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Opportunities for biological nitrogen fixation in rice and other non-legumesBook is availableOPPORTUNITIES FOR BIOLOGICAL NITROGEN FIXATION AND OTHER NON-LEGUMES A joint publication of IRRI and Kluwer Academic Publishers Edited by J.K. Ladha, F.J. de Bruijn, and K.A. Malik Introduction: Assessing opportunities for nitrogen fixation in rice - a frontier project J.K. Ladha1, F.J. de Bruijin2 and K.A. Malik3 1 International Rice Research Institute, DAPO Box 7777, Metro Manila Philippines; 2 DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan, U.S.A. and 3 National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Recent advances in understanding symbiotic Rhizobium-legume interactions at the molecular level, the discovery of endophytic interactions of nitrogen-fixing organisms with non-legumes, and the ability to introduce genes into rice by transformation have stimulated researchers world wide to harness opportunities for nitrogen fixation and improved N nutrition in rice. In a think-tank workshop organized by IRRI in 1992, the participants reaffirmed that such opportunities do exist for cereals and recommended that rice be used as a model system. Subsequently, IRRI developed a New Frontier Project to coordinate the worldwide collaborative efforts among research centers committed to reducing dependency of rice on mineral N resources. An international biological Nitrogen fixation (BNF) working group was established to review, share research results/materials and to catalyze research. The strategies of enabling rice to fix its own N are complex and of a long-term nature. However, if achieved, they could enhance rice productivity, resource conservation, and environmental security. The rate of obtaining success would, of course, benefit tremendously from concerted efforts from a critical mass of committed scientists around the world, as well as a constant and continued funding support from the "donor" community. O.C. Bøckman Norsk Hydro Research Centre, P.O. Box 2560, N-3901 Porsgrunn, Norway Biological nitrogen fixation (BNF) has an assured place in agriculture, mainly as a source of nitrogen for legumes. Legumes are currently grown mostly as a source of vegetable oil and as food for humans and animals, but not as nitrogen source. Other crops with BNF capability may be eventually be developed eventually. Such crops will also need mineral fertilizers to maintain a good status of soil nutrients, but their possible effects to the environment is also a concern. Fertilizers, however, will remain a necessary and sustainable input to agriculture to feed the present and increasing human population. It is not a case of whether BNF is better or worse than mineral fertilizers because both plays an important role in agriculture. Isolation of endophytic diazotrophic bacteria from wetland rice W.L. Barraquio, L. Revilla and J.K. Ladha Soil Microbiology Laboratory, SWSD, International Rice Research Institute, Los Baños, Laguna, Philippines. Endophytic nitrogen-fixing bacteria are believed to contribute substantial amounts of N to certain gramineous crops. We have been interested to find (a) a diazotroph(s) in rice which can aggressively and stably persist and fix nitrogen in interior tissues and (b) unique rice-diazotrophic endophyte combinations. To achieve these objectives, it has been essential to find an efficient method to surface sterilize rice tissues. The method described here consists of exposing tissues to 1% Chloramine T for 15 min followed by shaking with glass beads. It has proven very efficient since (a) surface bacterial populations on the root and culm were found to be reduced by more than 90%, (b) the number of the internal colonizers was found to be significantly higher than the number of surface bacteria, and (c) colonication of root but not subepidermal tissue by gusA-marked Herbaspirillum seropedicae Z67 bacteria was found to be virtually eliminated. Nitrogen-fixing putative endophytic populations (MPN g dry wt-1) in the root (7.94 x 107) and culm (2.57 x 106) on field-grown IR72 plants grown in the absence of N fertilizer was found to be significantly higher near heading stage. The corresponding total putative endophyte populations in the tissues of 25 highly diverse genotypes of rice and their relatives was found to range from 105-108 and 104-109, in