Differential phosphorylation of Ca2+-permeable channel CNGC20 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. Here 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 exhibited 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.

PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis.

Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48 (K48)-linked polyubiquitin chain are usually targeted to the 26S proteasome for degradation; however, other polyubiquitin chains, such as those attached to K63, usually regulate other protein properties. Here, we show that 2 PLANT U-BOX E3 ligases, PUB25 and PUB26, facilitate both K48- and K63-linked ubiquitination of the transcriptional regulator INDUCER OF C-REPEAT BINDING FACTOR (CBF) EXPRESSION1 (ICE1) during different periods of cold stress in Arabidopsis (Arabidopsis thaliana), thus dynamically modulating ICE1 stability. Moreover, PUB25 and PUB26 attach both K48- and K63-linked Ub chains to MYB15 in response to cold stress. However, the ubiquitination patterns of ICE1 and MYB15 mediated by PUB25 and PUB26 differ, thus modulating their protein stability and abundance during different stages of cold stress. Furthermore, ICE1 interacts with and inhibits the DNA-binding activity of MYB15, resulting in an upregulation of CBF expression. This study unravels a mechanism by which PUB25 and PUB26 add different polyubiquitin chains to ICE1 and MYB15 to modulate their stability, thereby regulating the timing and degree of cold stress responses in plants.

The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants.

The transient elevation of cytosolic free calcium concentration ([Ca2+ ]cyt ) induced by cold stress is a well-established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca2+ -permeable transporter ANNEXIN1 (AtANN1) mediates cold-triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold-induced [Ca2+ ]cyt increase and upregulation of the cold-responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold-induced [Ca2+ ]cyt elevation in the ost1-3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1-AtANN1 to cold-induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.

Plasma Membrane CRPK1-Mediated Phosphorylation of 14-3-3 Proteins Induces Their Nuclear Import to Fine-Tune CBF Signaling during Cold Response.

In plant cells, changes in fluidity of the plasma membrane may serve as the primary sensor of cold stress; however, the precise mechanism and how the cell transduces and fine-tunes cold signals remain elusive. Here we show that the cold-activated plasma membrane protein cold-responsive protein kinase 1 (CRPK1) phosphorylates 14-3-3 proteins. The phosphorylated 14-3-3 proteins shuttle from the cytosol to the nucleus, where they interact with and destabilize the key cold-responsive C-repeat-binding factor (CBF) proteins. Consistent with this, the crpk1 and 14-3-3κλ mutants show enhanced freezing tolerance, and transgenic plants overexpressing 14-3-3λ show reduced freezing tolerance. Further study shows that CRPK1 is essential for the nuclear translocation of 14-3-3 proteins and for 14-3-3 function in freezing tolerance. Thus, our study reveals that the CRPK1-14-3-3 module transduces the cold signal from the plasma membrane to the nucleus to modulate CBF stability, which ensures a faithfully adjusted response to cold stress of plants.

EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis.

OST1 (open stomata 1) protein kinase plays a central role in regulating freezing tolerance in Arabidopsis; however, the mechanism underlying cold activation of OST1 remains unknown. Here, we report that a plasma membrane-localized clade-E growth-regulating 2 (EGR2) phosphatase interacts with OST1 and inhibits OST1 activity under normal conditions. EGR2 is N-myristoylated by N-myristoyltransferase NMT1 at 22°C, which is important for its interaction with OST1. Moreover, myristoylation of EGR2 is required for its function in plant freezing tolerance. Under cold stress, the interaction of EGR2 and NMT1 is attenuated, leading to the suppression of EGR2 myristoylation in plants. Plant newly synthesized unmyristoylated EGR2 has decreased binding ability to OST1 and also interferes with the EGR2-OST1 interaction under cold stress. Consequently, the EGR2-mediated inhibition of OST1 activity is released. Consistently, mutations of EGRs cause plant tolerance to freezing, whereas overexpression of EGR2 exhibits decreased freezing tolerance. This study thus unravels a molecular mechanism underlying cold activation of OST1 by membrane-localized EGR2 and suggests that a myristoyl switch on EGR2 helps plants to adapt to cold stress.

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), ß-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.

BRASSINOSTEROID-INSENSITIVE2 Negatively Regulates the Stability of Transcription Factor ICE1 in Response to Cold Stress in Arabidopsis.

