Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses

1As sessile organisms, plants encounter various types of environmental stresses, which are generally classified into biotic stresses such as insect and pathogen attacks, and abiotic stresses such as unfavorable temperature, lack of or excessive amounts of water, salinity, heavy metal toxicity, chemical toxicity, and nutrient deficiency. On the other hand, the process of industrialization inevitably brings many detrimental effects to the environment. Poorly controlled release of wastes from industrial processes and human life not only adds various toxic chemicals to our water and soil but also release harmful gasses into the atmosphere. Obviously, human activities are creating environmental challenges, making sustained crop production difficult. Classic agricultural technologies such as irrigation, applications of fertilizer, insecticides, fungicides, and chemical phytoprotectants have helped to improve crop yield, but the effects are limited, the costs are high and the impacts on the ecosystems and human health are undesirable and dangerous. Understanding how plants perceive and respond to various environmental stresses provides the necessary platform to create crop varieties which could fit better into the challenging environments, and has become one of the most important tasks for plant scientists around the world.

2Calcium is one of the most abundant elements on earth. Ca2+ concentration outside the plasma membrane is usually at millimolar level. Since Ca2+ can form insoluble compounds with phosphate derivatives and complex with macromolecules, high levels of cytosolic Ca2+ are toxic to cells. Ca2+ concentration in the cytoplasm and nucleus is usually maintained at 50–100 nM under resting conditions. Ca2+ gradient across the plasma membrane as well as inner membrane system are involved in cell signaling process controlled by stimulus responsive Ca2+ permeable channels, Ca2+ pumps and Ca2+/H+ exchangers. Accumulating evidence reveals that various external stimuli such as gravity, light, cold, heat, drought, water-logging (hypoxia), salt, wind, touch, wounding, and pathogen attacks can quickly induce elevations in cytosolic Ca2+ concentration. Signal-induced nuclear Ca2+ changes have also been documented, but they are not as well studied as cytosolic Ca2+ transients. The excessive amount of Ca2+ in cytoplasm is quickly moved out of the cell or pumped back into the endogenous Ca2+ reservoirs such as vacuole and endoplasmic reticulum (ER) through the involvement of Ca2+ pumps and Ca2+/H+ exchangers distributed on the plasma membrane and inner membrane system. Interestingly, the transient changes of intracellular Ca2+ concentration triggered by various stimuli differ from each other in terms of amplitude, duration, frequency, and spatial distribution inside the cell; and these stimulus-specific Ca2+ transients are named calcium signatures by. Stimulus-specific signals are decoded by downstream effector proteins to generate specific or overlapping responses. These effectors include Ca2+ sensor proteins which are represented by three major types in plants, namely calmodulin (CaM)/CaM-like (CML) proteins, calcium-dependent protein kinases (CDPKs) and calcineurin B like (CBL) proteins. In this review, our primary focus will be limited to CaM/CMLs and their important roles in plant abiotic stress signaling and responses.

3Calmodulin is a ubiquitous Ca2+-binding protein which exists in all eukaryotes. It is a small acidic protein composed of two pairs of EF-hands located at both the N- and C-terminus. In Arabidopsis, seven genes encode four CaM isoforms (CaM1/4; CaM2/3/5; CaM6; CaM7), which differ only in one to five amino acid residues. It has been reported that different CaM isoforms differ in binding and regulating downstream effectors. The slight differences in their structural features may have considerable impacts on their binding to targets.

4In addition to canonical CaM, there are 50 genes coding for CaM-like proteins in the Arabidopsis genome which are made of varying number of EF hands and share at least 16% of overall sequence identity with canonical CaM. Similarly, five CaM and 32 CML genes, respectively are reported in the rice genome. Despite having four EF hands, most CMLs show low (less than 50%) overall similarity to CaMs. Several Arabidopsis CMLs, including CML37, 38, 39, and 42 displayed an electrophoretic mobility shift in the presence of Ca2+, indicating that, like CaMs, CMLs also act as Ca2+ sensors. Besides EF-hands, CaMs and CMLs do not carry any known functional domain, and hence usually have no enzymatic or biochemical functions. So far the only exception is CaM7 from Arabidopsis which was reported to specifically bind Z-/G-box in a Ca2+-dependent manner and act as a transcription factor to regulate light-responsive gene expression and light morphogenesis. Therefore, identifying CaM/CML targets and understanding the impacts of CaM/CML-binding on their functional behaviors are the major challenges in deciphering the functional significance of CaM/CMLs at molecular, biochemical, and physiological levels.

