Search

Mangrove ecosystems, where litter and organic components are degraded and converted into detrital materials, support rich coastal fisheries resources. Sesarmid (Grapsidae) crabs, which feed on mangrove litter, play a crucial role in material flow in carbon-rich and nitrogen-limited mangrove ecosystems; however, the process of assimilation and conversion into detritus has not been well studied. In this study, we performed microbiome analyses of intestinal bacteria from three species of mangrove crab and five sediment positions in the mud lobster mounds, including the crab burrow wall, to study the interactive roles of crabs and sediment in metabolism. Metagenome analysis revealed species-dependent intestinal profiles, especially in Neosarmatium smithi, while the sediment microbiome was similar in all positions, albeit with some regional dependency. The microbiome profiles of crab intestines and sediments were significantly different in the MDS analysis based on OTU similarity; however, 579 OTUs (about 70% of reads in the crab intestinal microbiome) were identical between the intestinal and sediment bacteria. In the phenotype prediction, cellulose degradation was observed in the crab intestine. Cellulase activity was detected in both crab intestine and sediment. This could be mainly ascribed to Demequinaceae, which was predominantly found in the crab intestines and burrow walls. Nitrogen fixation was also enriched in both the crab intestines and sediments, and was supported by the nitrogenase assay. Similar to earlier reports, sulfur-related families were highly enriched in the sediment, presumably degrading organic compounds as terminal electron acceptors under anaerobic conditions. These results suggest that mangrove crabs and habitat sediment both contribute to carbon and nitrogen cycling in the mangrove ecosystem via these two key reactions. [ABSTRACT FROM AUTHOR]

In January 2019, four cheetahs (Acinonyx jubatus) kept at a Japanese zoo intermittently showed respiratory signs following the incidence of seasonal influenza in animal caregivers. Respiratory materials (saliva, sputum and food tray swabs) were non‐invasively collected from the four cheetahs. Although we were unable to isolate the virus, the NP gene of influenza A virus was detected in three of the cheetahs but not in the fourth cheetah that had nearly recovered. From a food tray swab which tested weakly positive by a commercial influenza detection kit, we were able to obtain the whole‐genome sequence of the influenza A virus. Analysis of the genome, A/cheetah/Kanagawa/2/2019(H1N1), revealed that the virus was closely related to influenza A(H1N1)pdm09 viruses isolated from humans in Japan in the 2018–2019 winter. Production of haemagglutinin inhibition (HI) antibodies (64–128 HI) against an A(H1N1)pdm09 virus in plasma samples confirmed infection of all four cheetahs. The animals continued to produce antibodies for at least 314 days after disease onset. These findings strongly suggest that reverse zoonotic transmission of A(H1N1)pdm09 virus occurred from human to cheetah and subsequently from cheetah to cheetah in the zoo. We also show that specimens can be safely and non‐invasively collected from non‐domesticated animals and used to investigate respiratory infectious diseases. [ABSTRACT FROM AUTHOR]

SNAREs (soluble N -ethyl maleimide-sensitive factor attachment protein receptors) mediate membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s are the members of Qa-SNAREs in Lotus japonicus. Two isoforms, LjSYP132a and LjSYP132b , are generated by alternative splicing. Immunoblot analysis detected strong expression of LjSYP132s in infected root nodules and seeds by posttranscriptional modification. In either LjSYP132a or LjSYP132b silenced roots (RNAi-LjSYP132a, RNAi-LjSYP132b), the infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR showing that both genes were expressed at the early stage of infection. However, during the nodulation, only LjSYP132a was induced. LjSYP132s protein was observed in the Mesorhizobium loti -inoculated roots of mutants, nfr1 , castor and pollux , suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the peribacteroid membrane suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b , R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting that LjSYP132b has a role in pollen tube growth and nutrient transport in the plasma membrane of seeds. [ABSTRACT FROM AUTHOR]

SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins mediate membrane trafficking in eukaryotic cells. Both LjVAMP72a and LjVAMP72b are members of R-SNARE and belong to a symbiotic subgroup of VAMP72 in Lotus japonicus. Their sequences are closely related and both were induced in the root upon rhizobial inoculation. The expression level of LjVAMP72a in the nodules was higher than in the leaves or roots; however, LjVMAP72b was expressed constitutively in the leaves, roots, and nodules. Immunoblot analysis showed that not only LjVAMP72a but also LjVAMP72b were accumulated in a symbiosome-enriched fraction, suggesting its localization in the symbiosome membrane during nodulation. Since there was 89% similarity between LjVAMP72a and LjVAMP72b, knockdown mutant by RNAi suppressed both genes. The suppression of both genes impaired root nodule symbiosis (RNS). The number of bacteroids and the nitrogen fixation activity were severely curtailed in the nodules formed on knockdown roots (RNAi-LjVAMP72a/72b). Arbuscular mycorrhization (AM) was also attenuated in knockdown roots, indicating that LjVAMP72a and LjVAMP72b were required to establish not only RNS but also AM. In addition, transgenic hairy roots of RNAi-LjVAMP72a/72b suppressed the elongation of root hairs without infections by rhizobia or arbuscular mycorrhizal fungi. Amino acid alignment showed the symbiotic subclade of VAMP72s containing LjVAMP72a and LjVAMP72b were a conserved six amino acid region (HHQAQD) within the SNARE motif. Taken together, our data suggested that LjVAMP72a and LjVAMP72b positively controlled both symbioses and root hair formation by affecting the secretory pathway. [ABSTRACT FROM AUTHOR]

