Shiro Higashi

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma, respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1-overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1- and AfHb1-overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodules, whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.

Expression of a Class 1 Hemoglobin Gene and Production of Nitric Oxide in Response to Symbiotic and Pathogenic Bacteria in Lotus japonicus

Symbiotic nitrogen fixation by the collaboration between leguminous plants and rhizobia is an important system in the global nitrogen cycle, and some molecular aspects during the early stage of host-symbiont recognition have been revealed. To understand the responses of a host plant against various bacteria, we examined expression of hemoglobin (Hb) genes and production of nitric oxide (NO) in Lotus japonicus after inoculation with rhizobia or plant pathogens. When the symbiotic rhizobium Mesorhizobium loti was inoculated, expression of LjHb1 and NO production were induced transiently in the roots at 4 h after inoculation. In contrast, inoculation with the nonsymbiotic rhizobia Sinorhizobium meliloti and Bradyrhizobium japonicum induced neither expression of LjHb1 nor NO production. When L. japonicus was inoculated with plant pathogens (Ralstonia solanacearum or Pseudomonas syringae), continuous NO production was observed in roots but induction of LjHb1 did not occur. These results suggest that modulation of NO levels and expression of class 1 Hb are involved in the establishment of the symbiosis.

A Class 1 Hemoglobin Gene from Alnus firma Functions in Symbiotic and Nonsymbiotic Tissues to Detoxify Nitric Oxide

Actinorhizal symbiosis is as important in biological nitrogen fixation as legume-rhizobium symbiosis in the global nitrogen cycle. To understand the function of hemoglobin (Hb) in actinorhizal symbiosis, we characterized a Hb of Alnus firma, AfHb1. A cDNA that encodes nonsymbiotic Hb (nonsym-Hb) was isolated from a cDNA library of A. firma nodules probed with LjHb1, a nonsym-Hb of Lotus japonicus. No homolog of symbiotic Hb (sym-Hb) could be identified by screening in the cDNA library or by polymerase chain reaction (PCR) using degenerate primers for other sym-Hb genes. The deduced amino acid sequence of AfHb1 showed 92% sequence similarity with a class 1 nonsym-Hb of Casuarina glauca. Quantitative reverse transcriptase-PCR analysis showed that AfHb1 was expressed strongly in the nodules and enhanced expression was detected under cold stress but not under hypoxia or osmotic stress. Moreover, AfHfb1 was strongly induced by the application of nitric oxide (NO) donors, and the application of a NO scavenger suppressed the effect of NO donors. Acetylene reduction was strongly inhibited by the addition of NO donors. AfHb1 may support the nitrogen fixation ability of members of the genus Frankia as a NO scavenger.

Studies on cyclic β-1,2-glucan obtained from periplasmic space ofRhizobium trifolii cells

SummaryA glycan was obtained from the periplasmic space ofRhizobium trifolii cells by saccharose-osmotic shock method. This glycan was composed of only β-1,2-linked glucose, having no end groups (reducing and non-reducing) and branching points. It was concluded that this glucan has a structure of unbranched cyclic form.When the cyclic β-1,2-glucan was added to the seedlings of white clover as a host plant withR. trifolii, it promoted not only the infection thread formation but also the nodule formation. In the culture medium containing the cyclic β-1,2-glucan, cells were found agglutinated during the middle logarithmic phase of growth, and their morphological changes were recognized as cell elongation or swelling in the middle region of a cell when observed in scanning electron microscope (SEM).

Bioaccumulation of Arsenic by Freshwater Algae and the Application to the Removal of Inorganic Arsenic from an Aqueous Phase. Part II. By Chlorella vulgaris Isolated from Arsenic-Polluted Environment

Abstract Green algae, Chlorella vulgaris Beijerinck var. vulgaris, isolated from an arsenicpolluted environment, was examined for the effects of arsenic levels, arsenic valence, temperature, illumination intensity, phosphate levels, metabolism inhibitors, heat treatment on the growth, and arsenic bioaccumulation. The following conclusions were reached from the experimental results: (a) The growth of the cell increased with an increase of arsenic(V) levels of the medium up to 2,000 ppm, and the cell survived even at 10,000 ppm; (b) The arsenic bioaccumulation increased with an increase of the arsenic level. The maximum accumulation of arsenic was about 50,000 μg As/g dry cell; (c) The growth decreased with an increase of the arsenic(III) level and the cell was cytolyzed at levels higher than 40 ppm; (d) No arsenic(V) was bioaccumulated by a cell which had been pretreated with dinitrophenol (respiratory inhibitor) or with heat. Little effect of NaN3 (photosynthesis inhibitor) on the bioaccumulation was obse...