5It is well-documented that Ca2+-binding-induced conforma tional changes in CaMs and CMLs usually increase their binding affinity to downstream targets through hydrophobic and electrostatic interactions. A stretch of 16–35 amino acids in the target proteins called CaM-binding domain (CaMBD) is usually necessary and sufficient for its interaction with CaM. In some cases, CaM interacts with its target proteins in a Ca2+-independent manner, and this kind of interaction requires that the target proteins carry an IQ motif, a stretch of amino acids fitting a conserved pattern of IQXXX(R/K)GXXXR where I could be replaced with “FLV” and “X” represents any amino acid residue. CMLs could follow similar models to interact with their targets; however, this assumption requires experimental verification. Usually, CaMBDs are not conserved in their primary structure, however, most of the Ca2+-dependent CaMBD peptides share a conserved secondary structure, a basic amphipathic helix with hydrophobic residues arranged on one side and positively charged residues arranged on the other side. Hence, most CaM and CML target proteins have to be identified empirically.

6As mentioned above, the interactions between CaM/CMLs and target proteins are usually Ca2+-dependent; regular strategies used for detection of protein–protein interaction including yeast-two-hybrid and coimmunoprecipitation are not effective and fruitful in identifying CaM/CML-binding proteins. The majority of the CaM-binding proteins (CBPs) from plants were identified by screening cDNA expression libraries with labeled CaM as probes (usually 35S-labled;;; ). Another effective approach to identify CBPs is utilizing protein microarray; however, false positive identification is still a major concern and making protein chips with adequate coverage is currently a challenge. Accumulated results indicated that CaM bind to a variety of CBPs in plants, which include kinases, phosphatases, transcription factors, receptors, metabolic enzymes, ion channels and pumps, and cytoskeletal proteins. Hence, it is reasonable to conclude that, in most cases, CaMs and CMLs act as multifunctional regulatory proteins, and their functional significance is materialized through the actions of their downstream target proteins. CBPs with well-defined CaM-binding domain, CaM-binding property and involved in plant responses to abiotic stresses are listed in Table 1.

7Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (O2-), and hydroxyl radical (⋅OH) are usually produced in various physiological processes and serve as a class of second messengers. While controlled production of ROS is essential to signal appropriate actions to protect plants from various environmental stresses, excessive accumulation of ROS causes damages to plant cells. Oxidative stress is defined as disruption of the cellular redox balance, which could be triggered by a wide range of biotic and abiotic stimuli. Because of its long half-life and excellent permeability, H2O2 is broadly accepted as the major form of ROS in plant cells. It is well known that H2O2 can trigger increases in cytosolic Ca2+ by activating the Ca2+-permeable channels. On the other hand, H2O2 production during oxidative burst is also dependent on continuous Ca2+ influx, which activates not only the NADPH oxidase, an EF-hand containing enzyme on the plasma membrane, but also the CaM-binding NAD kinase (NADK), which supplies NADP cofactor for ROS production through NADPH oxidase.

8In addition, early studies from heat-stressed maize seedling suggested that ROS homeostasis and the entire antioxidant system including catalase, superoxide dismutase (SOD) and ascorbate peroxidase, could be regulated by Ca2+ influx and intracellular CaM. Later plant catalases, a class of H2O2 scavenger enzymes catalyzing its degradation to water and oxygen was found to bind CaM in a Ca2+-dependent manner. The activity of the Arabidopsis CAT3 is stimulated by Ca2+/CaM rather than Ca2+ or CaM alone, but catalases from other organisms such as Aspergillus niger, human and bovine, do not interact with CaM. A peroxidase from Euphorbia latex, was also reported to be a CBP activated by Ca2+/CaM. Evidence also suggests that another class of ROS-scavenging enzyme SOD could be regulated by CaM in maize, although the specific SOD gene has not been cloned. The critical role of Ca2+/CaM in balancing ROS actions was further supported by the observation that the oxidative damage caused by heat stress in Arabidopsis seedlings is exacerbated by pretreatment with CaM inhibitors.

