Calcium/calmodulin-mediated microbial symbiotic interactions in plants.

Cytoplasmic calcium (Ca2+) transients and nuclear Ca2+ oscillations act as hubs during root nodulation and arbuscular mycorrhizal symbioses. Plants perceive bacterial Nod factors or fungal signals to induce the Ca2+ oscillation in the nucleus of root hair cells, and subsequently activate calmodulin (CaM) and Ca2+/CaM-dependent protein kinase (CCaMK). Ca2+ and CaM-bound CCaMK phosphorylate transcription factors then initiate down-stream signaling events. In addition, distinct Ca2+ signatures are activated at different symbiotic stages: microbial colonization and infection; nodule formation; and mycorrhizal development. Ca2+ acts as a key signal that regulates a complex interplay of downstream responses in many biological processes. This short review focuses on advances in Ca2+ signaling-regulated symbiotic events. It is meant to be an introduction to readers in and outside the field of bacterial and fungal symbioses. We summarize the molecular mechanisms underlying Ca2+/CaM-mediated signaling in fine-tuning both local and systemic symbiotic events.

Interplay between Ca2+/Calmodulin-Mediated Signaling and AtSR1/CAMTA3 during Increased Temperature Resulting in Compromised Immune Response in Plants.

Changing temperatures are known to affect plant-microbe interactions; however, the molecular mechanism involved in plant disease resistance is not well understood. Here, we report the effects of a moderate change in temperature on plant immune response through Ca2+/calmodulin-mediated signaling. At 30 °C, Pst DC3000 triggered significantly weak and relatively slow Ca2+ influx in plant cells, as compared to that at 18 °C. Increased temperature contributed to an enhanced disease susceptibility in plants; the enhanced disease susceptibility is the result of the compromised stomatal closure induced by pathogens at high temperature. A Ca2+ receptor, AtSR1, contributes to the decreased plant immunity at high temperatures and the calmodulin-binding domain (CaMBD) is required for its function. Furthermore, both salicylic acid biosynthesis (ICS) and salicylic acid receptor (NPR1) are involved in this process. In addition to stomatal control, AtSR1 is involved in high temperature-compromised apoplastic immune response through the salicylic acid signaling pathway. The qRT-PCR data revealed that AtSR1 contributed to increased temperatures-mediated susceptible immune response by regulating SA-related genes in atsr1, such as PR1, ICS1, NPR1, as well as EDS1. Our results indicate that Ca2+ signaling has broad effects on the molecular interplay between changing temperatures as well as plant defense during plant-pathogen interactions.

Calmodulin-binding transcription activator AtSR1/CAMTA3 fine-tunes plant immune response by transcriptional regulation of the salicylate receptor NPR1.

Calcium (Ca2+ ) signalling regulates salicylic acid (SA)-mediated immune response through calmodulin-meditated transcriptional activators, AtSRs/CAMTAs, but its mechanism is not fully understood. Here, we report an AtSR1/CAMTA3-mediated regulatory mechanism involving the expression of the SA receptor, NPR1. Results indicate that the transcriptional expression of NPR1 was regulated by AtSR1 binding to a CGCG box in the NPR1 promotor. The atsr1 mutant exhibited resistance to the virulent strain of Pseudomonas syringae pv. tomato (Pst), however, was susceptible to an avirulent Pst strain carrying avrRpt2, due to the failure of the induction of hypersensitive responses. These resistant/susceptible phenotypes in the atsr1 mutant were reversed in the npr1 mutant background, suggesting that AtSR1 regulates NPR1 as a downstream target during plant immune response. The virulent Pst strain triggered a transient elevation in intracellular Ca2+ concentration, whereas the avirulent Pst strain triggered a prolonged change. The distinct Ca2+ signatures were decoded into the regulation of NPR1 expression through AtSR1's IQ motif binding with Ca2+ -free-CaM2, while AtSR1's calmodulin-binding domain with Ca2+ -bound-CaM2. These observations reveal a role for AtSR1 as a Ca2+ -mediated transcription regulator in controlling the NPR1-mediated plant immune response.