the roots and culms, respectively. Generally, the resident bacteria were found to be non-diazotrophic, although in isolated cases diazotrophs were found, for example in the roots and culm of IR72 rice plants, or the culm of Zizaniopsis villanensis plants. The size of populations of diazotrophic bacteria in different rice genotypes was found to be103-107 for the roots and 104-106 for the culms, respectively. The rice genera-related plant Potamophila parrifforai and Rhynchoryza subulata showed the highest levels. J.R. Stoltzfus1, R. So4, P.P. Malarvithi4, J.K. Ladha4 and F.J. de Bruijin1,2,3 1 MSU-DOE Plant Research Laboratory, 2 Department of Microbiology, 3 NSF Center for Microbial Ecology, Michigan StateUniversity, E. Lansing, MI 48824, U.S.A. and 4International Rice Research Institute, Los Baños, Philippines The extension of nitrogen-fixing symbioses to important crop plants such a the cereals has been a long-standing goal in the field of biological nitrogen fixation. One of the approaches that has been used to try to achieve this goal involves the isolation and characterization of stable endophytic bacteria from a variety of wild and cultivated rice species that either have a natural ability to fix nitrogen or can be engineered to do so. Here we present the results of our first screening effort for rice endophytes and their characterization using acetylene reduction assays (ARA), genomic fingerprinting with primers corresponding to naturally occurring repetitive DNA elements (rep-PCR), partial 16S rDNA sequence analysis and PCR mediated detection of nitrogen fixation (nif) genes with universal nif primers developed in our laboratory. We also describe our efforts to inoculate rice plants with the isolates obtained from the screening, in order to examine their invasiveness and persistence (stable endophytic maintenance). Lastly, we review our attempts to tag selected isolates with reporter genes/proteins, such as beta-glucuronidase (gus) or green fluorescent protein (gfp), in order to be able to track putative endophytes during colonization of rice tissues. K.A. Malik, Rakhshanda Bilal1, Samina Mehnaz, G. Rasul, M.S. Mirza and S. Ali National Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Faisalabad, Pakistan. 1Present address: PINSTECH, Rawalpindi, Pakistan Leptochloa fusca (L.) Kunth (Kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using 15N isotopic dilution were carried out in nutrient solution and saline soil. These studies indicated an agronomically significant amount of nitrogen being fixed in soil. Kallar grass has a similar growth habitat to rice. Therefore similar studies were carried out with rice after isolating various diazotrophs from the roots which were also screened for their ability to produce auxin (IAA). Five such strains namely Azospirillum lipoferum N-4, Azospirillum brasilense Wb-3, Azoarcus K-1, Pseudomonas 96-51, Zoogloea Ky-1 were selected for inoculating two rice varieties i.e. NIAB-6and BAS-370 under aseptic laboratory conditions. The nitrogen fixed was quantified using the 15N isotopic dilution method. Variety BAS-370 had nearly 70% nitrogen derived from atmosphere (Ndfa) when inoculated with Azospirillum N-4. Similar studies with the mixed inoculum using 15N fertilizer in the micro plots indicated that nearly 29% of plant nitrogen was derived from the atmosphere. G. Kirchhof1, V.M. Reis2, J.I. Baldani2, B. Eckert1, J. Döbereiner2 and A. Hartmann1 1GSF-National Research Center for Environment and Health, Institute of Soil Ecology, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany and 2EMBRAPA-Centro National de Pesquisa de Agrobiologia (CNPAB-EMBRAPA), Seropédica CEP 23851-970 Rio de Janeiro, Brazil. Endophytic diazotrophic bacteria could be isolated from the energy plants Pennisetum purpureum, Miscanthus sinensis, Miscanthus sacchariflorus and spartina pectinata using semisolid nitrogen free media. Higher levels of diazotrophic bacteria were found if no nitrogen fertilizer was applied. The bacteria were characterized on the basis of typical morphology, physiological tests, and the use of phylogenetic oligonucleotide probes. They belong partially to the species Azospirillum lipoferum and Herbaspirillum seropedicae while others supposedly represent a new species of Herbaspirillum. Using PCR-fingerprinting techniques a limited genetic diversity of these isolates