Fragrant rice has a potent flavor compound, 2-acetyl-1-pyrroline (2AP). A better understanding of the 2AP biosynthetic pathway is gained by proteomic analysis of two isogenic lines of Thai jasmine rice, Oryza sativa L. cv. Khao Dawk Mali 105, which differ only in the aromatic gene Os2AP. The protein profiles of two lines, from six growth stages, seedling to grain filling, had 41 identifiable protein spots. Four of these spots were betaine aldehyde dehydrogenase, a key enzyme responsible for 2AP production. This enzyme occurred in every growth stage of the non-aromatic rice line except smaller amount detected in the hard grain-filling stage of the aromatic line. Glyceraldehyde 3-phosphate dehydrogenase and aspartate aminotransferase, observed in the aromatic line, may involve in the metabolism of precursors for 2AP biosynthesis. In addition, glutamine synthetase and 1-cys peroxiredoxin A which function in ammonia reassimilation and hydrogen peroxide detoxification were unique in the aromatic line. However, proteins that correspond to photosynthesis and the nutrient reservoir were only detected in lower abundances. This possibly explains why the aroma rice grain weight is low. Our study proposed the possible role of these remarkable proteins which involved in 2AP biosynthesis in jasmine rice. [ABSTRACT FROM AUTHOR]

In leguminous root nodules, rhizobia differentiate into morphology specific to symbiosis, called bacteroids. As bacteroids are surrounded with peribacteroid membranes filled with peribacteroid solution (PBS), it is considered that PBS contains substances inducing differentiation of rhizobia into bacteroids. In this study, genome-wide expression profiles ofBradyrhizobium japonicumcells cultured in PBS purified from root nodule of soybean (Glycine maxL.) were compared with those of bacteroids using macroarray. PBS treatment preferentially induced regions in a large symbiosis island including various symbiosis relevant genes such asnod, fix, nolandnoe, in which 75% of regions were commonly induced in bacteroids, while general repressions outside of the symbiosis island seen in bacteroids were not observed in PBS treated cells. The present results suggest that PBS contained some, but not all, substances inducing expression of the genes which are involved in differentiation into bacteroids. [ABSTRACT FROM PUBLISHER]

Endophytic bacteria carrying out dinitrogen (N2) fixation and indole acetic acid (IAA) synthesis were firstly identified inC. alismatifolia, a globally important flower crop. Their potential as growth promoters to stimulate the rapid growth of host plant was also examined. It will be beneficial to reduce the propagation period of tissue culture plantlets, and also utilize as a biofertilizer for rhizome production in the field. Seven endophytic bacteria were isolated from the leaf, four isolates from the leaf base, and two from the rhizome. ECS203, a gram-negative bacterium with a round shape, showed the highest N2fixation at 4.2 nmol C2H4/106cells/hr, and ECS202 showed the highest IAA synthesis at 296 μL μg−1protein. Three selected isolates of N2-fixing and IAA synthesizing endophytic bacteria, i.e., ECS202, ECS203, and ECS204, isolated from the leaf base, were used to reinoculateCurcumaplantlets derived from tissue culture. Then, plants were grown in sterilized sand for 2 months and weekly supplied with N-free nutrient solution. Plant growth, colonization, nitrogen fixation, and IAA synthesis were measured at two months after planting. The inoculated plants clearly showed a better performance of plant growth and yield in terms of the plant height, plant weight, leaf area, and diameter of new rhizomes compared with uninoculated plants. The chlorophyll content and N concentration of leaves and roots also increased in inoculated plants. Endophytic bacteria from inoculated plants colonized the roots, rhizome, and leaf base. Partial sequence analysis using 16S rDNA indicated that the isolate ECS202 corresponded toSphingomonas pseudosanguinis(99.2% similarity over 1,371 bp), ECS203 toBacillus drentensis(99.4% similarity over 1,450 bp) and ECS204 toBacillus methylotrophicus(99.9% similarity over 13,06 bp). [ABSTRACT FROM PUBLISHER]