Calcium/Calmodulin-Mediated Defense Signaling: What Is Looming on the Horizon for AtSR1/CAMTA3-Mediated Signaling in Plant Immunity.

Calcium (Ca2+) signaling in plant cells is an essential and early event during plant-microbe interactions. The recognition of microbe-derived molecules activates Ca2+ channels or Ca2+ pumps that trigger a transient increase in Ca2+ in the cytoplasm. The Ca2+ binding proteins (such as CBL, CPK, CaM, and CML), known as Ca2+ sensors, relay the Ca2+ signal into down-stream signaling events, e.g., activating transcription factors in the nucleus. For example, CaM and CML decode the Ca2+ signals to the CaM/CML-binding protein, especially CaM-binding transcription factors (AtSRs/CAMTAs), to induce the expressions of immune-related genes. In this review, we discuss the recent breakthroughs in down-stream Ca2+ signaling as a dynamic process, subjected to continuous variation and gradual change. AtSR1/CAMTA3 is a CaM-mediated transcription factor that represses plant immunity in non-stressful environments. Stress-triggered Ca2+ spikes impact the Ca2+-CaM-AtSR1 complex to control plant immune response. We also discuss other regulatory mechanisms in which Ca2+ signaling activates CPKs and MAPKs cascades followed by regulating the function of AtSR1 by changing its stability, phosphorylation status, and subcellular localization during plant defense.

Distinct Molecular Pattern-Induced Calcium Signatures Lead to Different Downstream Transcriptional Regulations via AtSR1/CAMTA3.

Plants encrypt the perception of different pathogenic stimuli into specific intracellular calcium (Ca2+) signatures and subsequently decrypt the signatures into appropriate downstream responses through various Ca2+ sensors. Two microbe-associated molecular patterns (MAMPs), bacterial flg22 and fungal chitin, and one damage-associated molecular pattern (DAMP), AtPep1, were used to study the differential Ca2+ signatures in Arabidopsis leaves. The results revealed that flg22, chitin, and AtPep1 induced distinct changes in Ca2+ dynamics in both the cytosol and nucleus. In addition, Flg22 and chitin upregulated the expression of salicylic acid-related genes, ICS1 and EDS1, whereas AtPep1 upregulated the expression of jasmonic acid-related genes, JAZ1 and PDF1.2, in addition to ICS1 and EDS1. These data demonstrated that distinct Ca2+ signatures caused by different molecular patterns in leaf cells lead to specific downstream events. Furthermore, these changes in the expression of defense-related genes were disrupted in a knockout mutant of the AtSR1/CAMTA3 gene, encoding a calmodulin-binding transcription factor, in which a calmodulin-binding domain on AtSR1 was required for deciphering the Ca2+ signatures into downstream transcription events. These observations extend our knowledge regarding unique and intrinsic roles for Ca2+ signaling in launching and fine-tuning plant immune response, which are mediated by the AtSR1/CAMTA3 transcription factor.

A Ca2+/CaM-regulated transcriptional switch modulates stomatal development in response to water deficit.

Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis.

Soybean (Glycine max (L.) Merrill) is an important component of the human diet and animal feed, but soybean production is limited by abiotic stresses especially salinity. We recently found that rhizobia inoculation enhances soybean tolerance to salt stress, but the underlying mechanisms are unaddressed. Here, we used quantitative phosphoproteomic and metabonomic approaches to identify changes in phosphoproteins and metabolites in soybean roots treated with rhizobia inoculation and salt. Results revealed differential regulation of 800 phosphopeptides, at least 32 of these phosphoproteins or their homologous were reported be involved in flavonoid synthesis or trafficking, and 27 out of 32 are transcription factors. We surveyed the functional impacts of all these 27 transcription factors by expressing their phospho-mimetic/ablative mutants in the roots of composite soybean plants and found that phosphorylation of GmMYB183 could affect the salt tolerance of the transgenic roots. Using data mining, ChIP and EMSA, we found that GmMYB183 binds to the promoter of the soybean GmCYP81E11 gene encoding for a Cytochrome P450 monooxygenase which contributes to the accumulation of ononin, a monohydroxy B-ring flavonoid that negatively regulates soybean tolerance to salinity. Phosphorylation of GmMYB183 was inhibited by rhizobia inoculation; overexpression of GmMYB183 enhanced the expression of GmCYP81E11 and rendered salt sensitivity to the transgenic roots; plants deficient in GmMYB183 function are more tolerant to salt stress as compared with wild-type soybean plants, these results correlate with the transcriptional induction of GmCYP81E11 by GmMYB183 and the subsequent accumulation of ononin. Our findings provide molecular insights into how rhizobia enhance salt tolerance of soybean plants.