9In addition to these direct regulations on ROS homeostasis, Ca2+/CaM-mediated signaling is also well known to regulate ROS-related signal transduction at various stages. Maize CAP1 encoding a novel form of CaM-regulated Ca2+-ATPase was shown to be induced only during early anoxia, indicating its possible role in oxygen-deprived maize cells. CaM may also participate indirectly in regulating ROS content through the CaM-regulated -aminobutyrate (GABA) synthesis and the GABA shunt metabolic pathway. Recently, it was demonstrated that ZmCCaMK and OsDMI3 (also called OsCCaMK) from maize (Zea mays) and rice (Oryza sativa), respectively, play a critical role in ABA-induced antioxidant actions, suggesting a role for CCaMK in plant oxidative stress response. Arabidopsis MPK8 was found to be activated by CaM and activated MPK8 suppresses wound-induced ROS accumulation via transcriptional control of RbohD expression, revealing a novel mechanism for CaM-mediated signaling to fine-tune ROS homeostasis under wounding stress. Interestingly, some genes encoding CaM-binding transcription factors (CAMTAs) and co-factor (AtBTs) are responsive to H2O2, suggesting that CaM-mediated signaling could directly regulate gene expression in plant responses to oxidative cues.

10Prolonged high temperature is usually lethal to all organisms; fluctuations in temperature above optimal level, usually called heat shock (HS), impose major stress affecting plant growth and productivity. Almost all organisms including plants synthesize HS proteins (HSPs), a class of chaperons to assure normal function of various client proteins under adversely high temperature conditions. It was observed long ago that HS induced a quick and strong increase in cytosolic Ca2+ in tobacco. Expression of CaM in the maize coleoptiles was found to be remarkably induced during HS and was affected by Ca2+ level, suggesting that Ca2+ and CaM may be involved in the acquisition of HS-induced thermotolerance. observed an increase in intracellular Ca2+ within one min after wheat was subjected to 37°C HS. Expression of CaM mRNA and protein was both induced by HS in the presence of Ca2+, and expression of HSP26 and HSP70 was stimulated by exogenous application of Ca2+. HS-induced expression of CaM was 10 min earlier than that of HSPs, and both were suspended by pharmacological reagents which interfere with Ca2+ signaling. These results indicate that Ca2+ and CaM are directly involved in HS signaling. The Ca2+/CaM signaling system was also proposed to be involved in the induction of HSP genes in Arabidopsis. Using molecular and genetic tools, found that Arabidopsis AtCaM3 was involved in the Ca2+/CaM-mediated HS signal transduction pathway. atcam3 loss-of-function mutant showed a pronounced decrease in thermotolerance after 50 min of incubation at 45°C. The compromised thermotolerance of atcam3 mutant could be rescued by functional complementation with 35S promoter driven AtCaM3, and overexpression of AtCaM3 in wild-type (WT) background increased thermotolerance of the transgenic plants. Furthermore, the DNA-binding activity of HS transcription factors and the expression of tested HS genes at both mRNA and protein levels were shown to be down-regulated in atcam3 null mutant and up-regulated in its overexpressing lines upon HS treatment. A role for CaM in HS signaling was also demonstrated in rice. HS was reported to induce biphasic cytosolic Ca2+ transients, and this signature feature was found to be reflected in the HS-induced expression of OsCaM1-1. OsCaM1-1 was observed to localize to the nucleus and overexpression of OsCaM1-1 in Arabidopsis resulted in enhanced thermotolerance which coincided with elevated expression of HS-responsive AtCBK3, AtPP7, AtHSF, and AtHSP at a non-inducing temperature. Nitric oxide (NO) level in plants was found to be elevated by high temperatures, and exogenous application of NO donor provides effective protection to plants under heat stress. However, for a long time it was unknown how NO is involved in protecting plants from damage by HS. Recently, Arabidopsis CaM3 was reported to act as a downstream factor of NO in activation of HS transcription factors, accumulation of HSPs and establishment of thermotolerance.

