CALCIUM/CALMODULIN-MEDIATED SIGNALING IN PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1003221
• Project Start Date: Aug 1, 2014
• Project End Date: Mar 31, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001

Performing Department
Horticulture

Non Technical Summary
Transient changes in intracellular calcium have been well-known to act as important and ubiquitous cellular signals coupling numerous developmental and environmental stimuli to various physiological responses in eukaryotes. Environmental stimuli, including light, gravity, wounding, drought, temperature perturbations, pathogens, symbionts, insects, ROS generating chemicals and phytohormones are all documented to induce rapid increases in the cytoplasmic and/or nucleoplasmic calcium concentration via the coordinated actions of calcium permeable channels, pumps and antiporters, as well as calcium sequester proteins. Transient changes in intracellular calcium elevations are sensed by various calcium-binding/sensor proteins including AtSRs/CAMTAs, which usually contain the typical calmodulin binding domain. This research seeks to more fully elucidate the mechanisms and language of calcium signaling in plants, and identify opportunities to manage these natural plant responses to improve agricultural sustainability.

Animal Health Component

15%

Research Effort Categories

Basic

70%

Applied

15%

Developmental

15%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1848	1030	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1848 - Canola;

Field Of Science
1030 - Cellular biology;

Keywords

calcium

calmodulin

signaling

plant growth

immunity

plant defense

Goals / Objectives
Our long-term goal is to elucidate how intracellular Ca2+ transients are interpreted into various physiological responses through CaM in plants. The overarching objective of this proposed investigation is to study the Ca2+/CaM-mediated activation mechanism of AtSRs/CAMTAs, and investigate the molecular basis for AtSR1/CAMTA3's responses to specific calcium signatures. The main hypothesis of this proposed research is that the function of AtSR1/CAMTA3 as well as its homologs as transcription activators (or suppressor) is controlled by Ca2+/CaM-mediated signals. In addition, the full scope of AtSR1/CAMTA3 mediated regulations on plant responses to environmental stimuli and the functional significance of other AtSR/CAMTA homologs in Arabidopsis will also be explored in this proposed investigation. The obvious pleotropic phenotype of atsr1/camta3 mutant in defenses against pathogen and insects, its confirmed regulation on expression of EDS1 as a direct downstream target, provide us an excellent system to monitor the innate biochemical function of AtSR1/CAMTA3 at the cellular level or in planta. Hence, we are now in an ideal position to pursue our specific aims:Ca2+/CaM-mediated activation mechanism of AtSR1/CAMTA3.The overarching hypothesis is that the activities of AtSR1/CAMT3 are regulated through its interaction with Ca2+/CaM. We will test whether increasing its CaM-binding affinity through mutation or exchange of CaM-binding domain could result in a potentiated/hyperactivated version of AtSR1/CAMTA3. A related hypothesis is that AtSR1/CAMTA3 is regulated through the removal of autoinhibition, and we will test whether appropriate deletions could result in a constitutively activated form.2. Study the variation in target motifs recognized by the AtSRs/CaMTAs family and identify the direct downstream targets of AtSR1/CAMTA3.Our working hypothesis for this aim is that, although CGCG box could be the major recognition core of AtSRs/CAMTAs, certain degrees of variation exists between different members. The flanking sequences (or chromosomal landscaping) could also play some role in controlling AtSRs/CAMTAs:target promoter interaction. We will test this hypothesis by oligo-selection and ChIP-on-chip analysis of AtSR1/CAMTA3.3. Explore the functional significance of other AtSR/CAMTA family members using functionally activated mutants.Our working hypothesis for this aim is that normal function of AtSR/CAMTA homolog is dependent on its interaction with Ca2+/CaM, but could be activated/potentiated through mutation. We will generate constitutively activated mutants of other AtSRs/CAMTAs from Arabidopsis following the steps used for preparing a constitutively activated form of AtSR1/CAMTA3.Over expression of these functionally activated mutant rather than wild-type of a tested AtSR/CAMTA homolog will generate a detectable perturbation on its innate function.

