Katsura Izui

Molecular and physiological evaluation of transgenic tobacco plants expressing a maize phosphoenolpyruvate carboxylase gene under the control of the cauliflower mosaic virus 35S promoter

The expression of maize (Zea mays) phosphoenolpyruvate carboxylase (PEPC) gene constructs in transgenic tobacco plants (Nicotiana tabacum) was studied. Where transcription was under the control of a CaMV 35S promoter, maize PEPC transcripts of the correct size were detected. Western blot analysis indicated that the transgenic plants contained about twice as much PEPC as non-transformed plants. Furthermore, the enzymatic activity of PEPC in the leaves of these transgenic plants was up to twice as high as that in non-transformed plants. Two forms of PEPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form (the maize isoform) gave a high apparentKm value for phosphoenolpyruvate (PEP) and a high maximum activity, and the other (the tobacco isoform) exhibited a low apparentKm value for PEP and a low maximum activity. These biochemical differences resulted in several significant physiological changes in the transgenic plants: (1) the growth rate of the transgenic plants was lower than that of non-transgenic plants: (2) chlorophyll content per leaf area was relatively lower in the transgenic plants; and (3) the quantum yield of photosynthesis in the transgenic plants was not affected by changes in leaf temperature.

Isolation and characterization of cDNAs encoding mitochondrial phosphate transporters in soybean, maize, rice, and Arabidopsis

AbstractcDNA clones encoding mitochondrial phosphate transporters were isolated from four herbaceous plants. The cDNAs for the soybean, maize and rice transporters contained entire coding regions, whereas the Arabidopsis cDNA lacked the 5′ portion. The hydropathy profiles of the deduced amino acid sequences predicted the existence of six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. In soybeans, the mRNA level for the transporter was high in tissues containing dividing cells. It was suggested that there are multiple copies of transporter genes in both dicots and monocots. The soybean transporter was expressed as inclusion bodies in Escherichia coli, solubilized with detergents, and then reconstituted into liposomes. The resulting proteoliposomes exhibited high phosphate transport activity. The activity was inhibited by N-ethylmaleimide, like those of mammalian phosphate transporters.

Maize leaf phosphoenolpyruvate carboxylase: phosphorylation of Ser15 with a mammalian cyclic AMP-dependent protein kinase diminishes sensitivity to inhibition by malate

The so‐called light‐activation of phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) involved in C4 photosynthesis is known to be mediated by phosphorylation. A cyclic AMP‐dependent protein kinase from bovine heart was found to be able to phosphorylate PEPC. The phosphorylation was accompanied by the changes in kinetic properties, which were very similar to the reported light activation. The phosphorylated amino acid residue was identified as Ser and the position of this Ser on the primary structure [(1988) FEBS Lett. 229, 107‐110] was determined to be Ser15.

Transcriptome Profiling of Lotus japonicus Roots During Arbuscular Mycorrhiza Development and Comparison with that of Nodulation

Abstract To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT–PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs.

Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases.

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first step in the fixation of atmospheric CO(2) during C(4) photosynthesis. The crystal structure of C(4) form maize PEPC (ZmPEPC), the first structure of the plant PEPCs, has been determined at 3.0 A resolution. The structure includes a sulfate ion at the plausible binding site of an allosteric activator, glucose 6-phosphate. The crystal structure of E. coli PEPC (EcPEPC) complexed with Mn(2+), phosphoenolpyruvate analog (3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), and an allosteric inhibitor, aspartate, has also been determined at 2.35 A resolution. Dynamic movements were found in the ZmPEPC structure, compared with the EcPEPC structure, around two loops near the active site. On the basis of these molecular structures, the mechanisms for the carboxylation reaction and for the allosteric regulation of PEPC are proposed.

Lotus japonicus forms early senescent root nodules with Rhizobium etli

Mesorhizobium loti and Rhizobium etli are microsymbionts of the Lotus and Phaseolus spp., respectively, and secrete essentially the same Nod factors. Lotus japonicus efficiently formed root nodules with R. etli CE3, irrespective of the presence or absence of a flavonoid-independent transcription activator nodD gene. On a nitrogen-free medium, however, the host plant inoculated with R. etli showed a severe nitrogen deficiency symptom. Initially, the nodules formed with R. etli were pale pink and leghemoglobin mRNA was detectable at significant levels. Nevertheless, the nodules became greenish with time. Acetylene-reduction activity of nodules formed with R. etli was comparable with that formed by M. loti 3 weeks postinoculation, but thereafter it decreased rapidly. The nodules formed with R. etli contained much more starch granules than those formed with M. loti. R. etli developed into bacteroids in the L. japonicus nodules, although the density of bacteroids in the infected cells was lower than that in the nodules formed with M. loti. The nodules formed with R. etli were of the early senescence type, in that membrane structures were drastically disintegrated in the infected cells of the greenish nodules. Thus, L. japonicus started and then ceased a symbiotic relationship with R. etli at the final stage.

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.

cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases

Using degenerate oligonucleotide primers corresponding to conserved regions of the calcium-dependent protein kinase (CDPK) family, we carried out a polymerase chain reaction and obtained four distinct partial-length cDNAs from a maize leaf library. We then used these clones as probes for conventional screening and isolated 19 longer clones from another cDNA library of maize seedlings. These clones were classified into four groups based on their DNA cross-hybridization. Two full-length cDNAs, designated as ZmCDPK9 and ZmCDPK7, were sequenced and characterized. The predicted protein of each clone was a typical CDPK with eleven canonical subdomains of protein kinases, and four EF-hand calcium-binding motifs in its N-terminal and C-terminal halves, respectively. The catalytic and regulatory domains were linked by a well-conserved junction domain. The N-terminus of the protein also contained a consensus sequence for an N-myristoylation signal. Northern blot analysis showed that the transcription level of each gene was higher in roots and etiolated leaves than in green leaves. To confirm the calcium dependency of the maize enzymes, the entire coding region of ZmCDPK9 was subcloned into an expression vector so that it was in frame with the vector-encoded peptide tags. A cell-free extract of Escherichia coli transformed with the recombinant plasmid exhibited calcium-dependent phosphorylation activity, using casein as a substrate.

Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full‐length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615–1628]. Alignment of the sequence for the N‐terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3′‐untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.

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.