Mesorhizobium loti is a Gram negative bacterium that induces N-fixing root nodules on the model legume Lotus japonicus. Proteomic analysis in M. loti indicated that 3-phosphoglycerate dehydrogenase (EC. 1.1.1.95, PHGDH) protein content was 2.2 times higher in bacteroids than in cultured bacteria. A M. loti mutant (STM5) with a transposon insertion in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in minimal medium for growth. When L. japonicus plants were infected with STM5, the roots formed nodules in numbers comparable to those formed by wild type M. loti; however, the nodules showed very low acetylene reduction activity, and significant starch granule accumulation was observed in the uninfected cells. In such nodules, vast necrosis occurred in the central tissue of the nodules, although bacteroids were detected in the infected cell of the nodules. These data indicate that serine or glycine biosynthesis by PHGDH is important for maintaining symbiosis and nitrogen fixation in L. japonicus nodules. [ABSTRACT FROM AUTHOR]

During nodule development of legumes, together with the morphological alteration of plant cells, the rhizobia undergo marked biochemical and physiological changes and differentiate to bacteroids. Bacteroids invading a nodule are no longer free-growing cells, instead depending on the plant cell for all resources. To elucidate the molecular mechanism of the bacteroid differentiation of Bradyrhizobium japonicum, a time-course analysis of bacteroid protein profiles was studied by 2-D gel electrophoresis in soybean nodules. Using proteomic analysis, protein expressions in soybean nodule bacteroids 7, 10, 14, 28, and 49 days after inoculation (DAI) were monitored. The time points coincide with the early stage of nodule formation (7–10 DAI), the onset of nitrogen fixation (14–28 DAI), and nodule senescence (49 DAI). In this study, 275 annotated protein spots were successfully identified, and a cluster analysis of their expression was performed. A large portion of putative, upregulated proteins were observed in bacteroids at 7 or 10 DAI, and these upregulated proteins were mainly related to transcription, translation, protein folding, and degradation. In the later period (14–28 DAI) of bacteroid differentiation, a number of Nif and Fix proteins were upregulated. In addition, proteins related to the chaperonin and a synthetic enzyme of the poly-beta-hydroxybutyrate were expressed at high abundance. A number of proteins related to solute transporter were upregulated in the bacteroids and dominantly detected throughout nodule development. Changes of relative abundance of these proteins are discussed in relation to symbiosis. [ABSTRACT FROM AUTHOR]

Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium for Glycine max, has complex respiratory electron transport chains. Bll4880 contained a copper-binding motif for metallochaperone, H(M)X10MX21HXM. A mutant strain, Bj4880, induced nodules with lower acetylene reduction activity. A double mutant, Bj4880-1131, which had inserted mutations both in blr1131, a gene of the Sco1-like protein, and in bll4880, induced nodules of significant Fix− phenotype and low cytochrome c oxidase (Cco) activity in the bacteroid. Our data suggest that bll4880 protein is involved in copper ion delivery to Cco through blr1131 protein, and the expression of both proteins was induced under microaerobic conditions. [ABSTRACT FROM PUBLISHER]

We isolated cDNA clones for novel protein kinases by expression cloning from Lotus japonicus. The LNZ001, one of the isolated clones, encodes a protein of 547 amino acids with a predicted molecular weight of 63,349. Since the protein contains 12 highly conserved subdomains specific to Ser/Thr protein kinases, we designated it PKL01. When homology searches based on the PKL01 sequence were carried out, the protein was found to show sequence homology with nuclear Dbf2-related kinases (Ndr kinases). When PKL01 was produced using an Escherichia coli expression system and purified to homogeneity, it underwent intermolecular autophosphorylation. The major autophosphorylation site was identified as Ser-317 by using various point mutants, and phosphorylation at this site was found to be critical for the kinase activity. PKL01 was found to be widely distributed in the leaves, stems, roots and root nodules by northern hybridization experiments. When endogenous substrates were screened using fractionated preparations from various parts of plants, PKL01 preferentially phosphorylated basic proteins in tissue extracts. These results suggest that PKL01 is an Ndr kinase homolog in L. japonicus and may be involved in the regulation of cellular functions through phosphorylation of basic protein substrates such as histones. [ABSTRACT FROM PUBLISHER]

Homocitrate is a component of the iron–molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix- (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation. [ABSTRACT FROM AUTHOR]