11Calmodulin-binding proteins have also been shown to play a crucial role in mediating plant responses to heat stress. pTCB48 encoding a CBP was isolated by screening a cDNA expression library constructed from tobacco cell cultures subjected to HS, and its expression was strongly induced by HS treatment, suggesting a role in HS response. Maize cytosolic Hsp70 was identified to bind CaM in the presence of Ca2+ and could inhibit the activity of CaM-dependent NADK in a concentration-dependent manner, but its possible function in HS response has not been elucidated. Recently, DgHsp70, a homolog of cytosolic Hsp70 from orchardgrass (Dactylis glomerata) was also found to bind AtCaM2 in the presence of Ca2+, and the binding of Ca2+/CaM decreased the ATPase and foldase activities of this chaperon protein. PP7 is a ser/thr protein phosphatase which interacts with CaM in a Ca2+-dependent manner. Arabidopsis AtPP7 was induced by HS and its knockout mutant is impaired in thermotolerance, while the overexpression of AtPP7 results in increased thermotolerance and increased expression of AtHSP70 and AtHSP101 following HS treatment. Interestingly, AtPP7 was also found to interact with HS transcription factor AtHSF1 implying that AtPP7 could also regulate the expression of HSP genes via AtHSF1. However, the mechanistic detail by which AtPP7 dephosphorylates and regulates downstream substrates such as AtHSF1 is not clear. Arabidopsis AtCRK1 (CDPK-related protein kinase 1, also called AtCBK3) was identified as a Ca2+-dependent CBP. found that AtCBK3 activates HSFs which further regulate HS gene expression by binding to HS elements.

12Ca2+ has been recognized as a vital second messenger coupling cold stress to specific plant responses. Researchers found that cold shock and wind initiate Ca2+ transients in both cytosol and nucleus in transgenic tobacco (Nicotiana plumbaginifolia) seedlings expressing aequorin, and the expression of NpCaM-1 is induced by both cold shock and wind but mediated by distinct Ca2+ signaling pathways operating predominantly in the cytoplasm or in the nucleus. Transgenic Arabidopsis plants overexpressing CaM3 showed decreased levels of COR (cold regulated) transcripts suggesting CaM may function as a negative regulator of cold-induced gene expression. Genes encoding CMLs, such as AtCML24/TCH2 and OsMSR2 (O. sativa Multi-Stress-Responsive gene2, a novel CML gene), were also found to be induced by cold treatment and thus, likely participate in transducing cold-induced Ca2+ signals.

13In addition, as downstream effectors of Ca2+/CaM-mediated signaling, CBPs are also known to be involved in plant responses to cold stress. Ca2+/CaM-regulated receptor-like kinase CRLK1, which is mainly localized in the plasma membrane, was found to be involved in cold tolerance. CRLK1 carries two CaM-binding sites in both N- and C-termini with affinities for Ca2+/CaM of 25 and 160 nM, respectively. crlk1 knockout mutant plants grow and behave like WT plants under regular conditions, but are more sensitive to chilling and freezing treatments than WT plants. In addition, cold response genes such as CBF1, RD29A, COR15a, and KIN1 showed delayed responses to cold in crlk1, suggesting a positive role for CRLK1 in regulating cold tolerance. MEKK1, which is a member of the MAP kinase kinase kinase family, was shown to interact with CRLK1 both in vitro and in planta. Knockout mutation of CRLK1 abolished the cold-triggered MAP kinase activities, and altered cold-induced expression of genes involved in MAP kinase signaling. Therefore, Ca2+/CaM-regulated CRLK1 may modulate cold acclimation through MAP kinase cascade in plants. Other CaM-binding kinases are also suggested to be involved in cold acclimation. The expression of PsCCaMK in pea (Pisum sativa) roots was found to be up-regulated by low temperature or salinity stress, and the activity of the Ca2+/CaM-dependent NADK was found to be increased by cold shock.

14AtSRs/CAMTAs belong to one of the best characterized classes of CaM-binding transcription factors in plants and animals. In an attempt to understand transcriptional control of CBF2 (a critical regulator of cold acclimation), compared the promoter sequence of three CBFs and found seven conserved DNA motifs CM1 to 7 in their promoters. CM2 is a typical AtSR1/CAMTA3 recognition motif, and importantly, the expression of CBF2 was found to be positively regulated by AtSR1/CAMTA3. Although camta3 knockout mutant had no phenotypic change under cold stress, camta1/camta3 double mutant was found to have reduced freezing tolerance. Recently, AtCAMTA1, AtCAMTA2, and AtCAMTA3 were shown to participate in cold tolerance by cooperatively inducing CBF genes and repressing SA biosynthesis. These results filled a long-standing knowledge gap between cold induced Ca2+ transients and cold-regulated gene expression.

