Mutants of Lotus japonicus deficient in flavonoid biosynthesis.

Spatiotemporal features of anthocyanin accumulation in a model legume Lotus japonicus (Regel) K.Larsen were elucidated to develop criteria for the genetic analysis of flavonoid biosynthesis. Artificial mutants and wild accessions, with lower anthocyanin accumulation in the stem than the standard wild type (B-129 'Gifu'), were obtained by ethyl methanesulfonate (EMS) mutagenesis and from a collection of wild-grown variants, respectively. The loci responsible for the green stem of the mutants were named as VIRIDICAULIS (VIC). Genetic and chemical analysis identified two loci, namely, VIC1 and VIC2, required for the production of both anthocyanins and proanthocyanidins (condensed tannins), and two loci, namely, VIC3 and VIC4, required for the steps specific to anthocyanin biosynthesis. A mutation in VIC5 significantly reduced the anthocyanin accumulation. These mutants will serve as a useful system for examining the effects of anthocyanins and proanthocyanidins on the interactions with herbivorous pests, pathogenic microorganisms and nitrogen-fixing symbiotic bacteria, Mesorhizobium loti.

Isolation and characterization of arbuscules from roots of an increased-arbuscule-forming mutant of Lotus japonicus.

BACKGROUND AND AIMS: Previous methods for isolation of arbuscules from mycorrhizal roots are time-consuming, complex and expensive. Therefore, a simple, rapid and inexpensive method for the isolation of metabolically active arbuscules from plant root of an increased-arbuscule-forming mutant of Lotus japonicus (Ljsym78-2) is described. METHODS: Roots of the L. japonicus mutant plants Ljsym78-2 colonized by Glomus sp. were separated from soil, washed with water, immersed in CaSO(4) before being cut into 5-mm pieces and homogenized with a Waring blender at 6000 rpm for 30 s. The arbuscules were purified by separation from plant tissues with a 50-mum nylon mesh, finally collecting on a 30-mum nylon mesh. Enzyme histochemical staining showed that the collected arbuscules had succinate dehydrogenase, alkaline phosphatase and acid phosphatase activities. KEY RESULTS AND CONCLUSIONS: The enzymic activity of the arbuscules was not affected after the isolation process. The establishment of this simple, rapid and inexpensive method for the isolation of metabolically active arbuscules will be useful to clarify the biochemical processes occurring in nutrient exchange at the arbuscular interface.

Root, root hair, and symbiotic mutants of the model legume Lotus japonicus.

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod- (Ljsym70 to Ljsym73), Hist- (alb1-1, alb1-2, and Ljsym79), and Fix- (fen1, Ljsym75, and Ljsym81).

Characterization of NADPH-dependent oxidoreductase induced by auxin in rice.

An NADPH-dependent oxidoreductase (NADPH-DO) with an isoelectric point of 5.2 and a molecular mass of 35 kDa was isolated from suspension-cultured rice cells. NADPH-DO was inducible by auxin and gibberellin. NADPH-DO mRNA was expressed in roots and leaf sheaths of rice seedlings, but not in leaf blades. The levels of NADPH-DO mRNA and protein in suspension-cultured cells were increased by auxin; they were further raised by the application of zinc. In contrast, NADPH-DO mRNA and protein accumulation in intact roots was stimulated by auxin, but in this case the stimulation was inhibited by zinc. Auxin-induced callus growth and root formation in intact rice plants were further enhanced by zinc. These results indicate that high levels of NADPH-DO are necessary for auxin- and zinc-induced callus formation, and imply that zinc plays a role in the regulation of NADPH-DO induction by auxin.

Nitrogen Fixation and Translocation in Young Sugarcane (Saccharum officinarum L.) Plants Associated with Endophytic Nitrogen-Fixing Bacteria.

The tracer (15)N(2) was used to investigate sites of N(2) fixation and the possible translocation of the fixed N. Young sugarcane plants (Saccharum officinarum L.) from a stem cutting were exposed to (15)N(2)-labeled air in a 500 mL plastic cylinder. Plants fed (15)N(2) for 7 days were grown in normal air for a further chase period. After 21 days, about half of the N originating in the stem cutting had been transported to the shoot and roots, suggesting that the cutting played a role in supplying N for growth. After 3 days of feeding, the percentage of N derived from (15)N(2) was higher in the roots (2.22%) and stem cutting (0.271%) than the shoot (0.027%). Most of the fixed N was distributed in the 80% ethanol-insoluble fractions in each plant part, and the (15)N fixed either in the roots or in the stem cutting remained there and was not appreciably transported to the shoot. The results were quite different from the fate of fixed N in soybean nodules, which is rapidly transported from nodules to roots and shoots.