Simple Summary: The Tsushima leopard cat is an endangered wild felid that lives solely on Tsushima Island, Nagasaki, Japan. Approximately, only 100 Tsushima leopard cats can be found in the wild, and there are concerns that the population will further reduce due to habitat degradation and traffic accidents. In 1994, Japan's Ministry of the Environment (MOE) developed a conservation and breeding project for the Tsushima leopard cat. The MOE is working with the Japanese Association of Zoo and Aquariums for ex situ conservation of this species. However, considering genetic diversity, it is difficult to conduct captive breeding programs using only natural breeding; hence, assisted reproductive technologies are required. This study aimed to breed Tsushima leopard cats using artificial insemination (AI) by depositing sperms into the oviducts. Ovulation was artificially induced in two females, laparoscopically inseminated with fresh sperms into the oviducts. The pregnancies were monitored via fecal levels of progestogens and radiography. One female had spontaneous delivery of a female kitten 66 days post-AI. This is the first successful case of AI in a Tsushima leopard cat. The Tsushima leopard cat (Prionailurus bengalensis euptilurus) is an endangered wild felid that lives solely on Tsushima Island, Nagasaki, Japan. Japan's Ministry of the Environment is working with the Japanese Association of Zoo and Aquariums for ex situ conservation of this species. However, considering genetic diversity, it is difficult to conduct captive breeding programs by natural breeding alone; hence, assisted reproductive technologies are required. This study aimed to breed Tsushima leopard cats, which otherwise cannot be paired, using laparoscopic oviductal artificial insemination (AI). Female Tsushima leopard cats (female 1, aged 7 years; female 2, aged 6 years) were treated with 200 IU equine chorionic gonadotropin, followed by administration of 1000 IU porcine luteinizing hormone (pLH) after 96 h to induce ovulation. Laparoscopic AI was performed 32 h post-pLH administration. Females 1 and 2 were inseminated in the oviduct with 2.4 × 10⁶ and 3.3 × 10⁶ motile spermatozoa, respectively, collected from two males. Pregnancy was confirmed by radiography 45 and 51 days post-AI in females 1 and 2, respectively; one fetus was found in female 2. Moreover, female 2 had spontaneous delivery of a female kitten 66 days post-AI. This is the first successful case of AI in a Tsushima leopard cat. [ABSTRACT FROM AUTHOR]

BACKGROUND Recently, some investigators have shown that telmisartan, an angiotensin II (Ang II)-receptor blocker (ARB), is a partial agonist of the peroxisome proliferator-activated receptor-γ (PPAR-γ). We investigate whether telmisartan improves cardiovascular remodeling associated with the production of endothelial nitric oxide synthase (eNOS) through PPAR-γ, inhibits the Rho-kinase pathway, and suppresses oxidative stress in Dahl salt-sensitive (DS) hypertensive rats. METHODS Telmisartan (1 mg/kg per day) or telmisartan plus PPAR-γ inhibitor, GW9662 (1 mg/kg per day) was administered from the age of 6-11 weeks. Age-matched male Dahl salt-resistant (DR) rats served as a control group. RESULTS The levels of eNOS and PPAR-γ expression, and eNOS phosphorylation were significantly lower in DS rats than in DR rats. Chronic telmisartan treatment in DS rats significantly increased these parameters, but not telmisartan plus GW9662. Telmisartan effectively inhibited the vascular lesion formation such as medial thickness and perivascular fibrosis, but not telmisartan plus GW9662. Moreover, upregulated RhoA protein, Rho-kinase mRNA, and myosin light-chain phosphorylation in DS rats was decreased by telmisartan to a similar degree as observed after treatment with Y-27632, a selective Rho-kinase inhibitor. In addition, NAD(P)H oxidase p22phox, p47phox, gp91phox expression, and mitogen-activated protein kinase and its downstream effector p70 S6 kinase phosphorylation in DS rats was also inhibited by telmisartan. CONCLUSIONS These results suggest that the cardioprotective mechanism of telmisartan may be partly due to improvement of endothelial function associated with PPAR-γ-eNOS, oxidative stress, and Rho-kinase pathway. [ABSTRACT FROM AUTHOR]

In contrast to other cereals, typical barley cultivars have caryopses with adhering hulls at maturity, known as covered (hulled) barley. However, a few barley cultivars are a free-threshing variant called naked (hulless) barley. The covered/naked caryopsis is controlled by a single locus (nud) on chromosome arm 7HL. On the basis of positional cloning, we concluded that an ethylene response factor (ERF) family transcription factor gene controls the covered/naked caryopsis phenotype. This conclusion was validated by (I) fixation of the 17-kb deletion harboring the ERF gene among all 100 naked cultivars studied; (ii) two x-ray-induced nud alleles with a DNA lesion at a different site, each affecting the putative functional motif; and (iii) gene expression strictly localized to the testa. Available results indicate the monophyletic origin of naked barley. The Nud gene has homology to the Arabidopsis WIN1/SHN1 transcription factor gene, whose deduced function is control of a lipid biosynthesis pathway. Staining with a lipophilic dye (Sudan black B) detected a lipid layer on the pericarp epidermis only in covered barley. We infer that, in covered barley, the contact of the caryopsis surface, overlaid with lipids to the inner side of the hull, generates organ adhesion. [ABSTRACT FROM AUTHOR]