Mutual interplay of Ca2+ and ROS signaling in plant immune response.

Second messengers are cellular chemicals that act as "language codes", allowing cells to pass outside information to the cell interior. The cells then respond through triggering downstream reactions, including transcriptional reprograming to affect appropriate adaptive responses. The spatiotemporal patterning of these stimuli-induced signal changes has been referred to as a "signature", which is detected, decoded, and transmitted to elicit these downstream cellular responses. Recent studies have suggested that dynamic changes in second messengers, such as calcium (Ca2+), reactive oxygen species (ROS), and nitric oxide (NO), serve as signatures for both intracellular signaling and cell-to-cell communications. These second messenger signatures work in concert with physical signal signatures (such as electrical and hydraulic waves) to create a "lock and key" mechanism that triggers appropriate response to highly varied stresses. In plants, detailed information of how these signatures deploy their downstream signaling networks remains to be elucidated. Recent evidence suggests a mutual interplay between Ca2+ and ROS signaling has important implications for fine-tuning cellular signaling networks in plant immunity. These two signaling mechanisms amplify each other and this interaction may be a critical element of their roles in information processing for plant defense responses.

Calcium Signaling-Mediated Plant Response to Cold Stress.

Low temperatures have adverse impacts on plant growth, developmental processes, crop productivity and food quality. It is becoming clear that Ca2+ signaling plays a crucial role in conferring cold tolerance in plants. However, the role of Ca2+ involved in cold stress response needs to be further elucidated. Recent studies have shown how the perception of cold signals regulate Ca2+ channels to induce Ca2+ transients. In addition, studies have shown how Ca2+ signaling and its cross-talk with nitric oxide (NO), reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) signaling pathways ultimately lead to establishing cold tolerance in plants. Ca2+ signaling also plays a key role through Ca2+/calmodulin-mediated Arabidopsis signal responsive 1 (AtSR1/CAMTA3) when temperatures drop rapidly. This review highlights the current status in Ca2+ signaling-mediated cold tolerance in plants.

Ca2+/Calmodulin-Dependent AtSR1/CAMTA3 Plays Critical Roles in Balancing Plant Growth and Immunity.

During plant-pathogen interactions, plants have to relocate their resources including energy to defend invading organisms; as a result, plant growth and development are usually reduced. Arabidopsis signal responsive1 (AtSR1) has been documented as a negative regulator of plant immune responses and could serve as a positive regulator of plant growth and development. However, the mechanism by which AtSR1 balances plant growth and immunity is poorly understood. Here, we performed a global gene expression profiling using Affymetrix microarrays to study how AtSR1 regulates defense- and growth-related genes in plants with and without bacterial pathogen infection. Results revealed that AtSR1 negatively regulates most of the immune-related genes involved in molecular pattern-triggered immunity (PTI), effector-triggered immunity (ETI), and in salicylic acid (SA)- and jasmonate (JA)-mediated signaling pathways. AtSR1 may rigidly regulate several steps of the SA-mediated pathway, from the activation of SA synthesis to the perception of SA signal. Furthermore, AtSR1 may also regulate plant growth through its involvement in regulating auxin- and BRs-related pathways. Although microarray data revealed that expression levels of defense-related genes induced by pathogens are higher in wild-type (WT) plants than that in atsr1 mutant plants, WT plants are more susceptible to the infection of virulent pathogen as compared to atsr1 mutant plants. These observations indicate that the AtSR1 functions in suppressing the expression of genes induced by pathogen attack and contributes to the rapid establishment of resistance in WT background. Results of electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR assays suggest that AtSR1 acts as transcription factor in balancing plant growth and immunity, through interaction with the "CGCG" containing CG-box in the promotors of its target genes.