15Several Arabidopsis MADS box transcription factors were identified as putative CBPs by a high throughput proteomics approach. Expression of some of these MADS box genes, including AGL3, AGL8, AGL15, and AGL32, was reported to be suppressed by cold stress, implying a role for Ca2+ signal to regulate cold responses through the MADS proteins, however, whether and how Ca2+/CaM regulates MADS box transcription factors remain to be addressed. The expression of CBF2 is down-regulated in transgenic Arabidopsis plants constitutively expressing AGL15, in comparison to WT plants. GT factors are plant-specific transcription factors sharing a conserved trihelix DNA-binding domain that specifically interacts with GT cis-elements. Recently, AtGT2L, a classic member of the GT-2 subfamily, was identified to encode a Ca2+-dependent CBP and it is responsive to cold, salt and ABA treatments. Furthermore, overexpression of AtGT2L resulted in elevated expression levels of cold- and salt-specific marker genes RD29A and ERD10, both in basal and chilling- or salt-treated conditions. These results indicated that Ca2+/CaM-binding AtGT2L is involved in plant responses to cold and salt stresses.

16High salinity and drought are the major environmental stresses frequently experienced by plants, and both impose osmotic stress on plant cells. Osmotic stress induces a series of responses at the molecular and cellular levels and a primary event is an increase in the cytosolic Ca2+ concentration and subsequent transduction of Ca2+ signals that promotes appropriate cellular responses in an effort to mitigate potential damages. In addition to the well documented salt-overly-sensitive (SOS) pathway, CaM-mediated signaling is also actively involved in plant response to osmotic stress. Overexpression of a salt-induced CaM gene from soybean, GmCaM4, in Arabidopsis confers salt stress tolerance through the up-regulation of DNA-binding activity of a MYB transcription factor MYB2. Interestingly, MYB2 was also reported to interact with CaM in a Ca2+-dependent manner and regulate salt and dehydration responsive genes. AtCML8, an ortholog of GmCaM4, was also found to be induced by salt treatment. Another similar CML protein AtCML9 was found to be involved in osmotic stress tolerance through ABA-mediated pathways. AtCML9 was readily induced by abiotic stress and ABA; knock-out mutant atcml9 showed a hypersensitive response to ABA during seed germination and seedling growth stages, and exhibited enhanced tolerance to salt and dehydration stresses. Furthermore, expression of several stress and ABA-responsive genes including RAB18, RD29A, and RD20 was altered in atcml9. The rice CML gene OsMSR2 was also suggested to be involved in ABA-mediated salt and drought tolerance. As the most abundant vacuolar Na+-proton exchanger in Arabidopsis, Na+/H+ exchanger 1 (AtNHX1) regulates various cellular activities such as maintaining pH, ion homeostasis, and protein trafficking. found that AtCaM15 (also called AtCML18) is localized in the vacuolar lumen and interacts with the C-terminus of AtNHX1. The interaction between AtCaM15 and AtNHX1 is affected by both Ca2+ and pH, and the binding of AtCaM15 to AtNHX1 alters the Na+/K+ selectivity of the exchanger by decreasing its Na+/H+ exchange speed. The interaction between AtCaM15 and AtNHX1 suggests the presence of Ca2+-pH-dependent signaling components in the vacuole, which are involved in mediating plant responses to salt stress. In addition to the above mentioned CaM/CMLs, CML37, CML38, and CML39 are also responsive to various stimuli, including salt, drought, and ABA, but whether they are also involved in osmotic stress tolerance remains to be identified.

