molecular mechanisms of their responses or adaptations to low temperature stress. Two cold-responsive NAC genes, SgNAC1 and SgNAC2, were selected from a whole transcriptome profiling study of fine-stem stylo (S. guianensis var. intermedia) and further investigated for their roles in cold stress tolerance. Bioinformatic analysis indicated that SgNAC1 and SgNAC2 belonged to ATAF subgroup of NAC family, and shared highly conserved N-terminal A–E NAC subdomains and a EVQS[E/x]PK[W/I] motif with ATAF-like NAC transcription factors. Expression profiling, subcellular location and transactivation assay revealed that SgNAC1 and SgNAC2 encode nucleus-localized polypeptides with transactivation activities and responds promptly to cold stress. Phenotypic and physiological changes indicated that the transgenic tobacco plants overexpressing SgNAC1 and SgNAC2 were more tolerant to cold stress than the wild-type plants. Meanwhile, the expression of SgNAC1 and SgNAC2 were significantly enhanced at the early stages of cold treatment, indicating that overexpression of SgNAC1 and SgNAC2 is sufficient to confer cold tolerance to tobacco plants. These results suggest that SgNAC1 and SgNAC2 are promising candidate genes for cold tolerance improvement strategies in stylo.

Two HbHsfA1 and HbHsfB1 genes from the tropical woody plant rubber tree confer cold stress tolerance in Saccharomyces cerevisiae

Plant heat stress factors are abiotic stress-responsive regulators. In model or non-model plants, heat stress factors could be divided into different subgroups based on their amino acids through comprehensive bioinformatic analysis. In this study, two cold-inducible HbHsfA1 and HbHsfB1 genes in cold-resistant rubber tree clone ‘93-114’ were identified. Both genes were highly expressed in leaves and stems. Phylogenetic analysis revealed that HbHsfA1 and HbHsfB1 belong to class A and class B Hsf families. MEME analysis showed that different motifs were distributed in the HbHsfA1 and HbHsfB1 proteins. Both HbHsfA1 and HbHsfB1 were located in the nucleus, but only HbHsfA1 exhibited strong transcriptional activation activity in yeasts. Relatively higher expression levels in cold-tolerant rubber tree clone ‘93-114’ than that of cold-sensitive clone ‘Reken501’ were correlated with their roles in cold stress tolerance. The enhanced tolerance observed in eukaryotic yeasts, respectively overexpressed HbHsfA1 and HbHsfB1, suggested that these two Hsf transcription factors may be candidates for genetic engineering to improve cold stress tolerance of rubber trees.

Genome-Wide Identification, Characterization, and Expression Profiling of Glutathione S-Transferase (GST) Family in Pumpkin Reveals Likely Role in Cold-Stress Tolerance.

Plant growth and development can be adversely affected by cold stress, limiting productivity. The glutathione S-transferase (GST) family comprises important detoxifying enzymes, which play major roles in biotic and abiotic stress responses by reducing the oxidative damage caused by reactive oxygen species. Pumpkins (Cucurbita maxima) are widely grown, economically important, and nutritious; however, their yield can be severely affected by cold stress. The identification of putative candidate genes responsible for cold-stress tolerance, including the GST family genes, is therefore vital. For the first time, we identified 32 C. maxima GST (CmaGST) genes using a combination of bioinformatics approaches and characterized them by expression profiling. These CmaGST genes represent seven of the 14 known classes of plant GSTs, with 18 CmaGSTs categorized into the tau class. The CmaGSTs were distributed across 13 of pumpkin’s 20 chromosomes, with the highest numbers found on chromosomes 4 and 6. The large number of CmaGST genes resulted from gene duplication; 11 and 5 pairs of CmaGST genes were segmental- and tandem-duplicated, respectively. In addition, all CmaGST genes showed organ-specific expression. The expression of the putative GST genes in pumpkin was examined under cold stress in two lines with contrasting cold tolerance: cold-tolerant CP-1 (C. maxima) and cold-susceptible EP-1 (Cucurbita moschata). Seven genes (CmaGSTU3, CmaGSTU7, CmaGSTU8, CmaGSTU9, CmaGSTU11, CmaGSTU12, and CmaGSTU14) were highly expressed in the cold-tolerant line and are putative candidates for use in breeding cold-tolerant crop varieties. These results increase our understanding of the cold-stress-related functions of the GST family, as well as potentially enhancing pumpkin breeding programs.

Diverse roles of glycine-rich RNA-binding protein 7 in the response of camelina (Camelina sativa) to abiotic stress

Camelina sativa L. is an oilseed crop used as a potential low-cost biofuel resource. Despite the economic and agricultural benefits of this crop, studies demonstrating the physiological and genetic response of camelina to changing environmental conditions are limited. In this study, three stress-responsive glycine-rich RNA-binding proteins (GRPs) in camelina—named CsGRP7a, CsGRP7b, and CsGRP7c—were isolated, and their functional roles in stress responses were characterized. The three CsGRP7 genes had similar nucleotide and deduced amino acid sequences, and contained an N-terminal RNA-recognition motif and a C-terminal glycine-rich region. The CsGRP7 genes were ubiquitously expressed in all plant tissues, and CsGRP7 proteins were localized to both the cytoplasm and the nucleus. The expression of CsGRP7 genes was markedly upregulated by cold stress, whereas their expression was only slightly affected by salt or dehydration stress. Analysis of CsGRP7a-expressing transgenic Arabidopsis thaliana and camelina plants revealed that CsGRP7a plays a positive role in cold stress tolerance, but a negative role in salt or drought stress tolerance. All three CsGRP7s harbored RNA chaperone activity. Collectively, these data indicate that the stress-responsive CsGRP7s harbor RNA chaperone activity and play different roles in the plant response to abiotic stresses.