Nodule-specific (nodulin) genes are thought to play crucial roles during establishment of the nitrogen-fixing symbiosis between legume plants and Rhizobium bacteria. On the basis of a gene expression database for early stages of the nodulation process of Lotus japonicus, previously constructed by a cDNA macroarray analysis, we identified a novel nodulin gene, LjnsRING, which encodes a protein with a typical RING-H2 finger domain that is well conserved in a number of plant E3 ubiquitin ligases. LjnsRING transcripts were almost exclusively expressed in nodules, and very low expression was detected in roots and shoots. RNA interference (RNAi) knockdown of LjnsRING by hairy root transformation caused impaired root growth together with abortion of nodule formation. Examination with lacZ-labeled Mesorhizobium loti indicated that infection thread formation in the RNAi transgenic hairy roots was significantly inhibited. Analysis using a chimeric gene of LjnsRING promoter and β-glucuronidase (GUS) coding region demonstrated that LjnsRING transcription in nodules was restricted to the infected cells. These results suggest the requirement for LjnsRING in rhizobial infection and the subsequent nodule formation process. [ABSTRACT FROM PUBLISHER]

We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. In the L. japonicus expressed sequence tag (EST) clone databases of Kazusa DNA Research Institute, another syntaxin-related clone (LjSYP32-2) was also detected, and the nucleotide and amino acid sequences of these two clone are very similar to each other. Real-time PCR and promoter analysis indicated that expression of LjSYP32-1 was dominant compared with LjSYP32-2 in the various plant organs. Promoter analysis and in situ hybridization revealed that LjSYP32-1 was expressed significantly in the inner cortex cell layer surrounding the infected zone of young nodules and in the meristem area of developing lateral root. To explore the function and physiological role of LjSYP32-1 in nodules and other plant organs, stable transformation lines of L. japonicus expressing either sense or antisense LjSYP32-1 were prepared. The antisense plants showed a significantly retarded plant growth phenotype, suggesting a role for LjSYP32-1 in supporting plant growth. In the same transgenic lines, the plants were capable of forming nodules, but the acetylene reduction activity was reduced by around 50% per plant. The nodules were much smaller and some nodules were fused to each other by sharing the inner cortex. The rate of occurence of such irregular nodules was twice that observed in wild-type plants. The data suggest that LjSYP32-1 contributes to the support of plant growth and normal nodule tissue differentiation. [ABSTRACT FROM AUTHOR]

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is believed to play a significant role in supporting nitrogen fixation via anaplerotic CO2 fixation for recycling carbon in nodules. Using the antisense technique, we decreased the expression levels of the nodule-enhanced PEPC gene (Ljpepc1) in a determinate legume plant (Lotus japonicus) in order to look at the influence of the symbiotic phenotype and biochemical parameters. Three independent transgenic L. japonicus plants (designated as Asppc1, Asppc2 and Asppc3) were prepared using a Ljpepc1 DNA fragment which is under the control of the cauliflower mosaic virus 35S promoter. Extensive suppression of the Ljpepc1 transcript in nodules of Asppc plants (T3 homologous plants) was confirmed by RNA gel blot, Western blot and enzyme activity assays. In nodules of Asppc plants, PEPC activity was reduced to about 10% of that of non-transformants and the plants showed typical nitrogen-deficient symptoms without a supply of nitrogen nutrient, and returned to normal growth when nitrate was supplied at 2.5 mM. The acetylene reduction activity per fresh weight of nodules of these Asppc plants decreased by 29% at 35 dai (days after infection). Various enzyme activities and metabolite levels were surveyed using Asppc plants at 35 dai. Significant reduction of sucrose synthase and asparagine aminotransferase activities was observed in Asppc nodules. In addition, sucrose, succinate, asparagine, aspartate and glutamate contents also decreased in Asppc nodules. The data are discussed in terms of a role for PEPC in the carbon/nitrogen metabolic flux in nodules. [ABSTRACT FROM AUTHOR]

NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) are specifically expressed in bundle sheath cells (BSCs) in NADP-ME-type and PCK-type C4 plants, respectively. Unlike the high activities of these enzymes in the green leaves of C4 plants, their low activities have been detected in the leaves of C3 plants. In order to elucidate the differences in the gene expression system between C3 and C4 plants, we have produced chimeric constructs with the β-glucuronidase (GUS) reporter gene under the control of the maize NADP-Me (ZmMe) or Zoysia japonica Pck (ZjPck) promoter and introduced these constructs into rice. In leaves of transgenic rice, the ZmMe promoter directed GUS expression not only in mesophyll cells (MCs) but also in BSCs and vascular cells, whereas the ZjPck promoter directed GUS expression only in BSCs and vascular cells. Neither the ZjPck nor ZmMe promoters induced GUS expression due to light. In rice leaves, the endogenous NADP-Me (OsMe1) was expressed in MCs, BSCs and vascular cells, whereas the rice Pck (OsPck1) was expressed only in BSCs and vascular cells. Taken together, the results obtained from transgenic rice demonstrate that the expression pattern of ZmMe or ZjPck in transgenic rice was reflected by that of its counterpart gene in rice. [ABSTRACT FROM AUTHOR]