Cold acclimation is a crucial strategy for plant survival at freezing temperatures. C-REPEAT BINDING FACTOR (CBF) genes are rapidly and transiently induced by low temperature and play important roles in cold acclimation. However, the mechanism underlying the attenuation of CBF expression during the later stages of the cold stress response is obscure. Here, we show that the protein kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) interacts with and phosphorylates INDUCER OF CBF EXPRESSION1 (ICE1) in Arabidopsis (Arabidopsis thaliana) under prolonged cold stress, facilitating the interaction between ICE1 and the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 and thereby promoting ICE1 degradation. The kinase activity of BIN2 is inhibited during the early stages of the cold stress response and is subsequently restored, suggesting that BIN2 mainly downregulates ICE1 abundance when CBF expression is attenuated. A loss-of-function mutation of ICE1 partially suppresses the cold-induced expression of CBFs and compromises the enhanced freezing tolerance of bin2-3 bil1 bil2 These findings reveal an important role for BIN2 in fine-tuning CBF expression, and thus in balancing plant growth and the cold stress response.

EAR1 Negatively Regulates ABA Signaling by Enhancing 2C Protein Phosphatase Activity.

The reversible phosphorylation of proteins by kinases and phosphatases is an antagonistic process that modulates many cellular functions. Protein phosphatases are usually negatively regulated by inhibitor proteins. During abscisic acid (ABA) signaling, these inhibitor proteins comprise PYR1/PYL/RCAR ABA receptors, which inhibit the core negative regulators, the clade A type 2C protein phosphatases (PP2Cs). However, it is not known whether these PP2Cs are positively regulated by other proteins. Here, we identified an Arabidopsis thaliana ear1 (enhancer of aba co-receptor1) mutant that exhibits pleiotropic ABA-hypersensitive phenotypes. EAR1 encodes an uncharacterized protein that is conserved in both monocots and dicots. EAR1 interacts with the N-terminal inhibition domains of all six PP2Cs, ABA INSENSITIVE1 (ABI1), ABI2, HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABA-HYPERSENSITIVE GERMINATION1 (AHG1), and AHG3, during ABA signaling and enhances the activity of PP2Cs both in vitro and in vivo. ABA treatment caused EAR1 to accumulate in the nucleus. These results indicate that EAR1 is a negative regulator of ABA signaling that enhances the activity of PP2Cs by interacting with and releasing the N-terminal autoinhibition of these proteins.

Protein kinases in plant responses to drought, salt, and cold stress.

Protein kinases are major players in various signal transduction pathways. Understanding the molecular mechanisms behind plant responses to biotic and abiotic stresses has become critical for developing and breeding climate-resilient crops. In this review, we summarize recent progress on understanding plant drought, salt, and cold stress responses, with a focus on signal perception and transduction by different protein kinases, especially sucrose nonfermenting1 (SNF1)-related protein kinases (SnRKs), mitogen-activated protein kinase (MAPK) cascades, calcium-dependent protein kinases (CDPKs/CPKs), and receptor-like kinases (RLKs). We also discuss future challenges in these research fields.

Reciprocal regulation between the negative regulator PP2CG1 phosphatase and the positive regulator OST1 kinase confers cold response in Arabidopsis.

Protein phosphorylation and dephosphorylation have been reported to play important roles in plant cold responses. In addition, phospho-regulatory feedback is a conserved mechanism for biological processes and stress responses in animals and plants. However, it is less well known that a regulatory feedback loop is formed by the protein kinase and the protein phosphatase in plant responses to cold stress. Here, we report that OPEN STOMATA 1 (OST1) and PROTEIN PHOSPHATASE 2C G GROUP 1 (PP2CG1) reciprocally regulate the activity during the cold stress response. The interaction of PP2CG1 and OST1 is inhibited by cold stress, which results in the release of OST1 at the cytoplasm and nucleus from suppression by PP2CG1. Interestingly, cold-activated OST1 phosphorylates PP2CG1 to suppress its phosphatase activity, thereby amplifying cold signaling in plants. Mutations of PP2CG1 and its homolog PP2CG2 enhance freezing tolerance, whereas overexpression of PP2CG1 decreases freezing tolerance. Moreover, PP2CG1 negatively regulates protein levels of C-REPEAT BINDING FACTORs (CBFs) under cold stress. Our results uncover a phosphor/dephosphor-regulatory feedback loop mediated by PP2CG1 phosphatase and OST1 protein kinase in plant cold responses.

