Cold Tolerance In Arabidopsis Research Articles

The GDSL lipase CpGLIP1 from Chimonanthus praecox improves drought and cold tolerance in Arabidopsis and poplar

Different types of abiotic stress significantly affect plant growth and crop production even though plants have evolved complex responses to mitigate their effects. Some studies have shown that the GDSL (Gly-Asp-Ser-Leu) lipase proteins regulate the adaptation of plants to abiotic stresses. However, the functions of most GDSL lipases still remain unexplored in plants. To our knowledge, this study is the first to identify the GDSL lipase (GLIP) gene CpGLIP1 in Chimonanthus praecox, which is a hardy and important deciduous aromatic shrub that is native to China. CpGLIP1 was highly expressed in the young leaves and the sprout stage of flowers when there were easily damaged parts of C. praecox. Treatments of drought, cold, jasmonic acid, and gibberellic acid significantly increased the level of expression of CpGLIP1. Additionally, a β-glucuronidase histochemical analysis showed that the CpGLIP1 promoter activity was tissue-specific, and the inducibility of the CpGLIP1 promoter was consistent with the pattern of gene expression. Moreover, the overexpression of CpGLIP1 in Arabidopsis thaliana and poplar (Populus tremula×Populus alba) plants resulted in higher rates of growth and survival and better physiological indices than the wild-type (WT) plants after drought and cold treatment, which indicated that overexpression of CpGLIP1 improved tolerance to drought and cold. In addition, 22 upstream regulatory proteins were screened using a yeast one-hybrid (Y1H) system, and SRG (senescence-related gene) and HARBI1 (harbinger transposase derived 1) proteins were verified to bind the promoter region of CpGLIP1 gene, which suggests that their function is to enhance their regulation in drought and cold response by CpGLIP1. These findings comprehensively demonstrate the function of GDSL lipase in C. praecox. It also provides effective references to elucidate the potential molecular regulatory mechanism of GDSL lipases and provide candidate genes to improve the tolerance of crops, such as drought and cold stress, in future research.

VvJAZ13 Positively Regulates Cold Tolerance in Arabidopsis and Grape.

Cold stress adversely impacts grape growth, development, and yield. Therefore, improving the cold tolerance of grape is an urgent task of grape breeding. The Jasmonic acid (JA) pathway responsive gene JAZ plays a key role in plant response to cold stress. However, the role of JAZ in response to low temperatures in grape is unclear. In this study, VvJAZ13 was cloned from the 'Pinot Noir' (Vitis vinefera cv. 'Pinot Noir') grape, and the potential interacting protein of VvJAZ13 was screened by yeast two-hybrid (Y2H). The function of VvJAZ13 under low temperature stress was verified by genetic transformation. Subcellular localization showed that the gene was mainly expressed in cytoplasm and the nucleus. Y2H indicated that VvF-box, VvTIFY5A, VvTIFY9, Vvbch1, and VvAGD13 may be potential interacting proteins of VvJAZ13. The results of transient transformation of grape leaves showed that VvJAZ13 improved photosynthetic capacity and reduced cell damage by increasing maximum photosynthetic efficiency of photosystem II (Fv/Fm), reducing relative electrolyte leakage (REL) and malondialdehyde (MDA), and increasing proline content in overexpressed lines (OEs), which played an active role in cold resistance. Through the overexpression of VvJAZ13 in Arabidopsis thaliana and grape calli, the results showed that compared with wild type (WT), transgenic lines had higher antioxidant enzyme activity and proline content, lower REL, MDA, and hydrogen peroxide (H2O2) content, and an improved ability of scavenging reactive oxygen species. In addition, the expression levels of CBF1-2 and ICE1 genes related to cold response were up-regulated in transgenic lines. To sum up, VvJAZ13 is actively involved in the cold tolerance of Arabidopsis and grape, and has the potential to be a candidate gene for improving plant cold tolerance.

Decoding VaCOLD1 Function in Grapevines: A Membrane Protein Enhancing Cold Stress Tolerance.

