Plant Freezing Tolerance Research Articles

NtSAP9 confers freezing tolerance in Nicotiana tabacum plants

Abiotic stresses, such as extreme temperatures, drought, and salinity, significantly affect plant growth and productivity. Among these, cold stress is particularly detrimental, impairing cellular processes and leading to reduced crop yields. In recent years, stress-associated proteins (SAPs) containing A20 and AN1 zinc-finger domains have emerged as crucial regulators in plant stress responses. However, the functions of SAPs in tobacco plants remain unclear. Here, we isolated Nicotiana tabacum SAP9 (NtSAP9), whose expression was induced by cold treatment, based on RNA-sequences data. Knock down of NtSAP9 expression reduced freezing tolerance, while overexpression conferred freezing tolerance in transgenic tobacco plants, as indicated by relative electrolytic leakage and photosystem II photochemical efficiency. Untargeted metabolomics via liquid chromatography-tandem mass spectrometry revealed distinct metabolic profiles between WT and NtSAP9-overexpressing tobacco plants under normal and low temperature conditions. Upregulation of amino acids like D-Glutamine, DL-Glutamine, and O-Acetyl-L-serine suggests NtSAP9 enhances cold tolerance. Further expression analysis by quantitative real-time PCR indicated that NtSAP9 participates in cold stress response possibly through amino acid synthesis-related genes expression, such as glutamine synthetase and glutamate dehydrogenase. These findings improve our understanding of SAP proteins in tobacco's response to cold stress.

Molecular Mechanisms Underlying Freezing Tolerance in Plants: Implications for Cryopreservation.

Cryopreservation is a crucial technique for the long-term ex situ conservation of plant genetic resources, particularly in the context of global biodiversity decline. This process entails freezing biological material at ultra-low temperatures using liquid nitrogen, which effectively halts metabolic activities and preserves plant tissues over extended periods. Over the past seven decades, a plethora of techniques for cryopreserving plant materials have been developed. These include slow freezing, vitrification, encapsulation dehydration, encapsulation-vitrification, droplet vitrification, cryo-plates, and cryo-mesh techniques. A key challenge in the advancement of cryopreservation lies in our ability to understand the molecular processes underlying plant freezing tolerance. These mechanisms include cold acclimatization, the activation of cold-responsive genes through pathways such as the ICE-CBF-COR cascade, and the protective roles of transcription factors, non-coding RNAs, and epigenetic modifications. Furthermore, specialized proteins, such as antifreeze proteins (AFPs) and late embryogenesis abundant (LEA) proteins, play crucial roles in protecting plant cells during freezing and thawing. Despite its potential, cryopreservation faces significant challenges, particularly in standardizing protocols for a wide range of plant species, especially those from tropical and subtropical regions. This review highlights the importance of ongoing research and the integration of omics technologies to improve cryopreservation techniques, ensuring their effectiveness across diverse plant species and contributing to global efforts regarding biodiversity conservation.

Antisense transcription from stress-responsive transcription factors fine-tunes the cold response in Arabidopsis.

Transcription of antisense long noncoding RNAs (lncRNAs) occurs pervasively across eukaryotic genomes. Only a few antisense lncRNAs have been characterized and shown to control biological processes, albeit with idiosyncratic regulatory mechanisms. Thus, we largely lack knowledge about the general role of antisense transcription in eukaryotic organisms. Here, we characterized genes with antisense transcription initiating close to the poly(A) signal of genes (PAS genes) in Arabidopsis (Arabidopsis thaliana). We compared plant native elongation transcript sequencing (plaNET-seq) with RNA sequencing during short-term cold exposure and detected massive differences between the response in active transcription and steady-state levels of PAS gene-derived mRNAs. The cold-induced expression of transcription factors B-BOX DOMAIN PROTEIN28 (BBX28) and C2H2-TYPE ZINC FINGER FAMILY PROTEIN5 (ZAT5) was detected by plaNET-seq, while their steady-state level was only slightly altered due to high mRNA turnover. Knockdown of BBX28 and ZAT5 or of their respective antisense transcripts severely compromised plant freezing tolerance. Decreased antisense transcript expression levels resulted in a reduced cold response of BBX28 and ZAT5, revealing a positive regulatory role of both antisense transcripts. This study expands the known repertoire of noncoding transcripts. It highlights that native transcription approaches can complement steady-state RNA techniques to identify biologically relevant players in stress responses.

