Cold Signaling Pathway Research Articles

Exogenous Melatonin Promotes Cold Tolerance in Grape Seedlings: Physiological, Transcriptomic, and Functional Evidence.

Melatonin (MEL) is an antioxidant molecule that enhances plant tolerance to environmental stress. However, the mechanisms by which MEL regulates cold signaling pathways in grapes under cold stress remain elusive. Here, we investigated the physiological and transcriptomic changes in grape seedlings treated with exogenous MEL to determine their protective role under cold stress. Results showed that 150 μM MEL effectively attenuated cold-induced cell damage by reducing reactive oxygen species (ROS) and preserving the chloroplast structure and function. MEL also inhibited tannin degradation, which contributed to its protective effect. Exogenous MEL promoted the synthesis of endogenous MEL, abscisic acid, auxin, and cytokinin while inhibiting gibberellin. Transcriptomic profiling revealed 776 differentially expressed transcripts in MEL-treated samples compared to controls. Functional analysis of a candidate hub gene, VvHSFA6b, showed that its overexpression in grape calli enhances cold tolerance by activating jasmonic acid synthesis pathway genes, promoting JA accumulation, and inhibiting JAZ-repressed transcription factors.

Postharvest fruit quality of tomatoes influenced by an ethylene signaling component during long-term cold storage.

Ethylene production is essential for improving cold resistance of postharvest tomatoes. However, the role of ethylene signaling pathway in maintaining fruit quality during long-term cold storage remains poorly understood. Here, we demonstrated that a partial loss of function in ethylene signaling by mutation of Ethylene Response Factor 2 (SlERF2), worsened fruit quality during cold storage, as determined by visual characterization, and physiological analyses of membrane damage and reactive oxygen species metabolism. In addition, the transcriptions of genes related to abscisic acid (ABA) biosynthesis and signaling were also altered by SlERF2 gene in response to cold storage. Furthermore, mutation of SlERF2 gene compromised cold-induced expression of genes in the C-repeat/dehydration-responsive binding factor (CBF) signaling pathway. Therefore, it's concluded that an ethylene signaling component, SlERF2 contributed to the regulations of ABA biosynthesis and signaling, as well as CBF cold signaling pathway, ultimately affecting the fruit quality during long-term cold storage of tomatoes.

MdWRKY40is directly promotes anthocyanin accumulation and blocks MdMYB15L, the repressor of MdCBF2, which improves cold tolerance in apple

Cold stress is an important factor that limits apple production. In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337’ and ‘60-160’, which are resistant and sensitive to cold stress, respectively. The enriched pathways of differentially expressed genes (DEGs) and physiological changes in ‘M9T337’ and ‘60-160’ plantlets were clearly different after cold stress (1°C) treatment for 48 h, suggesting that they have differential responses to cold stress. The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance. When we overexpressed MdWRKY40is and MdWRKY48 in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2. Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter. Yeast two-hybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2 inhibitor MdMYB15L through the leucine zipper (LZ). When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L. In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance. These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

SlMAPK3 Positively Regulates the Ethylene Production of Postharvest Tomato Fruits and Is Involved in Ethylene-Mediated Cold Tolerance.

Mitogen-activated protein kinase (MAPK) cascades and ethylene are crucial for plant growth, development, and stress responses, but their potential mechanisms in cold resistance remain unclear. We revealed that SlMAPK3 transcript levels were dramatically induced by cold treatment in an ethylene-dependent manner. Under cold stress, the proline content of SlMAPK3-overexpression fruit was 96.5 and 115.9% higher than that of wild-type fruit (WT), respectively, while the ion leakage was 37.3 and 32.5% lower than that of WT. RNA sequencing revealed that overexpression of SlMAPK3 caused upregulation of genes that are enriched in the ethylene-activated signaling pathway (GO:0009873), cold signaling pathway (GO:0009409), and heat signaling pathway (GO:0009408). RT-qPCR demonstrated that the expression levels of SlACS2, SlACS4, SlSAHH, SlCBF1, SlDREB, SlGolS1, and SlHSP17.7 in the OE.MAPK3 fruits were consistent with the RNA sequencing results. Meanwhile, the knockout of SlMAPK3 reduced the ethylene content, ACC content, and ACS activity. Moreover, the knockout of SlMAPK3 reduced the positive effect of ethylene in cold stress, while suppressing the expression of SlICE1 and SlCBF1. In conclusion, our study demonstrated a novel mechanism by which SlMAPK3 positively regulates the ethylene production of postharvest tomato fruits and is involved in ethylene-mediated cold tolerance.