In globally cultivated grapevines, low-temperature stress poses a persistent challenge. Although COLD1 is recognized as a cold receptor in rice, its function in grapevine cold signaling is unclear. Here, we identified VaCOLD1, a transmembrane protein from the cold-tolerant Vitis amurensis Rupr, which is primarily located on plasma and endoplasmic reticulum membranes. Broadly expressed across multiple tissues, VaCOLD1 responds to various environmental stresses, particularly to cold. Its promoter contains distinct hormone- and stress-responsive elements, with GUS assays confirming widespread expression in Arabidopsis thaliana. Validation of interaction between VaCOLD1 and VaGPA1, together with their combined expression in yeast and grape calli, notably improved cold endurance. Overexpression of VaCOLD1 enhances cold tolerance in Arabidopsis by strengthening the CBF-COR signaling pathway. This is achieved through shielding against osmotic disturbances and modifying the expression of ABA-mediated genes. These findings emphasize the critical role of the VaCOLD1-VaGPA1 complex in mediating the response to cold stress via the CBF-COR pathway.

Orthogroup and phylotranscriptomic analyses identify transcription factors involved in the plant cold response: A case study of Arabidopsis BBX29

C-repeat binding factors (CBFs) are well-known transcription factors (TFs) that regulate plant cold acclimation. RNA sequencing (RNA-seq) data from diverse plant species provide opportunities to identify other TFs involved in the cold response. However, this task is challenging because gene gain and loss has led to an intertwined community of co-orthologs and in-paralogs between and within species. Using orthogroup (closely related homologs) analysis, we identified 10,549 orthogroups in five representative eudicots. A phylotranscriptomic analysis of cold-treated seedlings from eudicots identified 35 high-confidence conserved cold-responsive transcription factor orthogroups (CoCoFos). These 35 CoCoFos included the well-known cold-responsive regulators CBFs, HSFC1, ZAT6/10, and CZF1 among others. We used Arabidopsis BBX29 for experimental validation. Expression and genetic analyses showed that cold-induction of BBX29 is CBF- and abscisic acid-independent, and BBX29 is a negative regulator of cold tolerance. Integrative RNA-seq and Cleavage Under Targets and Tagmentation followed by sequencing analyses revealed that BBX29 represses a set of cold-induced TFs (ZAT12, PRR9, RVE1, MYB96, etc.). Altogether, our analysis yielded a library of eudicot CoCoFos and demonstrated that BBX29 is a negative regulator of cold tolerance in Arabidopsis.

Over-activation of cold tolerance in arabidopsis causes carbohydrate shortage compared with Chorispora bungeana

Many plants cope with cold stress by developing acquired freezing tolerance (AFT) through cold acclimation (CA), and some species have strong basal freezing tolerance (BFT) independent of CA. Although CA has been extensively studied, its potential in agricultural applications is still unclear. Here, carbohydrate metabolism and transcriptome in AFT plant Arabidopsis and BFT plant Chorispora bungeana were compared with each other. The results showed that, although both species were able to accumulate soluble sugars during CA, leaf starch accumulation in the daytime was almost blocked in Arabidopsis while it was greatly enhanced in C. bungeana, revealing that Arabidopsis experienced carbohydrate shortage during CA. Transcriptome and pathway enrichment analysis found that genes for photosynthesis antenna proteins were generally repressed by cold stress in both species. However, cold-up-regulated genes were enriched in protein translation in Arabidopsis, whilst they were enriched in carotenoid biosynthesis, flavonoid biosynthesis, and beta-amylases in C. bungeana. Furthermore, weighted gene co-expression network analysis (WGCNA) showed that the inhibition of starch accumulation was associated with down-regulation of genes for photosynthesis antenna proteins and up-regulation of genes for protein translation, DNA repair, and proteasome in Arabidopsis but not in C. bungeana. Taken together, our results revealed that over-activation of common tolerant mechanisms resulted in insufficient carbohydrate supplies in Arabidopsis during CA, and photoprotective mechanisms played important roles in cold adaptation of C. bungeana. These findings uncovered the drawback of CA in improving freezing tolerance and highlighted photoprotection as a possible solution for agricultural applications.

Identification of Brassica rapa BrEBF1 homologs and their characterization in cold signaling

EIN3-binding F-box 1 (EBF1) is involved in cold tolerance in Arabidopsis; however, its exact roles in cold signaling in Brassica rapa remain uncertain. Herein, we demonstrated that EBF1 homologs are highly conserved in Brassica species, but their copy numbers are diverse, with some motifs being species specific. Cold treatment activated the expression of EBF1 homologs BrEBF1 and BrEBF2 in B. rapa; however, their expression schemas were diverse in different cold-resistant varieties of the plant. Subcellular localization analysis revealed that BrEBF1 is a nuclear-localized F-box protein, and cold treatment did not alter its localization but induced its degradation. BrEBF1 overexpression enhanced cold tolerance, reduced cold-induced ROS accumulation, and enhanced MPK3 and MPK6 kinase activity in Arabidopsis. Our study revealed that BrEBF1 positively regulates cold tolerance in B. rapa and that BrEBF1-regulated cold tolerance is associated with ROS scavenging and MPK3 and MPK6 kinase activity through the C-repeat binding factor pathway.