The direct targets of CBFs: In cold stress response and beyond.

Cold acclimation in Arabidopsis thaliana triggers a significant transcriptional reprogramming altering the expression patterns of thousands of cold-responsive (COR) genes. Essential to this process is the C-repeat binding factor (CBF)-dependent pathway, involving the activity of AP2/ERF (APETALA2/ethylene-responsive factor)-type CBF transcription factors required for plant cold acclimation. In this study, we performed chromatin immunoprecipitation assays followed by deep sequencing (ChIP-seq) to determine the genome-wide binding sites of the CBF transcription factors. Cold-induced CBF proteins specifically bind to the conserved C-repeat (CRT)/dehydration-responsive elements (CRT/DRE; G/ACCGAC) of their target genes. A Gene Ontology enrichment analysis showed that 1,012 genes are targeted by all three CBFs. Combined with a transcriptional analysis of the cbf1,2,3 triple mutant, we define 146 CBF regulons as direct CBF targets. In addition, the CBF-target genes are significantly enriched in functions associated with hormone, light, and circadian rhythm signaling, suggesting that the CBFs act as key integrators of endogenous and external environmental cues. Our findings not only define the genome-wide binding patterns of the CBFs during the early cold response, but also provide insights into the role of the CBFs in regulating multiple biological processes of plants.

Advances and challenges in uncovering cold tolerance regulatory mechanisms in plants.

Contents Summary I. Introduction II. Cold stress and physiological responses in plants III. Sensing of cold signals in plants IV. Messenger molecules involved in cold signal transduction V. Cold signal transduction in plants VI. Conclusions and perspectives Acknowledgements References SUMMARY: Cold stress is a major environmental factor that seriously affects plant growth and development, and influences crop productivity. Plants have evolved a series of mechanisms that allow them to adapt to cold stress at both the physiological and molecular levels. Over the past two decades, much progress has been made in identifying crucial components involved in cold-stress tolerance and dissecting their regulatory mechanisms. In this review, we summarize recent major advances in our understanding of cold signalling and put forward open questions in the field of plant cold-stress responses. Answering these questions should help elucidate the molecular mechanisms underlying plant tolerance to cold stress.

Temperature-dependent autoimmunity mediated by chs1 requires its neighboring TNL gene SOC3.

Toll/interleukin receptor (TIR)-nucleotide binding site (NB)-type (TN) proteins are encoded by a family of 21 genes in the Arabidopsis genome. Previous studies have shown that a mutation in the TN gene CHS1 activates the activation of defense responses at low temperatures. However, the underlying molecular mechanism remains unknown. To genetically dissect chs1-mediated signaling, we isolated genetic suppressors of chs1-2 (soc). Several independent soc mutants carried mutations in the same TIR-NB-leucine-rich repeat (LRR) (TNL)-encoding gene SOC3, which is adjacent to CHS1 on chromosome 1. Expression of SOC3 was upregulated in the chs1-2 mutant. Mutations in six soc3 alleles and downregulation of SOC3 by an artificial microRNA construct fully rescued the chilling sensitivity and defense defects of chs1-2. Biochemical studies showed that CHS1 interacted with the NB and LRR domains of SOC3; however, mutated chs1 interacted with the TIR, NB and LRR domains of SOC3 in vitro and in vivo. This study reveals that the TN protein CHS1 interacts with the TNL protein SOC3 to modulate temperature-dependent autoimmunity.

The cbfs triple mutants reveal the essential functions of CBFs in cold acclimation and allow the definition of CBF regulons in Arabidopsis.