For NAD-malic enzyme (NAD-ME)-type C4 photosynthesis, two types of aspartate aminotransferase (AAT) are involved. We examined the expression pattern of the Panicum miliaceum mitochondrial Aat gene (PmAat) and P. miliaceum cytosolic Aat gene (PcAat) in transgenic rice plants, which were specifically expressed in bundle sheath cells (BSCs) and mesophyll cells (MCs), respectively. Expression of a β-glucuronidase (GUS) reporter gene under the control of the PcAat promoter was regulated in an organ-preferential and light-dependent manner in the transgenic rice plants. However, the PmAat promoter drove the GUS expression in all organs we tested without light dependency, and this non-preferential expression pattern was also observed in transgenic rice with introduction of the intact PmAat gene. The expression patterns of the rice counterpart Aat genes to PmAat or PcAat showed that the rice mitochondrial Aat (RmAat1) gene was expressed in all organs tested in a light-independent manner, while expression of the rice cytosolic Aat (RcAat1) gene showed an organ-preferential and light-dependent pattern. Taking these results together, we can generalize that the regulatory system of BSC-specific or light-dependent expression of mitochondrial Aat is not shared between P. miliaceum (C4) and rice (C3) and that the expression of the C4 genes introduced into rice mimics that of their counterpart genes in rice. [ABSTRACT FROM AUTHOR]

To investigate protein kinases expressed in Lotus japonicus, a cDNA expression library of the root-nodule of L. japonicus was immunologically screened with monoclonal antibodies directed to a highly conserved region in protein serine/threonine kinases (Ser/Thr kinases). Among 178 positive clones obtained from the λZAPII cDNA library, 164 clones were found to encode novel proteins possessing the subdomain VIB sequences characteristic of Ser/Thr kinases. By phylogenetic analysis on the basis of deduced amino acid sequences, the isolated clones could be classified into five different families of Ser/Thr kinases : the SnRK family, GSK-3 family, Ndr kinase family, Ark family, and receptor kinase family. These results suggest that this expression cloning using the kinase-specific antibodies will provide new clues for investigations of a wide variety of known and novel protein kinases in higher plants. [ABSTRACT FROM PUBLISHER]

Symbiotic interactions between legume plants and rhizobia induce specific metabolisms and intracellular organelles in nodules. For surveying symbiotic differentiation of a key organelle, mitochondria, protein constituents of soybean nodule and root mitochondria were compared after two-dimensional (2-D) electrophoresis, and the proteins were characterized in combination with matrix-assisted desorption/ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry and N-terminal amino acid sequencing. Of the proteins that were detected only in nodule mitochondria, phosphoserine aminotransferase, flavanone 3-hydroxylase, coproporphyrinogen III oxidase, one ribonucleoprotein and three unknown proteins were identified. Seven up-regulated, eight down-regulated and two strongly suppressed protein spots in nodule mitochondria were also assigned protein identities. The physiological roles of these differential expressions were discussed in relation to nodule-specific metabolisms in soybean nodules. [ABSTRACT FROM AUTHOR]

Betaine aldehyde dehydrogenase (BADH) is an important enzyme for Gly betaine synthesis. We isolated two types of BADH cDNAs (BBD1 and BBD2) from barley. As BBD1 contained the signal sequence (SKL) targeting to microbodies, BBD2 was more similar to previously reported genes coding for BADH in dicotyledons (chloroplast type) than those in monocotyledons (microbody type). The two barley BADH genes showed different expression patterns. The BBD1 transcript was more abundant in roots than leaves and was induced to higher levels by salt, drought and abscisic acid (ABA) treatment. BBD2 transcript was more abundant in leaves and induced by salt, drought, PEG and ABA treatment. To understand the processing of these BADH proteins, we partially purified both enzymes and determined their N-terminal sequences. Based on comparisons of the N-terminal sequences to their deduced amino acid sequence, neither BBD1 nor BBD2 is processed at the N-terminus. These results suggest that BBD2 codes for a new type of BADH, which is not localized in either chloroplasts or mitochondria. [ABSTRACT FROM AUTHOR]