Be sweet, be strong, and be tolerant: ERDL4 regulates sugar transport and promotes cold tolerance in Arabidopsis.

Be sweet, be strong, and be tolerant: ERDL4 regulates sugar transport and promotes cold tolerance in Arabidopsis.

AeWRKY32 from okra regulates anthocyanin accumulation and cold tolerance in Arabidopsis

Okra (Abelmoschus esculentus L.) is a tropical crop species, and its growth and development are severely affected by cold stress. Recent studies have identified a potential association between WRKY transcription factors and the cold response mechanism of crops. In this study, the AeWRKY32 transcription factor that encodes 482 amino acids was amplified from A. esculentus, and its expression level was found to be the highest in the okra flower. AeWRKY32 localized to the nucleus and displayed transcriptional activation capability. Under normal conditions, overexpression of AeWRKY32 induced anthocyanin accumulation, with higher expression levels of AtCHS1, AtCHI4, AtF3H1, and AtDFR2 in transgenic Arabidopsis. Under cold stress, anthocyanin levels were further elevated in transgenic Arabidopsis plants. At the same time, AeWRKY32 overexpression promoted ABA biosynthesis, inhibited H2O2 and O2− generation, induced stomatal closure, reduced electrolyte leakage, and thus improved the cold resistance of transgenic Arabidopsis. Furthermore, under cold stress, the expression profiles of AtCOR413, AtCOR15B, AtCBF1, and AtCBF2 were upregulated in transgenic Arabidopsis. Overall, our study provides evidence that AeWRKY32 serves as a crucial regulator in both anthocyanin accumulation and cold tolerance of transgenic Arabidopsis. Our findings could provide insights into the molecular mechanism linking AeWRKYs to plant cold tolerance.

An AP2/ERF transcription factor VvERF63 positively regulates cold tolerance in Arabidopsis and grape leaves

Cold stress adversely impacts grape growth, development, and yield. Thus, enhancing the cold tolerance of grape is a priority for grape breeding. Several APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors (TFs) play critical roles in plant responses to various abiotic stresses. However, only a few ERF TFs in grape have been identified under various stress. Here, we cloned a novel ERF gene, VvERF63 from ‘Cabernet Sauvignon’ ( Vitis vinifera L . ), which is strongly induced by cold treatment and exogenous abscisic acid (ABA) treatment, and has transcriptional activation activity in yeast. Overexpression of VvERF63 in Arabidopsis improved cold resistance at the seedling and mature stages. Moreover, VvERF63 -overexpressing and silencing grape leaves displayed improved and decreased cold tolerance, respectively. These results indicated that VvERF63 plays a positive role in cold tolerance by enhancing the photosynthetic capacity, and ameliorating cellular damage, which was evidenced by improved maximum photosynthetic efficiency of photosystem II (Fv/Fm), chlorophyll content, lower electrolyte leakage and malondialdehyde, as well as by increased antioxidant enzymes activities in the transgenic Arabidopsis . Additionally, the expression levels of stress- and antioxidant- related genes were upregulated in VvERF63 -overexpressing plants. Our results suggested that VvERF63 positively participated in the cold resistance response and has excellent potential as a candidate gene for improving the cold tolerance of plant. • VvERF63 was an ethylene response factor, the expression of which was induced by cold and ABA treatment. • VvERF63 could increase photosynthetic capacity and enhance membrane stabilization in VvERF63 -overexpressing grape leaves and Arabidopsis under cold stress. • VvERF63 plays a role in cold tolerance by increasing activities of antioxidant enzymes and expression levels of stress-responsive genes. • VvERF63 possibly mediates CBF-COR signal pathway to improve plant cold tolerance in transgenic Arabidopsis and transient transgenic grape leaves.

MdbHLH4 negatively regulates apple cold tolerance by inhibiting MdCBF1/3 expression and promoting MdCAX3L-2 expression.