A double-edged sword: Phosphorylation of Ca2+ channel CNGC20 fine-tunes plant freezing tolerance.

A double-edged sword: Phosphorylation of Ca2+ channel CNGC20 fine-tunes plant freezing tolerance.

Differential phosphorylation of Ca2+-permeable channel CYCLIC NUCLEOTIDE-GATED CHANNEL20 modulates calcium-mediated freezing tolerance in Arabidopsis.

Plants respond to cold stress at multiple levels, including increasing cytosolic calcium (Ca2+) influx and triggering the expression of cold-responsive genes. In this study, we show that the Ca2+-permeable channel CYCLIC NUCLEOTIDE-GATED CHANNEL20 (CNGC20) positively regulates freezing tolerance in Arabidopsis (Arabidopsis thaliana) by mediating cold-induced Ca2+ influx. Moreover, we demonstrate that the leucine-rich repeat receptor-like kinase PLANT PEPTIDE CONTAINING SULFATED TYROSINE1 RECEPTOR (PSY1R) is activated by cold, phosphorylating and enhancing the activity of CNGC20. The psy1r mutant exhibits decreased cold-evoked Ca2+ influx and freezing tolerance. Conversely, COLD-RESPONSIVE PROTEIN KINASE1 (CRPK1), a protein kinase that negatively regulates cold signaling, phosphorylates and facilitates the degradation of CNGC20 under prolonged periods of cold treatment, thereby attenuating freezing tolerance. This study thus identifies PSY1R and CRPK1 kinases that regulate CNGC20 activity and stability, respectively, thereby antagonistically modulating freezing tolerance in plants.

월동 배추의 추위 스트레스 반응성 ERF 유전자 발굴과 활용

To mitigate plant freeze damage caused by extreme weather events, we need to elucidate the freeze resistance mechanism of freeze-tolerant plants through molecular biological approaches. Our research focuses on identifying the cold stress responsive, ERF(Ethylene-Responsive transcription Factor) genes that confer freeze tolerance in overwintering Chinese cabbage. Additionally, we aim to explore the molecular basis for conferring freezing tolerance to other organisms through genetic transformation. Expression of the ERF, cold stress responsive gene in overwintering Chinese cabbage, leads to the synthesis of AFP(Antifreeze Protein), which results in cellular freezing tolerance. We identified and sequenced functionally important ERF domain regions within the ERF genes, which we named BrERF1 and BrERF2. Their responsiveness to cold stress was confirmed through RT-PCR and qRT-PCR. Furthermore, we propose strategies to introduce BrERF1 and BrERF2 insertion plasmids into other organisms for transformation. Significant improvements in the freezing tolerance of transgenic organisms are expected to improve the productivity of these target organisms. In particular, the development of permanently transgenic plants using the BrERFs gene offers the possibility of increasing plant production under cold stress.

Freezing treatment under light conditions leads to a dramatic enhancement of freezing tolerance in cold-acclimated Arabidopsis.

Overwintering plants survive subzero temperatures by cold acclimation (CA), wherein they acquire freezing tolerance through short-term exposure to low temperatures above 0°C. The freezing tolerance of CA plants increases when they are subsequently exposed to mild subzero temperatures, a phenomenon known as second-phase cold hardening (2PH). Here, we explored the molecular mechanism and physiological conditions of 2PH. The results show that, compared with supercooling, a freezing treatment during 2PH after CA enhanced the freezing tolerance of Arabidopsis. This required CA as a pretreatment, and was designated as second-phase freezing acclimation (2PFA). Light increased the effect of 2PFA to enhance freezing tolerance after CA. C-repeat binding factor and cold-regulatedgenes were downregulated by light during the 2PFA treatment, a different transcription profile from that during CA. The freezing tolerance of 2PFA plants was decreased by the presence of the photosynthetic electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylureaduring the 2PFA treatment. Compared with wild-type plants, phototropin1,2 and phyb mutants showed lower freezing tolerance after 2PFA treatment. These results show that exposure to freezing after CA increases freezing tolerance as a secondary process, and that freezing under light conditions further increases freezing tolerance via pathways involving photoreceptors and photosynthetic electron transfer.

