Tsuyoshi Furumoto

A plastidial sodium-dependent pyruvate transporter

Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids, terpenoids and branched-chain amino acids. In spite of the importance of pyruvate uptake into plastids (organelles within cells of plants and algae), the molecular mechanisms of this uptake have not yet been explored. This is mainly because pyruvate is a relatively small compound that is able to passively permeate lipid bilayers, which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C3 and C4 plants of the genera Flaveria and Cleome, here we have identified a novel gene that is abundant in C4 species, named BASS2 (BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2). The BASS2 protein is localized at the chloroplast envelope membrane, and is highly abundant in C4 plants that have the sodium-dependent pyruvate transporter. Recombinant BASS2 shows sodium-dependent pyruvate uptake activity. Sodium influx is balanced by a sodium:proton antiporter (NHD1), which was mimicked in recombinant Escherichia coli cells expressing both BASS2 and NHD1. Arabidopsis thaliana bass2 mutants lack pyruvate uptake into chloroplasts, which affects plastid-localized isopentenyl diphosphate synthesis, as evidenced by increased sensitivity of such mutants to mevastatin, an inhibitor of cytosolic isopentenyl diphosphate biosynthesis. We thus provide molecular evidence for a sodium-coupled metabolite transporter in plastid envelopes. Orthologues of BASS2 can be detected in all the genomes of land plants that have been characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import into plastids.

AGF1, an AT-Hook Protein, Is Necessary for the Negative Feedback of AtGA3ox1 Encoding GA 3-Oxidase

Negative feedback is a fundamental mechanism of organisms to maintain the internal environment within tolerable limits. Gibberellins (GAs) are essential regulators of many aspects of plant development, including seed germination, stem elongation, and flowering. GA biosynthesis is regulated by the feedback mechanism in plants. GA 3-oxidase (GA3ox) catalyzes the final step of the biosynthetic pathway to produce the physiologically active GAs. Here, we found that only the AtGA3ox1 among the AtGA3ox family of Arabidopsis (Arabidopsis thaliana) is under the regulation of GA-negative feedback. We have identified a cis-acting sequence responsible for the GA-negative feedback of AtGA3ox1 using transgenic plants. Furthermore, we have identified an AT-hook protein, AGF1 (for the AT-hook protein of GA feedback regulation), as a DNA-binding protein for the cis-acting sequence of GA-negative feedback. The mutation in the cis-acting sequence abolished both GA-negative feedback and AGF1 binding. In addition, constitutive expression of AGF1 affected GA-negative feedback in Arabidopsis. Our results suggest that AGF1 plays a role in the homeostasis of GAs through binding to the cis-acting sequence of the GA-negative feedback of AtGA3ox1.

Plant calcium-dependent protein kinase-related kinases (CRKs) do not require calcium for their activities

In plants, calcium‐dependent protein kinases (CDPKs) make up a large family that is characterized by a C‐terminal calmodulin(CaM)‐like domain. Recently, a novel carrot cDNA clone encoding an atypical CDPK, which has a significantly degenerate sequence in the CaM‐like domain, was found and named CDPK‐related protein kinase (CRK) [Lindzen, E. and Choi, J.H. (1995) Plant Mol. Biol. 28, 785–797]. We obtained two different cDNA clones from maize which encode CRKs. For the first enzymatic characterization of CRK, a maize cDNA clone was expressed in E. coli. The recombinant protein efficiently phosphorylated casein, a conventional protein substrate. Notably, in this in vitro phosphorylation assay, the kinase activity did not require calcium as an activator. Thus, CRKs were suggested to be novel calcium‐independent protein kinases having a degenerate CaM domain, the function of which remains to be elucidated.

Phosphoenolpyruvate carboxylase kinase involved in C4 photosynthesis in Flaveria trinervia: cDNA cloning and characterization1

In C4 plants, phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), a key enzyme in C4 photosynthesis, is controlled by reversible phosphorylation of a conserved Ser residue near the N‐terminus. We now report the first cloning of a cDNA from a C4 plant, Flaveria trinervia, which encodes the specific protein kinase (FtPEPC‐PK) involved in the phosphorylation of C4‐form PEPC. Several lines of supportive evidence are: strict substrate specificity of the recombinant enzyme, prominent light/dark response of the transcript level and abundant expression in leaves of C4 plant (F. trinervia) but very low expression in a C3 plant of the same genus (Flaveria pringlei). We also discuss the possibility that the FtPEPC‐PK gene has co‐evolved with the PEPC gene to participate in C4 photosynthesis.

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Not Essential for High Photosynthetic Rates in the C4 Species Flaveria bidentis

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C4 photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C4 photosynthetic CO2 fixation, we have transformed Flaveria bidentis (a C4 dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO2 and light response of CO2 assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C4 photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C4 photosynthesis.

