Search

GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for Dictyostelium species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation., This research was funded by the European Research Council under grant 742288 and by The Wellcome Trust under grant 100293/Z/12/Z; European Research Council [742288].

Gluconeogenesis is a key interface between organic acid/amino acid/lipid and sugar metabolism. The aims of this article are four-fold. First, to provide a concise overview of plant gluconeogenesis. Second, to emphasise the widespread occurrence of gluconeogenesis and its utilisation in diverse processes. Third, to stress the importance of the vacuolar storage and release of Krebs cycle acids/nitrogenous compounds, and of the role of gluconeogenesis and malic enzyme in this process. Fourth, to outline the contribution of fine control of enzyme activity to the coordinate-regulation of gluconeogenesis and malate metabolism, and the importance of cytosolic pH in this.

In grapevines, as in other plants, sucrose and its constituents glucose and fructose are fundamentally important and carry out a multitude of roles. The aims of this review are three-fold. First, to provide a summary of the metabolism and transport of sucrose in grapevines, together with new insights and interpretations. Second, to stress the importance of considering the compartmentation of metabolism. Third, to outline the key role of acid invertase in osmoregulation associated with sucrose metabolism and transport in plants.

In selective breeding, a central parameter in summarizing the proportion of variance due to genetics for the purpose of predicting gains from selection is realized heritability (h2). We applied the genome-wide complex trait analysis (GCTA) method to genome-wide SNP data obtained by 2b-RAD reduced-representation genotyping and phenotypic data for four traits (shell length, shell height, shell width and whole wet weight) with the aim of establishing the heritability for growth in Zhikong scallop (Chlamys farreri) in a selective breeding program. The GCTA-based heritabilities of 0.42 (S.E. 0.09) for shell length, 0.47 (S.E. 0.07) for shell height, 0.54 (S.E. 0.11) for shell width and 0.28 (S.E. 0.03) for whole wet weight, which were estimated with uncommon SNPs (26,471 SNPs with MAF >2%), were close to and strongly correlated (r = 0.957) with traditional estimates of realized heritability and in the moderate-to-high range, in line with values previously obtained for growth rates in bivalves. hGCTA2 with 20,000 and 10,000 SNPs were very close to estimates with uncommon SNPs and appeared relatively robust to SNP number. Removing causal SNPs had little effect on obtaining reliable estimates of hGCTA2, suggesting that causal SNPs is not necessary for accurate estimates of h2. Chromosome-wise heritability estimates suggested that the genetic contribution to growth complex traits is scattered across the genome and driven by many loci with small effect rather than a few causal loci with a large effect. These results indicate that the genome-wide complex trait analysis method may be useful for estimating realized heritability for growth in Zhikong scallop with SNPs obtained by reduced-representation genotyping approaches, which is less expensive and faster than full-genome sequencing for non-model species growing in natural environments.

Stone fruits of the Rosaceae family consist of several distinct parts, and these include the flesh, woody endocarp, and seed. To understand the metabolism of these fruits, it is necessary to have knowledge of both their structure and growth characteristics. The nitrogen metabolism of the different tissues of stone fruits is interlinked. For example, there is an import and storage of nitrogenous compounds in the endocarp that are then exported to the seed. Moreover, there are links between the metabolism of nitrogen and that of malic/citric acids. In this article, the structure and growth characteristics, together with the import/export, contents, metabolism, and functions of nitrogenous compounds and organic acids in the different parts of stone fruits and their seeds are reviewed. Para citar este articulo: Famiani F, Bonghi C, Chen Z-H, Drincovich MF, Farinelli D, Lara MV, Proietti S, Rosati A, Vizzotto G and Walker RP (2020) Stone Fruits: Growth and Nitrogen and Organic Acid Metabolism in the Fruits and Seeds—A Review. Front. Plant Sci. 11:572601. doi: 10.3389/fpls.2020.572601 Fil: Famiani, Franco. Università degli Studi di Perugia. Dipartimento di Scienze Agrarie, Alimentari e Ambientali; Italia. Fil: Bonghi, Claudio. University of Padova Agripolis. Department of Agronomy, Food, Natural Resources, Animals and Environment; Italy. Fil: Chen, Zhi-Hui. University of Dundee. College of Life Science; United Kingdom. Fil: Drincovich, María Fabiana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas Fil: Farinelli, Daniela. Università degli Studi di Perugia. Dipartimento di Scienze Agrarie, Alimentari e Ambientali; Italia. Fil: Lara, María V. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI -CONICET); Argentina. Fil: Proietti, Simona. Consiglio Nazionale delle Ricerche. Istituto di Ricerca sugli Ecosistemi Terrestri; Italia. Fil: Rosati, Adolfo. Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria (CREA). Centro di ricerca Olivicoltura, Frutticoltura e Agrumicoltura; Italia. Fil: Vizzotto, Giannina. University of Udine. Department of Agricultural, Food, Environmental, and Animal Sciences; Italy. Fil: Walker, Robert P. Università degli Studi di Perugia. Dipartimento di Scienze Agrarie, Alimentari e Ambientali; Italia.