Quantitative Phosphoproteomic and Metabolomic Analyses Reveal GmMYB173 Optimizes Flavonoid Metabolism in Soybean under Salt Stress.

Salinity causes osmotic stress to crops and limits their productivity. To understand the mechanism underlying soybean salt tolerance, proteomics approach was used to identify phosphoproteins altered by NaCl treatment. Results revealed that 412 of the 4698 quantitatively analyzed phosphopeptides were significantly up-regulated on salt treatment, including a phosphopeptide covering the serine 59 in the transcription factor GmMYB173. Our data showed that GmMYB173 is one of the three MYB proteins differentially phosphorylated on salt treatment, and a substrate of the casein kinase-II. MYB recognition sites exist in the promoter of flavonoid synthase gene GmCHS5 and one was found to mediate its recognition by GmMYB173, an event facilitated by phosphorylation. Because GmCHS5 catalyzes the synthesis of chalcone, flavonoids derived from chalcone were monitored using metabolomics approach. Results revealed that 24 flavonoids of 6745 metabolites were significantly up-regulated after salt treatment. We further compared the salt tolerance and flavonoid accumulation in soybean transgenic roots expressing the 35S promoter driven cds and RNAi constructs of GmMYB173 and GmCHS5, as well as phospho-mimic (GmMYB173S59D ) and phospho-ablative (GmMYB173S59A ) mutants of GmMYB173 Overexpression of GmMYB173S59D and GmCHS5 resulted in the highest increase in salt tolerance and accumulation of cyaniding-3-arabinoside chloride, a dihydroxy B-ring flavonoid. The dihydroxy B-ring flavonoids are more effective as anti-oxidative agents when compared with monohydroxy B-ring flavonoids, such as formononetin. Hence the salt-triggered phosphorylation of GmMYB173, subsequent increase in its affinity to GmCHS5 promoter and the elevated transcription of GmCHS5 likely contribute to soybean salt tolerance by enhancing the accumulation of dihydroxy B-ring flavonoids.

W342F Mutation in CCaMK Enhances Its Affinity to Calmodulin But Compromises Its Role in Supporting Root Nodule Symbiosis in Medicago truncatula.

The calcium/calmodulin-dependent protein kinase (CCaMK) is regulated by free Ca2+ and Ca2+-loaded calmodulin. This dual binding is believed to be involved in its regulation and associated physiological functions, although direct experimental evidence for this is lacking. Here we document that site-directed mutations in the calmodulin-binding domain of CCaMK alters its binding capacity to calmodulin, providing an effective approach to study how calmodulin regulates CCaMK in terms of kinase activity and regulation of rhizobial symbiosis in Medicago truncatula. We observed that mutating the tryptophan at position 342 to phenylalanine (W342F) markedly increased the calmodulin-binding capability of the mutant. The mutant CCaMK underwent autophosphorylation and catalyzed substrate phosphorylation in the absence of calcium and calmodulin. When the mutant W342F was expressed in ccamk-1 roots, the transgenic roots exhibited an altered nodulation phenotype. These results indicate that altering the calmodulin-binding domain of CCaMK could generate a constitutively activated kinase with a negative role in the physiological function of CCaMK.

Autophosphorylation of calcium/calmodulin-dependent protein kinase (CCaMK) at S343 or S344 generates an intramolecular interaction blocking the CaM-binding.

The Ca2+ and Ca2+/calmodulin-dependent protein kinase (CCaMK) is an important effector protein of Ca2+/calmodulin-mediated signaling, and in legumes, it is a critical regulator of plant-rhizobia and mycorrhizal symbioses. CCaMK contains a kinase domain, a calmodulin-binding/autoinhibitory domain and a visinin-like domain. Previous studies revealed the presence of 2 phosphorylation sites, S343 and S344, in the calmodulin-binding domain. Mutations at these sites affected the kinase activity and downstream rhizobium and mycorrhizal symbioses, which highlighted the importance of these residues in regulating protein activity. This addendum further clarifies the regulation of CCaMK by identifying an intramolecular interaction between residue(s) in the kinase domain and phosphorylation sites S343 and S344. This interaction turns off the substrate phosphorylation capacity of CCaMK.

