Tianbao Yang

Nodulation independent of rhizobia induced by a calcium-activated kinase lacking autoinhibition

Legumes, such as Medicago truncatula, form mutualistic symbiotic relationships with nitrogen-fixing rhizobial bacteria. This occurs within specialized root organs—nodules—that provide the conditions required for nitrogen fixation. A rhizobium-derived signalling molecule, Nod factor, is required to establish the symbiosis. Perception of Nod factor in the plant leads to the induction of Ca2+ oscillations, and the transduction of this Ca2+ signal requires DMI3 (refs 2, 3), which encodes the protein kinase Ca2+/calmodulin-dependent protein kinase (CCaMK). Central to the regulation of CCaMK is an autoinhibitory domain that negatively regulates kinase activity. Here we show that the specific removal of the autoinhibition domain leads to the autoactivation of the nodulation signalling pathway in the plant, with the resultant induction of nodules and nodulation gene expression in the absence of bacterial elicitation. This autoactivation requires nodulation-specific transcriptional regulators in the GRAS family. This work demonstrates that the release of autoinhibition from CCaMK after calmodulin binding is a central switch that is sufficient to activate nodule morphogenesis. The fact that a single regulation event is sufficient to induce nodulation highlights the possibility of transferring this process to non-legumes.

Ca2+/calmodulin regulates salicylic-acid-mediated plant immunity

Intracellular calcium transients during plant–pathogen interactions are necessary early events leading to local and systemic acquired resistance. Salicylic acid, a critical messenger, is also required for both of these responses, but whether and how salicylic acid level is regulated by Ca2+ signalling during plant–pathogen interaction is unclear. Here we report a mechanism connecting Ca2+ signal to salicylic-acid-mediated immune response through calmodulin, AtSR1 (also known as CAMTA3), a Ca2+/calmodulin-binding transcription factor, and EDS1, an established regulator of salicylic acid level. Constitutive disease resistance and elevated levels of salicylic acid in loss-of-function alleles of Arabidopsis AtSR1 suggest that AtSR1 is a negative regulator of plant immunity. This was confirmed by epistasis analysis with mutants of compromised salicylic acid accumulation and disease resistance. We show that AtSR1 interacts with the promoter of EDS1 and represses its expression. Furthermore, Ca2+/calmodulin-binding to AtSR1 is required for suppression of plant defence, indicating a direct role for Ca2+/calmodulin in regulating the function of AtSR1. These results reveal a previously unknown regulatory mechanism linking Ca2+ signalling to salicylic acid level.

Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin

Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H2O2) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H2O2 into water and oxygen, is the major H2O2-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H2O2 and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H2O2. Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H2O2 levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H2O2 homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

A Calcium/Calmodulin-regulated Member of the Receptor-like Kinase Family Confers Cold Tolerance in Plants

Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulin-regulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.Cold is a limiting environmental factor that adversely affects plant growth and productivity. Calcium/calmodulin-mediated signaling is believed to play a pivotal role in plant response to cold stress, but its exact role is not clearly understood. Here, we report that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase, is crucial for cold tolerance in plants. CRLK1 has two calmodulin-binding sites with different affinities as follows: one located at residues 369–390 with a Kd of 25 nm, and the other located at residues 28–112 with a Kd of 160 nm. Calcium/calmodulin stimulated the kinase activity, but the addition of chlorpromazine, a calmodulin antagonist, blocked its stimulation. CRLK1 is mainly localized in the plasma membrane, and its expression is stimulated by cold and hydrogen peroxide treatments. Under normal growth conditions, there is no noticeable phenotypic difference between wild-type and crlk1 knock-out mutant plants. However, as compared with wild-type plants, the crlk1 knock-out mutants exhibited an increased sensitivity to chilling and freezing temperatures. Northern analysis showed that the induction of cold-responsive genes, including CBF1, RD29A, COR15a, and KIN1 in crlk1 mutants, is delayed as compared with wild-type plants. These results indicate that CRLK1 is a positive regulator of cold tolerance in plants. Furthermore, our results suggest that CRLK1 plays a role in bridging calcium/calmodulin signaling and cold signaling.