Low temperature affects the yield and quality of crops. Inducer of CBF expression 1 (ICE1) plays a positive role in plant cold tolerance by promoting the expression of CRT binding factor (CBF) and cold-responsive (COR) genes. Several ICE1-interacting transcription factors (TFs) that regulate plant cold tolerance have been identified. However, how these TFs affect the function of ICE1 and CBF expression under cold conditions remains unclear. Here, we identified the MYC-type TF MdbHLH4, a negative regulator of cold tolerance in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) plants. Under cold conditions, MdbHLH4 inhibits the expression of MdCBF1 and MdCBF3 by directly binding to their promoters. It also interacts with MdICE1L, a homolog of AtICE1 in apple, and inhibits the binding of MdICE1L to the promoters of MdCBF1/3 and thus their expression. We showed that MdCAX3L-2, a Ca2+/H+ exchanger (CAX) family gene that negatively regulates plant cold tolerance, is also a direct target of MdbHLH4. MdbHLH4 reduced apple cold tolerance by promoting MdCAX3L-2 expression. Moreover, overexpression of either MdCAX3L-2 or MdbHLH4 promoted the cold-induced ubiquitination and degradation of MdICE1L. Overall, our results reveal that MdbHLH4 negatively regulates plant cold tolerance by inhibiting MdCBF1/3 expression and MdICE1L promoter-binding activity, as well as by promoting MdCAX3L-2 expression and cold-induced MdICE1L degradation. These findings provide insights into the mechanisms by which ICE1-interacting TFs regulate CBF expression and ICE1 function and thus plant cold tolerance.

MRNA N6 -methyladenosine is critical for cold tolerance in Arabidopsis.

SUMMARYPlants respond to low temperatures by altering the mRNA abundance of thousands of genes contributing to numerous physiological and metabolic processes that allow them to adapt. At the post‐transcriptional level, these cold stress‐responsive transcripts undergo alternative splicing, microRNA‐mediated regulation and alternative polyadenylation, amongst others. Recently, m6A, m5C and other mRNA modifications that can affect the regulation and stability of RNA were discovered, thus revealing another layer of post‐transcriptional regulation that plays an important role in modulating gene expression. The importance of m6A in plant growth and development has been appreciated, although its significance under stress conditions is still underexplored. To assess the role of m6A modifications during cold stress responses, methylated RNA immunoprecipitation sequencing was performed in Arabidopsis seedlings esposed to low temperature stress (4°C) for 24 h. This transcriptome‐wide m6A analysis revealed large‐scale shifts in this modification in response to low temperature stress. Because m6A is known to affect transcript stability/degradation and translation, we investigated these possibilities. Interestingly, we found that cold‐enriched m6A‐containing transcripts demonstrated the largest increases in transcript abundance coupled with increased ribosome occupancy under cold stress. The significance of the m6A epitranscriptome on plant cold tolerance was further assessed using the mta mutant in which the major m6A methyltransferase gene was mutated. Compared to the wild‐type, along with the differences in CBFs and COR gene expression levels, the mta mutant exhibited hypersensitivity to cold treatment as determined by primary root growth, biomass, and reactive oxygen species accumulation. Furthermore, and most importantly, both non‐acclimated and cold‐acclimated mta mutant demonstrated hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis.

Heterologous expression of the cysteine proteinase gene VaCP17 from the cold-adapted grapevine Vitis amurensis increases cold tolerance in Arabidopsis

Cysteine proteinases (thiol) carry out diverse and critical functions in plants through their ability to hydrolyze peptide bonds in target proteins. Here, we cloned a cysteine proteinase gene designated VaCP17 from a highly cold-resistant wild Vitis amurensis accession ‘Shuangyou’, and then its potential function in cold resistance was investigated. The results showed that the CDS of VaCP17 is 1404 bp, encoding 467 amino acids, the VaCP17 protein localized to the cell membrane. Expression of CP17 was highly distinctive among different structures of ‘Shuangyou’ and the cold-sensitive Vitis vinifera cultivar ‘Red Globe’, with the highest expression in the stem of ‘Shuangyou’ and the flower of ‘Red Globe’. Arabidopsis plants constitutively expressing a VaCP17-GREEN FLUORESCENT PROTEIN fusion (35S::VaCP17-GFP) showed increased survival after transient exposure to freezing (-6 °C), and showed lower electrolyte leakage and MDA content, higher soluble sugar content and SOD, POD and CAT activities, as compared with non-transgenic Arabidopsis controls. The expression of nine cold-resistance related genes (CBF1, CBF2, CBF3, RD29A, COR15A, KIN1, NCED3, AOC1 and JAZ10) in 35S::VaCP17-GFP plants was increased under cold treatment at 4 °C, relative to control plants. Using a yeast two-hybrid system, we identified VaNAC72, VaCAM7 and VaDi19 as potential interactors of VaCP17, and their interactions were demonstrated by a bimolecular fluorescence complementation assay. In conclusion, we revealed that VaCP17 can enhance cold resistance by influencing physiology and biochemistry and the expression of cold resistance related genes under cold stress.