Cyclic di-GMP, an important prokaryote second messenger is used by the eukaryote Dictyostelium discoideum as a secreted signal to trigger stalk formation in fruiting bodies. Cyclic di-GMP is synthesized by a prokaryote-type diguanylate cyclase DgcA, but its mode of action was unknown. Transcriptional profiling yielded several target genes for cyclic di-GMP, which were tested for cyclic di-GMP induced expression in mutants with similar phenotypes as dgca-. A mutant with reduced PKA activity showed defective cyclic di-GMP induced stalk gene expression. Cyclic di-GMP increased cAMP levels in wild-type cells, but not in a mutant that lacked adenylate cyclase A (ACA) activity in slugs. This mutant also did not show cyclic di-GMP induced stalk gene expression. The stalk-less dgca- mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is the intermediate for cyclic di-GMP in normal development. ACA is expressed at the tip of emerging fruiting bodies, where it produces the cAMP pulses that organize morphogenetic movement. The tip is also the site where stalk differentiation initiates. Our finding that cyclic di-GMP acts on tip-expressed ACA explains why the Dictyostelium stalk is always formed at the morphogenetic organizer.

The complete chloroplast genome of Firmina danxiaensis, an important deciduous tree, was identified and sequenced in this study. The genome size is 161,205 bp, the GC content is 36.88%. A total of 129 genes were identified, including 84 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. 10 plastome accessions from Sterculiaceae were selected to assess the phylogenetic placement of genus and the result showed that F. danxiaensis is most closely related to F. simplex.

BackgroundDictyostelid social amoebas self-organize into fruiting bodies, consisting of spores and up to four supporting cell types in the phenotypically most complex taxon group 4. High quality genomes and stage- and cell-type specific transcriptomes are available for representative species of each of the four taxon groups. To understand how evolution of gene regulation in Dictyostelia contributed to evolution of phenotypic complexity, we analysed conservation and change in abundance, functional domain architecture and developmental regulation of their transcription factors (TFs).ResultsWe detected 440 sequence-specific TFs across 33 families, of which 68% were upregulated in multicellular development and about half conserved throughout Dictyostelia. Prespore cells expressed two times more TFs than prestalk cells, but stalk cells expressed more TFs than spores, suggesting that gene expression events that define spores occur earlier than those that define stalk cells. Changes in TF developmental expression, but not in TF abundance or functional domains occurred more frequently between group 4 and groups 1–3, than between the more distant branches formed by groups 1 + 2 and 3 + 4.ConclusionsPhenotypic innovation is correlated with changes in TF regulation, rather than functional domain- or TF acquisition. The function of only 34 TFs is known. Of 12 TFs essential for cell differentiation, 9 are expressed in the cell type for which they are required. The information acquired here on conserved cell type specifity of 120 additional TFs can effectively guide further functional analysis, while observed evolutionary change in TF developmental expression may highlight how genotypic change caused phenotypic innovation.

Selective breeding is a common and effective approach for genetic improvement of aquaculture stocks with parental selection as the key factor. Genomic selection (GS) has been proposed as a promising tool to facilitate selective breeding. Here, we evaluated the predictability of four GS methods in Zhikong scallop (Chlamys farreri) through real dataset analyses of four economical traits (e.g., shell length, shell height, shell width, and whole weight). Our analysis revealed that different GS models exhibited variable performance in prediction accuracy depending on genetic and statistical factors, but non-parametric method, including reproducing kernel Hilbert spaces regression (RKHS) and sparse neural networks (SNN), generally outperformed parametric linear method, such as genomic best linear unbiased prediction (GBLUP) and BayesB. Furthermore, we demonstrated that the predictability relied mainly on the heritability regardless of GS methods. The size of training population and marker density also had considerable effects on the predictive performance. In practice, increasing the training population size could better improve the genomic prediction than raising the marker density. This study is the first to apply non-linear model and neural networks for GS in scallop and should be valuable to help develop strategies for aquaculture breeding programs.

