BdGUCD1 and Cyclic GMP Are Required for Responses of Brachypodium distachyon to Fusarium pseudograminearum in the Mechanism Involving Jasmonate.

Guanosine 3',5'-cyclic monophosphate (cGMP) is an important signaling molecule in plants. cGMP and guanylyl cyclases (GCs), enzymes that catalyze the synthesis of cGMP from GTP, are involved in several physiological processes and responses to environmental factors, including pathogen infections. Using in vitro analysis, we demonstrated that recombinant BdGUCD1 is a protein with high guanylyl cyclase activity and lower adenylyl cyclase activity. In Brachypodium distachyon, infection by Fusarium pseudograminearum leads to changes in BdGUCD1 mRNA levels, as well as differences in endogenous cGMP levels. These observed changes may be related to alarm reactions induced by pathogen infection. As fluctuations in stress phytohormones after infection have been previously described, we performed experiments to determine the relationship between cyclic nucleotides and phytohormones. The results revealed that inhibition of cellular cGMP changes disrupts stress phytohormone content and responses to pathogen. The observations made here allow us to conclude that cGMP is an important element involved in the processes triggered as a result of infection and changes in its levels affect jasmonic acid. Therefore, stimuli-induced transient elevation of cGMP in plants may play beneficial roles in priming an optimized response, likely by triggering the mechanisms of feedback control.

Brachypodium distachyon ERECTA-like1 protein kinase is a functional guanylyl cyclase.

The plant proteins called ERECTA family play important role in inflorescence architecture, stomatal patterning and phloem-xylem organization. ERECTA proteins belong to the moonlighting proteins family containing the guanylyl cyclase (GC) catalytic center embedded within the intracellular kinase domain. This characteristic architecture of ERECTA proteins prompted us to experimentally confirm of enzymatic activity of one of these, BdERL1 (ERECTA-like1 from Brachypodium distachyon). We have shown that BdERL1 is dual-function protein with both kinase and GC activity. Moreover, our mutagenesis studies also revealed the catalytic roles of key conserved amino acid residues at the GC center and importantly, probing of the kinase and GC with Ca2+ and/or cGMP, shed light on the intramolecular regulations of BdERL1.

In Vitro Characterization of Guanylyl Cyclase BdPepR2 from Brachypodium distachyon Identified through a Motif-Based Approach.

In recent years, cyclic guanosine 3',5'-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules-BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP.

Cross Talk Between Cyclic Nucleotides and Calcium Signaling Pathways in Plants-Achievements and Prospects.

A variety of plant cellular activities are regulated through mechanisms controlling the level of signal molecules, such as cyclic nucleotides (cNMPs, e.g., cyclic adenosine 3':5'-monophosphate, cAMP, and cyclic guanosine 3':5'- monophosphate, cGMP) and calcium ions (Ca2+). The mechanism regulating cNMP levels affects their synthesis, degradation, efflux and cellular distribution. Many transporters and the spatiotemporal pattern of calcium signals, which are transduced by multiple, tunable and often strategically positioned Ca2+-sensing elements, play roles in calcium homeostasis. Earlier studies have demonstrated that while cNMPs and Ca2+ can act separately in independent transduction pathways, they can interact and function together. Regardless of the context, the balance between Ca2+ and cNMP is the most important consideration. This balance seems to be crucial for effectors, such as phosphodiesterases, cyclic nucleotide gated channels and cyclase activity. Currently, a wide range of molecular biology techniques enable thorough analyses of cellular cross talk. In recent years, data have indicated relationships between calcium ions and cyclic nucleotides in mechanisms regulating specific signaling pathways. The purpose of this study is to summarize the current knowledge on nucleotide-calcium cross talk in plants.

Brachypodium distachyon triphosphate tunnel metalloenzyme 3 is both a triphosphatase and an adenylyl cyclase upregulated by mechanical wounding.

Proteins with a CyaB, thiamine triphosphatase domain (CYTH domain) may play a central role at the interface between nucleotide and polyphosphate metabolism. One of the plant CYTH domain-containing proteins from Brachypodium distachyon, BdTTM3, is annotated in NCBI databases as an 'adenylyl cyclase (AC)' or a 'triphosphate tunnel metalloenzyme'. The divergent nomenclature and the search for plant ACs induced us to experimentally confirm the enzymatic activity of BdTTM3. Based on in vitro analysis, we have shown that the recombinant form of BdTTM3 is a protein with high triphosphatase activity (binding both tripolyphosphate and ATP) and low AC activity. Furthermore, the analysis of BdTTM3 transcriptional activity indicates its involvement in the mechanism underlying responses to wounding stress in B. distachyon leaves.

Cyclic nucleotide gated channels (CNGCs) in plant signalling-Current knowledge and perspectives.