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.

Structural studies on the extracellular acidic polysaccharide from Rhizobium trifolii 4S

Abstract The structure of an extracellular acidic polysaccharide produced by Rhizobium trifolii 4S was studied by fragmentation with phage-induced depolymerase, methylation analysis, digestion with exo-β- d -glycanases, and n.m.r. spectroscopy. The polysaccharide is composed of d -glucose, d -glucuronic acid, pyruvic acid, and acetic acid in the molar ratio of 5:2:1:2. The repeating unit is a heptasaccharide containing ( a ) terminal 4,6- O -(1-carboxyethylidene)- d -glucose (1 residue), ( b ) (1 → 3)-linked d -glucose (1 residue), ( c ) (1 → 4)-linked d -glucose (2 residues), ( d ) (1 → 4)-linked d -glucoronic acid (2 residues), and ( e ) (1 → 4), (1 → 6)-linked d -glucose (1 residue). A possible structure for the polysaccharide is proposed. The phage-induced depolymerase hydrolyzes linkages of 4- O -α- d -glucosyl-β- d -glucuronic acid adjacent to branching points.

Suppression of root nodule formation by artificial expression of the TrEnodDR1 (coat protein of White clover cryptic virus 1) gene in Lotus japonicus.

TrEnodDR1 (Trifolium repens early nodulin downregulation 1) encodes a coat protein of White clover cryptic virus 1. Its expression in white clover was down-regulated at the time when root nodules formed. We surmised that its artificial expression would interfere with root nodulation. Therefore, we investigated the effects of its artificial expression on the growth and root nodulation of Lotus japonicus (a model legume). Transformants were prepared by Agrobacterium spp.-mediated transformation. The growth of transformants was reduced and the number of root nodules per unit root length was greatly decreased relative to control. The concentration of endogenous abscisic acid (ABA), which controls nodulation, increased in plants containing TrEnodDR1. These phenotypes clearly were canceled by treatment with abamine, a specific inhibitor of ABA biosynthesis. The increase in endogenous ABA concentration explained the reduced stomatal aperture and the deformation of root hairs in response to inoculation of transgenic L. japonicus with Mesorhizobium loti. Transcriptome comparison between TrEnodDR1 transformants and control plants showed clearly enhanced expression levels of various defense response genes in transformants. These findings suggest that TrEnodDR1 suppresses nodulation by increasing the endogenous ABA concentration, perhaps by activating the plants innate immune response. This is the first report of the suppression of nodulation by the artificial expression of a virus coat protein gene.

Split-root study of autoregulation of nodulation in the model legume Lotus japonicus

We used a split-root system to determine the timing for induction of the autoregulation of nodulation (AUT) in Lotus japonicus (Regel) Larsen after inoculation with Mesorhizobium loti. The signal took at least five days for full induction of AUT and inhibition of infection thread formation. Strain ML108 (able to nodulate but unable to fix nitrogen) induced full AUT, but ML101 (unable to nodulate or to fix nitrogen) did not induce autoregulation. These results indicate that Nod factor-producing strains induce AUT, but that the nitrogen fixed by rhizobia and supplied to the plant as ammonia does not elicit the AUT in L. japonicus.

Bioaccumulation of Arsenic by Freshwater Algae and the Application to the Removal of Inorganic Arsenic from an Aqueous Phase. Part I. Screening of Freshwater Algae Having High Resistance to Inorganic Arsenic

Abstract Several freshwater alga having resistance to arsenic were screened from microorganisms which had been sampled at sites polluted with arsenic from a geothermal electric power plant and old mines and smelters of arsenic ores. The alga thus screened could grow in the liquid medium (Modified-Detmer culture medium) containing sodium arsenate at levels up to 2000 ppm as elemental arsenic concentration. Some mixed systems of alga grew rapidly in the media at the higher levels of arsenic ranging from 50 to 2000 ppm. The mixed systems of alga screened included predominantly blue-green algae, green algae, and diatom, and also included protozoa, rotifera, and bacteria as minor components. One pure algal culture was obtained by means of an agar plate culture, and the algae isolated was identified as Chlorella vulgaris Beijerinck var. vulgaris. The growth of C. vulgaris in a pure culture was unaffected by 100 ppm of arsenic.