Grape CIPK18 acts as a positive regulator of CBF cold signaling pathway by modulating ROS homeostasis

Cold temperatures trigger a diverged Ca 2+ decoding paradigm that includes the proteins CBL (Calcineurin B-like proteins) and CIPK (CBL-interacting protein kinases). Here, putative CBL and CIPK genes were surveyed in cold-tolerant wild Amur grape ( Vitis amurensis ), designated as VaCBLs and VaCIPKs , respectively. The transcripts of 8 VaCBLs and 19 VaCIPKs encoded in this genome were confirmed, among which VaCIPK18 physically interacted with all 8 VaCBLs through the C-terminal regulatory domain (NAF), indicating that it may serve as a node regulator. VaCIPK18 was ubiquitously expressed in various tissues and was highly induced by cold, drought, salt, and ABA . The NAF domain of VaCIPK18 was required for functional homodimerization in yeast as well as in planta . Complementation tests confirmed that VaCIPK18 rescued the cold sensitivity of ArabidopsisAtcipk3 mutant, in which the expressions of CBFs were suppressed and is sensitive to cold and freezing stresses. In addition, overexpression of VaCIPK18 enhanced the cold tolerance by upregulating the C-repeat binding transcription factor (CBF) transcriptional pathway under normal or cold conditions, and reducing reactive oxygen species production. Thus, our study demonstrates that VaCIPK18 positively regulates cold signaling at least partly through the direct transcriptional control of cold-regulated CBFs and modulation of ROS homeostasis . Moreover, VaCIPK18 was found to interact with VaMYB4a in vivo and in vitro . Our findings provide evidence that VaCIPK18 functions as a key node in the CBL-CIPK network and may interact with VaMYB4a to contribute to cold response in grape plants. • An Amur grape VaCIPK18 could function as a node regulator of CBL-CIPK network. • VaCIPK18 modulates cold tolerance by directly controlling CBFs and ROS homeostasis. • 'VaCIPK18-VaMYB4a' module may regulate cold tolerance.

CsMYB Transcription Factors Participate in Jasmonic Acid Signal Transduction in Response to Cold Stress in Tea Plant (Camellia sinensis).

Low-temperature stress is an increasing problem for the cultivation of tea (Camellia sinensis), with adverse effects on plant growth and development and subsequent negative impacts on the tea industry. Methyl jasmonate (MeJA), as a plant inducer, can improve the cold-stress tolerance in tea plants. R2R3-MYB transcription factors (TFs) are considered potentially important regulators in the resistance to cold stress in plants. However, the molecular mechanisms, by which MYB TFs via the jasmonic acid pathway respond to cold stress in the tea plant, remain unknown. In this study, physiological and biochemical assays showed that exogenous MeJA application could effectively promote ROS scavenging in the tea plant under cold stress, maintaining the stability of the cell membrane. Sixteen R2R3-MYB TFs genes were identified from the tea plant genome database. Quantitative RT-PCR analysis showed that three CsMYB genes were strongly induced under a combination of MeJA and cold-stress treatment. Subcellular localization assays suggest CsMYB45, CsMYB46, and CsMYB105 localized in the nucleus. Exogenous MeJA treatment enhanced the overexpression of CsMYB45, CsMYB46, and CsMYB105 in E. coli and improved the growth and survival rates of recombinant cells compared to an empty vector under cold stress. Yeast two-hybrid and bimolecular fluorescence complementation experiments confirmed that CsMYB46 and CsMYB105 interacted with CsJAZ3, CsJAZ10, and CsJAZ11 in the nucleus. Taken together, these results highlight that CsMYB45, CsMYB46, and CsMYB105 are not only key components in the cold-stress signal response pathway but also may serve as points of confluence for cold stress and JA signaling pathways. Furthermore, our findings provide new insight into how MYB TFs influence cold tolerance via the jasmonic acid pathway in tea and provide candidate genes for future functional studies and breeding.

