Shingo Hata

Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development.

We took advantage of a subtractive hybridization procedure to isolate a set of cDNA clones of nodule-specific genes (nodulin genes) from developing soybean root nodules. Single-standed 32P-labelled cDNA synthesized from nodule poly(A)+ RNA was hybridized with a large excess of uninfected root poly(A)+ RNA. Unhybridized cDNA was selected and used to screen nodule cDNA libraries. By this procedure we isolated several novel nodulin cDNA clones together with most of the nodulin cDNAs previously described. Four novel nodulin genes, which were expressed long before the onset of nitrogen fixation, were further characterized. GmN # 36 and GmN # 93 transcripts appeared in the roots less than 3 days after sowing and inoculation with Bradyrhizobium, but GmN # 36 transcripts were also detected at very low levels in the stems of uninfected plants. Transcripts of GmN # 315 and GmN # 70 first appeared at 6–7 days, just before nodule emergence. Amino acid sequences of the predicted products of GmN # 36, GmN # 93 and GmN # 70 exhibited no significant homology to proteins identified so far. The GmN # 315 encoded protein has a limited but significant homology to some plant cyanins, suggesting that it is a metal-binding glycoprotein. In situ hybridization studies revealed that GmN # 36 transcripts first appeared in the pericycle cells of the root stele near the infected site. During nodule emergence they were found in a few cell layers surrounding the vascular strands connecting the nodule meristem with the root stele, and in mature nodules they were present specifically in the pericycle cells in vascular bundles. These observations led us to hypothesize that GmN # 36 gene products play a role in the transport and/or degradation of photosynthate. On the other hand, GmN # 93 transcripts first appeared in the primary nodule meristem just below the root epidermis. In mature nodules they were only present in the infected cells.

Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

Genetics of Symbiosis in Lotus japonicus: Recombinant Inbred Lines, Comparative Genetic Maps, and Map Position of 35 Symbiotic Loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

Induction and signaling of an apoptotic response typified by DNA laddering in the defense response of oats to infection and elicitors.

Cells in the primary leaves of oats displayed internucleosomal DNA cleavage in response to incompatible crown rust infection. DNA laddering also was evident in leaves treated with calcium ionophore A23187, nonspecific elicitors such as chitin and chitosan oligomers, and victorin, which functions as a specific elicitor in Pc-2/Vb containing oat leaves. The nuclei in a victorin-treated susceptible oat line were positive for the TUNEL assay. These elicitors clearly induced a 28-kDa nuclease (p28) in addition to three constitutive nucleases of 33, 24, and 22 kDa. Activation of p28 preceded the appearance of DNA laddering and possibly was mediated by de novo synthesis and/or cysteine protease activity. Pharmacological studies showed that the induction of DNA laddering was associated with oxidative stress, Ca2+ influx, and serine and cysteine proteases. Protein kinase and calmodulin activities did not seem to be involved in the induction of DNA laddering by victorin, whereas kinase-mediated signals were involved in DNA laddering induced by A23187. Protein kinase, calmodulin, G-protein activities, and Ca2+ influx, however, are involved in phytoalexin production. Our results imply that p28 is a possible nuclease candidate responsible for the induction of DNA laddering. The results also demonstrated that the mediators involved in the induction of apoptosis depended on the type of stimuli, whereas p28 and serine and cysteine proteases commonly are associated with each elicitor-induced apoptosis.

Isolation and characterization of cDNA clones encodingcdc2 homologues fromOryza sativa: a functional homologue and cognate variants

SummaryUsing probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2+/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.

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.

cDNA cloning of a novel cdc2+/CDC28-related protein kinase from rice

A cDNA clone, named R2, has been isolated by screening a rice cell cDNA library with a redundant oligonucleotide probe derived from the conserved ATP binding site of cdc2+/CDC28 protein kinases. The cDNA contained the entire coding sequence for a 424 amino acid polypeptide with a molecular mass of 47.6 kDa. The R2 mRNA, 2.1 kb in size, was expressed in both cultured rice cells and rice seedings at similar levels. The predicted R2 protein has canonical motifs for ATP binding and catalysis, and is significantly homologous (up to 47%) to members of the cdc2+/ CDC28 subfamily of serine/threonine protein kinase. The R2 protein is a novel member of the subfamily.

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.

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.