Cell signaling is an evolutionarily conserved mechanism that responds and adapts to various internal and external factors. Generally, a signal is mediated by various signaling molecules and is transferred to a cascade of effector proteins. To date, there is significant evidence that cyclic nucleotides (cNMPs), e.g., adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), may represent important elements of many signaling pathways in plants. However, in contrast to the impressive progress made in understanding cyclic nucleotide signaling in mammalian hosts, only few studies have investigated this topic in plants. Existing evidence indicates that cNMPs participate in growth and developmental processes, as well as the response to various stresses. Once synthesized by adenylyl or guanylyl cyclases, these signals are transduced by acting through a number of cellular effectors. The regulatory effects of cNMPs in eukaryotes can be mediated via various downstream effector proteins, such as protein kinases, Exchange Protein directly Activated by cAMP (EPAC), and Cyclic Nucleotide-Gated ion Channels (CNGC). These proteins sense changes in intracellular cNMP levels and regulate numerous cellular responses. Moreover, the amplitude of cNMP levels and the duration of its signal in the cell is also governed by phosphodiesterases (PDEs), enzymes that are responsible for the breakdown of cNMPs. Data collected in recent years strongly suggest that cyclic nucleotide gated channels are the main cNMP effectors in plant cells. These channels are important cellular switches that transduce changes in intracellular concentrations of cyclic nucleotides into changes in membrane potential and ion concentrations. Structurally, these channels belong to the superfamily of pore-loop cation channels. In this review, we provide an overview of the molecular properties of CNGC structure, regulation and ion selectivity, and subcellular localization, as well as describing the signal transduction pathways in which these channels are involved. We will also summarize recent insights into the role of CNGC proteins in plant growth, development and response to stressors.

Downstream Targets of Cyclic Nucleotides in Plants.

Efficient integration of various external and internal signals is required to maintain adaptive cellular function. Numerous distinct signal transduction systems have evolved to allow cells to receive these inputs, to translate their codes and, subsequently, to expand and integrate their meanings. Two of these, cyclic AMP and cyclic GMP, together referred to as the cyclic nucleotide signaling system, are between them. The cyclic nucleotides regulate a vast number of processes in almost all living organisms. Once synthesized by adenylyl or guanylyl cyclases, cyclic nucleotides transduce signals by acting through a number of cellular effectors. Because the activities of several of these effectors are altered simultaneously in response to temporal changes in cyclic nucleotide levels, agents that increase cAMP/cGMP levels can trigger multiple signaling events that markedly affect numerous cellular functions. In this mini review, we summarize recent evidence supporting the existence of cNMP effectors in plant cells. Specifically, we highlight cAMP-dependent protein kinase A (PKA), cGMP-dependent kinase G (PKG), and cyclic nucleotide phosphodiesterases (PDEs). Essentially this manuscript documents the progress that has been achieved in recent decades in improving our understanding of the regulation and function of cNMPs in plants and emphasizes the current gaps and unanswered questions in this field of plant signaling research.

The Hippeastrum hybridum PepR1 gene (HpPepR1) encodes a functional guanylyl cyclase and is involved in early response to fungal infection.

It is generally known that cyclic GMP widespread in prokaryotic and eukaryotic cells, is involved in essential cellular processes and stress signal transduction. However, in contrast to animals the knowledge about plant guanylyl cyclases (GCs) which catalyze the formation of cGMP from GTP is still quite obscure. Recent studies of plant GCs are focused on identification and functional analysis of a new family of membrane proteins called "moonlighting kinases with GC activity" with guanylyl cyclase catalytic center encapsulated within intracellular kinase domain. Here we report identification and characterization of plasma membrane receptor of peptide signaling molecules - HpPepR1 in Hippeastrum hybridum. Both bioinformatic analysis of amimo acid sequence and in vitro studies revealed that the protein can act as guanylyl cyclase. The predicted amino acid sequence contains highly conserved 14 aa-long search motif in the catalytic center of GCs from lower and higher eukaryotes. Here, we provide experimental evidence to show that the intracellular domain of HpPepR1 can generate cGMP in vitro. Moreover, it was shown that the accumulation of HpPepR1 transcript was sharply increased after Peyronellaea curtisii (=Phoma narcissi) fungal infection, whereas mechanical wounding has no influence on expression profile of studied gene. These results may indicate the participation of cGMP-dependent pathway in rapid, alarm plant reactions induced by pathogen infection.

Transcriptional response of a novel HpCDPK1 kinase gene from Hippeastrum x hybr. to wounding and fungal infection.

Calcium dependent protein kinases (CDPK) are well established plant sensor and effectors for calcium ions and participate in regulation of multiple abiotic and biotic stress responses in plant cells. Here we present the identification and characterization of a new CDPK kinase gene from bulbous plant Hippeastrum x hybr. and examine the role of this kinase in stress responses leading to phytoalexin (PA) production in plant tissues. In the previous research, it was shown that Hippeastrum bulbs mechanically wounded or infected with Peyronellaea curtisii (=Phoma narcissi) are inducted to an antifungal red substance synthesis. In this research, we demonstrated Ca2+ dependence of the phytoalexin production by wounded bulbs. Furthermore, the isolated HpCDPK1 cDNA for ORF was found to be 1596bp long and encoded 531 amino acid protein with CDPK kinase activity, as was shown by recombinant GST-HpCDPK1 enzyme production and analysis. HpCDPK1 transcript was present in all vegetative and chosen generative organs of Hippeastrum plant. The dynamics of the observed HpCDPK1 mRNA changes in bulbs depended on stressor type. The mechanical injury caused one wave of transcript increase while more complex transcript changes were observed within 48h after Peyronellaea inoculation. In plant bulbs already accumulating red phytoalexin, increases in HpCDPK1 mRNA level were observed at certain intervals within 48h whereas, in the case of fungal infection, only one big increment in the transcript amount at the 10th minute after inoculation was detected. The observed transcriptional response of HpCDPK1 gene to wounding and pathogen infection stress suggests a positive correlation with phytoalexin synthesis and maintenance in bulb tissues and puts more light on CDPK kinase role in the plant stress response regulation. This also bears some potential for understanding the mechanism of a phytoalexin formation.