Yiting Shi

Ethylene Signaling Negatively Regulates Freezing Tolerance by Repressing Expression of CBF and Type-A ARR Genes in Arabidopsis

This work examines the function of ethylene in freezing in Arabidopsis thaliana, finding that ethylene negatively regulates freezing stress signaling through the direct repression of cold-regulated CBF and type-A Arabidopsis response regulator genes targeted by EIN3. The phytohormone ethylene regulates multiple aspects of plant growth and development and responses to environmental stress. However, the exact role of ethylene in freezing stress remains unclear. Here, we report that ethylene negatively regulates plant responses to freezing stress in Arabidopsis thaliana. Freezing tolerance was decreased in ethylene overproducer1 and by the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid but increased by the addition of the ethylene biosynthesis inhibitor aminoethoxyvinyl glycine or the perception antagonist Ag+. Furthermore, ethylene-insensitive mutants, including etr1-1, ein4-1, ein2-5, ein3-1, and ein3 eil1, displayed enhanced freezing tolerance. By contrast, the constitutive ethylene response mutant ctr1-1 and EIN3-overexpressing plants exhibited reduced freezing tolerance. Genetic and biochemical analyses revealed that EIN3 negatively regulates the expression of CBFs and type-A Arabidopsis response regulator5 (ARR5), ARR7, and ARR15 by binding to specific elements in their promoters. Overexpression of these ARR genes enhanced the freezing tolerance of plants. Thus, our study demonstrates that ethylene negatively regulates cold signaling at least partially through the direct transcriptional control of cold-regulated CBFs and type-A ARR genes by EIN3. Our study also provides evidence that type-A ARRs function as key nodes to integrate ethylene and cytokinin signaling in regulation of plant responses to environmental stress.

A mutant CHS3 protein with TIR-NB-LRR-LIM domains modulates growth, cell death and freezing tolerance in a temperature-dependent manner in Arabidopsis.

Low temperature is one of environmental factors that restrict plant growth homeostasis and plant-pathogen interactions. Recent studies suggest a link between temperature responses and defense responses; however, the underlying molecular mechanisms remain unclear. In this study, the chilling sensitive 3 (chs3-1) mutant in Arabidopsis was characterized. chs3-1 plants showed arrested growth and chlorosis when grown at 16 degrees C or when shifted from 22 to 4 degrees C. chs3-1 plants also exhibited constitutively activated defense responses at 16 degrees C, which were alleviated at a higher temperature (22 degrees C). Map-based cloning of CHS3 revealed that it encodes an unconventional disease resistance (R) protein belonging to the TIR-NB-LRR class with a zinc-binding LIM domain (Lin-11, Isl-1 and Mec-3 domains) at the carboxyl terminus. The chs3-1 mutation in the conserved LIM-containing domain led to the constitutive activation of the TIR-NB-LRR domain. Consistently, the growth and defense phenotypes of chs3-1 plants were completely suppressed by eds1, sgt1b and rar1, partially by pad4 and nahG, but not by npr1 and ndr1. Intriguingly, chs3-1 plants grown at 16 degrees C showed enhanced tolerance to freezing temperatures. This tolerance was correlated with growth defect and cell death phenotypes caused by activated defense responses. Other mutants with activated defense responses, including cpr1, cpr5 and slh1 also displayed enhanced freezing tolerance. These findings revealed a role of an unconventional mutant R gene in plant growth, defense response and cold stress, suggesting a mutual interaction between cold signaling and defense responses.

Cold signal transduction and its interplay with phytohormones during cold acclimation

Cold stress is a major environmental factor that affects plant growth, development, productivity and distribution. In higher plants, the known major cold signaling pathway is the C-repeat (CRT)-binding factor/dehydration-responsive element (DRE) binding factor (CBF/DREB)-mediated transcriptional regulatory cascade, which is essential for the induction of a set of cold responsive (COR) genes. Recent studies indicate that various plant hormones are also involved in responses to cold stress. This review summarizes recent progress in cold signaling and our understanding of phytohormone signaling in the regulation of plant responses to cold stress.