Disulfide reductase activity of thioredoxin-h2 imparts cold tolerance in Arabidopsis

Many thioredoxin-h (Trx-h) proteins, cytosolic isotypes of Trxs, have been functionally characterized in plants; however, the physiological function of Arabidopsis Trx-h2, which harbors two active site cysteine (Cys) residues and an N-terminal extension peptide containing a fatty acid acylation site, remains unclear. In this study, we investigated the physiological function of Trx-h2 by performing several abiotic stress treatments using trx-h1–3 knockout mutant lines, and found that the reductase function of Trx-h2 is critical for cold resistance in Arabidopsis. Plants overexpressing Trx-h2 in the trx-h2 mutant background (Trx-h2OE/trx-h2) showed strong cold tolerant phenotypes compared with Col-0 (wild type) and trx-h2 mutant plants. By contrast, Trx-h2(C/S)OE/trx-h2 plants expressing a variant Trx-h2 protein, in which both active site Cys residues were substituted by serine (Ser) residues, showed high cold sensitivity, similar to trx-h2 plants. Moreover, cold-responsive (COR) genes were highly up-regulated in Trx-h2OE/trx-h2 plants but not in trx-h2 and Trx-h2(C/S)OE/trx-h2 plants under cold conditions. These results explicitly suggest that the cytosolic Trx-h2 protein relays the external cold stress signal to downstream cold defense signaling cascades through its protein disulfide reductase function.

Overexpression of CpSIZ1, a SIZ/PIAS-Type SUMO E3 Ligase from Wintersweet (Chimonanthus praecox), Delays Flowering, Accelerates Leaf Senescence and Enhances Cold Tolerance in Arabidopsis

Wintersweet (Chimonanthus praecox L.) is a traditional winter-flowering plant in China and a popular cut flower in winter. Its unique flowering characteristics under cold stress may involve the regulation of a large number of proteins. Protein post-translational modification is an important regulating type for the gene function. However, little is known about the post-translational modification in wintersweet. SUMOylation is an important post-translational modification in eukaryotes. Small ubiquitin-like modifier (SUMO) E3 ligases perform multiple functional regulatory activities in plants via SUMOylation. Here, we cloned and identified a SIZ/PIAS-type SUMO E3 ligase, CpSIZ1, from wintersweet. CpSIZ1 shared high similarity with other SIZ1 proteins. Quantitative real-time PCR (qRT-PCR) indicated that CpSIZ1 was expressed in all tissues tested, with the highest expression in flower wither period of stage 6, and followed by mature leaves except for different flower development stages. The ectopic expression of CpSIZ1 in Arabidopsis, including the CpSIZ1 overexpression in siz1-2 mutant (HB line) and CpSIZ1 overexpression in WT (OE line), not only promoted vegetative growth, delayed flowering and accelerated leaf senescence, but also improve the cold tolerance in Arabidopsis. Therefore, our studies have enrich the understanding of function of SIZ1 gene in woody plant, and provide a good foundation for further research on the post-translational modification regulation mechanism in this winter-flowering plant.

Overexpression of Arabidopsis ICE1 enhances yield and multiple abiotic stress tolerance in indica rice