Project Methods
Objective 1: Ca2+/CaM-mediated activation mechanism of AtSR1/CAMTA3Overview: Documented mechanisms by which CaM activates/regulates its target proteins falls basically into three categories: relief from autoinhibition, structural rearrangement into active form and assembly of monomers into an active dimer of target proteins through CaM-binding (Hoeflich and Ikura, 2002; Yang and Poovaiah, 2003). There have been a few structure-function dissections of AtSRs/CAMTAs (Bouche et al., 2002; Song et al., 2006), however, all of them were not in a position to address the critical question of how normal functions of AtSRs/CAMTAs are regulated at the organism level, especially by Ca2+/CaM. The major and common weakness in these experiments is that they are not performed using an innate target promoter of AtSRs/CAMTAs and carried out in planta. However, results from these preliminary studies favor a hypothesis that the function of AtSRs/CAMTAs could be activated through the relief of autoinhibition. Earlier studies have indicated that AtSR/CAMTA members could function as either a positive(Bouche et al., 2002; Han et al., 2006; Song et al., 2006) or negative (Doherty et al., 2009b; Du et al., 2009) regulator of transcription. While fused to LexA DNA-binding domain in an effort to analyze the ability of transcriptional activation on LexA promoter-driven lacZ gene in yeast cell, a fragment (TAD) between CG-1 and TIG domains of AtSR2/CAMTA1 was found to be required for the activation of LacZ (Bouche et al., 2002). The fragment of AtSR2/CAMTA1 with the highest activating capacity is a deletion of the C-terminal portion covering the CaMBD and its vicinity (Bouche et al., 2002). Dissection of MmCAMTA2 also showed that the transcriptional activation capacity of the TAD fragment (aa 285-468) on a Gal4-derived promoter is two orders of magnitude higher than that of the full-length MmCAMTA2 when they were tested as fusions with Gal4 DNA-binding domain. Deletion (Δ639-1116) of the ANK domain and part of the IQ motif from MmCAMTA2 resulted in a highly activated version (over 2-fold more active than full-length MmCAMTA2) when tested on the transcriptional control of the endogenous ANF promoter in cultured COS cells (Song et al., 2006). Whether these MmCAMTA2 deletion mutants are overly activated and able to complement MmCAMTA2 function in animals has not been tested. However, these results support a hypothesis that an intramolecular autoinhibitory mechanism exists in members of AtSR/CAMTA. On the other hand, deletion of the C-terminal 80 aa of MmCAMTA2 which contains a nuclear localization signal and part of the IQ domain resulted in the exclusion of the modified protein from entering the nucleus, and therefore failed to activate the ANF promoter (Song et al., 2006). Therefore, we cannot exclude the possibility that CaM-binding to the C-terminus of MmCAMTA2 is a mechanism that controls its subcellular localization and normal function. Therefore, the project leader and research assistants will use AtSR1/CAMTA3 as a model to test how its normal function in suppressing plant immunity and EDS1 expression is regulated in planta, at the molecular and cellular level.Objective 2: Downstream targets of AtSRs/CAMTAsOverview: Recognition of and binding to a specific target DNA motif are important characteristics of transcription factors. Although AtSR1/CAMTA3 was documented to bind the conserved (A/C/G)CGCG(T/C/G) motif, and does not tolerate any mutation in the CGCG core sequence (Yang and Poovaiah, 2002), and dCAMTA from fruit fly was confirmed to recognize this conserved motif, some evidence indicated that the target motifs recognized by various AtSRs/CAMTAs might be broader than (A/C/G)CGCG(T/C/G) recognized by AtSR1(Yang and Poovaiah, 2002).A rice AtSR/CAMTA homolog OsCBT was reported to tolerate T/C variation in the third position of CGCG core sequence (Choi et al., 2005).On the other hand, our data showed that AtSR1/CAMTA3 recombinant protein carrying CG-1 domain binds to the EDS1 promoter fragment containing a canonical ACGCGT core sequence with strong signal (Fig. 4b).However, the corresponding CG-1 domain of AtSR6 binds weakly to this EDS1 promoter fragment (Fig. 5c), and the CG-1 domain from AtSR5 almost did not bind.These results support a hypothesis that certain degrees of variation exist in the recognition sequences of different members of AtSRs/CAMTAs.On the other hand, CAMTA2 was reported not to bind to the promoter of target ANF gene and only to act as a transcription cofactor in assisting homeodomain transcription factor Nkx2-5 to activate the expression of ANF, indicating targets of AtSR/CAMTA could include those which do not have a CGCG box in their promoter (Song et al., 2006).Recent research on direct downstream targets showed that a transcription factor may not necessarily bind all the genes whose promoters carry corresponding recognition motif(s) (Oh et al., 2009).Although a recognition motif is very useful in identifying a potential target of AtSR/CAMTA, the flanking sequences of recognition motifs or even the chromosomal landscape have an impact on AtSR/CAMTA-target interaction in the real situation; therefore direct downstream targets must be determined empirically.Objective 3: Explore the functional significance of other AtSR/CAMTA family members using functionally activated mutants. Overview: Using AtSR1/CAMTA3 as a model, we will explore two aspects related to the regulation and function of AtSR1/CAMTA3, namely, Ca2+/CaM-mediated activation, and direct downstream targets of AtSR1/CAMTA3. AtSR/CAMTAs are conserved Ca2+/CaM-binding proteins found in multicellular eukaryotic organisms. It is generally accepted that conserved structure implies conserved biochemical function and regulatory mechanism among AtSR/CAMTA members, and this hypothesis is supported by the following observations.To date, all the characterized members from plants, insects and mammals are found to carry the ability to regulate transcription of target genes.The conserved Ca2+/CaM-binding has been shown to be required for several AtSR/CAMTA members to exercise their function in transcription regulation in rice (Choi et al., 2005), fruit fly (Han et al., 2006), and Arabidopsis (Du et al., 2009). Hence, it is reasonable to expect that the Ca2+/CaM-mediated activation model of AtSR1/CAMTA3 is applicable to other AtSR/CAMTA members. We will extend research on the Ca2+/CaM-mediated activation mechanism to other members of the AtSR/CAMTA family to explore their functional significance.

