The CBL–CIPK signaling module in plants: a mechanistic perspective

In a given environment, plants are constantly exposed to multitudes of stimuli. These stimuli are sensed and transduced to generate a diverse array of responses by several signal transduction pathways. Calcium (Ca²⁺) signaling is one such important pathway involved in transducing a large number of stimuli or signals in both animals and plants. Ca²⁺ engages a plethora of decoders to mediate signaling in plants. Among these groups of decoders, the sensor responder complex of calcineurin B‐like protein (CBL) and CBL‐interacting protein kinases (CIPKs) play a very significant role in transducing these signals. The signal transduction mechanism in most cases is phosphorylation events, but some structural role for the pair has also come to light recently. In this review, we discuss the structural nature of the sensor‐responder duo; their mechanism of substrate phosphorylation and also their structural role in modulating targets. Moreover, the mechanism of complex formation and mechanistic role of protein phosphatases with CBL–CIPK module has been mentioned. A comparison of CBL–CIPK with other decoders of Ca²⁺ signaling in plants also signifies the relatedness and diversity in signaling pathways. Further an attempt has been made to compare this aspect of Ca²⁺ signaling pathways in different plant species to develop a holistic understanding of conservation of stimulus–response‐coupling mediated by this Ca²⁺–CBL–CIPK module.     

The origin and function of calmodulin regulated Ca2+ pumps in plants

While Ca²⁺ signaling plays an important role in both plants and animals, the machinery that codes and decodes these signals have evolved to show interesting differences and similarities. For example, typical plant and animal cells both utilize calmodulin (CaM)-regulated Ca²⁺ pumps at the plasma membrane to help control cytoplasmic Ca²⁺ levels. However, in flowering plants this family of pumps has evolved with a unique structural arrangement in which the regulatory domain is located at the N-terminal instead of C-terminal end. In addition, some of the plant isoforms have evolved to function at endomembrane locations. For the 14 Ca²⁺ pumps present in the model plant Arabidopsis, molecular genetic analyses are providing exciting insights into their function in diverse aspects of plant growth and development.     

Effects of Endogenous Salicylic Acid During Calcium Deficiency-Induced Tipburn in Chinese Cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

By cultivating tipburn-susceptible plants in modified Hoagland’s medium containing of gradient exogenous calcium (Ca²⁺), we have shown that Ca²⁺ deficiency is one of the main causes of tipburn in Chinese cabbage (Brassica rapa L. ssp. pekinensis). The effect of endogenous plant Ca²⁺ concentrations on tipburn was also studied in a doubled haploid (DH) population consisting of 100 individuals, but no correlation was found. We then examined the expression of 12 Ca²⁺ transporter genes that function in cytosolic Ca²⁺ homeostasis in both tipburn-susceptible and tipburn-resistant plants under normal and tipburn-inducing conditions. Expression patterns for most of these genes differed between the two types of plants. Salicylic acid (SA) accumulated in response to conditions of calcium deficiency in our study, and both total SA and SA β-glucoside (SAG) in tipburn-susceptible plants was ～3-fold higher than it was in resistant plants following Ca²⁺ deficiency treatment. Also, the changes observed in SA levels correlated well with cell death patterns revealed by trypan blue staining. Therefore, we speculate that the cytoplasmic Ca²⁺ fluctuation-induced downstream signaling events, as well as SA signaling or other biological events, are involved in the plant defense response to tipburn in Chinese cabbage.     

Stress responses mediated by the CBL calcium sensors in plants

Calcium ions (Ca²⁺) are involved as second messenger in plant responses to a broad array of environmental stimuli, including biotic and abiotic stresses. Therefore, understanding Ca²⁺-signaling mechanisms may lead to the development of transgenic crops with enhanced tolerance to adverse environmental conditions. In order to initiate the signaling cascades and give rise to relevant cellular and physiological responses, changes in the parameters of Ca²⁺ transients should be first detected by appropriate Ca²⁺ sensors in plant cells. In this regard, elucidations of plant Ca²⁺ sensors and their target molecules are critical steps for unraveling the Ca²⁺ signal transduction pathways. Recent studies have revealed that plants possess many Ca²⁺-binding proteins with different properties, which can serve as distinct Ca²⁺ sensors. This present review mainly focuses on a family of calcineurin B-like Ca²⁺ sensors which has been most recently identified from higher plants including Arabidopsis, rice, maize and pea.     

