The Galpha subunit BCG1, the phospholipase C (BcPLC1) and the calcineurin phosphatase co-ordinately regulate gene expression in the grey mould fungus Botrytis cinerea

The Gα subunit BCG1 is essential for pathogenicity of the grey mould fungus Botrytis cinerea . Several processes such as the transition from primary infection to secondary invasive growth and the production of the phytotoxin botrydial are regulated by BCG1 via a cAMP-independent pathway. Our recent finding that the botrydial biosynthesis genes belong to the group of Ca²⁺/calcineurin-dependent genes suggested for the first time a connection between this Gα subunit and the calcineurin signalling pathway. To investigate whether this co-regulation of genes by BCG1 and calcineurin is a common feature, a cDNA macroarray approach was used to compare the gene expression pattern of the wild-type and the Δ bcg1 mutant, non-treated or treated with the calcineurin inhibitor cyclosporin A. We identified three sets of genes whose expression was regulated either by both BCG1 and calcineurin, or only by one of them. Among the BCG1/calcineurin-co-regulated genes, we found a new gene cluster coding for a yet unknown polyketide secondary metabolite. Furthermore, we show for the first time in a phytopathogenic fungus that the phospholipase C (BcPLC1) is a component of the BCG1- and calcineurin-dependent signalling pathway as several BCG1- and calcineurin-dependent genes were downregulated in bcplc1 knock-down mutants.     

Opposing functions of calcineurin and CaMKII regulate G-protein signaling in egg-laying behavior of C.elegans

Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.     

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase is involved in the tolerance of Arabidopsis seedlings to salt and osmotic stresses

In plants, pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) is a regulatory enzyme that participates in glycolysis and gluconeogenesis. Arabidopsis contains two PFPα subunit genes (PFPα1 and PFPα2) and two PFPβ subunit genes (PFPβ1 and PFPβ2). The single-knockout mutants of the PFP subunit genes were isolated, and double and quadruple pfp mutants were generated by crossing the single mutants. To elucidate the role of PFP in stress tolerance, the responses of the double and quadruple pfp knockout mutants to stress conditions, including osmotic and salt stresses, were examined. The seedling growth of the pfpα1/α2 and pfpβ1/β2 double mutants and the pfpα1/α2/β1/β2 quadruple mutant was severely retarded under salt and osmotic stress conditions compared with that of the wild type. The expression of PFP subunit genes increased in response to salt and osmotic stresses. In contrast, the vegetative growth of the wild type and pfp mutants after the seedling stage was similarly affected by salt and osmotic stresses. These findings suggest that PFP plays a role in the adaptation of Arabidopsis seedlings to salt and osmotic stresses.     

Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.

By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.     

Casein kinase II α subunits affect multiple developmental and stress-responsive pathways in Arabidopsis

Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory β subunits. The Arabidopsis genome has four α subunit and four β subunit genes, and members of both the α and β subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis.     

The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis

Ascorbic acid (AsA) is an important antioxidant in plants, and its biosynthesis is finely regulated through developmental and environmental cues; however, the regulatory mechanism remains unclear. In this report, the knockout and knockdown mutants of Arabidopsis AtERF98 decreased the AsA level, whereas the overexpression of AtERF98 increased it, which suggests that AtERF98 plays an important role in regulating AsA biosynthesis. AtERF98-overexpressing plants showed enhanced expression of AsA synthesis genes in the d-mannose/l-galactose (d-Man/l-Gal) pathway and the myo-inositol pathway gene MIOX4, as well as of AsA turnover genes. In contrast, AtERF98 mutants showed decreased expression of AsA synthesis genes in the d-Man/l-Gal pathway but not of the myo-inositol pathway gene or AsA turnover genes. In addition, the role of AtERF98 in regulating AsA production was significantly impaired in the d-Man/l-Gal pathway mutant vtc1-1, but the expression of the myo-inositol pathway gene or AsA turnover genes was not affected, which indicates that the regulation of AtERF98 in AsA synthesis is primarily mediated by the d-Man/l-Gal pathway. Transient expression and chromatin immunoprecipitation assays further showed that AtERF98 binds to the promoter of VTC1, which indicates that AtERF98 modulates AsA biosynthesis by directly regulating the expression of the AsA synthesis genes. Moreover, the knockout mutant aterf98-1 displayed decreased salt-induced AsA synthesis and reduced tolerance to salt. The supplementation of exogenous AsA increased the salt tolerance of aterf98-1; coincidently, the enhanced salt tolerance of AtERF98-overexpressing plants was impaired in vtc1-1. Thus, our data provide evidence that the regulation of AtERF98 in AsA biosynthesis contributes to enhanced salt tolerance in Arabidopsis.     