Calcium signatures and signaling events orchestrate plant-microbe interactions.

Calcium (Ca2+) acts as an essential second messenger connecting the perception of microbe signals to the establishment of appropriate immune and symbiotic responses in plants. Accumulating evidence suggests that plants distinguish different microorganisms through plasma membrane-localized pattern recognition receptors. The particular recognition events are encoded into Ca2+ signatures, which are sensed by diverse intracellular Ca2+ binding proteins. The Ca2+ signatures are eventually decoded to distinct downstream responses through transcriptional reprogramming of the defense or symbiosis-related genes. Recent observations further reveal that Ca2+-mediated signaling is also involved in negative regulation of plant immunity. This review is intended as an overview of Ca2+ signaling during immunity and symbiosis, including Ca2+ responses in the nucleus and cytosol.

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

Transient changes in intracellular Ca(2+) concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca(2+) sensors, playing critical roles in interpreting encrypted Ca(2+) signals. Ca(2+)-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes, and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca(2+) signal and overview of Ca(2+) signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca(2+)/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca(2+)/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of reactive oxygen species signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca(2+)/CaM-mediated signaling warrant further investigation. Ca(2+)/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca(2+) signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca(2+)/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops.

Calcium signaling and biotic defense responses in plants.

Calcium (Ca(2+)) acts as an important second messenger in plant cells. Cytosolic free Ca(2+) concentration in plant cells changes rapidly and dynamically in response to various endogenous or environmental cues. Elevation in calcium concentration in plant cells is an essential early event during plant defense responses. Alterations in the Ca(2+) concentration are sensed by Ca(2+)-binding proteins, including calmodulin, calcium-dependent protein kinases and calcineurin B-like proteins, which relay or decode the encoded Ca(2+) signals into specific cellular and physiological responses in order to survive challenges by pathogens. Genetic and functional studies have revealed that Ca(2+) signaling plays both positive and negative roles in regulating the establishment of defense responses. Furthermore, recent studies revealed that actions of Ca(2+)-mediated signaling could be regulated by other cell signaling systems such as the ubiquitin-proteasome system to mount precise and prompt plant defense responses.

Regulation of plant immunity through ubiquitin-mediated modulation of Ca(2+) -calmodulin-AtSR1/CAMTA3 signaling.

Transient changes in intracellular Ca(2+) concentration are essential signals for activation of plant immunity. It has also been reported that Ca(2+) signals suppress salicylic acid-mediated plant defense through AtSR1/CAMTA3, a member of the Ca(2+) /calmodulin-regulated transcription factor family that is conserved in multicellular eukaryotes. How plants overcome this negative regulation to mount an effective defense response during a stage of intracellular Ca(2+) surge is unclear. Here we report the identification and functional characterization of an important component of ubiquitin ligase, and the associated AtSR1 turnover. The AtSR1 interaction protein 1 (SR1IP1) was identified by CytoTrap two-hybrid screening. The loss-of-function mutant of SR1IP1 is more susceptible to bacterial pathogens, and over-expression of SR1IP1 confers enhanced resistance, indicating that SR1IP1 acts as a positive regulator of plant defense. SR1IP1 and AtSR1 act in the same signaling pathway to regulate plant immunity. SR1IP1 contains the structural features of a substrate adaptor in cullin 3-based E3 ubiquitin ligase, and was shown to serve as a substrate adaptor that recruits AtSR1 for ubiquitination and degradation when plants are challenged with pathogens. Hence, SR1IP1 positively regulates plant immunity by removing the defense suppressor AtSR1. These findings provide a mechanistic insight into how Ca(2+) -mediated actions are coordinated to achieve effective plant immunity.