Ca2+ signaling in prokaryotes

The role of Ca²⁺ ions in the regulation of motility, cell cycle, and division of prokaryotes is discussed, as well as their involvement in the pathogenesis of some infectious diseases. The structural and functional organization of the prokaryotic Ca²⁺ signaling system and the mechanisms of Ca²⁺ membrane transport and homeostasis are described. Special attention is paid to the role of Ca²⁺ cation channels, Ca²⁺ transporters, and Ca²⁺-binding proteins in the regulation of the intercellular Ca²⁺ concentration.     

Calcium Signaling Network in Plants: An Overview

Calcium ion (Ca²⁺) is one of the very important ubiquitous intracellular second messenger molecules involved in many signal transduction pathways in plants. The cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been found to increased in response to many physiological stimuli such as light, touch, pathogenic elicitor, plant hormones and abiotic stresses including high salinity, cold and drought. This Ca²⁺ spikes normally result from two opposing reactions, Ca²⁺ influx through channels or Ca²⁺ efflux through pumps. The removal of Ca²⁺ from the cytosol against its electrochemical gradient to either the apoplast or to intracellular organelles requires energized ‘active’ transport. Ca²⁺-ATPases and H⁺/Ca²⁺ antiporters are the key proteins catalyzing this movement. The increased level of Ca²⁺ is recognised by some Ca²⁺-sensors or calcium-binding proteins, which can activate many calcium dependent protein kinases. These kinases regulate the function of many genes including stress responsive genes, resulted in the phenotypic response of stress tolerance. Calcium signaling is also involved in the regulation of cell cycle progression in response to abiotic stress. The regulation of gene expression by cellular calcium is also crucial for plant defense against various stresses. However, the number of genes known to respond to specific transient calcium signals is limited. This review article describes several aspects of calcium signaling such as Ca²⁺ requiremant and its role in plants, Ca²⁺ transporters, Ca²⁺-ATPases, H⁺/ Ca²⁺-antiporter, Ca²⁺-signature, Ca²⁺-memory and various Ca²⁺-binding proteins (with and without EF hand).Key Words: Calcium binding proteins, Ca²⁺ channel, Ca²⁺-dependent protein kinases, Ca²⁺/H⁺ antiport, calcium memory, calcium sensors, calcium signatures, Ca²⁺-transporters, EF hand motifs, plant signal transduction     

MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots

Ca²⁺ signaling is central to plant development and acclimation. While Ca²⁺-responsive proteins have been investigated intensely in plants, only a few Ca²⁺-permeable channels have been identified, and our understanding of how intracellular Ca²⁺ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca²⁺ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca²⁺ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca²⁺ transport in planta and that this mechanism is the major route for fast Ca²⁺ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca²⁺ imaging in living root tips and the stimulation of Ca²⁺ transients of different amplitudes, we demonstrated that mitochondrial Ca²⁺ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca²⁺ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca²⁺ entry route in planta and link mitochondrial Ca²⁺ transport with phytohormone signaling.

Monitoring of subcellular Ca²⁺ dynamics in living Arabidopsis roots reveals that MCU proteins provide the dominant mitochondrial Ca²⁺ uptake mechanism in vivo.     

Ca2+ signals: The versatile decoders of environmental cues

Plants are often subjected to various environmental stresses that lead to deleterious effects on growth, production, sustainability, etc. The information of the incoming stress is read by the plants through the mechanism of signal transduction. The plant Ca²⁺ serves as secondary messenger during adaptations to stressful conditions and developmental processes. A plethora of Ca²⁺ sensors and decoders functions to bring about these changes. The cellular concentrations of Ca²⁺, their subcellular localization, and the specific interaction affinities of Ca²⁺ decoder proteins all work together to make this process a complex but synchronized signaling network. In this review, we focus on the versatility of these sensors and decoders in the model plant Arabidopsis as well as plants of economical importance. Here, we have also thrown light on the possible mechanism of action of these important components.     

Ca2+ signals in plant immunity

Calcium ions function as a key second messenger ion in eukaryotes. Spatially and temporally defined cytoplasmic Ca²⁺ signals are shaped through the concerted activity of ion channels, exchangers, and pumps in response to diverse stimuli; these signals are then decoded through the activity of Ca²⁺‐binding sensor proteins. In plants, Ca²⁺ signaling is central to both pattern‐ and effector‐triggered immunity, with the generation of characteristic cytoplasmic Ca²⁺ elevations in response to potential pathogens being common to both. However, despite their importance, and a long history of scientific interest, the transport proteins that shape Ca²⁺ signals and their integration remain poorly characterized. Here, we discuss recent work that has both shed light on and deepened the mysteries of Ca²⁺ signaling in plant immunity.     