TaMAPK3 phosphorylates TaCBF and TaICE and plays a negative role in wheat freezing tolerance

Freezing temperature during overwintering often kills plants; plants have thus, developed a defense mechanism called ‘cold acclimation’, in which a number of genes are involved in increasing cell protection and gene expression. Mitogen-activated protein kinase (MAPK) controls proteins' activities by phosphorylation and is involved in numerous metabolic pathways. In this study, we identified the protein interaction between TaMAPK3 and the proteins in the cold response pathway, ICE41, ICE87, and CBFIVd-D9. The subcellular localization and bimolecular fluorescence complement (BiFC) assays revealed that these proteins interact in the nucleus or in the plasma membrane. Furthermore, MAPK3-mediated phosphorylation of ICE41, ICE87, and CBFIVd-D9 was verified using an in vitro phosphorylation assay. TaMAPK3-overexpressing transgenic Brachypodium showed a lower survival rate upon freezing stress and lower proline content during cold acclimation, compared to wild-type plants. Furthermore, cold response gene expression analysis revealed that the expression of these genes was suppressed in the transgenic lines under cold treatment. It was further elucidated that MAPK3 mediates the degradation of ICE and CBF proteins, which implies the negative impact of MAPK3 on the freezing tolerance of plants. This study will help to elucidate the molecular mechanisms of cold tolerance and the activity of MAPK3 in wheat.

Structural changes in cell wall pectic polymers contribute to freezing tolerance induced by cold acclimation in plants

Subzero temperatures are often lethal to plants. Many temperate herbaceous plants have a cold acclimation mechanism that allows them to sense a drop in temperature and prepare for freezing stress through accumulation of soluble sugars and cryoprotective proteins. As ice formation primarily occurs in the apoplast (the cell wall space), cell wall functional properties are important for plant freezing tolerance. Although previous studies have shown that the amounts of constituent sugars of the cell wall, in particular those of pectic polysaccharides, are altered by cold acclimation, the significance of this change during cold acclimation has not been clarified. We found that β-1,4-galactan, which forms neutral side chains of the acidic pectic rhamnogalacturonan-I, accumulates in the cell walls of Arabidopsis and various freezing-tolerant vegetables during cold acclimation. The gals1 gals2 gals3 triple mutant, which has reduced β-1,4-galactan in the cell wall, exhibited impaired freezing tolerance compared with wild-type Arabidopsis during initial stages of cold acclimation. Expression of genes involved in the galactan biosynthesis pathway, such as galactan synthases and UDP-glucose 4-epimerases, was induced during cold acclimation in Arabidopsis, explaining the galactan accumulation. Cold acclimation resulted in a decrease in extensibility and an increase in rigidity of the cell wall in the wild type, whereas these changes were not observed in the gals1 gals2 gals3 triple mutant. These results indicate that the accumulation of pectic β-1,4-galactan contributes to acquired freezing tolerance by cold acclimation, likely via changes in cell wall mechanical properties.

Strigolactones promote plant freezing tolerance by releasing the WRKY41-mediated inhibition of CBF/DREB1 expression.

Cold stress is a major abiotic stress that adversely affects plant growth and crop productivity. The C-REPEAT BINDING FACTOR/DRE BINDING FACTOR 1 (CBF/DREB1) transcriptional regulatory cascade plays a key role in regulating cold acclimation and freezing tolerance in Arabidopsis (Arabidopsis thaliana). Here, we show that max (more axillary growth) mutants deficient in strigolactone biosynthesis and signaling display hypersensitivity to freezing stress. Exogenous application of GR245DS , a strigolactone analog, enhances freezing tolerance in wild-type plants and strigolactone-deficient mutants and promotes the cold-induced expression of CBF genes. Biochemical analysis showed that the transcription factor WRKY41 serves as a substrate for the F-box E3 ligase MAX2. WRKY41 directly binds to the W-box in the promoters of CBF genes and represses their expression, negatively regulating cold acclimation and freezing tolerance. MAX2 ubiquitinates WRKY41, thus marking it for cold-induced degradation and thereby alleviating the repression of CBF expression. In addition, SL-mediated degradation of SMXLs also contributes to enhanced plant freezing tolerance by promoting anthocyanin biosynthesis. Taken together, our study reveals the molecular mechanism underlying strigolactones promote the cold stress response in Arabidopsis.