ABSTRACT ICE1 (Inducer of CBF Expression 1), a MYC-type bHLH transcription factor, is a regulator of cold tolerance in Arabidopsis. Indica rice, which occupies the major rice cultivated area, is highly sensitive to cold stress. Hence in this study, Arabidopsis ICE1 (AtICE1) was overexpressed in indica rice to analyze its role in reproductive stage cold and other abiotic stress tolerance to indica rice. AtICE1 was overexpressed by using stress inducible AtRD29A promoter in mega rice cv. MTU1010. Under cold stress conditions, AtICE1 overexpression lines showed lower accumulation of MDA and H2O2, higher membrane stability, and thus higher seedling survival rate than the WT plants. Expression levels of OsDREB1A, OsMYB3R2, and OsTPP1 were significantly higher in transgenics as compared with WT under cold stress conditions. AtICE1 transgenic rice plants produced 44–60% higher grain yield as compared with WT plants under control conditions in three independent experiments. Of the three AtICE1 overexpression lines, two lines produced significantly higher grain yield as compared with WT plants after recovery from cold, salt and drought stresses. AtICE1 overexpression lines showed significantly higher stomatal density and conductance under non-stress conditions. qRT-PCR analysis showed that expression levels of stomatal pathway genes viz., OsSPCH1, OsSPCH2, OsSCR1, OsSCRM1, OsSCRM2 and OsMUTE were significantly higher in AtICE1 transgenics as compared with WT plants. The components of water use viz., stomatal conductance, photosynthesis, and instantaneous WUE were higher in transgenics as compared with WT plants. The results showed that AtICE1 confers multiple stress tolerance to indica rice, and the role of ICE1 in stress tolerance and stomatal development is conserved across species.

TRANSTHYRETIN-LIKE and BYPASS1-LIKE co-regulate growth and cold tolerance in Arabidopsis

BackgroundCold stress inhibits normal physiological metabolism in plants, thereby seriously affecting plant development. Meanwhile, plants also actively adjust their metabolism and development to adapt to changing environments. Several cold tolerance regulators have been found to participate in the regulation of plant development. Previously, we reported that BYPASS1-LIKE (B1L), a DUF793 family protein, participates in the regulation of cold tolerance, at least partly through stabilizing C-REPEAT BINDING FACTORS (CBFs). In this study, we found that B1L interacts with TRANSTHYRETIN-LIKE (TTL) protein, which is involved in brassinosteroid (BR)-mediated plant growth and catalyses the synthesis of S-allantoin, and both proteins participate in modulating plant growth and cold tolerance.ResultsThe results obtained with yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that B1L directly interacted with TTL. Similar to the ttl-1 and ttl-2 mutants, the b1l mutant displayed a longer hypocotyl and greater fresh weight than wild type, whereas B1L-overexpressing lines exhibited a shorter hypocotyl and reduced fresh weight. Moreover, ttl-1 displayed freezing tolerance to cold treatment compared with WT, whereas the b1l mutant and TTL-overexpressing lines were freezing-sensitive. The b1l ttl double mutant had a developmental phenotype and freezing tolerance that were highly similar to those of ttl-1 compared to b1l, indicating that TTL is important for B1L function. Although low concentrations of brassinolide (0.1 or 1 nM) displayed similarly promoted hypocotyl elongation of WT and b1l under normal temperature, it showed less effect to the hypocotyl elongation of b1l than to that of WT under cold conditions. In addition, the b1l mutant also contained less amount of allantoin than Col-0.ConclusionOur results indicate that B1L and TTL co-regulate development and cold tolerance in Arabidopsis, and BR and allantoin may participate in these processes through B1L and TTL.

RhNAC31, a novel rose NAC transcription factor, enhances tolerance to multiple abiotic stresses in Arabidopsis

This work identifies a new rose NAM/CUC subgroup member, RhNAC31. Overexpression of RhNAC31 in Arabidopsis confers salt, cold, and drought tolerance along with enhanced ABA sensitivity. Plant-specific NAM, ATAF1/2, and CUC (NAC) transcription factors serve essential functions in plant development and plant responses to environmental cues. Yet, transcription factors specific to the rose (Rosa hybrida) NAM/CUC3 subfamily are poorly understood. Here, we identify a novel NAM/CUC3-subfamily transcription factor, RhNAC31, that is associated with flower opening and can be induced by increased salt, cold, and dehydration stress treatment. RhNAC31 has a transactivation region in its C-terminal region, and its overexpression is associated with enhanced cold tolerance in Arabidopsis, conferring a higher survival rate and reduced reactive oxygen (H2O2 and O2−) levels. Under salt stress conditions, plants overexpressing RhNAC31 displayed increased germination rates and lower levels of H2O2, malondiadehyde (MDA), peroxidase (POD), and superoxide dismutase (SOD). Moreover, RhNAC31 conferred enhanced drought resistance by reducing the rate of water loss through leaves. Further characterization revealed a higher sensitivity of RhNAC31 transgenic plants to abscisic acid (ABA) both during and post-germination, causing lower germination and root inhibition rates under ABA treatment. Quantitative PCR experiments showed that numerous abiotic stress-related genes were activated by RhNAC31 overexpression. Our results highlight RhNAC31 as a positive transcriptional regulator of tolerance to multiple abiotic pressures, and we conclude that RhNAC31 has potential for use in the molecular breeding of stress-tolerant crops.