The interaction of CsWRKY4 and CsOCP3 with CsICE1 regulates CsCBF1/3 and mediates stress response in tea plant (Camellia sinensis)

Low temperature stress limits the geographical distribution of tea plants (Camellia sinensis) and the yield and quality of tea. CsICE1 is a crucial regulator of tea plant’s cold signaling pathway; however, the regulatory mechanism of CsICE1 is still unclear. In this study, we found that the N-terminus of CsICE1 contains a transcriptional activation domain and a region of protein interaction. Yeast two-hybrid and bimolecular fluorescence complementation experiments confirmed that CsWRKY4 and CsOCP3 interacted with CsICE1 in the nucleus. Like CsICE1, CsWRKY4 and CsOCP3 were localized in the nucleus, and CsOCP3 was also targeted to the chloroplast. The expression of CsWRKY4 and CsOCP3 was downregulated under cold stress, unlike that of CsICE1. Under biotic stresses and salicylic acid treatment, the expression of all three genes was increased, whereas jasmonic acid treatment resulted in decreased expression of all three genes. Dual-luciferase transient assays revealed that CsICE1 promoted the expression of CsCBF1 and CsCBF3, whereas CsWRKY4 and CsOCP3 inhibited the expression of CsCBF1 and CsCBF3 and attenuated their induction by CsICE1. Taken together, these results suggest that CsWRKY4 and CsOCP3 interact with CsICE1 and regulate CsCBF1/3, thus mediating the stress response in tea plant. Furthermore, these results highlight that CsICE1 is not only a key component in the low temperature signal response pathway, but it may also serve as a point of confluence for cold and other signaling pathways.

CPK28-NLP7 module integrates cold-induced Ca2+ signal and transcriptional reprogramming in Arabidopsis.

Exposure to cold triggers a spike in cytosolic calcium (Ca2+) that often leads to transcriptional reprogramming in plants. However, how this Ca2+ signal is perceived and relayed to the downstream cold signaling pathway remains unknown. Here, we show that the CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) initiates a phosphorylation cascade to specify transcriptional reprogramming downstream of cold-induced Ca2+ signal. Plasma membrane (PM)–localized CPK28 is activated rapidly upon cold shock within 10 seconds in a Ca2+-dependent manner. CPK28 then phosphorylates and promotes the nuclear translocation of NIN-LIKE PROTEIN 7 (NLP7), a transcription factor that specifies the transcriptional reprogramming of cold-responsive gene sets in response to Ca2+, thereby positively regulating plant response to cold stress. This study elucidates a previously unidentified mechanism by which the CPK28-NLP7 regulatory module integrates cold-evoked Ca2+ signal and transcriptome and thus uncovers a key strategy for the rapid perception and transduction of cold signals from the PM to the nucleus.

Genetic Mechanisms of Cold Signaling in Wheat (Triticum aestivum L.).

Cold stress is a major environmental factor affecting the growth, development, and productivity of various crop species. With the current trajectory of global climate change, low temperatures are becoming more frequent and can significantly decrease crop yield. Wheat (Triticum aestivum L.) is the first domesticated crop and is the most popular cereal crop in the world. Because of a lack of systematic research on cold signaling pathways and gene regulatory networks, the underlying molecular mechanisms of cold signal transduction in wheat are poorly understood. This study reviews recent progress in wheat, including the ICE-CBF-COR signaling pathway under cold stress and the effects of cold stress on hormonal pathways, reactive oxygen species (ROS), and epigenetic processes and elements. This review also highlights possible strategies for improving cold tolerance in wheat.

Osa-miR1320 targets the ERF transcription factor OsERF096 to regulate cold tolerance via JA-mediated signaling.