In this study the occurrence of a number of enzymes involved in gluconeogenesis was investigated in both tomato fruits and leaves during their development and senescence and in some other fruits. The enzymes studied were phosphoenolpyruvate carboxykinase (PEPCK), pyruvate orthophosphate dikinase (PPDK) and glyoxysomal isocitrate lyase (ICL). PPDK was detected in the ripe flesh of tomato, and much smaller amounts were detected in the flesh of both peach and pepper, whereas it was not detected (not present or at very low abundance) in the other fruits which were investigated (apricot, aubergine, blackberry, blueberry, cherry, grape, plum, raspberry and red current). By contrast PEPCK was present in the flesh of all the fruits investigated. Very small amounts of ICL were detected in ripe tomato flesh. PEPCK was present in the skin, flesh, locular gel and columella of tomato fruit, and in these its abundance increased greatly during ripening. PPDK showed a similar distribution, however, its abundance did not increase during ripening. PEPCK was not detected in tomato leaves at any stage of their development or senescence. The content of PPDK g(-1) fresh weight (FW) increased in tomato leaves as they matured, however, it declined during their senescence. In tomato leaves the content of ICL g(-1) FW increased until the mid-stage of development, then decreased as the leaf matured, and then increased during the latter stages of senescence. In the flesh of tomato fruits the contents of PPDK and PEPCK g(-1) FW decreased during senescence. The results suggest that in fruits other than tomato the bulk of any gluconeogenic flux proceeds via PEPCK, whereas in tomato both PEPCK and PPDK could potentially be utilised. Further, the results indicate that the conversion of pyruvate/acetyl-CoA to malate by the glyoxylate cycle, for which ICL is necessary, is not a major pathway utilised by gluconeogenesis in fruits under normal conditions of growth. Finally, the results contribute to our understanding of the role of several enzymes in the senescence of both leaves and fruits.

We used a template-assisted approach to develop synthetic antimicrobial peptides, which differ from naturally occurring antimicrobial peptides that can compromise host natural defenses. Previous researches have demonstrated that symmetrical distribution patterns of amino acids contribute to the antimicrobial activity of natural peptides. However, there is little research describing such design ideas for synthetic α-helical peptides. Therefore, here, we established a centrosymmetric α-helical sequence template (y + hhh + y)n (h, hydrophobic amino acid; +, cationic amino acid; y, Gly or hydrophobic amino acid), which contributed to amphipathicity and a series of centrosymmetric peptides was designed with pairs of small amino acids (Ala and Gly), which were utilized to modulate the biological activity. The centrosymmetric peptides with 3 repeat units exhibited strong antimicrobial activity; in particular, the Gly-rich centrosymmetric peptide GG3 showed stronger selectivity for gram-negative bacteria without hemolysis. Furthermore, beyond our expectation, fluorescence spectroscopy and electron microscopy analyses indicated that the GG3, which possessed poor α-helix conformation, dramatically exhibited marked membrane destruction via inducing bacterial membrane permeabilization, pore formation and disruption, even bound DNA to further exert antimicrobial activity. Collectively, the Gly-rich centrosymmetric peptide GG3 was an ideal candidate for commercialization as a clinical therapeutic to treat gram-negative bacterial infections.

In this study some aspects of organic and amino acid metabolism in cherry endocarp and seed were investigated during their development. The abundance and location of a number of enzymes involved in these processes were investigated. These enzymes were aspartate aminotransferase (AspAT; EC:2.6.1.1), glutamine synthetase (GS; EC:6.3.1.2), phosphoenolpyruvate carboxylase (PEPC; EC:4.1.1.31), phosphoenolpyruvate carboxykinase (PEPCK; EC:4.1.1.49), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC:4.1.1.39). There was a transient and massive accumulation of vegetative storage proteins in the endocarp. These proteins were remobilised as the endocarp lignified and at the same time that proteins were accumulated in the seed. This raised the possibility that a proportion of imported amino acids were temporarily stored in the endocarp as protein, and that these were later utilised by the seed when it started to accumulate storage proteins. Rubisco was present in the embryo and integuments of the seed although no chlorophyll was present. This is the first time that Rubisco has been detected in non-green seeds. The maximum abundance of Rubisco in the seed coincided with the deposition of seed storage proteins. A possible function for Rubisco in cherry seed is discussed. PEPCK was located in the integuments and appeared when seed storage proteins were being accumulated. In the integuments and embryo AspAT, GS, PEPC and Rubisco also appeared, or greatly increased in abundance, when seed storage proteins were being deposited.