The chloroplastic NADP-malic enzyme (NADP-ME) is a key enzyme of the C4 photosynthesis pathway in NADP-ME type C4 plants such as maize. To express the chloroplastic NADP-ME in leaves of a C3 plant, rice, full-length cDNAs encoding the rice C3-specific isoform and the maize C4-specific isoform of the enzyme were expressed under the control of the rice Cab promoter. Transformants carrying the rice cDNA showed the NADP-ME activities in the leaves less than several-fold that of non-transformants, while those carrying the maize cDNA showed activities up to 30-fold that of non-transformants or about 60% of the NADP-ME activity of maize leaves. These results indicate that expression of the rice C3-specific NADP-ME is suppressed at co- and/or post-transcriptional levels by some regulation mechanisms intrinsic to rice, while that of the foreign C4-specific isoform can escape from such suppression. The accumulation of the maize C4-specific NADP-ME led to bleaching of leaf color and growth hindrance in rice plants under natural light. These deteriorative effects resulted from enhanced photoinhibition of photosynthesis due to an increase in the level of NADPH inside the chloroplast by the action of the maize enzyme. [ABSTRACT FROM PUBLISHER]

nodules are reviewed, and current research data on Nod-35 (uricase) gene, including that of Lotus japonicus as an example of a late nodulin gene are presented. [ABSTRACT FROM AUTHOR]

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene. [ABSTRACT FROM AUTHOR]

Summary In a previous study, we identified the C4-like pyruvate, orthophosphate dikinase gene (Pdk) in the C3 plant rice, with a similar structure to the C4-type Pdk in the C4 plant maize. In order to elucidate the differences between C4-type and C4-like Pdk genes in C4 and C3 plants, we have produced chimeric constructs with the β-glucuronidase (GUS) reporter gene under the control of the Pdk promoters. In transgenic rice, both rice and maize promoters directed GUS expression in photosynthetic organs in a light-dependent manner. However, the maize promoter exhibited a much higher transcriptional activity than the rice promoter did. These results indicate that the rice C4-like Pdk gene resembles the maize C4-type Pdk gene in terms of regulation of expression. We also tested the activity of the rice promoter in transgenic maize. GUS activity was seen in both photosynthetic and non-photosynthetic organs. Thus, the rice promoter does not confer a strict organ-specific gene expression, as the maize promoter does. Moreover, the rice promoter directed GUS expression not only in mesophyll cells but also in bundle sheath cells, whereas the maize promoter directed expression only in mesophyll cells. Taken together, the results obtained from both transgenic maize and rice demonstrate that the rice and maize promoters differ not only quantitatively, but also qualitatively, in terms of their cell- and organ-specificity. Experiments with swapped promoters using the rice and maize promoters further demonstrated that a limited sequence region from -330 to -76 of the maize promoter confers light-regulated, high-level expression to the rice promoter in maize mesophyll protoplasts. We conclude the gain of cis-acting elements conferring high-level expression and mesophyll cell specificity was necessary for establishment of a C4-type Pdk gene during the course of evolution from C3 to C4 plants. [ABSTRACT FROM AUTHOR]

Eleven clones from five species of the planktonic microalgae, (Chattonella antiqua, Chattonella marina, Heterosigma akashiwo, Alexandrium catenella, and Scrippsiella trochoidea), which were collected from the Seto Inland Sea in Japan and from Thailand, were subjected to nucleotide sequence analysis of the D1/D2 domain of the large subunit (LSU) of their ribosomal RNA genes. After amplification by polymerase chain reaction using degenerated primers, whole-nucleotide sequences for the D1/D2 domains of the LSU rRNA gene of 11 microalgae were analyzed. Phylogenic tree analysis using these nucleotide sequences showed each species located in a cluster corresponding to its morphological classification. The nucleotide sequence data for Chattonella spp. suggest that multiple clones of both Chattonella antiqua and Chattonella marina are present in the Seto Inland Sea and that red tide blooms of Chattonella spp. in different years may have contained different clones. [ABSTRACT FROM AUTHOR]

Two cDNA clones, MsU2 and MsU9 encoding uricase (EC 1.7.3.3, Nodulin-35) were isolated from a cDNA library prepared from nodule tissues of alfalfa, Medicago sativa, plants. Both MsU2 and MsU9 encoded 308 amino acid polypeptides with a difference of 5 amino acids, and the deduced amino acid sequences shared 98% homology. Between these two cDNA clones and uricase genes of soybean which were designated as Nod-35s, more than 80% identity was observed in nucleotides and deduced amino acid sequences, suggesting that these MsU2 and MsU9 are homologs of Nod-35.Using the reverse transcription-PCR technique, we detected the transcripts of these two genes in almost all tissues of alfalfa. The operation of uricase genes was confirmed by the presence of ureide in the xylem sap and uricase activity in the nodules. In situ hybridization analysis revealed that MsU2 and MsU9 were expressed only in uninfected cells of the infected zone of the nodule tissue. The cell specific-expression of the two uricase genes was observed in an identical manner to that of Nod-35 in soybean nodules. [ABSTRACT FROM AUTHOR]

Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO[SUB2] in C[SUB4] plants into the C[SUB3] crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O[SUB2] (inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C[SUB4] pathway of photosynthesis in C[SUB3] plants. [ABSTRACT FROM AUTHOR]

C3 plants including many agronomically important crops exhibit a lower photosynthetic efficiency due to inhibition of photosynthesis by O2 and the associated photorespiration. C4 plants had evolved the C4 pathway to overcome low CO2 and photorespiration. This review first focuses on the generation of a system for high level expression of the C4-specific gene for pyruvate, orthophosphate dikinase ( Pdk), one of the key enzyme in C4 photosynthesis. Based on the results with transgenic rice plants, we have demonstrated that the regulatory system controlling the Pdk expression in maize is not unique to C4 plants but rice (C3 plant) posses a similar system. Second, we discussed the possibility of the high level expression of maize C4-specific genes in transgenic rice plants. Introduction of the maize intact phosphoenolpyruvate carboxylase gene ( Ppc) caused 30–100 fold higher PEPC activities than non-transgenic rice. These results demonstrated that intact C4-type genes are available for high level expression of C4 enzymes in rice plants. [ABSTRACT FROM AUTHOR]

Synechococcus sp. PCC7942 cells transformed with Escherichia coli bet genes accumulated glycinebetaine (to about 80 raM) and acquired an increased tolerance to salt stress. Ribulose 1,5-bisphosphate carboxylase activity in the extract from control cells was found to decrease more rapidly than that of either PSI or PSII under salt stress. We examined levels of the transcript, the protein and the enzyme activity of ribulose 1,5-bisphosphate carboxylase to determine at which level the enzyme is affected at the early stages of salt stress. The levels of rbcL mRNA in both control and def-containing cells increased around two-fold under salt stress compared to those under non-stress. Bef-containing cells showed slightly reduced levels of rbcL mRNA under both non-stress and salt stress compared to control cells. The protein levels of the enzyme in the control and bet-containing cells were almost the same and were little changed by salt stress. On the other hand, salt stress drastically decreased the enzyme activity in control cells, but not as much in the bet-containing cells. Interestingly the lowered enzyme activity in extracts from control cells grown under salt stress, as well as acid-denatured enzyme, was partially reactivated by glycinebetaine. [ABSTRACT FROM AUTHOR]

In barley plants exposed to stepwise salt-stress (up to 200 mM NaCl), sodium and chloride ions accumulated preferentially in old rather than in young leaf blades. Furthermore, the level of glycinebetaine in young leaf blades was approximately three times that in old leaf blades. [ABSTRACT FROM AUTHOR]

Background: Facial skin care (FSC) is an important routine for Japanese women. Hand motions during FSC physically affect psychological state. However, it is very difficult to evaluate hand motions during personal and complex FSC. The objective of this study was to find out objective and quantitative parameters for hand motions during facial skin care (FSC). Women who enjoy and soothe during FSC (Enjoyment group (E group), n = 20) or not (non-enjoyment group (NE group), n = 19) were recruited by an advance questionnaire. The same lotion, emulsion, and cream were provided to all subjects, and they used sequentially in the same way as the women's daily FSC. The motion of the marker on the back side of the right middle finger during FSC was tracked by a motion capture system. The heart rate variability (HRV) was also measured before and after FSC for evaluating psychological effect. Results: The averaged acceleration (Avg. ACC), approximate entropy (ApEn), and power law scaling exponent (Rest γ) of the cumulative duration of slow motion from the sequential data of acceleration were evaluated. Compared to the NE group, the E group showed a lower Avg. ACC when using emulsion (p = 0.005) and cream (p = 0.007), a lower ApEn when using emulsion (p = 0.003), and a lower Rest γ (p = 0.024) when using all items, suggesting that compared to the NE group, the E group had more tender and regular motion, and sustainable slow motions, especially in the use of emulsion. In the E group, the low/high-frequency component of HRV decreased significantly after FSC, suggesting suppression of sympathetic activity (p = 0.045). NE group did not. For all subjects, ApEn and Rest γ showed significantly positive correlation with the increase in the low/high-frequency component of HRV after FSC (p < 0.01). ApEn showed significantly negative correlation with the increase in the high-frequency component of HRV after FSC (p < 0.05). Avg. ACC did not show significant correlation with them. These results suggested that the behavior of FSC influences the autonomic nerve system. Conclusions: ApEn and Rest γ are useful parameters for evaluating quality of hand motions during FSC. [ABSTRACT FROM AUTHOR]