MicroRNAs play key roles in abiotic stress response. Rice (Oryza sativa L.) miR1320 is a species-specific miRNA that contributes to miR168-regulated immunity. However, it is still unknown whether miR1320 is involved in rice response to abiotic stress. In this study, we illustrated that the miR1320 precursor generated two mature miR1320s, miR1320-3p, and miR1320-5p, and they both displayed decreased expression under cold stress. Genetic evidence showed that miR1320 overexpression resulted in increased cold tolerance, while miR1320 knock down (KD) reduced cold tolerance. Furthermore, an APETALA2/ethylene-responsive factor (ERF) transcription factor OsERF096 was identified as a target of miR1320 via 5'-RACE and dual luciferase assays. OsERF096 expression was altered by miR1320 overexpression and KD and exhibited an opposite pattern to that of miR1320 in different tissues and under cold stress. Consistently, OsERF096 negatively regulated cold stress tolerance. Furthermore, we suggested that OsERF096 could bind to the GCC and DRE cis-elements and act as a transcriptional activator in the nucleus. Based on RNA-sequencing and targeted metabolomics assays, we found that OsERF096 modified hormone content and signaling pathways. Finally, phenotypic and reverse transcription-quantitative PCR assays showed that jasmonic acid (JA) methyl ester application recovered the cold-sensitive phenotype and JA-activated expression of three Dehydration Responsive Element Binding genes in the OsERF096-OE line. Taken together, our results strongly suggest that the miR1320-OsERF096 module regulates cold tolerance by repressing the JA-mediated cold signaling pathway.

CabHLH79 Acts Upstream of CaNAC035 to Regulate Cold Stress in Pepper.

Cold stress is one of the main restricting factors affecting plant growth and agricultural production. Complex cold signaling pathways induce the expression of hundreds of cold-sensitive genes. The NAC transcription factor CaNAC035 has previously been reported to significantly influence the response of pepper to cold stress. Here, using Yeast one-hybrid (Y1H) library screened to search for other relevant molecular factors, we identified that CabHLH79 directly binds to the CaNAC035 promoter. Different basic helix–loop–helix (bHLH) transcription factors (TFs) in plants significantly respond to multiple plant stresses, but the mechanism of bHLHs in the cold tolerance of pepper is still unclear. This study investigated the functional characterization of CabHLH79 in the regulation of cold resistance in pepper. Down-regulation of CabHLH79 in pepper by virus-induced gene silencing (VIGS) increased its sensitivity to low temperature, whereas overexpression of CabHLH79 in pepper or Arabidopsis enhanced cold resistance. Compared with control plants, VIGS mediated of CabHLH79 had lower enzyme activity and related gene expression levels, accompanied by higher reactive oxygen species (ROS) accumulation, relative electrolyte leakage (REL), and malondialdehyde accumulation (MDA) contents. Transient overexpression of CabHLH79 pepper positively regulated cold stress response genes and ROS genes, which reduced REL and MDA contents. Similarly, ectopic expression of CabHLH79 in Arabidopsis showed less ROS accumulation, and higher enzymes activities and expression levels. These results indicated that CabHLH79 enhanced cold tolerance by enhancing the expression of ROS-related and other cold stress tolerance-related genes. Taken together, our results showed a multifaceted module of bHLH79-NAC035 in the cold stress of pepper.

Functional Identification of ICE Transcription Factors in Rubber Tree

ICE (inducer of CBF expression) is a positive regulator of cold signaling pathway in plants. Identification of ICE transcription factors is important for the sustainable development of the natural rubber planting industry in nontraditional regions where sudden cold waves often occur. In this study, five ICE genes were isolated from genome of rubber tree (Hevea brasiliensis Muell. Arg.) for analysing tolerance to cold stress. They shared an ICE-specific region in the highly conserved bHLH-ZIP domain and were localized in the nucleus. The HbICEs were different in transcript abundance and expression patterns in response to cold and drought stresses and among different rubber tree clones. Generally, the expression level of HbICEs was significantly higher in the cold-tolerant rubber tree clones than that in the cold-sensitive rubber tree clones. Overexpression of HbICE1, HbICE2, and HbICE4 significantly enhanced the cold tolerance of transgenic Arabidopsis and tobacco, which showed a significant increase in chlorophyll content and decrease in relative water content and conductivity at the early stage of cold stress in comparison with wild-type plants. Furthermore, overexpression of HbICE2 and HbICE4, but also HbICE1 enhanced drought tolerance in transgenic Arabidopsis. The cold tolerance of rubber tree clones is positively controlled by the expression level of HbICE1, HbICE2, and HbICE4.