Cyclic AMP acting on protein kinase A controls sporulation and encystation in social and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R (ACR), is essential for spore encapsulation. In addition to its cyclase (AC) domain, ACR harbors seven transmembrane helices, a histidine kinase domain, and two receiver domains. We investigated the role of these domains in the regulation of AC activity. Expression of an ACR-YFP fusion protein in acr(-) cells rescued their sporulation defective phenotype and revealed that ACR is associated with the nuclear envelope and endoplasmic reticulum. Loss of the transmembrane helices (ΔTM) caused a 60% reduction of AC activity, but ΔTM-ACR still rescued the acr(-) phenotype. The isolated AC domain was properly expressed but inactive. Mutation of three essential ATP-binding residues in the histidine kinase domain did not affect the AC activity or phenotypic rescue. Mutation of the essential phosphoryl-accepting aspartate in receivers 1, 2, or both had only modest effects on AC activity and did not affect phenotypic rescue, indicating that AC activity is not critically regulated by phosphorelay. Remarkably, the dimerizing histidine phosphoacceptor subdomain, which in ACR lacks the canonical histidine for autophosphorylation, was essential for AC activity. Transformation of wild-type cells with an ACR allele (ΔCRA) that is truncated after this domain inhibited AC activity of endogenous ACR and replicated the acr(-) phenotype. Combined with the observation that the isolated AC domain was inactive, the dominant-negative effect of ΔCRA strongly suggests that the defunct phosphoacceptor domain acquired a novel role in enforcing dimerization of the AC domain.

The hypothesis that positive links exist among plant taxonomic diversity, belowground microbial taxonomic and metabolic diversities was tested for four secondary vegetation successional stages (tussock (T), shrub (S), secondary forest (SF) and primary forest (PF)) in Huanjiang county, SW China. Soil bacterial communities were characterized by DNA fingerprinting and metabolic profiling. Along the succession, Shannon diversity indices followed the order SF>PF>S>T for plant taxonomic diversity, T>SF>PF>S for bacterial operational taxonomic diversity, SF>T>S>PF for fungal operational taxonomic diversity, and SF>PF>S>T for bacterial metabolic diversity. Significant positive correlations were found between bacterial and fungal taxonomic diversities. However, there was no significant correlation between soil microbial taxonomic diversity and bacterial metabolic diversity. Two-way ANOVA revealed that vegetation and season, as well as their interaction, had significant effects on soil microbial (fungal and bacterial) taxonomic diversities, but that there were no seasonal effects on metabolic diversity. However, PCA and MANOVA revealed highly significant differences among the bacterial community-level physiological profiles, reflecting the successional sequence. The findings from this survey support the notion that there are strong interactions between aboveground and belowground communities and suggest that bacterial metabolic and plant taxonomic diversities, but not microbial taxonomic and metabolic diversities, can be correlated.

P(i) (inorganic phosphate) limitation severely impairs plant growth and reduces crop yield. Hence plants have evolved several biochemical and morphological responses to P(i) starvation that both enhance uptake and conserve use. The mechanisms involved in P(i) sensing and signal transduction are not completely understood. In the present study we report that a previously uncharacterized transcription factor, BHLH32, acts as a negative regulator of a range of P(i) starvation-induced processes in Arabidopsis. In bhlh32 mutant plants in P(i)-sufficient conditions, expression of several P(i) starvation-induced genes, formation of anthocyanins, total P(i) content and root hair formation were all significantly increased compared with the wild-type. Among the genes negatively regulated by BHLH32 are those encoding PPCK (phosphoenolpyruvate carboxylase kinase), which is involved in modifying metabolism so that P(i) is spared. The present study has shown that PPCK genes are rapidly induced by P(i) starvation leading to increased phosphorylation of phosphoenolpyruvate carboxylase. Furthermore, several Arabidopsis proteins that regulate epidermal cell differentiation [TTG1 (TRANSPARENT TESTA GLABRA1), GL3 (GLABRA3) and EGL3 (ENHANCER OF GL3)] positively regulate PPCK gene expression in response to P(i) starvation. BHLH32 can physically interact with TTG1 and GL3. We propose that BHLH32 interferes with the function of TTG1-containing complexes and thereby affects several biochemical and morphological processes that respond to P(i) availability.