LncRNA MtCIR2 positively regulates plant-freezing tolerance by modulating CBF/DREB1 gene clusters.

Emerging evidence suggests that long noncoding RNAs (lncRNAs) are involved in regulation of plant response to environmental stress. CBF/DREB1s are highly conserved transcription factors that regulate response to cold stress in plants. However, very few lncRNAs were found to regulate expression of CBFs and cold tolerance in plant. Here, we identified a cold-responsive long intergenic noncoding RNA (MtCIR2) of CBF/DREB1 genes that were located in a major freezing tolerance QTL region of legume Medicago truncatula. We found that response of MtCIR2 transcription was more rapid than that of MtCBF/DREB1s during cold treatment. MtCIR2 positively regulated M. truncatula freezing tolerance, such that overexpression of MtCIR2 led to higher survival rate and lower cell membrane damage than wild-type plants, while mutation of MtCIR2 rendered the mutants more sensitive to cold stress. In addition, expression levels of MtCBF/DREB1s were up-regulated in the MtCIR2 overexpressing lines and down-regulated in the mutants. Among the MtCIR2-regulated genes, the strongest enriched genes were those involved in polysaccharide metabolic processes. In addition, we demonstrated that overexpression of MtCIR2 led to increases in contents of soluble sugars. These results highlight that MtCIR2 positively regulates tolerance to freezing by regulating MtCBF/DREB1s expression and glycometabolism in M. truncatula.

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

Protease inhibitor ASP enhances freezing tolerance by inhibiting protein degradation in kumquat.

Cold acclimation is a complex biological process leading to the development of freezing tolerance in plants. In this study, we demonstrated that cold-induced expression of protease inhibitor FmASP in a Citrus-relative species kumquat [Fortunella margarita (Lour.) Swingle] contributes to its freezing tolerance by minimizing protein degradation. Firstly, we found that only cold-acclimated kumquat plants, despite extensive leaf cellular damage during freezing, were able to resume their normal growth upon stress relief. To dissect the impact of cold acclimation on this anti-freezing performance, we conducted protein abundance assays and quantitative proteomic analysis of kumquat leaves subjected to cold acclimation (4°C), freezing treatment (-10°C) and post-freezing recovery (25°C). FmASP (Against Serine Protease) and several non-specific proteases were identified as differentially expressed proteins induced by cold acclimation and associated with stable protein abundance throughout the course of low-temperature treatment. FmASP was further characterized as a robust inhibitor of multiple proteases. In addition, heterogeneous expression of FmASP in Arabidopsis confirmed its positive role in freezing tolerance. Finally, we proposed a working model of FmASP and illustrated how this extracellular-localized protease inhibitor protects proteins from degradation, thereby maintaining essential cellular function for post-freezing recovery. These findings revealed the important role of protease inhibition in freezing response and provide insights on how this role may help develop new strategies to enhance plant freezing tolerance.

Freezing transcriptome analysis showed that GhZAT10 regulates freezing tolerance through a partially CBF-dependent pathway in upland cotton (Gossypium hirsutum L.)

Cotton is an important economic crop worldwide, which is mainly distributed in the tropics and subtropics; therefore, it is highly sensitive to low temperatures. ZAT and CBF play important roles in plant growth and stress responses. However, few studies have investigated the role of these genes in freezing tolerance of cotton, and the interaction between these two remains unclear. In this study, KN27–3 cotton with strong freezing tolerance was selected from 17 cotton varieties. RNA-sequencing analysis showed that KN27–3 cotton presented 6747 differentially expressed genes (DEGs), which were mostly upregulated at the early time points after the treatments. Weighted gene co-expression network analysis and Gene Ontology enrichment analysis showed that various DEGs were involved in plant abiotic stress resistance, including SRK2I, ELF3, WRKY70, and TPS6. Moreover, 31 early upregulated transcription factors, including CBF and ZAT, were identified after 15 min of freezing treatment. Such significant upregulation at the early stage suggested that GhCBF4 (Gh_A12G2357) and GhZAT10 (Gh_D05G2011) play important roles in cotton freezing stress. Virus-induced gene silencing indicated that GhCBF4- and GhZAT10-silenced plants exhibit significant freezing-sensitive phenotypes. Biofilm interferometry and dual luciferase experiments showed that GhCBF4 regulated transcription by directly binding to a CRT/DRE motif within the GhZAT10 promoter. In summary, GhZAT10 and GhCBF4 regulated plant freezing tolerance, and GhZAT10 expression was at least partially regulated by CBF. These findings expand our current understanding of the mechanisms underlying GhZAT10- and GhCBF4-mediated freezing stress reactions in cotton, thereby establishing a foundation for further research on the molecular mechanisms underlying cotton freezing tolerance.