In Arabidopsis, the C-repeat binding factors (CBFs) have been extensively studied as key transcription factors in the cold stress response. However, their exact functions in the cold response remains unclear due to the lack of a null cbf triple mutant. In this study, we used CRISPR/Cas9 technology to mutate CBF1 or CBF1/CBF2 in a cbf3 T-DNA insertion mutant to generate cbf1,3 double and cbf1 cbf2 cbf3 (cbfs) triple mutants. The response of the cbfs triple mutants to chilling stress is impaired. Furthermore, no significant difference in freezing tolerance was observed between the wild-type and the cbf1,3 and cbfs mutants without cold acclimation. However, the cbfs mutants were extremely sensitive to freezing stress after cold acclimation, and freezing sensitivity ranking was cbfs > cbf1,3 > cbf3. RNA-Seq analysis showed that 134 genes were CBF regulated, of which 112 were regulated positively and 22 negatively by CBFs. Our study reveals the essential functions of CBFs in chilling stress response and cold acclimation, as well as defines a set of genes as CBF regulon. It also provides materials for the genetic dissection of components in CBF-dependent cold signaling.

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.

Surviving and thriving: How plants perceive and respond to temperature stress.

The dramatic temperature fluctuations spurred by climate change inhibit plant growth and threaten crop productivity. Unraveling how plants defend themselves against temperature-stress-induced cellular impairment is not only a crucial fundamental issue but is also of critical importance for agricultural sustainability and food security. Here, we review recent developments in elucidating the molecular mechanisms used by plants to sense and respond to cold and heat stress at multiple levels. We also describe the trade-off between plant growth and responses to high and low temperatures. Finally, we discuss possible strategies that could be used to engineer temperature-stress-tolerant, high-yielding crops.

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.

RAF22, ABI1 and OST1 form a dynamic interactive network that optimizes plant growth and responses to drought stress in Arabidopsis.

Plants adapt to their ever-changing environment via positive and negative signals induced by environmental stimuli. Drought stress, for instance, induces accumulation of the plant hormone abscisic acid (ABA), triggering ABA signal transduction. However, the molecular mechanisms for switching between plant growth promotion and stress response remain poorly understood. Here we report that RAF (rapidly accelerated fibrosarcoma)-LIKE MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 22 (RAF22) in Arabidopsis thaliana physically interacts with ABA INSENSITIVE 1 (ABI1) and phosphorylates ABI1 at Ser416 residue to enhance its phosphatase activity. Interestingly, ABI1 can also enhance the activity of RAF22 through dephosphorylation, reciprocally inhibiting ABA signaling and promoting the maintenance of plant growth under normal conditions. Under drought stress, however, the ABA-activated OPEN STOMATA1 (OST1) phosphorylates the Ser81 residue of RAF22 and inhibits its kinase activity, restraining its enhancement of ABI1 activity. Taken together, our study reveals that RAF22, ABI1, and OST1 form a dynamic regulatory network that plays crucial roles in optimizing plant growth and environmental adaptation under drought stress.

Rice functional genomics: decades' efforts and roads ahead.

Rice (Oryza sativa L.) is one of the most important crops in the world. Since the completion of rice reference genome sequences, tremendous progress has been achieved in understanding the molecular mechanisms on various rice traits and dissecting the underlying regulatory networks. In this review, we summarize the research progress of rice biology over past decades, including omics, genome-wide association study, phytohormone action, nutrient use, biotic and abiotic responses, photoperiodic flowering, and reproductive development (fertility and sterility). For the roads ahead, cutting-edge technologies such as new genomics methods, high-throughput phenotyping platforms, precise genome-editing tools, environmental microbiome optimization, and synthetic methods will further extend our understanding of unsolved molecular biology questions in rice, and facilitate integrations of the knowledge for agricultural applications.

Molecular Regulation of Plant Responses to Environmental Temperatures.

Temperature is a key factor governing the growth and development, distribution, and seasonal behavior of plants. The entire plant life cycle is affected by environmental temperatures. Plants grow rapidly and exhibit specific changes in morphology under mild average temperature conditions, a response termed thermomorphogenesis. When exposed to chilling or moist chilling low temperatures, flowering or seed germination is accelerated in some plant species; these processes are known as vernalization and cold stratification, respectively. Interestingly, once many temperate plants are exposed to chilling temperatures for some time, they can acquire the ability to resist freezing stress, a process termed cold acclimation. In the face of global climate change, heat stress has emerged as a frequent challenge, which adversely affects plant growth and development. In this review, we summarize and discuss recent progress in dissecting the molecular mechanisms regulating plant thermomorphogenesis, vernalization, and responses to extreme temperatures. We also discuss the remaining issues that are crucial for understanding the interactions between plants and temperature.