Infection, Multiplication and Evaluation of the Nitrogen-Fixing Ability of Herbaspirillum sp. Strain B501gfp1 in Sugarcane Stems Inoculated by the Vacuum Infiltration Method.

The present study was conducted with the aim of finding a suitable delivery method for introducing endophytic bacteria into new sugarcane stalks. Stem pieces of cultivars NiF8 and Ni15 were inoculated with Herbaspirillum sp. strain B501gfp1 by the vacuum infiltration method. The inoculated bacteria showed higher levels of nitrogenase activity in cultivar Ni15 than NiF8. The population density immediately after inoculation (10(7) CFU g [fresh weight](-1)) showed that strain B501gfp1 successfully entered the stem tissues of Ni15, where its presence was confirmed by fluorescence microscopic observations. Colonization was observed in the intercellular spaces and between the cell layers. The population of inoculated bacteria remained stable at 5 days after inoculation. The inoculated stems also showed adequately strong acetylene reduction activity compared to uninoculated stems in the absence of indigenous nitrogen-fixing bacteria.

Broad Distribution and Phylogeny of Anaerobic Endophytes of Cluster XIVa Clostridia in Plant Species Including Crops.

Endophytic clostridia present on various plants as obligate anaerobes were surveyed by terminal restriction fragment length polymorphism (TRFLP) analysis specific to the clostridial 16S rRNA gene. Endophytic clostridia were detected in 10 plant types: sugarcane, cultivated rice, corn, tobacco, soybean, bermuda grass, tall fescue, and three mangrove species. Phylogenetically, cluster XIVa clostridia were detected more frequently than cluster I clostridia in aerial parts. Isolation of clostridia from surface-sterilized sugarcane stem validated the TRFLP results. Plant-derived clostridia occupied two unique phylogenetic positions (groups I and II) within cluster XIVa. Most of cluster XIVa clostridia from other sources (e.g., human, animal, and insect intestines) were located outside these groups. Thus two unique groups of cluster XIVa clostridia are widely distributed in plants, including crops. In field-grown soybeans, TRFLP analysis revealed clostridia only in a non-nodulating mutant. Ribosomal intergenic spacer analysis (RISA) showed that the bacterial community in soybean shoot depended partly on the soybean nodulation genotype.

A proteomic approach to analyze auxin- and zinc-responsive protein in rice.

Auxin and zinc are involved in callus and root formation in rice. However, details of the mechanism underlying this process and functional relation between zinc and auxin are unclear. In this study, proteins induced by auxin and zinc in rice were analyzed by a proteomic approach. Root formation on rice seedlings was promoted by 0.45 microM 2,4-D treatment and was further promoted by addition of 260 microM Zn. Microscopic observation revealed that the number of root primodia formed was significantly increased in 2,4-D- and Zn-treated seedlings than that of the control. A total of seven proteins, as analyzed by 2D-PAGE, were increased, and one protein was decreased by 2,4-D and Zn treatment. Expression of elongation factor-1beta' (EF-1beta') both at transcriptional and translational levels was particular abundant in callus and basal parts of young seedlings, and the accumulation of EF-1beta' was consistent with root formation induced by 2,4-D and Zn. Results indicate that higher level of EF-1beta' expression is necessary for auxin- and zinc-induced root formation in rice.

Proteome approach to characterize the methylmalonate-semialdehyde dehydrogenase that is regulated by gibberellin.

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths ofslr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA(3). Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice.

Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice.

Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO4 and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc.

HAR1 mediates systemic regulation of symbiotic organ development.

Symbiotic root nodules are beneficial to leguminous host plants; however, excessive nodulation damages the host because it interferes with the distribution of nutrients in the plant. To keep a steady balance, the nodulation programme is regulated systemically in leguminous hosts. Leguminous mutants that have lost this ability display a hypernodulating phenotype. Through the use of reciprocal and self-grafting studies using Lotus japonicus hypernodulating mutants, har1 (also known as sym78), we show that the shoot genotype is responsible for the negative regulation of nodule development. A map-based cloning strategy revealed that HAR1 encodes a protein with a relative molecular mass of 108,000, which contains 21 leucine-rich repeats, a single transmembrane domain and serine/threonine kinase domains. The har1 mutant phenotype was rescued by transfection of the HAR1 gene. In a comparison of Arabidopsis receptor-like kinases, HAR1 showed the highest level of similarity with CLAVATA1 (CLV1). CLV1 negatively regulates formation of the shoot and floral meristems through cell-cell communication involving the CLV3 peptide. Identification of hypernodulation genes thus indicates that genes in leguminous plants bearing a close resemblance to CLV1 regulate nodule development systemically, by means of organ-organ communication.