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth,development, and pathogenesis

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca²⁺-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca²⁺ (i.e., “Ca²⁺ signature”), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca²⁺ biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca²⁺ ([Ca²⁺]_c) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca²⁺ signature. Furthermore, occurrence of pulsatile Ca²⁺ signatures was age and development dependent, and major [Ca²⁺]_c transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell–cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca²⁺-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca²⁺ signaling across eukaryotic kingdoms.     

Chemical signaling under abiotic stress environment in plants

Many chemicals are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses by modulating gene expression machinery and regulating various transporters/pumps and biochemical reactions. These chemicals include calcium (Ca²⁺), cyclic nucleotides, polyphosphoinositides, nitric oxide (NO), sugars, abscisic acid (ABA), jasmonates (JA), salicylic acid (SA) and polyamines. Ca²⁺ is one of the very important ubiquitous second messengers in signal transduction pathways and usually its concentration increases in response to the stimuli including stress signals. Many Ca²⁺ sensors detect the Ca²⁺ signals and direct them to downstream signaling pathways by binding and activating diverse targets. cAMP or cGMP protects the cell with ion toxicity. Phosphoinositides are known to be involved both in transmission of signal across the plasma membrane and in intracellular signaling. NO activates various defense genes and acts as a developmental regulator in plants. Sugars affect the expression of many genes involved in photosynthesis, glycolysis, nitrogen metabolism, sucrose and starch metabolism, defense mechanisms and cell cycle regulation. ABA, JA, SA and polyamines are also involved in many stress responses. Cross-talk between these chemical signaling pathways is very common in plant responses to abiotic and bitotic factors. In this article we have described the role of these chemicals in initiating signaling under stress conditions mainly the abiotic stress.Key words: ABA, abiotic stress, Ca²⁺ binding proteins, calcium signaling, cyclic nucleotides, nitric oxide, phosphoinositides signaling, signal transduction, sugar signaling     

钙调素结合蛋白参与调控植物逆境胁迫的研究进展

Ca²⁺是植物体内重要的第二信使,当植物受到各种环境刺激时,细胞内的Ca²⁺浓度瞬间产生变化,并被Ca²⁺信号效应器识别,通过与下游的靶蛋白结合并调节其活性,参与调控植物各种生理活动。钙调素结合蛋白以依赖Ca²⁺或不依赖Ca²⁺的方式结合钙调素。对目前已经鉴定的植物钙调素结合蛋白结构特点进行了综述,并着重介绍了钙调素结合蛋白是如何参与调节植物对生物胁迫和非生物胁迫的反应,为提高作物抗病抗逆能力研究提供理论基础。     

Cytosolic calcium and pH signaling in plants under salinity stress

Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH_cyt, either individually, or in coordination with changes in cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca²⁺ and protons. The variety and complexity of Ca²⁺ and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca²⁺]_cyt concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca²⁺]_cyt and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca²⁺]_cyt and pH_cyt at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.Key words: cytosolic calcium, ionic toxicity, osmotic stress, pH, salinity stress, salt tolerance, signaling     

Calcium signaling in plant immunity: a spatiotemporally controlled symphony

Calcium ions (Ca²⁺) are prominent intracellular messengers in all eukaryotic cells. Recent studies have emphasized the crucial roles of Ca²⁺ in plant immunity. Here, we review the latest progress on the spatiotemporal control of Ca²⁺ function in plant immunity. We discuss discoveries of how Ca²⁺ influx is triggered upon the activation of immune receptors, how Ca²⁺-permeable channels are activated, how Ca²⁺ signals are decoded inside plant cells, and how these signals are switched off. Despite recent advances, many open questions remain and we highlight the existing toolkit and the new technologies to address the outstanding questions of Ca²⁺ signaling in plant immunity.     