The glutamate carboxypeptidase AMP1 mediates abscisic acid and abiotic stress responses in Arabidopsis

ALTERED MERISTEM PROGRAM1 (AMP1) encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis. We isolated a new mutant allele of AMP1, amp1-20, from a screen for abscisic acid (ABA) hypersensitive mutants and characterized the function of AMP1 in plant stress responses. amp1 mutants displayed ABA hypersensitivity, while overexpression of AMP1 caused ABA insensitivity. Moreover, endogenous ABA concentration was increased in amp1-20- and decreased in AMP1-overexpressing plants under stress conditions. Application of ABA reduced the AMP1 protein level in plants. Interestingly, amp1 mutants accumulated excess superoxide and displayed hypersensitivity to oxidative stress. The hypersensitivity of amp1 to ABA and oxidative stress was partially rescued by reactive oxygen species (ROS) scavenging agent. Furthermore, amp1 was tolerant to freezing and drought stress. The ABA hypersensitivity and freezing tolerance of amp1 was dependent on ABA signaling. Moreover, amp1 had elevated soluble sugar content and showed hypersensitivity to high concentrations of sugar. By contrast, the contents of amino acids were changed in amp1 mutant compared to the wild-type. This study suggests that AMP1 modulates ABA, oxidative and abotic stress responses, and is involved in carbon and amino acid metabolism in Arabidopsis.

PIF3 is a negative regulator of the CBF pathway and freezing tolerance in Arabidopsis

Significance PHYTOCHROME-INTERACTING FACTORS (PIFs) are central integrators of plants’ responses to various environmental signals. In this study, we show that PIF3 acts as a negative regulator of plant cold acclimation by directly repressing the expression of CBF genes, whereas its protein stability is negatively regulated by two F-box proteins, EBF1 and EBF2, via the 26S proteasome pathway. Moreover, EBF1 and EBF2 are degraded under cold stress, which enhances the stability of PIF3 protein. Collectively, our study establishes an important regulatory paradigm for PIF3 in preventing runaway expression of the CBF genes at low temperature, which allows plants to adapt to and withstand harsh environments. Light and temperature are major environmental factors that coordinately control plant growth and survival. However, how plants integrate light and temperature signals to better adapt to environmental stresses is poorly understood. PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), a key transcription factor repressing photomorphogenesis, has been shown to play a pivotal role in mediating plants’ responses to various environmental signals. In this study, we found that PIF3 functions as a negative regulator of Arabidopsis freezing tolerance by directly binding to the promoters of C-REPEAT BINDING FACTOR (CBF) genes to down-regulate their expression. In addition, two F-box proteins, EIN3-BINDING F-BOX 1 (EBF1) and EBF2, directly target PIF3 for 26S proteasome-mediated degradation. Consistently, ebf1 and ebf2 mutants were more sensitive to freezing than were the wild type, and the pif3 mutation suppressed the freezing-sensitive phenotype of ebf1. Furthermore, cold treatment promoted the degradation of EBF1 and EBF2, leading to increased stability of the PIF3 protein and reduced expression of the CBF genes. Together, our study uncovers an important role of PIF3 in Arabidopsis freezing tolerance by negatively regulating the expression of genes in the CBF pathway.

ABI4 represses the expression of type-A ARRs to inhibit seed germination in Arabidopsis.

Summary The plant hormone abscisic acid (ABA) plays a crucial role in regulating seed germination and post‐germination growth. ABSCISIC ACID INSENSITIVE4 (ABI4), an APETALA2 (AP2)‐type transcription factor, is required for the ABA‐mediated inhibition of seed germination. Cytokinins promote seed germination and seedling growth by antagonizing ABA signaling. However, the interaction between ABA and cytokinin signaling during seed germination remains unclear. Here, we report that ABA signaling downregulates Arabidopsis response regulators (ARRs), a class of cytokinin‐inducible genes, during seed germination and cotyledon greening. We found that the application of exogenous ABA repressed the expression of type‐A ARRs in Arabidopsis seeds and seedlings. Among the type‐A ARR family members, the expression of ARR6, ARR7 and ARR15 was upregulated in ABA‐deficient mutants, indicating that the transcriptional inhibition of type‐A ARRs requires the ABA signaling pathway. Single and multiple mutations of these ARRs resulted in increased ABA sensitivity during germination and cotyledon greening; overexpression of ARR7 or ARR15 led to an ABA‐insensitive phenotype. These observations suggest that type‐A ARRs inhibit the ABA response during seed germination and cotyledon greening. Further analysis showed that ABI4 negatively regulated the transcription of ARR6, ARR7 and ARR15 by directly binding to their promoters. Genetic analysis showed that loss‐of‐function mutations of ARR7 and ARR15 partially rescued the ABA insensitivity of abi4‐1. Thus, this study revealed that ABI4 plays a key role in ABA and cytokinin signaling by inhibiting the transcription of type‐A ARRs to inhibit seed germination and cotyledon greening. Significance Statement The plant hormone abscisic acid (ABA) suppresses seed germination, while cytokinin promotes seed germination and seedling growth. The detailed molecular mechanisms underlying ABA and cytokinin antagonism during seed germination remain elusive. Here we report that the transcription factor ABI4 negatively regulates the expression of Arabidopsis response regulators (ARRs), a class of cytokinin‐inducible genes. Thus ABI4 is a key node that integrates ABA and cytokinin signalling to modulate seed germination and cotyledon greening.