HbSnRK2.6 Functions in ABA-Regulated Cold Stress Response by Promoting HbICE2 Transcriptional Activity in Hevea brasiliensis.

Low temperature remarkably limits rubber tree (Hevea brasiliensis Muell. Arg.) growth, latex production, and geographical distribution, but the underlying mechanisms of Hevea brasiliensis cold stress response remain elusive. Here, we identified HbSnRK2.6 as a key component in ABA signaling functions in phytohormone abscisic acid (ABA)-regulated cold stress response in Hevea brasiliensis. Exogenous application of ABA enhances Hevea brasiliensis cold tolerance. Cold-regulated (COR) genes in the CBF pathway are upregulated by ABA. Transcript levels of all five HbSnRK2.6 members are significantly induced by cold, while HbSnRK2.6A, HbSnRK2.6B, and HbSnRK2.6C can be further activated by ABA under cold conditions. Additionally, HbSnRK2.6s are localized in the cytoplasm and nucleus, and can physically interact with HbICE2, a crucial positive regulator in the cold signaling pathway. Overexpression of HbSnRK2.6A or HbSnRK2.6B in Arabidopsis extensively enhances plant responses to ABA and expression of COR genes, leading to increased cold stress tolerance. Furthermore, HbSnRK2.6A and HbSnRK2.6B can promote transcriptional activity of HbICE2, thus, increasing the expression of HbCBF1. Taken together, we demonstrate that HbSnRK2.6s are involved in ABA-regulated cold stress response in Hevea brasiliensis by regulating transcriptional activity of HbICE2.

The CRY2-COP1-HY5-BBX7/8 module regulates blue light-dependent cold acclimation in Arabidopsis.

Light and temperature are two key environmental factors that coordinately regulate plant growth and development. Although the mechanisms that integrate signaling mediated by cold and red light have been unraveled, the roles of the blue light photoreceptors cryptochromes in plant responses to cold remain unclear. In this study, we demonstrate that the CRYPTOCHROME2 (CRY2)-COP1-HY5-BBX7/8 module regulates blue light-dependent cold acclimation in Arabidopsis thaliana. We show that phosphorylated forms of CRY2 induced by blue light are stabilized by cold stress and that cold-stabilized CRY2 competes with the transcription factor HY5 to attenuate the HY5-COP1 interaction, thereby allowing HY5 to accumulate at cold temperatures. Furthermore, our data demonstrate that B-BOX DOMAIN PROTEIN7 (BBX7) and BBX8 function as direct HY5 targets that positively regulate freezing tolerance by modulating the expression of a set of cold-responsive genes, which mainly occurs independently of the C-repeat-binding factor pathway. Our study uncovers a mechanistic framework by which CRY2-mediated blue-light signaling enhances freezing tolerance, shedding light on the molecular mechanisms underlying the crosstalk between cold and light signaling pathways in plants.

Ethylene increases the cold tolerance of apple via the MdERF1B-MdCIbHLH1 regulatory module.

Cold stress has always been a major abiotic factor affecting the yield and quality of temperate fruit crops. Ethylene plays a critical regulatory role in the cold stress response, but the underlying molecular mechanisms remain elusive. Here, we revealed that ethylene positively modulates apple responses to cold stress. Treatment with 1-aminocyclopropane-1-carboxylate (an ethylene precursor) and aminoethoxyvinylglycine (an ethylene biosynthesis inhibitor) respectively increased and decreased the cold tolerance of apple seedlings. Consistent with the positive effects of ethylene on cold stress responses, a low-temperature treatment rapidly induced ethylene release and the expression of MdERF1B, which encodes an ethylene signaling activator, in apple seedlings. Overexpression of MdERF1B significantly increased the cold tolerance of apple plant materials (seedlings and calli) and Arabidopsis thaliana seedlings. A quantitative real-time PCR analysis indicated that MdERF1B upregulates the expression of the cold-responsive gene MdCBF1 in apple seedlings. Moreover, MdCIbHLH1, which functions upstream of CBF-dependent pathways, enhanced the binding of MdERF1B to target gene promoters as well as the consequent transcriptional activation. The stability of MdERF1B-MdCIbHLH1 was affected by cold stress and ethylene. Furthermore, MdERF1B interacted with the promoters of two genes critical for ethylene biosynthesis, MdACO1 and MdERF3. The resulting upregulated expression of these genes promoted ethylene production. However, the downregulated MdCIbHLH1 expression in MdERF1B-overexpressing apple calli significantly inhibited ethylene production. These findings imply that MdERF1B-MdCIbHLH1 is a potential regulatory module that integrates the cold and ethylene signaling pathways in apple.