Progress 08/01/14 to 03/31/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We have a series of manuscripts at various stages of preparation.

Impacts
What was accomplished under these goals? During this short timeframe, we continued to work on the goals iterated above.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Plant scientists and other interested groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trained agraduate student and a postdoc. How have the results been disseminated to communities of interest?There have been four refereed journal publications during this reporting period. What do you plan to do during the next reporting period to accomplish the goals?We will continue our studies on the mechanistic aspects of transcription factor (AtSR1) and its role in plant immune/defense response. This area is of major agricultural significance. Specifically, (1) we will continue to evaluate fluctuations in intra-cellular calcium in response to microbial signal and study how the signal is amplified and produces the final response; (2) we will continue our studies on NPR1:AtSR1 interactions and study the role in immune response; (3) we will explore the primary regulon of AtSR1 by combining the powers of EMSA-seq and expression profiling; and (4) we will also study calcium signaling-mediated plant response to abiotic stresses such as cold since it is becoming clear that calcium signaling plays a crucial role in conferring cold tolerance in plants.

Impacts
What was accomplished under these goals? We studied calcium transients (signatures) and signaling events that orchestrate plant-microbe interactions. Calcium acts as a second messenger connecting the perception of microbe signals to the establishment of appropriate immune 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 calcium signatures, which are sensed by diverse intracellular calcium-binding proteins. The calcium signatures are eventually decoded into distinct downstream responses through transcriptional reprogramming of the defense-related genes. Calcium-mediated signal networks involved in plant immunity: Calcium transients have been observed to be one of the early cellular responses required for the establishment of immunity after plants are challenged by pathogens. How calcium signals are coupled to the subsequent immune responses is just beginning to unfold. Transcriptional reprograming is a generally accepted final step in decoding calcium signaling. Several calcium/CaM-binding transcription factors are involved in transcriptional control of critical genes in the salicylic acid-mediated plant defense. AtSR1, a calcium/CaM-regulated transcription factor, plays a negative role in the expression of both Enhanced Disease Susceptibility 1 (EDS1) and no disease resistance-1 (NDR1) genes which are required for the NLR-mediated immunities and associated programmed cell death. These genes are required for the activation of Isochorismate Synthase 1 (ICS1), the rate-limiting enzyme for SA biosynthesis. Salicylic acid (SA) alone is capable of inducing the expression of a number of pathogen-related genes (PR genes) and the establishment of local and systemic acquired resistance; hence it is called the defense hormone in plants. A loss-of-function mutation in AtSR1 gene depresses the immune signaling cascade and consequently gives rise to spontaneous cell death and elevated SA accumulation. Further work has revealed that CAMTA3/AtSR1 could be temporarily turned over following pathogen attack. CBP60g is a CaM-binding transcription factor which plays a positive role in plant immunity. CaM relays pathogen-triggered calcium signaling to induce conformational change by binding to CBP60g. CaM-modified CBP60g recognizes the promoter and induces the expression of isochorismate synthase 1 (ICS1), facilitating the launch of defense against pathogen attack. Further studies demonstrated that CBP60g acts to decode early calcium signaling and contributes to the accumulation of SA at an early point in the plant immune response. A homolog of CBP60g without a CaM-binding domain, SARD1 (systemic-acquired resistance deficient 1), also targets the ICS1 promoter and contributes to the production of SA at later stages after decreased [Ca2+]cyt. These findings revealed that calcium/CaM is involved in both positive and negative regulation of plant immunity, providing novel insights into how calcium signals are integrated to fine tune the activation of plant immune responses. Calcium signaling in plant autoimmunity and a guard model for AtSR1/CAMTA3-mediated immune response: We proposed a model to summarize our work on AtSR1/CAMTA3-mediated immune response and calcium's role in it. It is becoming clear that calcium signals play a fundamental role in plant immune responses. Various calcium sensors have been characterized to relay and/or decode pathogen-induced calcium signals. Autoimmune mutants are helpful resources for the dissection of plant immune systems which have allowed researchers to access in-depth mechanisms of defense signaling. Recent discoveries in this rapidly advancing field have revealed new elements that could help in the understanding of mediation by the nucleotide-binding oligomerization domain-like receptors (NLRs). We used calcium signaling mutants that exhibit autoimmune phenotypes in this study. We then focused on AtSR1/CAMTA3-regulated immune signaling, especially how this target of calcium sensor acts as a guardee monitored by NLR proteins. Calcium/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)/CAMTA3 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. 