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.