The calcineurin inhibitor gossypol impairs growth, cell signalling and development in Dictyostelium discoideum

The Dictyostelium genome harbors single copy genes for both the catalytic and regulatory subunits of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Since molecular genetic approaches to reduce the expression of these genes have failed so far, we attempted to pharmacologically target calcineurin activity in vivo by using the recently described calcineurin inhibitor, gossypol. Up-regulation of expression of the gene for the Ca2+-ATPase PAT1 in conditions of Ca2+ stress was reduced by gossypol. Dictyostelium wild-type cells treated with 12.5-100 microM gossypol showed reduced growth rates and impaired development. In addition, cell signalling was affected. A cell line that overproduces the catalytic subunit of calcineurin was more resistant to gossypol.     

Substrate specificities and kinetic properties of two (1→3), (1→4)-β-d-glucan endo-hydrolases from germinating barley (Hordeum vulgare)

Two β-d-glucan endo-hydrolases purified from germinating barley (Hordeum vulgare) hydrolyse (1→4)-β linkages in (1→3),(1→4)-β-d-glucans where the d-glucosyl residue is substituted at O-3, but will not hydrolyse (1→3)-β-d-glucans or (1→4)-β-d-glucans. Methylation analysis of hydrolytic products released from barley (1→3),(1→4)-β-d-glucan indicates that 3-O-β-cellobiosyl-d-glucose and 3-O-β-cellotriosyl-d-glucose are the major oligomers formed. The enzymes exhibit characteristic endo-hydrolase action-patterns on this substrate. Both enzyme can therefore be classified as (1→3),(1→4)-β-d-glucan 4-glucanohydrolases (EC 3.2.1.73). The reduced, pneumococcal polysaccharide RS III, which consists of alternating (1→3)- and (1→4)-linked β-d-glucosyl residues, is hydrolysed by the enzymes to release laminaribiose as a major oligomeric product. Although the kinetic parameters of the two enzymes are similar, one hydrolyses barley (1→3),(1→4)-β-d-glucan at a significantly higher rate than the other and is more stable at elevated temperatures.     

A zinc finger-containing glycine-rich RNA-binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress conditions

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to changing environmental conditions, the reports demonstrating their biological roles are severely limited. Here, we examined the functional roles of a zinc finger-containing GRP, designated atRZ-1a, in Arabidopsis thaliana under drought or salt stress conditions. Transgenic Arabidopsis plants overexpressing atRZ-1a displayed retarded germination and seedling growth compared with the wild-type plants under salt or dehydration stress conditions. In contrast, the loss-of-function mutants of atRZ-1a germinated earlier and grew faster than the wild-type plants under the same stress conditions. Germination of the transgenic plants and mutant lines was influenced by the addition of ABA or glucose, implying that atRZ-1a affects germination in an ABA-dependent way. H(2)O(2) was accumulated at higher levels in the transgenic plants compared with the wild-type plants under stress conditions. The expression of several germination-responsive genes was modulated by atRZ-1a, and proteome analysis revealed that the expression of different classes of genes, including those involved in reactive oxygen species homeostasis and functions, was affected by atRZ-1a under dehydration or salt stress conditions. Taken together, these results suggest that atRZ-1a has a negative impact on seed germination and seedling growth of Arabidopsis under salt or dehydration stress conditions, and imply that atRZ-1a exerts its function by modulating the expression of several genes under stress conditions.     

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.