Integration of chromatin accessibility and gene expression reveals new regulators of cold hardening to enhance freezing tolerance in Prunus mume.

Low temperature is one of the most important abiotic factors limiting the growth, development and geographical distribution of plants. Prunus mume is an attractive woody ornamental plant that blooms in early spring in Beijing. However, the molecular mechanisms underlying cold hardening to enhance freezing tolerance in Prunus genus remains elusive. This study examined the dynamic physiological responses induced by cold hardening, and identified freezing-tolerance genes by RNA-seq and ATAC-seq analyses. Cold hardening elevated the content of soluble substances and enhanced freezing resistance in P. mume. Transcriptome analysis indicated that the candidate differentially expressed genes (DEGs) were those enriched in Ca2+ signalling, mitogen-activated protein kinase (MAPK) cascade, abscisic acid signalling, and inducer of CBF expression 1 (ICE)-C-repeat binding factor (CBF) signalling pathways. The openness of gene chromatin positively correlated with the expression level of these genes. Thirteen motifs were identified in the open chromatin regions in the treatment group subjected to freezing after cold hardening. The chromatin opening of transcription start site at the proximal -177 region of cold-shock protein CS120-like (PmCSL) was markedly increased, while the expression level of PmCSL was significantly up-regulated. Overexpression of PmCSL in Arabidopsis significantly improved the freezing tolerance of transgenic plants. These findings provide new insights into the regulatory mechanism of freezing tolerance to improve breeding of cold-hardy P. mume plants.

Gesneriads, a Source of Resurrection and Double-Tolerant Species: Proposal of New Desiccation- and Freezing-Tolerant Plants and Their Physiological Adaptations.

Gesneriaceae is a pantropical family of plants that, thanks to their lithophytic and epiphytic growth forms, have developed different strategies for overcoming water scarcity. Desiccation tolerance or "resurrection" ability is one of them: a rare phenomenon among angiosperms that involves surviving with very little relative water content in their tissues until water is again available. Physiological responses of desiccation tolerance are also activated during freezing temperatures, a stress that many of the resurrection gesneriads suffer due to their mountainous habitat. Therefore, research on desiccation- and freezing-tolerant gesneriads is a great opportunity for crop improvement, and some of them have become reference resurrection angiosperms (Dorcoceras hygrometrica, Haberlea rhodopensis and Ramonda myconi). However, their difficult indoor cultivation and outdoor accessibility are major obstacles for their study. Therefore, this review aims to identify phylogenetic, geoclimatic, habitat, and morphological features in order to propose new tentative resurrection gesneriads as a way of making them more reachable to the scientific community. Additionally, shared and species-specific physiological responses to desiccation and freezing stress have been gathered as a stress response metabolic basis of the family.

Analysis of genetic diversity of valuable walnut species collected at N.V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences Using SSR markers

Walnut is one of the most economically significant nut crops. Evaluation of the genetic structure of the domestic walnut gene plasma using modern molecular genetic approaches is a relevant research task. The walnut samples collected by Tsitsin Main Moscow Botanical Garden of Academy of Sciences (MBG RAS) are of particular importance for breeding practice aimed at increasing winter freezing tolerance of plants. The seed material for this collection was introduced from different regions of the former Soviet Union, including Tajikistan, Kyrgyzstan, Ukraine, Belarus, as well as regions of Russia. The MBG RAS collection presents interest as a breeding material for mobilizing the genetic resources and replenishing the gene pool of the South of Russia with new, economically valuable walnut varieties. This work aims to analyze the genetic diversity of a J. regia genotype sample, which includes the most valuable forms from the MBG RAS collection, in order to establish their genetic relationships with samples representing the walnut gene pool of the South of Russia. The genetic analysis of the studied walnut species and varieties was carried out using eight SSR markers: WGA001, WGA376, WGA069, WGA276, WGA009, WGA202, WGA089, and WGA054. The polymorphism of microsatellite DNA markers established during genotyping indicated a high heterogeneity between the MBG RAS walnut sample and genetic resources in other regions. An analysis of genetic relationships using UPGMA and PCoA clustering methods revealed the genetic isolation of most samples in the MBG RAS collection from walnut varieties in the South of Russia. The most genetically distant samples in the MBG RAS collection were found to be 199, 196, 236, 256, 106, and 134. Therefore, these samples should be introduced in the gene pool of North-Caucasus Federal Scientific Center of Horticulture, Viticulture, and Wine-making and Nikitsky Botanical Garden with the purpose of increasing the heterogeneity of their gene pools.