Maize C4-form phosphoenolpyruvate carboxylase engineered to be functional in C3 plants : mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity

Introducing a C(4)-like pathway into C(3) plants is one of the proposed strategies for the enhancement of photosynthetic productivity. For this purpose it is necessary to provide each component enzyme that exerts strong activity in the targeted C(3) plants. Here, a maize C(4)-form phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was engineered for its regulatory and catalytic properties so as to be functional in the cells of C(3) plants. Firstly, amino acid residues Lys-835 and Arg-894 of maize PEPC, which correspond to Lys-773 and Arg-832 of Escherichia coli PEPC, respectively, were replaced by Gly, since they had been shown to be involved in the binding of allosteric inhibitors, malate or aspartate, by our X-ray crystallographic analysis of E. coli PEPC. The resulting mutant enzymes were active but their sensitivities to the inhibitors were greatly diminished. Secondly, a Ser residue (S780) characteristically conserved in all C(4)-form PEPC was replaced by Ala conserved in C(3)- and root-form PEPCs to decrease the half-maximal concentration (S(0.5)) of PEP. The double mutant enzyme (S780A/K835G) showed diminished sensitivity to malate and decreased S(0.5)(PEP) with equal maximal catalytic activity (V(m)) to the wild-type PEPC, which will be quite useful as a component of the C(4)-like pathway to be introduced into C(3) plants.

cDNA cloning and expression analysis of a non-photosynthetic ferredoxin gene in morning glory (Pharbitis nil)

A full-length cDNA encoding a non-photosynthetic ferredoxin was isolated from apical buds of morning glory (Pharbitis nil), a short-day plant, by differential screening under flower-inducing and non-inducing conditions. Northern analysis and in situ hybridization showed that the transcript was abundant in shoot apices and root tips. The transcript level in the apical buds decreased with the flower-inducing light treatment.

In Vivo Phosphorylation of Arabidopsis thaliana 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase

Fructose 2,6-bisphosphate (F2,6BP) is a non-metabolic sugar-phosphate ubiquitously found in eukaryotes. This compound regulates the balance of glycolysis and gluconeogenesis. In plants, its physiological functions are thought to be an inhibition of sucrose synthesis in source leaves and to be an activation of glycolysis in sink organs. Both synthesis and degradation of F2,6BP are catalyzed by a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (PFK 2/FBPase 2; EC 2.7.1.105/EC 3.1.3.46). In animals and yeast, it is well known that several isozymes are regulated by protein phosphorylation. In plant enzymes, on the other hand, it had been thought that the protein phosphorylation is not involved in the enzymatic regulation. An Arabidopsis cDNA for PFK 2/FBPase 2 was isolated and expressed in E. coli. Antisera prepared from the recombinant enzyme identified 96-kD and 92-kD protein bands of plant crude protein extracts. Results of in vitro calf intestine alkaline phosphatase treatment and an immunoprecipitation of the crude extracts from [32P]-labeled whole plants revealed that the 96-kD protein was phosphorylated in vivo. Immunoblot analysis showed that the 96-kD protein was enriched in younger leaves than in older leaves. These results indicate that Arabidopsis enzyme is phosphorylated. Now we try to identify the biochemical effects of this phosphorylation, especially whether this phosphorylation can change the enzymatic activities or not.

Cloning of Cell-Type Specific Genes in Maize Leaves and Characterization of PEP Carboxykinase in Bundle-Sheathstrands

C4 plants are adapted well to grow under high temperature and intense light conditions. Because they have a unique mechanism for CO2 concentration, their photorespiration is negligible. The C4 photosynthesis functions under the cooperation of mesophyll cells (MC) and bundle-sheath-cells (BSC). Primary CO2 fixation reaction by PEP carboxylase occurs in MC. The C4 acids, malate and aspartate, are transported into BSC and decarboxylated. There CO2 is refixed into the Calvin cycle via ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) [1]. Most enzymes involved in the C4 metabolism have been investigated well about their cell-type localization, regulation of gene expression [2], post-translational regulation [3], and so on. However, many other proteins indispensable for C4 photosynthesis remain to be elucidated. To isolate cDNAs for the proteins functioning specifically in MC and BSC, we carried out differential screening and isolated a number of MC-specific- and bundle sheath strands (BSS)-specific clones. During the course of analysis of these clones, we found a phosphoenolpyruvate carboxykinase (PCK) [EC4.1.1.49] gene as a member of BSS-specific genes. In this report, we describe novel genes, which were expressed specifically in MC or in BSS, and characterization of maize PCK gene.