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 pathogen-derived molecular pattern-triggered immunity (PTI), effector-triggered immunity (ETI), and in salicylic acid (SA)- and jasmonate (JA)-mediated signaling pathways. AtSR1 also regulates several steps in the SA-mediated pathway, from the activation of SA synthesis to the perception of the SA signal. Furthermore, AtSR1 could also regulate plant growth through its involvement in controlling 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 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.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: New Phytologist. 2018. 217:1598-1609
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mol Cell Proteomics. 2018. mcp.RA117.000417. doi:10.1074/mcp.RA117.000417
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Molecular Plant. 2018. 11, 637-639.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Int. J. Mol. Sci. 2018, 19(6), 1764; https://doi.org/10.3390/ijms19061764

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Plant scientists and other interested parties. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trained graduate students and postdocs. How have the results been disseminated to communities of interest?There have been three refereed journalpublications in 2017. What do you plan to do during the next reporting period to accomplish the goals?We will continue our studies on the mechanistic aspects of transcription factor (AtSR1) and its role in plant immune/defense, and the role of CCaMK in symbioses. These areas are of major agricultural significance. Specifically, (1) we will continue to evaluate fluctuations in intra-cellular calcium in response to microbial signaling; (2) we will continue our studies on NPR1:AtSR1 interactions and their role in immune response; (3) we will study the practical applications of our research by studying fire blight in apple, which is a major bacterial disease caused by the bacterial pathogen, Erwinia amylovora. This disease can kill young trees or cause permanent structural damage to mature trees. Young plantings of susceptible scion cultivars on susceptible rootstocks further increase the potential of fire blight damage. Furthermore, recent increases in organic apple production in Washington state call for new approaches to apple blight control. It is becoming clear from our studies that plant hormone salicylic acid is responsible for priming the plant's immune/defense response. Accumulating results have shown an inverse relationship between disease resistance and plant growth. The goal of this project is to fine-tune the immune response in apple cultivars to avoid the negative impact of defense-related salicylic acid accumulation to maximize production.

Impacts
What was accomplished under these goals? We studied calcium signatures and signaling events that orchestrate plant-microbe interactions. Calcium acts as a 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 calcium signatures, which are sensed by diverse intracellular calcium-binding proteins. The calcium signatures are eventually decoded to distinct downstream responses through transcriptional reprogramming of the defense or symbiosis-related genes. Recent observations further reveal that calcium-mediated signaling is also involved in negative regulation of plant immunity. This study was intended as an overview of calcium signaling during immunity and symbiosis, including calcium responses in the nucleus and cytosol. In a related study, we investigated the autophosphorylation of calcium/calmodulin-dependent protein kinase (CCaMK) at S343 or S344 which generates an Intramolecular interaction blocking the CaM-binding and the response. The calcium and calcium/calmodulin-dependent protein kinase (CCaMK) is an important effector protein of calcium/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 two 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. Our results documented that this interaction turns off the substrate phosphorylation capacity of CCaMK. We have also investigated the W342F mutation in CCaMK and found that it 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 calcium and calcium-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. The practical implications of this study to maximize nitrogen fixation are currently being investigated.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Current Opinion in Plant Biology 2017, 38:173-183
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Plant Signaling and Behavior, http://dx.doi.org/10.1080/15592324.2017.1343779
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Frontiers in Plant Science, https://doi.org/10.3389/fpls.2017.01921