Plasma membrane proteomic changes of Arabidopsis DRP1E during cold acclimation in association with the enhancement of freezing tolerance.

The freezing tolerance of plants that live in cold regions increases after exposure to low temperature, a process termed cold acclimation (CA). During CA, restructuring of the plasma membrane (PM) is important to enhance freezing tolerance. We have previously shown that the function of DYNAMIN-RELATED PROTEIN 1 E (DRP1E), which regulates endocytosis by pinching vesicles from the PM, is associated with the enhancement of freezing tolerance during CA in Arabidopsis. DRP1E is predicted to play a role in reconstituting the PM composition during CA. In this study, to test the validity of this hypothesis, we studied the changes in PM proteome patterns induced by drp1e mutation. In a detailed physiological analysis, after 3 days of CA, only young leaves showed significantly less increase in freezing tolerance in the mutant than in the wild type (WT). Using nano-liquid chromatography-tandem mass spectrometry, 496 PM proteins were identified. Among these proteins, 81 or 71 proteins were specifically altered in the WT or the mutant, respectively, in response to CA. Principal component analysis showed that the proteomic pattern differed between the WT and the mutant upon cold acclimation (CA), suggesting that DRP1E contributes to reconstruction of the PM during CA. Cluster analysis revealed that proteins that were significantly increased in the mutant after CA were biased toward glycosylphosphatidylinositol-anchored proteins, such as fasciclin-like arabinogalactan proteins. Thus, a primary target of DRP1E-associated PM reconstruction during CA is considered to be glycosylphosphatidylinositol-anchored proteins, which may be removed from the PM by DRP1E in young leaves after 3 days of CA.

Pre-stress salicylic-acid treatment as an intervention strategy for freeze-protection in spinach: Foliar versus sub-irrigation application and duration of efficacy

Exogenous application of salicylic acid (SA) to plant tissues has been shown to confer tolerance against various abiotic stresses. Recently, SA application through sub-irrigation was shown to improve plant freezing tolerance (FT). For SA treatment to be employable as an effective intervention strategy for frost protection under field conditions, it is important to study its effect on FT when applied as a foliar spray to whole plants. It is also important to determine for how long the FT-improvement by SA lasts. Present study was conducted to compare SA-induced FT of spinach (Spinacia oleracea L. ‘Reflect’) seedlings following SA-application by foliar spray vs. sub-irrigation. Durability of FT-promotive effect of SA was evaluated using three freeze-tests over a 4-d period, i.e., at 10-d, 12-d, and 14-d after the SA application. Freezing stress was applied using a temperature-controlled freeze-thaw protocol, and FT was assessed by visual observations (leaf flaccidness vs. turgidity) as well as ion-leakage assay. Data indicated that both foliar spray and sub-irrigation methods improved FT of the seedlings against a relatively moderate (−5.5 °C) as well as severe stress (−6.5 °C). Moreover, improved FT against moderate stress was sustained over a 4-d period, whereas such benefit waned somewhat against the severe stress. SA-treated leaves' growth performance was similar to the non-treated control based on dry weight, fresh weight, leaf area, and dry weight/leaf area parameters. Our results suggest that SA application as a foliar spray can potentially be used to protect field-grown transplants against episodic frosts.

Temperature modulation of CAMTA3 gene induction activity is mediated through the DNA binding domain.

The calmodulin-binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C-REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3334 , which includes the N-terminal CG-1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN-LIKE A1 (EXPL1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold-treated plants. Rather, domain-swapping experiments indicated that the CAMTA3 CG-1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG-1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C-terminal calmodulin-binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature.