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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.

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.

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.

Under the field conditions crop plants interact with diverse microorganisms. These include beneficial (symbiotic) and phytopathogenic microorganisms, which jointly affect growth and productivity of the plants. In last decades, production of potato (Solanum tuberosum L.) suffers from increased incidence of potato virus Y (PVY), which is one of most important potato pests. Arbuscular mycorrhizal fungi (AMF) are common symbionts of potato, however the impact of mycorrhizal symbiosis on the progression of PVY-induced disease is scarcely known. Therefore, in the present study we investigated the effect of joint PVY infection and mycorrhizal colonization by Rhizophagus irregularis on growth traits of the host potato plant (cv. Pirol). The tested PVY isolate belonged to N-Wilga strain group, which is considered to be predominant in Europe and many other parts of the world. The viral particles were concentrated in the leaves, but decreased the root growth. Furthermore, the infection with PVY evoked prolonged oxidative stress reflected by increased level of endogenous H2O2. AMF alleviated oxidative stress in PVY-infected host plants by a substantial decrease in the level of shoot- and root-derived H2O2, but still caused asymptomatic growth depression. It was assumed that mycorrhizal symbiosis of potato might mask infection by PVY in field observations.

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.

Phytopathogenic fungi use a variety of strategies to infect hosts and have a diverse sensitivity to the natural defence mechanisms of the host plant, e.g. synthesis of phytoalexins. Many important pathogens of crops belong to the problematic genus Phoma, including species recently reallocated to Peyronellaea. The work presents: (1) molecular identification of five pathogenic strains of Peyronellaea (syn. Phoma) based on two loci, actin (ACT) and β-tubulin (TUB), and morphological observations; (2) differentiation of these strains based on their enzymatic activity; and (3) their effect on biosynthesis of phytoalexin in Hippeastrum scales. Phylogenetic analysis showed a close relationship between our strains and Peyronellaea curtisii (syn. Phoma narcissi) species. This was also supported by morpho-anatomical analysis although its results were less conclusive. Some phenotypic traits of the strains overlapped with characteristics of other Phoma s.l. species, allowing for misidentification based on morphology alone. Tested cellulolytic, pectolytic and amylolytic activities were strain specific. A high level of amylase activity was positively correlated with cellulase activity but negatively correlated with pectolytic activity. Moreover, some strains suppressed phytoalexin production in Hippeastrum scales and were resistant to various concentrations of phytoalexin added to the culture medium. Based on these results, we suggest that pathogenicity in P. curtisii strains is complex and that it may be associated with their interactive physiological potential for enzymatic attack and defence capabilities against phytoalexin.

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.

Potato plants can be colonized by various viruses and by symbiotic, saprophytic and pathogenic fungi. However, the significance of interactions of viral infection and fungal colonization is hardly known. This work presents a model experiment in which the influence of three different types of fungal associations on the growth and physiology of the potato variety Pirol was tested individually or in combination with infection by PVY. It was hypothesized that simultaneous viral and fungal infections increase the biotic stress of the host plant, but mutualistic plant-fungal associations can mask the impact of viral infection. In the present study, a symbiotic arbsucular mycorrhizal fungus, Glomus intraradices, significantly stimulated the growth of plants infected with PVY. In contrast, two saprophytic Trichoderma spp. strains either did not influence or even inhibited the growth of PVY-infected plants. Also, inoculation of PVY-infected potato plants with a pathogenic strain of Colletotrichum coccodes did not inhibit the plant growth. Growth of the PVY-free potato plants was not promoted by the symbiotic fungus, whereas T. viride, T. harzianum and C. coccodes had an evident inhibitory effect. The strongest growth inhibition and highest concentration of H2O2, as an indicator of biotic stress, was observed in PVY-free potato plants inoculated with T. harzianum and C. coccodes strains. Surprisingly, ultrastructural analysis of PVY-infected plant roots colonized by G. intraradices showed virus-like structures in the arbuscules. This pointed to the possibility of mycorrhizal-mediated transmission of virus particles and has to be further examined by testing with immunoassays and real transmission to uninfected plants. In conclusion, although mycorrhiza formation might decrease the impact of PVY infection on plants, a possible role of mycorrhizal fungi as virus vectors is discussed.

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.

Mechanical damage to scales of Hippeastrum × hybr. bulbs leads to the formation of phytoalexin-like compounds which redden the wounded tissue. The reaction is accompanied by an increase in methyl jasmonate (JA-Me). Applying 2-(4-isobutylphenyl) propionic acid, a jasmonate biosynthesis inhibitor, decreases the level of endogenous jasmonates and decreases the plant's ability to produce the red pigment. Experimental results indicate that jasmonates are involved in the defense response to wounding in Hippeastrum, which is manifested in the formation of red pigment, a compound of chalcones and flavans with phytoalexin-like properties.

Adenylyl cyclases (ACs) are enzymes that generate cyclic AMP, which is involved in different physiological and developmental processes in a number of organisms. Here, we report the cloning and characterization of a new plant adenylyl cyclases (AC) gene, designated HpAC1 , from Hippeastrum x hybridum . This gene encodes a protein of 206 amino acids with a calculated molecular mass of 23 kD and an isoelectric point of 5.07. The predicted amino acid sequence contains all the typical features of and shows high identity with putative plant ACs. The purified, recombinant HpAC1 is able to convert ATP to cAMP. The complementation test that was performed to analyze the ability of HpAC1 to compensate for the AC deficiency in the Escherichia coli SP850 strain revealed that HpAC1 functions as an adenylyl cyclase and produces cyclic AMP. Moreover, it was shown that the transcript level of HpAC1 and cyclic AMP concentration changed during certain stress conditions. Both mechanical damage and Phoma narcissi infection lead to two sharp increases in HpAC1 mRNA levels during a 72-h test cycle. Changes in intracellular cAMP level were also observed. These results may indicate the participation of a cAMP-dependent pathway both in rapid and systemic reactions induced after disruption of symplast and apoplast continuity.

The aim of this work was to determine if there is any relationship between an endogenous phyA level and photoperiodic flower induction. The level of phyA was characterised with polyclonal antibodies directed to phyA from pea. At first it was detected that phyA level is predominant in cotyledons, whereas in roots and stems the concentration of labile phytochrome is rather low. So cotyledons were used for later experiments. In these cotyledons exposed to light illumination a rapid destruction of phyA has been observed. The loss of extractable phyA chromoprotein occurs already after 60 min of irradiation. Pharbitis nil is a short-day plant and a single 16-hours-long dark period is fully inductive. We assessed that phyA level is extremely low during a long inductive night and an immunodetectable phytochrome appears only after 24 hours of darkness. The obtained results suggest that labile phytochrome is not taking part in the direct control of the photoperiodic flower induction.

Signaling pathways, and specifically the signaling pathway of calcium, have been widely implicated in the regulation of a variety of signals in plants. Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways, the functional characterization of which is of great interest because they play important roles during growth and in response to a wide range of environmental and developmental stimuli. Here, we report the first evidence of transient and specific elevation of PnCDPK1 transcript level and enzyme activity following conversion of a leaf bud to a flower bud, as well as participation of PnCDPK1 in evocation and flower morphogenesis in Pharbitis nil. Fluorescence microscopy immunolocalization and biochemical analysis confirmed the presence of CDPK in shoot apexes. The protein level was low in leaves, vegetative apexes and increased significantly in apexes after a flowering long-induction night. In the vegetative apex, a very weak PnCDPK1 protein signal was accumulated prominently in the zone of the ground meristem and in external layers of tissues of the cortex. After the dark treatment, the signal in cells of the ground meristem was still present, but a significantly stronger signal appeared in epidermal cells, cortex tissue, and leaf primordium. At the onset of flower meristem development, the PnCDPK1 level diverged significantly. PnCDPK1 mRNA, protein level and enzyme activity were very low at the beginning of flower bud development and gradually increased in later stages, reaching the highest level in a fully open flower. Analysis of flower organs revealed that PnCDPK1 was accumulated mainly in petals and sepals rather than in pistils and stamens. Our results clearly indicate that PnCDPK1 is developmentally regulated and may be an important component in the signal transduction pathways for flower morphogenesis. Findings from this research are important for further dissecting mechanisms of flowering and functions of CDPKs in flowering plants.

The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei. Electronic supplementary material The online version of this article (doi:10.1007/s00425-012-1640-z) contains supplementary material, which is available to authorized users.

Calcium-dependent protein kinases (CDPKs) are central calcium signal decoders. Here we report the isolation and characterization of the Japanese Morning Glory’s (Pharbitis nil) CDPK gene, termed PnCDPK1. The full-length cDNA of 1943 bp contains an open reading frame for PnCDPK1 consisting of 514 amino acid residues and a calculated molecular mass of 57.9 kDa. The deduced amino acid sequence suggests that this protein contains the kinase domain at the amino terminus and autoregulatory and calmodulin-like domain at the carboxy terminus. Sequence alignment indicated that PnCDPK1 shared high similarities with other CDPKs. Biochemical analyses showed that bacterially expressed recombinant protein was catalytically active and was able to phosphorylate the histone III-S in a calcium-dependent manner. Besides the identification of PnCDPK1 as a member of the CDPK family, it was shown that a transcript of PnCDPK1 was modulated during germination and seedling growth. Moreover, PnCDPK1 mRNA was present in every tested organ, including root, hypocotyl, and cotyledon of the light- and dark-grown plant; however, the higher expression level was found in dark-treated ones. The PnCDPK1 mRNA level in the cotyledons exhibited a significant increase after moving seedlings into the dark, peaking 2 and 8 h after dark exposure, and then it gradually decreased. Expression was significantly changed by exposure to red light after 8 h in the dark. These data unequivocally identify the product of the PnCDPK1 gene as a calcium-dependent protein kinase and emphasize the potential that this gene can be an element of light signal transduction involved in growth and development of P. nil.

Guanylyl cyclases (GCs) are enzymes involved in the biosynthesis of cyclic guanosine monophosphate (cGMP). Here we report the cloning and characterization of a new guanylyl cyclase, designated PnGC-1, from Pharbitis nil. This gene encodes a protein of 286 amino acids, with a calculated molecular mass of 32 kDa. The predicted amino acid sequence contains all typical features and shows high identity with known plant GCs. The GST-PnGC-1 was catalytically active in E. coli cells and the purified, recombinant PnGC-1 was able to convert GTP to cGMP in the presence of Mn2+. Moreover, the enzyme activity was strongly inhibited by a specific sGC inhibitor, NS2028, whereas in the case of nitric oxide, an animal sGC stimulator, no positive effect was observed. Besides the identification of the PnGC-1 as a guanylyl cyclase, it was shown that a transcript of PnGC-1 was present in every tested organ of the light- or dark-grown plants; however, the highest expression level was found in dark-treated plants. The PnGC-1 mRNA level in the cotyledons exhibited diurnal oscillations under short-day conditions (8/16-h photoperiod). Meanwhile, monitoring of transcript levels in cotyledons exposed to a special photoperiodic regime (24 h light of low intensity then 24 h long night with or without far-red light before the night) revealed that a stabile phytochrome is involved in this process. These data unequivocally identify the product of the PnGC-1 gene as a guanylyl cyclase and emphasize the potential that soluble GC can be an element of light signal transduction.

It is known that the level of cGMP is modulated in plant cells in response to a number of stimuli but intracellular events dependent on cGMP metabolism are not clear. Guanylyl cyclases (GCs) are enzymes which are responsible for synthesis of cGMP in eukaryotic and prokaryotic cells. To collect evidence for the participation of cGMP in light signal transduction we isolated enzyme with guanylyl cyclase activity from Pharbitis nil and analysed its level and activity during photoperiodic flower induction. Soluble proteins were isolated from seedlings of a model short-day plant P. nil, partly purified and identified by in vivo and in vitro enzyme assay. In green plants enzyme activity amounted to 484 nmol cGMP/min/mg protein, whereas in etiolated plants it was three times lower (158 nmol cGMP/min/mg protein). Analyse cyclase consists of a single polypeptide of Mr 40 kDa. In order to determine if changes in guanylyl cyclase activity occurred in response to a long, inductive night, we measured enzyme activity in 4-h intervals and observed its increase at 4, 8 and 16 h of darkness. This pattern also fits well with changes in the endogenous cGMP level during a 16 h long flower inductive night. Immunocytochemical analysis confirmed these observations and revealed that changes in the GC level during light/dark conditions appeared. During 16 h long inductive night the strongest signal was observed in cotyledons after 4 and 16 h of the darkness. A high level of fluorescence was generally distributed in mesophyll, however, it was also observed in guard cells. Staining was apparently absent in the veins and cotyledon body. Furthermore, the location inside the cell was analysed. The protein was immunolocalized preferentially in the cytosol, chloroplasts and peroxysomes. Taken together, these data demonstrate in Pharbitis nil the presence of an enzyme which is able to convert GTP to cGMP. Because its level and activity are affected by light we believe that GC/cGMP play a substantial role in light/dark dependent process in plants, such as photoperiodic flower induction.

It was revealed that cGMP is involved in the control of photoperiodic flower induction. Further insight into the signalling function of cGMP is likely to be obtained by analysis of its effectors. Therefore, in the present study, we used various agents that cause changes in cGMP-dependent kinase (PKG) activity and examined their effects on the activity of kinase isolated from Pharbitis nil and flower induction. It was found that exogenous applications of PKG activators (cGMP, 8-pCPT-cGMP, 8-Br-cGMP, 8-pCPT-PET-cGMP) to cotyledons which were exposed to a 12-h-long subinductive night significantly increased flowering response. From among the many antagonists of cGMP-dependent protein kinase Rp-8-Br-PET-cGMPS, Rp-8-pCPT-cGMP and the synthetic heptapeptide inhibitor of PKG were used for our analysis. When Rp-8-Br-PET-cGMPS and Rp-8-pCPT-cGMP were applied during a 16-h-long inductive night, significant reduction in the number of flower buds was observed, whereas synthetic heptapeptide did not change the intensity of flowering. The influence of the analysed chemicals on protein kinase activity was also examined in vitro. With the exception of synthetic heptapeptide, which seems ineffective, the enzyme activity was stimulated by all agonists and significantly reduced by all antagonists. The activity of protein kinase was assayed in P. nil soluble protein fractions from plants grown under flower-inducing and non-inducing conditions. In vitro phosphorylation was slightly greater in the soluble fraction obtained from plants grown under the flower-inducing condition, reaching 1.05 nmol/min/mg protein, when compared to the control 0.81 nmol/min/mg protein. In relation to the results described above, we can conclude that cGMP as a mediator participating in photoperiodic flower induction may govern this process by the phosphorylation mechanism via its influence on cGMP-dependent protein kinase activity.

Guanosine 3',5'-cyclic monophosphate (cGMP) is a critical component of many (patho)physiological processes in plants whilst guanylyl cyclases (GCs) which catalyse the formation of cGMP from GTP have remained somewhat elusive. Consequently, the two major aims are the discovery of novel guanylyl cyclases and the identification of GC/cGMP mediated processes. To identify a novel GC from Hippeastrum hybridum plant and facilitate the preparation of guanylyl cyclase in an amount sufficient for further crystallographic studies, we have constructed an overproduction system for this enzyme. This gene encodes a protein of 256 amino acids, with a calculated molecular mass of 28kD. The predicted amino acid sequence contains all the typical features and shows a high identity to other plant GCs. The GST-HpGC1 was catalytically active in Escherichia coli cells and the purified, recombinant HpGC1 was able to convert GTP to cGMP in the presence of divalent cations. The used overexpression system yields a guanylyl cyclase as 6% of the bacterial cytosolic protein. Besides the identification of HpGC1 as a guanylyl cyclase, the study has shown that the level of HpCG1 mRNA changed during stress conditions. Both mechanical damage and a Peyronellaea curtisii (=Phoma narcissi) fungi infection led to an initial decrease in the HpGC1 transcript level, followed by a substantial increase during the remainder of the 48-h test cycle. Moreover, significant changes in cyclic GMP level were observed, taking the form of oscillations. In conclusion, our data unequivocally identified the product of the HpGC1 gene as a guanylyl cyclase and demonstrates that such an overproduction system can be successfully used in enzyme synthesis. Furthermore, they indicate a link between the causing stimulus (wounding, infection) and guanylyl cyclase expression and the increase in cGMP amplitude. Therefore, it is concluded that appearance of cyclic GMP as a mediator in defense and wound-healing mechanisms provides a clue to the regulation of these processes.

CDPK kinases are a unique class of calcium sensor/responders that regulate many growth and developmental processes as well as stress responses of plants. PnCDPK1 kinase from Pharbitis nil is regulated by light and contributes to seed germination, seedling growth and flower formation. Following an earlier work in which we identified the PnCDPK1 coding sequence and a 330bp long 3'UTR (untranslated region), we present for the first time the genomic organization of PnCDPK1, including intron analysis and the gene copy number designation. We completed the research by identifying the 5'-flanking region of PnCDPK1 and analyzed it in silico, which led to the discovery of several cis-regulatory elements involved in light regulation, embryogenesis and seed development. The functional analysis of P. nil CDPK showed characterization of the PnCDPK1 transcript and PnCDPK protein level during seed formation and fruit maturation. The greatest amount of PnCDPK1 mRNA was present in the last stages of seed maturation. Moreover, two PnCDPK proteins of different molecular masses were discovered during fruit development, showing various protein accumulation and activity profile. The 56kDa protein dominated in the early stages of fruit development, whereas the smaller protein (52kDa) was prominent in the latter stages.

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from NAD+ by ADP-ribosyl cyclases described for several animal cells. Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release channels. There is also information about the sub-micromolar concentration of cADPR in plant cells. Whether cADPR can act as a Ca2+-mobilizing intracellular messenger in plant tissue is an unresolved question. Despite the obvious importance of monitoring cADPR cellular levels under various physiological conditions in plants, its measurement has been technically difficult and requires specialized reagents. In the present study a widely applicable sensitivity assay for cADPR is described. We show that Pharbitis nil tissue from cotyledons contains a certain cADPR level. To explain the possible roles of this second messenger in photoperiodic flower induction, some physiological experiments were also performed. The exogenous applications of cADPR to Pharbitis nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. Nevertheless 8-Br-cADPR inhibited flowering when these compounds were applied during a 16-h-long inductive night. The effect of ruthenium red, a calcium channel blocker and ryanodine, a calcium channel stimulator, on the photoperiodic induction of flowering was also studied. Ruthenium red, when applied before and during an inductive 16-h dark period, slightly inhibited flowering, whereas ryanodine, when applied before and during a 12-h long subinductive night, stimulated flower bud formation. We also confirmed evidence that Ca2+ ions are involved in the photoperiodic induction of flowering. Thus, the obtained results may suggest the involvement of cyclic ADPR-activated Ca2+ mobilization in the photoperiodic flower induction process in Pharbitis nil.

A protein, identifiable as calmodulin (CaM), has been isolated from the seedling tissue of Pharbitis nil. The method has been developed to isolate a high quality protein from plant tissue containing the high content of polyphenols. This protein was relatively heat-stable and bound to hydrophobic resin in calcium-dependent manner. It was recognized by the antibody against pea and carrot, but did not bind to antibody against Dictyostelium discoideum. This protein had Mr of 15 kDa and 18.5 kDa in the presence and absence of Ca2+, respectively, and was able to stimulate calmodulin-deficient cAMP phosphodiesterase. Based on its migration on SDS-PAGE gels, Mr and binding to anti-CaM antibodies it was deduced that calmodulin from P. nil is essentially identical to calmodulin isolated from other plants.

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 Mr 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 α and β 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.

Little work was done so far with phytochrome from Pharbitis nil. Purification of phyA from this plant has been exceptionally difficult. Labile phytochrome was presented in too small amount to obtain either absorption spectra or enough protein to produce antibodies. Monoclonal antibodies mAP5, MAC 50, 52, 198 recognized Pharbitis nil labile phytochrome poorly, so it was necessary to develop independently an antiserum against labile phytochrome. The antiserum was prepared against proteolytically undegraded phytochrome obtained from etiolated Avena and Pisum seedlings using conventional methods. The antiserum to phytochrome from each of the above mentioned plants was prepared by injecting purified phytochrome into rabbits. The newly produced polyclonal antibodies to phyA from Avena and Pisum were used to characterize phytochrome from etiolated seedlings of Pharbitis nil. The cross reaction was tested by immunobloting. Both kinds of PAbs recognised phyA from Pharbitis nil, however IgG against the labile phytochrome from Pisum gave stronger reaction. The recognized peptide had the molecular weight of about 120-kDa.

A new enzymatic activity, which catalyses formation in vitro of di-O-(indole-3-acetyl)-D-glucose from 1-O-(indole-3-acetyl)-beta-D-glucose has been found in extracts of Zea mays seedlings. The structure of di-O-(indole-3-acetyl)-D-glucose, not as yet described, has been assigned by GC-MS, 1H NMR and ammonolysis.

Cyclic nucleotides (cAMP and cGMP) play an essential role in many important cellular processes in prokaryotic and eukaryotic organisms. They are produced by purine nucleotide cyclases: adenylyl and guanylyl cyclases. They are classified as one of two distinct forms: soluble and bound to membranes. Beside the differences in enzyme localization, the domain structure and regulation of enzymes activity are also diverse. However, all cyclases possess three groups of important residues: substrate specifying residue, metal binding residues and transition state stabilization residues. The natural occurrence of cyclic nucleotides in plants is now established. It was shown that in higher plants cNMPs act as a second messengers in a large number of (patho)physiological responses. However, it is only recently that the first plant enzymes with AC and GC activity of the unique structure have been identified and functionally characterized. In this study a systematic analysis of all the known prokaryotic, fungal and animal cyclases was done and direct evidences for the presence AC and GC in plant cells were shown.

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 Km 0.5 mM for GTP. A plot of 1/v versus 1/[GTP] was linear and Vmax 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.

Summary Light is one of the most important environmental factors influencing the induction of flowering in plants. Light is absorbed by specific photoreceptors – the phytochromes and cryptochromes system – which fulfil a sensory and a regulatory function in the process. The absorption of light by phytochromes initiates a cascade of related biochemical events in responsive cells, and subsequently changes plant growth and development. Induction of flowering is controlled by several paths. One is triggered by the guanosine-3′:5′-cyclic monophosphate (cGMP) level. Thus, the aim of our study was to investigate the role of cGMP in phytochrome-controlled flowering. It is best to conduct such research on short-day plants because the photoperiodic reactions of only these plants are totally unequivocal. The most commonly used plant is the model short-day plant Pharbitis nil. The seedlings of P. nil were cultivated under special photoperiodic conditions: 72-h-long darkness, 24-h-long white light with low intensity and 24-h-long inductive night. Such light conditions cause a degradation of the light-labile phytochrome. Far red (FR) treatment before night causes inactivation of the remaining light-stable phytochrome. During the 24-h-long inductive darkness period, the total amount of cGMP in cotyledons underwent fluctuations, with maxima at the 4th, 8th and 14th hours. When plants were treated with FR before the long night, fluctuations were not observed. A red light pulse given after FR treatment could reverse the effect induced by FR, and the oscillation in the cGMP level was observed again. Because the intracellular level of cGMP is controlled by the opposite action of guanylyl cyclases (GCs) and phosphodiesterases (PDEs), we first tested whether accumulation of the nucleotide in P. nil tissue may be changed after treatment with a GC stimulator or PDE inhibitor. Accumulation of the nucleotide in P. nil cotyledons treated with a stimulator of cGMP synthesis (sodium nitroprusside) was markedly (approximately 80%) higher. It was highest in the presence of dipyridamole, whereas 3-isobutyl-1-methylxanthine did not significantly affect cGMP level. These results show that the analysed compounds were able to penetrate the cotyledons’ tissue, and that they influenced enzyme activity and cGMP accumulation. FR light applied at the end of the 24-h-long white light period inhibited flowering. Exogenous cGMP added on cotyledons could reverse the effect of FR, especially when the compound was applied in the first half of the long night. Flowering was also promoted by exogenous application of guanylyl cyclase activator and phosphodiesterase inhibitors, and in particular dipyridamole. The results obtained suggest that an endogenous cGMP system could participate in the mechanism of a phytochrome-controlled flowering in P. nil .

Calcium signals play an important role in many aspects of plant growth and development, including plant response to biotic and abiotic stress. The stimulus characteristic intracellular Ca2+ signals are generated in plant cells by a variety of stimuli, including changes in environmental conditions, interaction with microbes and growth and development processes. Cytoplasmatic calcium brings about responses by interacting with target proteins, like calcium-dependent kinases. In plant there are at least five classes of protein kinases (CDPK, CRK, CCaMK, CaMK and SnRK3), which activity is regulated by calcium ions. In this article the structure, regulation and function of calcium stimulated protein kinases are briefly reviewed.

Summary The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil .

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.

In this study using biochemical approaches we identified two calcium-dependent protein kinases (CDPKs) named PnCDPK52 and PnCDPK56 in soluble protein extracts from seedlings of Pharbitis nil. Both enzymes phosphorylated the specific substrate histone III-S in the presence of Ca2+ and cross-reacted with antibodies against the CDPK. PnCDPKs exhibited quite different activity and protein levels during germination and successive stages of seedling growth. PnCDPK52 protein level was high in seeds and during germination, whereas PnCDPK56 increased in the next stages of seedling growth, being the dominant enzymes in mature seedlings, of the light- and dark-grown plant. In all cases both activity and accumulation of protein PnCDPK56 was higher in dark grown plants whereas exposure to light reduced both factors. When etiolated cotyledons were exposed to light, the activity of PnCDPK56 was reduced to the basal level within 5 h. Conversely, increasing activity of PnCDPK56 in cotyledons of green plants shifted to darkness was extremely rapid, reaching the maximum level after just 1 h of darkness and then gradually decreased. Further lengthening of the darkness to 16 h resulted in a strong increase in activity at 12 h. These data indicate that at least two isoforms of CDPK are involved in germination and seedling growth of P. nil. The differences in PnCDPKs strongly argue for the pleiotropic role of these isoforms. It seems that PnCDPK52 is associated with the germination process and PnCDPK56 with seedling growth. Moreover it suggests that activity of PnCDPK56 is controlled by light via the photoreceptor-dependent pathway.

It has been previously suggested that 1-O-IAGIuc growth stimulation occurs as the effect of its hydrolysis into a free IAA. In present experiments castanospermine, a known β-glucosidase inhibitor, was included. 1-O-IAGluc in the presence of castanospermine stimulated growth of corn coleoptiles segments even stronger then free IAA. So, it seems that 1-O-IAGluc itself, is responsible for the observed stimulation of corn coleoptile segments elongation.