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in different tissues of Arabidopsis thaliana throughout its vegetative and reproductive growth. The A. thaliana genome contains two PEPCK genes (PCK1 and PCK2), and these are predicted to generate 73,404 and 72,891 Da protein products, respectively. Both genes were transcribed in a range of tissues; however, PCK1 mRNA appeared to be more abundant and was present in a wider range of tissues. PEPCK protein was present in flowers, fruit, developing seed, germinating seed, leaves, stems and roots. Two PEPCK polypeptides, of approximately 74 and approximately 73 kDa were detected by immunoblotting, and these may arise from PCK1 and PCK2, respectively. PEPCK was abundant in cotyledons during post-germinative growth, and this is consistent with its well established role in gluconeogenesis. PEPCK was also abundant in sink tissues, such as young leaves, in developing flowers, fruit and seed. Immunohistochemistry and in situ hybridization showed that PEPCK was present in the nectaries, stigma, endocarp of the fruit wall and in tissues involved in the transfer of assimilates to the developing ovules and seeds, such as the vasculature and seed coat. The potential functions of PEPCK in A. thaliana are discussed.

Dictyostelid social amoebas represent one of several groups of genetically divergent lineages that display aggregative multicellularity. In this chapter, we describe the evolution of developmental complexity in Dictyostelia and discuss the signalling mechanisms that control the developmental programme of the model organism Dictyostelium discoideum. We also reconstruct the evolutionary history of these developmental control mechanisms from environmental sensing in the unicellular ancestors of Dictyostelia. Finally, we explore the parameters that define the boundary between uni- and multicellularity.

In cucumber ( Cucumis sativus L.), phosphoenolpyruvate carboxykinase (PEPCK) was shown by activity measurements and immunoblots to be present in leaves, stems, roots, flowers, fruit and seed. However, immunolocalisation showed that it was present only in certain cell types. PEPCK was present in the companion cells of the adaxial phloem of minor veins, the adaxial and abaxial phloem of larger veins, the internal and external phloem of vascular bundles in petioles and stems, the phloem in roots and the extra-fascicular phloem in leaves, cotyledons, petioles and stems. Immunohistochemical evidence suggests that both the extra-fascicular phloem and the adaxial phloem are involved in the transport of amino acids. In roots and stems, the abundance of PEPCK was greatly increased by watering plants with a solution of ammonium chloride at low, but not at high pH. PEPCK also increased in leaves, but not roots or stems, of seedlings grown in an atmosphere containing 5% CO(2), and in roots and stems of seedlings watered with butyric acid. All these treatments are known to lower the pH of plant cells. Amino acid metabolism in the phloem may produce an excess of carbon skeletons, pH perturbations and an imbalance in the production/utilisation of NADH. This raises the possibility that PEPCK may function in the conversion of these carbon skeletons to PEP, which, depending on the energy requirements of the phloem, is subsequently utilised by either gluconeogenesis or the Krebs cycle, which both consume protons.

The effect of Mn2+/Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+/Mg2+ similar to those found in the cytosol (low micromolar Mn2+ and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+/Mg2+ led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The K m(CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 μmCO2, comparable with the K m(CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems.

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plantUrochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.

A light-regulated adenylyl cyclase, mPAC, was previously identified from the cyanobacterium Microcoleus chthonoplastes PCC7420. MPAC consists of a flavin-based blue light-sensing LOV domain and a catalytic domain. In this work, we expressed mPAC in an adenylate cyclase A null mutant (aca-) of the eukaryote Dictyostelium discoideum and tested to what extent light activation of mPAC could restore the cAMP-dependent developmental programme of this organism. Amoebas of Dictyostelium, a well-established model organism, generate and respond to cAMP pulses, which cause them to aggregate and construct fruiting bodies. mPAC was expressed under control of a constitutive actin-15 promoter in D. discoideum and displayed low basal adenylyl cyclase activity in darkness that was about five-fold stimulated by blue light. mPAC expression in aca- cells marginally restored aggregation and fruiting body formation in darkness. However, more and larger fruiting bodies were formed when mPAC expressing cells were incubated in light. Extending former applications of light-regulated AC, these results demonstrate that mPAC can be used to manipulate multicellular development in eukaryotes in a light dependent manner.

Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

Background In our previous study we found that the expression of stlA showed peaks both in the early and last stages of development and that a product of SteelyA, 4-methyl-5-pentylbenzene-1,3-diol (MPBD), controlled Dictyostelium spore maturation during the latter. In this study we focused on the role of SteelyA in early stage development. Principal Findings Our stlA null mutant showed aggregation delay and abnormally small aggregation territories. Chemotaxis analysis revealed defective cAMP chemotaxis in the stlA null mutant. cAMP chemotaxis was restored by MPBD addition during early stage development. Assay for cAMP relay response revealed that the stlA null mutant had lower cAMP accumulation during aggregation, suggesting lower ACA activity than the wild type strain. Exogenous cAMP pulses rescued the aggregation defect of the stlA null strain in the absence of MPBD. Expression analysis of cAMP signalling genes revealed lower expression levels in the stlA null mutant during aggregation. Conclusion Our data indicate a regulatory function by SteelyA on cAMP signalling during aggregation and show that SteelyA is indispensable for full activation of ACA.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2. The role of the enzyme in gluconeogenesis and anaplerotic reactions in a range of organisms is discussed, along with the important function in C4 and CAM photosynthesis in higher plants. In addition, new data are presented indicating that PEPCK may play a key role in amino acid metabolism. It is proposed that PEPCK is involved in the conversion of the carbon skeleton of asparagine/aspartate (oxaloacetate) to that of glutamate/glutamine (2-oxoglutarate). This metabolism is particularly important in the transport system, seeds and fruits of higher plants.

The aim of this study was to investigate whether gluconeogenesis catalysed by phosphoenolpyruvate carboxykinase (PEPCK) occurs during leaf senescence. This was addressed by determining changes in the abundance and intercellular location of enzymes necessary for gluconeogenesis during the senescence of barley leaves and cucumber cotyledons. PEPCK was never present in barley leaves, despite the presence of large amounts of isocitrate lyase (ICL), a key enzyme of the glyoxylate cycle, and of its product, glyoxylate. Although PEPCK was present in non-senescent cucumber cotyledons, its abundance declined during senescence. Throughout senescence, PEPCK was only present in the trichomes and vasculature, whereas ICL was located in mesophyll cells. Pyruvate,Pi dikinase (PPDK) which, in concert with NAD(P)-malic enzyme, is also capable of catalysing gluconeogenesis, was present in non-senescent barley leaves and cucumber cotyledons, but in both plants its abundance decreased greatly during senescence. The abundance of ICL was greatly reduced in senescing detached barley leaves by either illumination or by co-incubation with sucrose, and greatly increased in darkened attached barley leaves. These results argue against the large-scale occurrence of gluconeogenesis during senescence catalysed either by PEPCK or PPDK. In cucumber cotyledons, PEPCK may play a role in metabolic processes linked to the export of amino acids, a role in which phosphoenolpyruvate carboxylase may also be involved. The amount of ICL was increased by starvation and during senescence may function in the conversion of lipids to organic acids, which are then utilised in the mobilisation of amino acids from leaf protein.

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Técsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459–468). In the present study, we investigated the role of PEPCK in C4 photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-14C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn2+, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-14C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn2+ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK.

In this study the abundance and location of phosphoenolpyruvate carboxykinase (PEPCK) was determined in the flesh and skin of the sweet cherry (Prunus avium L.) cultivar Durone Nero II during development. PEPCK was not present in young fruit but appeared in both tissues as the fruit increased in size. In these there was no net dissimilation of malic acid, which accounts for the bulk of their organic acid contents when PEPCK was present. To assist in understanding the function of PEPCK, the abundance of a number of other enzymes was determined. These enzymes were aspartate aminotransferase (AspAT), glutamine synthetase (GS), phosphoenolpyruvate carboxylase (PEPC), pyruvate, orthophosphate dikinase (PPDK), and ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). A potential role for PEPCK in the regulation of pH and the utilization of malate in gluconeogenesis in the flesh and skin of cherries is presented.