Recent Advances in Calcium/Calmodulin-mediated Signaling with an Emphasis on Plant:Microbe Interactions

Calcium/calmodulin-mediated signaling contributes in diverse roles in plant growth, development, and response to environmental stimuli.

Calcium/Calmodulin Up-regulates a Cytoplasmic Receptor-like Kinase in Plants

Calcium/calmodulin-dependent kinases play an important role in protein phosphorylation in eukaryotes. However, not much is known about calcium/calmodulin-dependent protein phosphorylation and its role in signal transduction in plants. By using a protein-protein interaction-based approach, we have isolated a novel plant-specific calmodulin-binding receptor-like cytoplasmic kinase (CRCK1) from Arabidopsis thaliana, as well as its ortholog from Medicago sativa (alfalfa). CRCK1 does not show high homology to calcium/calmodulin-dependent protein kinases in animals. In contrast, it shows high homology in the kinase domain to serine/threonine receptor-like kinases in plants. However, it contains neither a transmembrane domain nor an extracellular domain. Calmodulin binds to CRCK1 in a calcium-dependent manner with an affinity of ∼20.5 nm. The calmodulin-binding site in CRCK1 is located in amino acids 160–183, which overlap subdomain II of the kinase domain. CRCK1 undergoes autophosphorylation in the presence of Mg2+ at the threonine residue(s). The Km and Vmax values of CRCK1 for ATP are 1 μm and 33.6 pmol/mg/min, respectively. Calcium/calmodulin stimulates the kinase activity of CRCK1, which increases the Vmax of CRCK1 ∼9-fold. The expression of CRCK1 is increased in response to stresses such as cold and salt and stress molecules such as abscisic acid and hydrogen peroxide. These results indicate the presence of a calcium/calmodulin-regulated receptor-like cytoplasmic kinase in plants. Furthermore, these results also suggest that calcium/calmodulin-regulated protein phosphorylation involving CRCK1 plays a role in stress signal transduction in plants.

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

Abstract. Chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca2+ and Ca2+/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca2+-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the β-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by β-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca2+ concentration in target cells, thereby controlling developmental events in the anther.

Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants

Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulin-regulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.

Concept of redesigning proteins by manipulating calcium/calmodulin-binding domains to engineer plants with altered traits

The importance of calcium and calcium-binding proteins such as calmodulin in plant growth and development as well as plant response to environmental stimuli has been recognised for some time. However, it is only recently that the underlying mechanisms have begun to be unravelled. A variety of intracellular calcium signatures have been observed in response to various stimuli. However, how these changes induce downstream actions and how one can manipulate these events to alter plant response is an area of major interest. Here we discuss the recent advances on three intriguing calcium/calmodulin-regulated proteins: a calcium/calmodulin-regulated metabolic enzyme (DWF1); a chimeric calcium/calmodulin-dependent protein kinase (CCaMK); and a family of calcium/calmodulin-regulated transcription factors (AtSRs or CAMTAs). These proteins play critical roles in plant growth, plant : microbe interactions and plant response to multiple environmental signals. The identification and manipulation of calcium-binding and calmodulin-binding sites in these proteins have provided direct evidence for the role of calcium-binding and calmodulin-binding to the proteins, as well as providing new ways to rebuild the proteins and engineer plants to obtain desired traits.

Novel Calcium/Calmodulin-modulated Proteins

Ca2+/CaM-regulated protein phosphorylation is believed to play a pivotal role in amplifying and diversifying the action of Ca2+-mediated signals (Poovaiah and Reddy 1993). Although Ca2+/CaM-dependent protein phosphorylation is implicated in regulating a number of cellular processes in plants, not much is known about Ca2+/CaM-dependent protein kinases and their role in Ca2+ signaling. A chimeric Ca2+/CaM-dependent protein kinase (CCaMK) gene with a neural visinin-like Ca2+-binding domain was cloned and characterized (Patil et al 1995). CCaMK is characterized by the presence of a catalytic domain, a CaM-binding domain, and a neural visinin-like Ca2+-binding domain in a single polypeptide, making it distinctly different from other protein kinases (Takezawa et al 1996a, Ramachandiran et al 1997, Poovaiah et al 1999)