17A few CaMBPs are involved in the signaling pathways triggered by salt, drought or osmotic stresses. Wheat (Triticum aestivum) TaCCaMK was down-regulated by ABA, salt and PEG treatments, and overexpression of TaCCaMK reduces ABA sensitivity of Arabidopsis, indicating that TaCCaMK is a negative regulator of ABA-mediated signaling. Arabidopsis AtACA4 encoding a CaM-regulated Ca2+-ATPase was found to be localized to small vacuoles, which is similar to PMC1, the yeast vacuolar Ca2+-ATPase, and AtACA4 confers tolerance against osmotic stresses imposed by high NaCl, KCl, and mannitol, when expressed in the yeast K616 strain lacking Ca2+ transporter PMC1. A CaM-regulated Ca2+-ATPase gene from soybean, SCA1, was found to be induced by salt stress. Methylglyoxal (MG), a byproduct of carbohydrate and lipid metabolism and a potent mutagenic chemical known to arrest growth, reacts with DNA and protein and increases sister chromatid exchange; and glyoxalase enzymes, including glyoxalase I (gly-I) and glyoxalase II (gly-II), catalyze the detoxification of MG with the involvement of glutathione (GSH; ). Glyoxalase I from Brassica juncea (BjGly-I) was reported to be a Ca2+/CBP, and its enzymatic function is significantly stimulated by Ca2+/CaM binding. The expression of BjGly-I is induced by salt, dehydration and heavy metal stresses; ectopic expression of BjGly-I in tobacco conferred remarkable tolerance to exogenous MG and high salt stress. AtCaMBP25 was identified to be a CaM-binding nuclear protein and is induced by dehydration, low temperature or high salinity. Overexpression of AtCaMBP25 compromised the tolerance of transgenic plants to osmotic stress, and silencing AtCaMBP25 via antisense approach increased plant tolerance to osmotic stress. These results suggested that AtCaMBP25 functions as a negative regulator in plant tolerance to osmotic stress, revealing a connection coupling Ca2+ signals to plant responses to osmotic stresses.

18Ca2+/CaM-regulated transcription factors are also involved in plant response to salt and drought stresses. A few CAMTA genes from Arabidopsis and soybean are up-regulated by salt and dehydration treatments. Arabidopsis CAMTA1 is involved in drought stress response. Knockout mutant camta1 was shown to be more sensitive to drought stress, and expression of many drought responsive genes was affected in this mutant. Similar to AtCAMTA1, tomato CAMTA homolog SlSR1L was also positively involved in drought stress tolerance. In addition to regulating salicylic acid (SA)-induced defense response and systemic acquired resistance, AtCBP60g, a CaM-binding transcription factor from Arabidopsis was found to positively regulate drought stress response. Transgenic plants overexpressing CBP60g displayed hypersensitivity to ABA and enhanced tolerance to drought stress. AtGTL1 (GT-2 LIKE1), a CaM-binding member of the GTL transcription factor family, was found to be a negative regulator of drought tolerance. AtGTL1 expression was down-regulated by dehydration stress, and loss-of-function mutant gtl1 showed better survival under drought stress by reducing transpiration, due to lower stomata density on the abaxial surface and higher expression of SDD1, which is a negative regulator of stomatal development and is repressed by AtGTL1. Similarly, PtaGTL1 identified from Populus tremula × P. alba could bind to CaM and regulate water use efficiency and drought tolerance. Another transcription factor AtABF2/AREB1, which was identified as CBP through protein microarray analysis, was also found to be up-regulated by ABA, dehydration, and salinity stresses. Single and multiple mutants of ABF2, 3, and 4 showed varying degrees of reduced survival rate under drought conditions, implying functional redundancy among these three ABFs and Ca2+/CaM could regulate drought tolerance through ABF2/AREB1.