Characterization and Cloning of Grape Circular RNAs Identified the Cold Resistance-Related Vv-circATS1.

Circular RNAs (circRNAs) are widely distributed and play essential roles in a series of developmental processes, although none have been identified or characterized in grapevines (Vitis vinifera). In this study, we characterized the function of grape circRNA and uncovered thousands of putative back-splicing sites by global transcriptome analysis. Our results indicated that several reported circRNA prediction algorithms should be used simultaneously to obtain comprehensive and reliable circRNA predictions in plants. Furthermore, the length of introns flanking grape circRNAs was closely related to exon circularization. Although the longer introns flanking grape circRNAs appeared to circularize more efficiently, a 20- to 50-nt region seemed large enough to drive grape circRNA biogenesis. In addition, the endogenous introns flanking circularized exon(s) in conjunction with reverse complementary sequences could support the accurate and efficient circularization of various exons in grape, which constitutes a new tool for exploring the functional consequences caused by circRNA expression. Finally, we identified 475 differentially expressed circRNAs in grape leaves under cold stress. Overexpression of Vv-circATS1, a circRNA derived from glycerol-3-P acyltransferase, improved cold tolerance in Arabidopsis (Arabidopsis thaliana), while the linear RNA derived from the same sequence cannot. These results indicate the functional difference between circRNA and linear RNA, and provide new insight into plant abiotic stress resistance.

The glutamate receptors AtGLR1.2 and AtGLR1.3 increase cold tolerance by regulating jasmonate signaling in Arabidopsis thaliana

Plant glutamate-like receptors (GLRs), which are homologs of mammalian ionotropic glutamate receptors (iGluRs), are thought to be involved in plant growth, development, and environmental stress responses. In this study, we demonstrated that two members of Arabidopsis glutamate-like receptors, AtGLR1.2 and AtGLR1.3, play positive roles in the plant response to cold stress. Genetic and biochemical experiments revealed that exogenous jasmonate could attenuate the cold sensitivity of glr1.2 and glr1.3 mutants, and the overexpression of GLR1.2 or GLR1.3 enhanced cold tolerance by increasing endogenous jasmonate levels under cold stress. In addition, the expression of genes in the CBF/DREB1 signaling pathway was decreased in the glr1.2 and glr1.3 mutants, but was promoted in GLR1.2-OE and GLR1.3-OE transgenic plants compared with the wild-type during cold treatment. Further investigation revealed that AtGLR1.2 and AtGLR1.3 independently drove similar functions without directly interacting. Together, our findings suggest that AtGLR1.2 and 1.3 positively enhance cold tolerance in Arabidopsis by activating endogenous jasmonate accumulation and subsequently promoting the downstream CBF/DREB1 cold response pathway during cold stress.

Overexpression of ethylene response factors VaERF080 and VaERF087 from Vitis amurensis enhances cold tolerance in Arabidopsis

Low temperature is a vital environmental factor that affects plant growth, development and crop yields. Ethylene response factor (ERF) is a plant specific transcription factor with important roles in stress adaptation. However, the regulatory mechanism on how grapevine responds to cold stress is still unclear. In this study, we isolated and characterized two ERF genes from Vitis amurensis designated as VaERF080 and VaERF087. The function of these two genes was investigated in transgenic Arabidopsis. We found that both VaERF080 and VaERF087 were induced by cold stress and ethylene. The overexpression of these genes in Arabidopsis resulted in enhanced cold tolerance. The VaERF080- and VaERF087-overexpressing (OE) Arabidopsis lines showed lower levels of malondialdehyde (MDA) content and higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress compared to wild-type (WT) Arabidopsis. Nine representative cold-responsive genes, CBF1, CBF2, ICE1, ZAT12, KIN1, SIZ1, RD29A, COR15A, and COR47, were significantly upregulated in OE Arabidopsis. These results revealed that both VaERF080 and VaERF087 may improve the cold tolerance of transgenic Arabidopsis through increasing antioxidant enzyme activities and regulating the expression of cold-related genes.