The Arabidopsis gene At5g21040 encodes a protein containing both WD40 and F-box motifs, termed FBX2. A T-DNA insertional mutant in this gene was obtained. Analysis of this mutant line showed that FBX2 is a negative regulator of several P(i) starvation responses. FBX2 interacts with BHLH32, another negative regulator of P(i) starvation responses. We suggest that FBX2 may be part of an SCF-like complex that recruits BHLH32 and its partners, potentially targeting the latter for proteolysis.

Phosphoenolpyruvate carboxylase (PEPC) is thought to play many roles in C(3) plants including the provision of biosynthetic precursors and control of pH during N assimilation. Its activity is controlled via phosphorylation catalysed by PEPC kinases, which are encoded by PPCK genes. We examined PPCK expression in response to changes in the supply of N or C, and to changes in intracellular pH, using cultured Arabidopsis cells and seedlings. The results show that expression of both PPCK1 and PPCK2 is increased by C availability, but does not respond to N availability. Expression of the two PPCK genes and the phosphorylation state of PEPC are increased in response to increasing intracellular pH. Elevated pH also reduces the repression of PPCK gene expression by P(i). Expression of phosphoenolpyruvate carboxykinase (PEPCK), which catalyses the decarboxylation of oxaloacetate, is decreased in response to increasing intracellular pH. pH homeostasis may be mediated at least partly by reciprocal changes in the expression of PPCK genes and PEPCK.

Previous studies of grapes and tomatoes have shown that the abundance of phosphoenolpyruvate carboxykinase (PEPCK) increases in their flesh at the start of ripening, and that this coincides with a decrease in its citrate and/or malate content. Thus, PEPCK might function in the catabolism of organic acid anions during the ripening of these fruits. In the present study, the abundance of PEPCK was determined in the flesh of blueberries, raspberries, red currants, and strawberries at different stages of their development. In addition, changes in the amounts of citrate, malate, soluble sugars, isocitrate lyase, NADP-malic enzyme, phosphoenolpyruvate carboxylase, and pyruvate, orthophosphate dikinase in the flesh were determined. PEPCK was not detected in strawberry flesh, in which there was no dissimilation of malate or citrate. In the flesh of the other fruits, the abundance of PEPCK increased during ripening to an amount that was similar to that in grapes and tomatoes. In the flesh of blueberries and red currants, PEPCK was most abundant when there was dissimilation of malate. In the flesh of raspberries, PEPCK was most abundant when there was dissimilation of malate and citrate. These results are consistent with PEPCK playing a role in the dissimilation of citrate and/ or malate in the flesh of these fruits during ripening. However, PEPCK was also present in the flesh of blueberries, raspberries, and red currants when there was no dissimilation of malate or citrate, and this raises the possibility that PEPCK might have additional functions. Dissection of blueberries provided evidence that both PEPCK and phosphoenolpyruvate carboxylase were present in the same cells, and possible functions for this are discussed.

The aim of this article is to outline our understanding of the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Although emphasis is placed on the enzyme derived from flowering plants, other organisms are also considered, because comparative studies provide invaluable information. The following points are considered in detail. Firstly, the possibility that PEPCK in all organisms arose from a common ancestor, and that the extension of about 12 kDa at the N-terminus of PEPCK-ATP from flowering plants, which is not possessed by PEPCK-ATP from other organisms, is homologous to the N-terminal region of PEPCK-GTP. Secondly, the regulation of PEPCK activity in flowering plants by reversible protein phosphorylation is described. Phosphorylation of the N-terminal extension possessed by PEPCK from flowering plants reduces its catalytic velocity several-fold at physiological concentrations of oxaloacetate. How this is likely to contribute to regulation of PEPCK in vivo is described. Thirdly, it is proposed that in flowering plants PEPCK plays a widespread role as a component of a mechanism that counteracts intracellular acidification. The proposed role of PEPCK in this mechanism is similar to that in the kidney during acidosis.

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation., Highlights • Amoebas differentiate into dormant encapsulated cysts when exposed to environmental stress • Encystation renders pathogenic amoebas resistant to antibiotics and biocides • The social amoeba Polysphondylium pallidum is amenable to genetic approaches to resolve encystation mechanisms • The cAMP phosphodiesterase RegA and the sensor histidine kinases that regulate RegA activity are deeply conserved • RegA regulates encystation in P.pallidum and in the pathogen Acanthamoeba castellani