19Elevated concentration of both essential (e.g., Cu and Zn) and non-essential (e.g., Cd, Hg, Pb, and Ni) heavy metals in the soil can cause toxicity and inhibit plant growth. It was reported that Ca2+/CaM is involved in radish (Raphanus sativus L.) responses to Cd2+ toxicity during the early phases of seed germination. Ca2+ added in the medium could partially reverse the Cd2+-induced growth inhibition of the germinating embryo, and this coincides with decreased Cd2+ uptake. An equilibrium dialysis study revealed that Cd2+ compete with Ca2+ for CaM-binding, hence Cd2+ could significantly reduce the binding of Ca2+/CaM to its target proteins. Apparently, supplementation of Ca2+ in the medium counteracts the toxicity of Cd2+ by restoring the Ca2+-dependent interaction between CaM and its targets during the radish seed germination. A tobacco (N. tabacum) cyclic nucleotide gated ion channel (CNGC) called NtCBP4 was identified to be a CBP through protein–protein interaction-based library screening, and shown to be localized to plasma membrane. Transgenic tobacco plants with elevated expression of NtCBP4 displayed tolerance to Ni2+ and hypersensitivity to Pb2+, and consistently showed decreased Ni2+ and increased Pb2+ accumulation, suggesting that NtCBP4 is involved in heavy metal uptake across the plant plasma membrane. However, transgenic plants expressing a truncated version of NtCBP4 lacking the C-terminal stretch covering the CaMBD and part of the putative cyclic nucleotide-binding domain showed improved tolerance to Pb2+ and lower accumulation of Pb2+, and loss-of-function mutation of AtCNGC1, a homolog of NtCBP4 in Arabidopsis, also resulted in Pb2+ tolerance. These results suggested that CaM-binding is required for the normal function of both AtCNGC1 and NtCBP4 for the transport of heavy metals.

20Ca2+ is a critical second messenger coupling diverse stimuli to various physiological responses in plants. CaM, as well as CML, is one of the most extensively studied Ca2+ sensors, which mediate interpretation of Ca2+ signals in all aspects of plant life, especially in responses to environmental stresses, through interaction with and regulation of various downstream target proteins. Figure 1 depicts an overview of generation and interpretation of Ca2+ signals which are regulated by CaM/CMLs during plant responses to abiotic stresses. One of the most actively regulated class of target proteins are calcium permeable channels, pumps, and antiporters which are actively involved in the generation of intracellular Ca2+ transients. This indicates that the preciseness and accuracy of Ca2+ signal itself is closely monitored by CaM-mediated regulation. Although more than 50 proteins from different plant species have been identified as CBPs with well-defined CaM-binding properties (Table 1), the CaM-mediated regulations of these target proteins are frequently presumptive including SAURs, PCBP, AtBTs, and WRKYIIds. Only a few cases of CaM-mediated regulation in planta are supported with empirical evidences, such as GAD, CCaMK, AtCAT3, MLO, DWF1, AtSRs/CaMTAs, CRLK1, and CBP60g. Hence, more emphasis should be placed on studying the CaM-mediated regulation of target proteins to further improve our understanding of CaM-mediated signaling. Currently, most of the CBPs are targets of canonic CaMs which count for only 10% of the CaM/CML family. The targets of most of the CMLs are not reported yet, let alone the CML-mediated regulation of downstream targets and associated signal transduction. Identification of novel target proteins of CaMs and CMLs especially those interact with CMLs deserve special attentions. Environmental cues are known to trigger stimulus specific Ca2+ transients. In an effort to explain how the simple Ca2+ ion could act as a messenger to couple various environmental stimuli to appropriate physiological responses with astonishing accuracy, proposed the theory of “Ca2+ signature” which hypothesized that stimulus-triggered increases in intracellular Ca2+ concentration vary in terms of duration, frequency, amplitude, and spatial distribution, and these carry specific information when they are interpreted into different physiological responses. An obvious support for this hypothesis is that the different Ca2+ spikes triggered by Nod factor from rhizobia and Myc factor from arbuscular mycorrhizal fungi could be interpreted through the action of the same Ca2+, Ca2+/CaM dependent protein kinase, CCaMK, into different physiological responses to support the establishment of root nodulation symbiosis or arbuscular mycorrhization. Although exciting progress on how Ca2+ signals are interpreted into various physiological responses has been made in the last decade, what we know so far may be very limited in scope when one considers the complicated Ca2+ signaling network. Many issues such as specificity, preference and flexibility of interaction between various CaM/CMLs and target proteins in planta are barely understood. The dynamics of Ca2+/CaM mediated regulation, the mechanistic details by which a particular effector detects a difference in Ca2+ signature and initiates distinct signaling pathways, are basically unknown. Progress in addressing these issues will help in understanding the most amazing properties, the versatility, efficiency and accuracy of Ca2+-mediated signaling in plant responses to environmental stresses.

21The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.