Exogenous DA-6 Improves the Low Night Temperature Tolerance of Tomato Through Regulating Cytokinin.

Low night temperature (LNT) causes environmental stress and has a severe and negative impact on plant growth and productivity. Synthetic elicitors can regulate plant growth and induce defense mechanisms from this type of stress. Here, we evaluated the effect of the exogenous growth regulator diethyl aminoethyl hexanoate (DA-6) in tomato leaf response to LNT stress. Our results showed that exogenous DA-6 activates the expression of chlorophyll synthesis and photosystem-related genes, and results in higher photosynthetic activity and chlorophyll production. Furthermore, DA-6 can regulate the synthesis of endogenous cytokinin (CTK) and the expression of decomposition genes to stabilize chloroplast structure, reduce oxidative damage, and maintain the photochemical activity of tomato leaves under LNT stress. DA-6 maintains a high level of ABA content and induces the expression of CBF genes, indicating that DA-6 may participate in the cold response signaling pathway and induce the expression of downstream low temperature response genes and accumulation of compatible osmolytes. This study unravels a mode of action by which plant growth regulators can improve low temperature tolerance and provides important considerations for their application to alleviate the harmful effects of cold stress.

Transcriptome analysis of Chongyi wild mandarin, a wild species more cold-tolerant than Poncirus trifoliata, reveals key pathways in response to cold

Much effort has been made on the dissection of cold response in citrus, but relevant study in wild germplasm has been rare. Based on the result of the membership function value analysis with 12 physiological indexes, we reported a wild citrus germplasm, Chongyi wild mandarin (Citrus reticulata), which was more cold-tolerant than a cold-hardy rootstock, Poncirus trifoliata. To gain an overview of transcriptome changes under cold, leaves of Chongyi wild mandarin were sampled at 0, 1, 5 d after 0 °C treatment, and sequenced using an Illumina sequencing platform. Totally, 3921 and 5995 differentially expressed genes (DEGs) were identified in 1 d and 5 d, respectively. And then, RNA-seq results were further validated via real-time quantitative PCR with 17 DEGs. Many DEGs related to various cold signaling pathways were identified, especially those related to brassinosteroid cascade. Around 10 % of the total DEGs were transcription factors from 67 families, suggesting that transcriptional regulation, both C-repeat/dehydration responsive element-binding factor-dependent/independent pathways, was crucial for the cold tolerance of Chongyi wild mandarin. Our data also revealed that the transcriptional levels of many DEGs related to anti-oxidant enzymes and osmoprotectants metabolism were altered, which were consistent with the results of physiological experiments. Moreover, a number of DEGs involved in ABA, BR, GA metabolisms were isolated. Accordingly, the levels of ABA and BR were significantly increased under cold, but GAs were decreased. Taken together, this work provided a comprehensive view of physiological and transcriptional alterations in cold response of Chongyi wild mandarin.

A chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) provides insights into disease resistance, cold tolerance and genome evolution in Citrus.