OST1‐mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses

Cold stress is a major environmental factor that negatively affects plant growth and survival. OST1 has been identified as a key protein kinase in plant response to cold stress; however, little is known about the underlying molecular mechanism. In this study, we identified BTF3 and BTF3L (BTF3‐like), β‐subunits of a nascent polypeptide‐associated complex (NAC), as OST1 substrates that positively regulate freezing tolerance. OST1 phosphorylates BTF3 and BTF3L in vitro and in vivo, and facilitates their interaction with C‐repeat‐binding factors (CBFs) to promote CBF stability under cold stress. The phosphorylation of BTF3L at the Ser50 residue by OST1 is required for its function in regulating freezing tolerance. In addition, BTF3 and BTF3L proteins positively regulate the expression of CBF genes. These findings unravel a molecular mechanism by which OST1‐BTF3‐CBF module regulates plant response to cold stress.

Molecular Regulation of CBF Signaling in Cold Acclimation

Cold stress restricts plant growth, development, and distribution. Understanding how plants transduce and respond to cold signals has long been a topic of interest. Traditional genetic and molecular analyses have identified C-repeat/DREB binding factors (CBFs) as key transcription factors that function in cold acclimation. Recent studies revealed the involvement of pivotal protein kinases and transcription factors in CBF-dependent signaling, expanding our knowledge of cold signal transduction from perception to downstream gene expression events. In this review, we summarize recent advances in our understanding of the molecular regulation of these core components of the CBF cold signaling pathway. Knowledge of the mechanism underlying the ability of plants to survive freezing temperatures will facilitate the development of crop plants with increased freezing tolerance.

Long-chain base kinase1 affects freezing tolerance in Arabidopsis thaliana

Long-chain base kinases (LCBKs) phosphorylate sphingolipid-derived long-chain base lipids and participate in the regulation of stress responses in plants. Here, we isolated a novel Arabidopsis thaliana mutant, lcbk1-2, which was extremely sensitive to freezing temperatures with or without cold acclimation. Physiological assays revealed that concentrations of osmolytes (proline and soluble sugars) and the activity of superoxide dismutase were significantly decreased in the lcbk1-2 mutant, compared with wild type. Also, the balance of reactive oxygen species (ROS) was disrupted in the lcbk1-2 mutant with or without cold treatment and, consistent with this, gene expression profiling analysis showed that the expression of cold-responsive ROS-scavenging genes was substantially decreased in the lcbk1-2 mutant. The expression of membrane lipid-related genes, which are linked to freezing tolerance in plants, was also impaired in the lcbk1-2 mutant. Furthermore, transgenic lines overexpressing LCBK1 showed enhanced freezing tolerance with over-accumulation of osmolytes. Collectively, our results suggested that LCBK1 functions as a novel positive regulator of freezing tolerance in Arabidopsis and may participate in the accumulation of osmolytes, the regulation of ROS homeostasis and lipid metabolism.

Insights into the regulation of C-repeat binding factors in plant cold signaling: Regulation of CBF cold signaling

Cold temperatures, a major abiotic stress, threaten the growth and development of plants, worldwide. To cope with this adverse environmental cue, plants from temperate climates have evolved an array of sophisticated mechanisms to acclimate to cold periods, increasing their ability to tolerate freezing stress. Over the last decade, significant progress has been made in determining the molecular mechanisms underpinning cold acclimation, including following the identification of several pivotal components, including candidates for cold sensors, protein kinases, and transcription factors. With these developments, we have a better understanding of the CBF-dependent cold-signaling pathway. In this review, we summarize recent progress made in elucidating the cold-signaling pathways, especially the C-repeat binding factor-dependent pathway, and describe the regulatory function of the crucial components of plant cold signaling. We also discuss the unsolved questions that should be the focus of future work.