Heat stress is a major environmental constraint for crop production worldwide. To respond to and cope with heat stress, plants synthesize heat shock proteins (HSPs), which are often molecular chaperones and are under the control of heat stress transcription factors (HSFs). Very little is known about the upstream regulators of HSFs. In a forward genetic screen for regulators of C-REPEAT BINDING FACTOR (CBF) gene expression (RCFs), we identified RCF2 and found that it is allelic to CPL1/FIERY2, which encodes a homolog of C-terminal domain phosphatase. Our results also showed that, in addition to being critical for cold stress tolerance, RCF2 is required for heat stress-responsive gene regulation and thermotolerance, because, compared with the wild type, the rcf2-1 mutant is hypersensitive to heat stress and because the reduced thermotolerance is correlated with lower expression of most of the 21 HSFs and some of the HSPs in the mutant plants. We found that RCF2 interacts with the NAC transcription factor NAC019 and that RCF2 dephosphorylates NAC019 in vivo. The nac019 mutant is more sensitive to heat stress than the wild type, and chromatin immunoprecipitation followed by quantitative PCR analysis revealed that NAC019 binds to the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1. Overexpression of RCF2 or NAC019 in Arabidopsis thaliana increases thermotolerance. Together, our results suggest that, through dephosphorylation of NAC019, RCF2 is an integrator of high-temperature signal transduction and a mechanism for HSF and HSP activation.

Salt stress is an important environmental factor that significantly limits crop productivity worldwide. Studies on responses of plants to salt stress in recent years have identified novel signaling pathways and have been at the forefront of plant stress biology and plant biology in general. Thus far, research on salt stress in plants has been focused on cytoplasmic signaling pathways. In this study, we discovered a nuclear calcium-sensing and signaling pathway that is critical for salt stress tolerance in the reference plant Arabidopsis. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor. We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na+/H+ antiporter essential for salt tolerance. Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.

Chilling (0–18°C) and freezing (<0°C) are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation) mediates the adaptation to cold stresses in nature (e.g., in alpine regions). Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. Chorispora bungeana is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h). To disentangle how C. bungeana copes with these intricate cold stresses through epigenetic modifications, plants of C. bungeana were treated at 4°C (chilling) and -4°C (freezing) over five periods of time (0–24 h). Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of C. bungeana in response to chilling and freezing. It was found that the alterations in DNA methylation of C. bungeana largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h). Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that C. bungeana responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for C. bungeana to adapt to the intricate cold stresses in the alpine areas.

A ω-3 fatty acid desaturase gene from Chorispora bungeana confers multi-stress tolerance by the integrated regulation of membrane, [Ca2+]cyt, ROS, and stress-responsive genes in tobacco

As a result of global warming, vegetation suffers from repeated freeze-thaw cycles caused by more frequent short-term low temperatures induced by hail, snow, or night frost. Therefore, short-term freezing stress of plants should be investigated particularly in light of the current climatic conditions. Alcohol dehydrogenase (ADH) plays a central role in the metabolism of alcohols and aldehydes and it is a key enzyme in anaerobic fermentation. ADH1 responds to plant growth and environmental stress; however, the function of ADH1 in the response to short-term freezing stress remains unknown. Using real-time quantitative fluorescence PCR, the expression level of ADH1 was analyzed at low temperature (4°C). The lethal temperature was calculated based on the electrolyte leakage tests for both ADH1 deletion mutants (adh1) and wild type (WT) plants. To further investigate the relationship between ADH1 and cold tolerance in plants, low-Mr polar metabolite analyses of Arabidopsis adh1 and WT were performed at cold temperatures using gas chromatography-mass spectrometry. This investigation focused on freezing treatments (cold acclimation group: −6°C for 2 h with prior 4°C for 7 d, cold shock group: −6°C for 2 h without cold acclimation) and recovery (23°C for 24 h) with respect to seedling growth at optimum temperature. The experimental results revealed a significant increase in ADH1 expression during low temperature treatment (4°C) and at a higher lethal temperature in adh1 compared to that in the WT. Retention time indices and specific mass fragments were used to monitor 263 variables and annotate 78 identified metabolites. From these analyses, differences in the degree of metabolite accumulation between adh1 and WT were detected, including soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine). In addition, the correlation-based network analysis highlighted some metabolites, e.g., melibiose, fumaric acid, succinic acid, glycolic acid, and xylose, which enhanced connectedness in adh1 network under cold chock. When considered collectively, the results showed that adh1 possessed a metabolic response to freezing stress and ADH1 played an important role in the cold stress response of a plant. These results expands our understanding of the short-term freeze response of ADH1 in plants.

Chorispora bungeana Fisch. and C.A. Mey (Chorispora bungeana) is a rare alpine subnival plant species that is highly tolerant to environmental stress. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth, development and stress responses. We have isolated and partially characterized a full-length cDNA encoding PLD from the calluses of Chorispora bungeana (C. bungeana), with the aim of furthering our understanding of the role of PLD at the molecular level. The C. bungeana PLD was found to encode a 903-amino acid protein with moderate to high nucleotide sequence similarity to previously reported plant PLD beta genes. The predicted CbPLD sequence showed the characteristic C2 domain and the phospholipase domains conferring calcium sensitivity and the enzyme activity, respectively. The transcripts of CbPLD accumulated highly when Chorispora bungeana was treated with cold (4 and -4°C), abscisic acid (ABA), salinity, hydrogen peroxide (H 2 O 2 ) and heat. These results indicate that the CbPLD may play an important role in response to stresses in Chorispora bungeana.

No abstract

Abscisic acid responsive element binding factors (ABFs) play crucial roles in plant responses to abiotic stress. However, little is known about the roles of ABFs in alpine subnival plants, which can survive under extreme environmental conditions. Here, we cloned and characterized an ABF1 homolog, CbABF1 , from the alpine subnival plant Chorispora bungeana . Expression of CbABF1 was induced by cold, drought, and abscisic acid. Subcellular localization analysis revealed that CbABF1 was located in the nucleus. Further, CbABF1 had transactivation activity, which was dependent on the N-terminal region containing 89 residues. A Snf1-related protein kinase, CbSnRK2.6, interacted with CbABF1 in yeast two-hybrid analysis and bimolecular fluorescence complementation assays. Transient expression assay revealed that CbSnRK2.6 enhanced the transactivation of CbABF1 on ABRE cis -element. We further found that heterologous expression of CbABF1 in tobacco improved plant tolerance to freezing and drought stress, in which the survival rates of the transgenic plants increased around 40 and 60%, respectively, compared with wild-type plants. Moreover, the transgenic plants accumulated less reactive oxygen species, accompanied by high activities of antioxidant enzymes and elevated expression of stress-responsive genes. Our results thus suggest that CbABF1 is a transcription factor that plays an important role in cold and drought tolerance and is a candidate gene in molecular breeding of stress-tolerant crops.