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists/researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We are currently training two graduate students. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Please note: I was a department chair during this reporting period. This responsibility slowed my research progress. Currently there are three projects in my lab showing promising results that should result in publishable data. We will continue our studies on the mechanistic aspects of transcription factor (AtSR1) and its role in plant immune/defense. This is an area of major agricultural importance. We will also continue our work on CCaMK, a protein kinase involved in symbiotic nitrogen fixation, an area that is also of major agricultural significance.

Impacts
What was accomplished under these goals? his project is focused on understanding the role of calcium/calmodulin-mediated signaling in plant response to changes in the environment. Plants, unlike animals, cannot run away when there is a change in environmental conditions. They have to perceive their surroundings and rapidly adapt to these changes in order to survive. In 2009, our team discovered that a calcium/calmodulin-binding transcription factor (AtSR1) plays a central role in plant defense (Du et al., Nature 457:1154-1158, 2009; highlighted in Cell 136:195, 2009; patent received 2015). Calcium signals triggered by invading pathogens are essential for plants to marshal both local and systemic defense mechanisms. We are now investigating the mechanistic aspects of this calcium/calmodulin-mediated signal pathway. Arabidopsis thaliana signal responsive 1 (AtSR1) protein, also known as calmodulin-binding transcription activator 3 (CaMTA3), is a transcriptional factor regulated by calcium/calmodulin (CaM). AtSR1 and its homologs share a conserved domain structure CG-1 DNA-binding domain. In addition, AtSR proteins possess two different recognition motifs for CaM interaction: IQ motif and CaM-binding domain (CaMBD). It is accepted that calmodulin acts as a major calcium-sensor protein that interprets encrypted messages and regulates the functions of various effector proteins involved in physiological responses. In recent years, our research has documented that calcium/calmodulin plays critical roles in regulating plant growth (Du and Poovaiah, 2005, Nature 437: 741-745), nodule development during symbiotic nitrogen fixation (Gleason et al., 2006, Nature, 441:1149-1152) and plant immunity/defense (Du et al., 2009, Nature, 457:1154-1158). These findings have opened some exciting possibilities for further research based on the discovery that calcium/calmodulin-mediated signaling acts as a "master switch" in regulating diverse aspects of plant growth and development. To further understand the significance of calcium/calmodulin-mediated signaling, we focused on the calcium/calmodulin-dependent transcription regulator (AtSR1/CAMTA3). Our lab has generated substantial new information revealing how AtSR1/CAMTA3 is involved in regulating numerous downstream genes in the signal transcription process. Further research is currently under way to study the mechanistic aspects of this regulation. We have also reported that a calcium/calmodulin-dependent protein kinase (CCaMK) is involved in symbiotic nitrogen fixation. This dual regulation by calcium and calmodulin is critical for the activation of CCaMK and its interaction with substrate protein. Furthermore, CCaMK contains a regulatory area such as the calmodulin-binding domain in which the calmodulin binds and promotes substrate phosphorylation. Further investigations in understanding the structure:function relationships of different domains within CCaMK and their roles in symbiotic nitrogen fixation is critical to apply this knowledge in modern agriculture in creating self-fertilizing plants.

Publications

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience for this project are scientists working in the field; faculty and graduate students at universities; extension personnel and field researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and postdocs were trained. How have the results been disseminated to communities of interest?Scientific publication and patent. What do you plan to do during the next reporting period to accomplish the goals?This work will continue and the role of AtSR in disease resistance and immunity will be further investigated by identifying the downstream genes in the pathway. It is expected that a large number of genes are involved in this signal cascade.