Disturbance of chlorophyll biosynthesis at Mg branch affects the chloroplast ROS homeostasis and Ca<Superscript>2+</Superscript> signaling in <Emphasis Type="Italic">Pisum sativum</Emphasis>

Reactive oxygen species (ROS) and calcium (Ca²⁺), two crucial intracellular signaling molecules, have been reported to play important roles in chlorophyll biosynthesis. In this study, we aimed to investigate whether disturbance of chlorophyll synthesis affects chloroplast ROS and Ca²⁺ homeostases. Chlorophyll biosynthesis was inhibited at the Mg branch by virus-induced gene silencing (VIGS) of CHLI gene encoding the Mg chelatase CHLI subunit in pea (Pisum sativum). Subsequently, ROS and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in these chlorophyll-deficient pea plants were evaluated by histochemical and fluorescent staining assays. The results showed that the superoxide anion and hydrogen peroxide were predominantly generated in chloroplasts of the yellow leaves of pea VIGS-CHLI plants. The expression of genes encoding chloroplast antioxidant enzymes (CuZn-superoxide dismutase, ascorbate peroxidase, glutathione reductase, phospholipid glutathione peroxidase, peroxiredoxin and thioredoxins) were also decreased in the leaves of VIGS-CHLI plants compared with the control plants. Additionally, the [Ca²⁺]_i were significantly reduced in the yellow leaves of VIGS-CHLI plants compared with the green leaves of VIGS-GFP control plants. The expression of genes encoding Ca²⁺ signaling related proteins (thylakoid Ca²⁺ transporter, calmodulins and calcineurin B-like protein) was down-regulated in yellow VIGS-CHLI leaves. These results indicate that inhibition of chlorophyll biosynthesis at the Mg branch by silencing CHLI affects chloroplast ROS homeostasis and Ca²⁺ signaling and down-regulates the expression of ROS scavenging genes and Ca²⁺ signaling related genes.     

Delineating Calcium Signaling Machinery in Plants: Tapping the Potential through Functional Genomics

Plants have developed calcium (Ca²⁺) signaling as an important mechanism of  regulation of  stress perception,  developmental cues, and  responsive gene  expression. The  post-genomic era has witnessed the successful unravelling of the functional characterization of genes and the creation of large datasets of molecular information. The major elements of Ca²⁺ signaling machinery include Ca²⁺ sensors and responders such as Calmodulins (CaMs), Calmodulin-like proteins (CMLs), Ca²⁺/CaM-dependent protein kinases (CCaMKs), Ca²⁺-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) as well as transporters, such as Cyclic nucleotide-gated channels (CNGCs), Glutamate-like receptors (GLRs), Ca²⁺-ATPases, Ca²⁺/H⁺ exchangers (CAXs) and mechanosensitive channels. These elements play an important role in the regulation of physiological processes and plant responses to various stresses. Detailed genomic analysis can help us in the identification of potential molecular targets that can be exploited towards the development of stress-tolerant crops. The information sourced from model systems through omics approaches helps in the prediction and simulation of regulatory networks involved in responses to different stimuli at the molecular and cellular levels. The molecular delineation of Ca²⁺ signaling pathways could be a stepping stone for engineering climate-resilient crop plants. Here, we review the recent developments in Ca²⁺ signaling in the context of transport, responses, and adaptations significant for crop improvement through functional genomics approaches.     

Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling

Calcium is a crucial messenger in many growth and developmental processes in plants. The central mechanism governing how plant cells perceive and respond to environmental stimuli is calcium signal transduction, a process through which cellular calcium signals are recognized, decoded, and transmitted to elicit downstream responses. In the initial decoding of calcium signals, Ca²⁺ sensor proteins that bind Ca²⁺ and activate downstream signaling components are implicated, thereby regulating specific physiological and biochemical processes. After calcineurin B-like proteins (CBLs) sense these Ca²⁺ signatures, these proteins interact selectively with CBL-interacting protein kinases (CIPKs), thereby forming CBL/CIPK complexes, which are involved in decoding calcium signals. Therefore, specificity, diversity, and complexity are the main characteristics of the CBL-CIPK signaling system. However, additional CBLs, CIPKs, and CBL/CIPK complexes remain to be identified in plants, and the specific functions of their abiotic and biotic stress signaling will need to be further dissected. Therefore, a much-needed synthesis of recent findings is important to further the study of CBL-CIPK signaling systems. Here, we review the structure of CBLs and CIPKs, discuss the current knowledge of CBL–CIPK pathways that decode calcium signals in Arabidopsis, and link plant responses to a variety of environmental stresses with specific CBL/CIPK complexes. This will provide a foundation for future research on genetically engineered resistant plants with enhanced tolerance to various environmental stresses.