SUMMARYTrifoliate orange (Poncirus trifoliata), a deciduous close relative of evergreen Citrus, has important traits for citrus production, including tolerance/resistance to citrus greening disease (Huanglongbing, HLB) and other major diseases, and cold tolerance. It has been one of the most important rootstocks, and one of the most valuable sources of resistance and tolerance genes for citrus. Here we present a high‐quality, chromosome‐scale genome assembly of P. trifoliata. The 264.9‐Mb assembly contains nine chromosomal pseudomolecules with 25 538 protein‐coding genes, covering 97.2% of the estimated gene space. Comparative analyses of P. trifoliata and nine Citrus genomes revealed 605 species‐specific genes and six rapidly evolving gene families in the P. trifoliata genome. Poncirus trifoliata has evolved specific adaptation in the C‐repeat/DREB binding factor (CBF)‐dependent and CBF‐independent cold signaling pathways to tolerate cold. We identified candidate genes within quantitative trait loci for HLB tolerance, and at the loci for resistance to citrus tristeza virus and citrus nematode. Genetic diversity analysis of Poncirus accessions and Poncirus/Citrus hybrids shows a narrow genetic base in the US germplasm collection, and points to the importance of collecting and preserving more natural genetic variation. Two phenotypically divergent Poncirus accessions are found to be clonally related, supporting a previous conjecture that dwarf Flying Dragon originated as a mutant of a non‐dwarfing type. The high‐quality genome reveals features and evolutionary insights of Poncirus, and it will serve as a valuable resource for genetic, genomic and molecular research and manipulation in citrus.

Varying Atmospheric CO2 Mediates the Cold-Induced CBF-Dependent Signaling Pathway and Freezing Tolerance in Arabidopsis.

Changes in the stomatal aperture in response to CO2 levels allow plants to manage water usage, optimize CO2 uptake and adjust to environmental stimuli. The current study reports that sub-ambient CO2 up-regulated the low temperature induction of the C-repeat Binding Factor (CBF)-dependent cold signaling pathway in Arabidopsis (Arabidopsis thaliana) and the opposite occurred in response to supra-ambient CO2. Accordingly, cold induction of various downstream cold-responsive genes was modified by CO2 treatments and expression changes were either partially or fully CBF-dependent. Changes in electrolyte leakage during freezing tests were correlated with CO2′s effects on CBF expression. Cold treatments were also performed on Arabidopsis mutants with altered stomatal responses to CO2, i.e., high leaf temperature 1-2 (ht1-2, CO2 hypersensitive) and β-carbonic anhydrase 1 and 4 (ca1ca4, CO2 insensitive). The cold-induced expression of CBF and downstream CBF target genes plus freezing tolerance of ht1-2 was consistently less than that for Col-0, suggesting that HT1 is a positive modulator of cold signaling. The ca1ca4 mutant had diminished CBF expression during cold treatment but the downstream expression of cold-responsive genes was either similar to or greater than that of Col-0. This finding suggested that βCA1/4 modulates the expression of certain cold-responsive genes in a CBF-independent manner. Stomatal conductance measurements demonstrated that low temperatures overrode low CO2-induced stomatal opening and this process was delayed in the cold tolerant mutant, ca1ca4, compared to the cold sensitive mutant, ht1-2. The similar stomatal responses were evident from freezing tolerant line, Ox-CBF, overexpression of CBF3, compared to wild-type ecotype Ws-2. Together, these results indicate that CO2 signaling in stomata and CBF-mediated cold signaling work coordinately in Arabidopsis to manage abiotic stress.

The N-terminal and third transmembrane domain of PsCor413im1 are essential for targeting to chloroplast envelope membrane

Cold-regulated (COR) genes, located downstream of the C-repeat binding factors (CBFs) in cold signaling pathways, play a central role in plant response to cold stress. In our previous studies, a Cor413 chloroplast envelope membrane protein, PsCor413im1, was identified from the cold-tolerant plant Phlox subulata. Its overexpression enhanced cold tolerance and altered AtCor15 expression in Arabidopsis. In the present study, the function of PsCor413im1 was further investigated. Transmission electron microscope observation showed that the chloroplast envelope membrane of cold-treated transgenic Arabidopsis seedlings was more stable than that of cold-treated wild-type seedlings. Subcellular localization of green fluorescent protein as a marker revealed that the N-terminal and putative third transmembrane domain (TMD) of PsCor413im1 were essential for its targeting of the chloroplast envelope membrane. Furthermore, overexpression of PsCor413im1 fragments containing N-terminal and third TMD also altered the expression of AtCor15 genes in Arabidopsis. Overall, our results suggest that PsCor413im1 may stabilize the chloroplast envelope membrane under cold stress, and its N-terminal and third TMD are important for its targeting capability and function.