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.

Effect of light on soluble guanylyl cyclase activity in Pharbitis nil seedlings.

Cyclic GMP acts as a chemical switch in plant cells to modulate cellular reactions. However, its metabolism has not been extensively explored and is still poorly understood. Previous experiments suggest that an endogenous cGMP system could participate in the mechanism of phytochrome controlled photoperiodic flower induction in Pharbitis nil. In order to gain further information on the role of cGMP, we have begun to study the enzyme of cGMP synthesis. In this article, the presence of the enzyme with guanylyl cyclase (GC) activity in soluble protein fractions of P. nil is reported. A large portion of the enzymatic activity is present in the cotyledons, where enzyme activity amounted to 0.45 pmol cGMP/min/mg protein. The enzyme exhibited a K(m) 0.5mM for GTP. A plot of 1/v versus 1/[GTP] was linear and V(max) was 0.74 pmol cGMP/min/mg protein. It was shown that the anti-sGC antibody recognise a 40 kDa protein. Moreover, the NO-donor, sodium nitroprusside (SNP) and YC-1, as a NO-independent stimulator, enhanced enzyme activity. The NS 2028 (a potent GC inhibitor) treatments provoked a 3-fold reduction of the enzyme activity in comparison to the untreated fractions. Furthermore, the influence of light on GC activity was analysed. It was noted that cGMP level increased in cool white light, and darkness inhibited enzyme activity. Exposure to blue light acts to stimulate cGMP formation, whereas in red light a rapid decrease in GC activity was observed that returned to the high level when far-red light was applied after the red light treatment. The results presented in this work strongly argue that an enzyme with guanylyl cyclase activity is present in P. nil organs and its activity is controlled by light via the photoreceptors-dependent pathways.

Biochemical evidence for a cGMP-regulated protein kinase in Pharbitis nil.

It is known that the level of cGMP is modulated in response to a number of stimuli in plant cells but intracellular events distal to cGMP metabolism are not clear. Cyclic GMP-dependent protein kinase (Pk-G) is a major effector of cGMP action in animals and yeasts. We wanted to determine whether such kinase is present in plant cells. A soluble protein kinase was isolated from seedlings of Pharbitis nil and purified following purification methods including anion-exchange and affinity-chromatography. The enzyme consists of a single polypeptide of M(r) 70 kDa as determined by SDS-PAGE. From conventional modulators only cyclic GMP, when applied in low concentration, was able to accelerate the enzyme activity in the presence of histones. The enzyme autophosphorylated on serine and threonine residues and phosphorylated some substrates only on serine residues. Mixture of histones and histones H2B, H3 were the best phosphate acceptors. The process of autophosphorylation was accelerated by a low concentration of cGMP and reduced by high concentration of this second messenger. Antibodies raised against catalytic domain of animals Pk-G I alpha and beta cross-reacted with protein kinase from Pharbitis nil tissue. These data, taken together, demonstrate the presence of functional enzyme, which activity is regulated by cGMP and allow to classify this protein kinase as a member of the second messenger regulated group of enzymes.

Biochemical evidence for a calcium-dependent protein kinase from Pharbitis nil and its involvement in photoperiodic flower induction.

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.