Impacts
What was accomplished under these goals? Scientific Publication: Transient changes in intracellular Ca2 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 Ca2 sensors, playing critical roles in interpreting encrypted Ca2 signals. Ca2-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 Ca2 signal and overview of Ca2 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 Ca2 /CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2 /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 Ca2 /CaM-mediated signaling warrant further investigation. Ca2 /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 Ca2 signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2 /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. Patent: Provided are methods for enhancing plant cell disease resistance, comprising (1) generating a homozygous gene modification of AtSR1 (or AtSR1 ortholog or homolog) in a plant or plant cell characterized by sialic acid-mediated systemic acquired resistance (SA-mediated SAR), wherein said gene modification reduces or eliminates the calmodulin-binding activity of the respective AtSR1 or AtSR1 ortholog or homolog; or (2) expression of a recombinant or mutant AtSR1 sequence (or AtSR1 gene ortholog or homolog sequence) encoding a modified AtSR1, or AtSR1 ortholog or homolog protein, in a plant or plant cell, wherein said protein modification reduces or eliminates the calmodulin-binding activity of the respective AtSR1 or AtSR1 ortholog or homolog protein. Plants and/or plant cells comprising said modified AtSR1, or AtSR1 ortholog or homolog proteins, and/or said expression means (e.g., recombinant expression vector or expressible recombinant and/or mutant sequences), along with nucleic acids encoding said modified proteins are provided. Other results: AtSR1/CAMTA3 is a calmodulin-binding transcription activator. Previous studies in this laboratory have revealed a loss-of-function mutation of AtSR1, which resulted in elevated defense against a virulent strain of Pst DC3000 bacteria (elevated basal resistance). Further investigations have revealed that AtSR1 contributed to plant defense by binding to the "vCGCGb" box in the promoter of its target gene EDS1 and suppressed its expression and subsequent activation of salicylic acid-mediated plant immunity. Therefore, AtSR1 plays a negative role in the regulation of basal resistance. However, we recently observed that hypersensitive response (HR) is compromised in atsr1. When infiltrated with an avirulent strain of Pst DC3000 (Pst DC 3000 avrRpt2), HR was observed in wild-type (WT) plants but not in atsr1 mutant plants. Disease resistance tests showed that growth of Pst DC3000 carrying avrRpt2 is significantly increased in atsr1 as compared to WT plants. Furthermore, in WT plants the transcription of AtSR1 was not affected during basal resistance (BR), but was significantly induced during HR. Hence, this induced expression of AtSR1 could favor the establishment of HR. These results document that AtSR1 plays a dual role in plant defense/immunity. Furthermore, these results also show that AtSR1 is a suppressor of BR and an activator of HR. In recent years, it has become clear that calcium/calmodulin-regulated transcription factor AtSR1/CAMTA3 has a broader role in regulating plant responses to biotic and abiotic stresses and it is also involved in controlling plant growth and development. Our focus has been to investigate AtSR1/CAMTA3-regulated signaling network and study the AtSR1/CAMTA3-mediated regulation of NPR1, a critical regulator of SA-mediated immune responses in plants. Investigation of AtSR1 regulon, a collection of genes directly regulated by AtSR1/CAMTA3: we compared the transcriptomes of atsr1 null mutant and its wild-type. Considering salicylic acid (SA) is significantly increased in atsr1 mutant, and elevated SA level could change the expression of many genes at the genome level, we believe it is necessary to eliminate the secondary impact caused by increased endogenous SA. ICS1 is a gene involved in the synthesis of SA and is responsible for over 90% of the stress-induced synthesis of salicylic acid. Our published results confirmed that introducing ics1 mutation in the background of atsr1 resulted in a double mutant which fails to accumulate SA when the plants were grown at about 20°C. To better understand this process, we carried out the following studies. We performed parallel mRNA sequence analysis at the whole genome level, atsr1-ics1 double mutant and ics1 single mutant plants in an effort to eliminate the impact of SA. These plants were grown at 20°C, five-week-old plants were treated with Pseudomonas syringae pv. Pst DC3000 and samples were collected at different time points. Total RNA was isolated from all the samples. The mRNA was purified from these samples and sequenced. Homologs of AtSR1/CAMTA3 were found to bind to both vcgcgb and vcgtgb. We further performed oligo selection for three rounds and the selected DNA fragments were sequenced using ion-torrent technology. Functional studies of AtSR1-mediated regulation of NPR1: Our earlier studies confirmed that AtSR1 protein is able to interact with the promoter of NPR1 in vitro. Chromatin immunoprecipitation-PCR (Chip-PCR) assay further confirmed that AtSR1 protein interacts with the NPR1 promoter in planta. The npr1 single mutant, as well as the atsr1 npr1 double mutant, were more sensitive to Pst DC3000 (OD600=0.001) than the wild-type. AtSR1 has been known to include two different calmodulin (CaM)-binding sites: IQ motif and CaM-binding domain. The CaM-binding domain has been reported to have a high affinity for CaM in the presence of free calcium, while the IQ motif is confirmed to interact with CaM in the absence of free calcium. In plant cells, CaM may cycle between calcium-free and calcium-bound states, and bind to different sites in the AtSR1 protein in each state to trigger specific defense mechanisms. Fluorescence-based calcium sensor, R-GECO1, is select to report pathogen-induced calcium signal. Inoculation with Pst DC3000 (OD600=0.04) induced a clear transient increase of calcium signal in plant epidermal cells. Our goal is to explore the network regulated by AtSR1/CAMTA3, and understand how AtSR1 regulates the function of selected direct downstream target genes, such as NPR1. Further studies to understand the mechanistic aspects of this regulation involving downstream target genes would be of major interest.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Frontiers in Plant Science, 10.3389/fpls.2015.00600

Progress 08/01/14 to 09/30/14

Outputs
Target Audience: Researchers, extension agents, students, general public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have trained postdocs, visiting scientists, graduate and undergraduate students How have the results been disseminated to communities of interest? The results of our work were primarily disseminated through publications and presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Our primary focus will be on studying the interactions between calmodulin and AtSR1/CAMTA3, and observing the changes in plant response to pathogens and evaluate the effect on plant growth and development.

Impacts
What was accomplished under these goals? This project is focused on understanding the role of calcium/calmodulin-mediated signaling in plant response to changes in the environment. It is generally accepted that calmodulin acts as a major calcium-sensor protein that interprets encrypted messages and regulates the functions of various effector proteins involved in physiological responses. In recent years, our research has documented that calcium/calmodulin plays critical roles in regulating plant growth (Du and Poovaiah, 2005, Nature 437: 741-745), nodule development during symbiotic nitrogen fixation (Gleason et al., 2006, Nature, 441:1149-1152) and plant immunity/defense (Du et al., 2009, Nature, 457:1154-1158). These results have opened some exciting possibilities for further research based on the finding that calcium/calmodulin-mediated signaling acts as a “master switch” in regulating many aspects of plant growth and development. To further understand the significance of calcium/calmodulin-mediated signaling, we investigated protein kinases that were cloned and characterized in this laboratory with an emphasis on calcium/calmodulin-dependent protein kinase (CCaMK) and calcium/calmodulin-dependent transcription regulator (AtSR1/CAMTA3). Recently we reported a novel regulatory mechanism of Medicago truncatula CCaMK (MtCCaMK) through the autophosphorylation of S344 in the calmodulin-binding/autoinhibitory domain. Results revealed a novel mechanism of CCaMK which enables it to “turn off” its function through autophosphorylation. Furthermore, results also revealed a central role for CCaMK in bacterial/fungal symbioses. In addition, our work has also revealed that plant immunity/defense is controlled through ubiquitin-mediated modulation of AtSR1/CAMTA3.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Guoping Wang, Houqing Zeng, Xiaoyan Hu, Yiyong Zhu, Yang Chen, Chenjia Shen, Huizhong Wang, B. W. Poovaiah and Liqun Du. 2014. Identification and expression analyses of calmodulin-binding transcription activator genes in soybean. Plant and Soil, 10.1007/s11104-014-2267-6
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, L. Du, L. Shen, C., Yang Y. and Poovaiah, B.W. 2014. Regulation of Plant Immunity through Ubiquitin-mediated Modulation of Ca2 -Calmodulin-AtSR1/CAMTA3 Signaling. Plant Journal, 78:265-281.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, L., Du, L. and Poovaiah B.W. 2014. Calcium Signaling and Biotic Defense Responses in Plants. Plant Signaling and Behavior, DOI:10.4161/15592324.2014.973818.
• Type: Other Status: Published Year Published: 2014 Citation: Zhang, L., Du, L. and Poovaiah, B.W. 2014. CytoTrap Two-Hybrid Screening Assay. Bio-protocol, Vol 4: issue 23