was found which may indicate an adaptation to the specific conditions of the interior of these plants. Azoarcus spp. and their interactions with grass roots B. Reinhold-Hurek and T. Hurek Max-Planck-Institut für terrestrische Mikrobiologie, Arbeitsgruppe symbioseforschung, Karl-von-Frisch-Straße, D-35043 Marburg, Germany The current knowledge on the divergence within the genus Azoarchus and about interactions with grasses is summarized. Grass-associated members of this genus of diazotrophs have only been isolated from a salt-and flood-tolerant pioneer plant in Pakistan, Kallar grass (Leptochloa fusca (L.) Kunth). Members of these bacteria belong to the beta subclass of the Proteobacteria, most closely related to purple bacteria such as Rhodocyclus purpureus. The isolates from one single host plant showed a surprising divergence, consisting of five groups of Azoarcus distinct at the species level. Molecular diagnostic tests, which are based on 16S ribosomal RNA sequences, allowed prelimnary assignment of isolates to Azoarcus by PCR amplification and sequencing of PCR products. Moreover, the molecular tests enabled us to detect an unculturable strain in Kallar grass roots, stressing that classical cultivation techniques at times fail to detect some groups of the microbial population. Using similar techniques, sequences rooting in the Azoarchus clade were also detected in field-grown rice, indicating that the natural host range might extend to rice. In gnotobiotic laboratory cultures, a member of Azoarcus is able to colonize rice roots endophytically: bacteria invade the roots in the zone of elongation and differentiation, colonize the cortex intra- and inter-cellularly, and penetrate deeply into the vascular system, entering xylem vessels, allowing systemic spreading into the rice shoot. Recently, we detected expression of nitrogenase of Azoarcus cells inside roots of rice seedlings, a result encouraging us to analyze interactions with rice in detail. I.R. Kennedy, L.L. Pereg-gerk, C. Wood, R. Deaker, K. Gilchrist and S. Katupitiya SUNFix Centre for Nitrogen Fixation, Department of Agricultural chemistry and Soil Science, University of Sydney, NSW 2006, Australia Recent advances towards achieving significant nitrogen fixation by diazotrophs in symbioses with cereals are reviewed, referring to the literature on the evolution of effective symbioses involving rhizobia and Frankia as microsymbionts. Data indicating that strains of Acetobacter and Herbaspirillum colonizing specific cultivars of sugarcane as endophytes make a significant contribution to the nitrogen economy of this crop improves the prospects that similar associative systems may be developed for other gramineous species such as rice and wheat. By contract, the transfer of nodulation genes similar to those in legumes or Parasponia to achieve nodulation in crops like rice and wheat is considered to be a more ambitious and distant goal. Progress in developing an effective associative system for cereals has been materially assisted by the development of genetic tools based on the application of lacZ and gusA fusions with the promoters of genes associated with nitrogen fixation. These reporter genes have provided clear evidence that 'crack-entry' at the points of emergence of lateral roots or of 2,4-D induced para-nodules is the most significant eoute of endophytic colonization. Furthermore, using the laboratory model of para-nodulated wheat, there is now evidence that the ability of azospirilla and other nitrogen fixing bacteria to colonize extensively as endophytes can be genetically controlled. The most successful strain of Azospirillum brasilense (Sp7-S) for endophytic colonization and nitrogen fixation in wheat seedlings is a mutant with reduced exopolysaccharide production. Most other strains of azospirilla do not colonize as endophytes and it is concluded that though these are poorly adapted to providing nitrogen for the host plant, they are well adapted for survival and persistence in soil. A research program combining the study of endophytic colonization by azospirilla with an examination of the factors controlling the effectiveness of association (oxygen tolerance and nitrogen transfer) is now being pursued. It is proposed that a process of facilitated evolution of para-nodulated wheat involving the stepwise genetic improvement of both the prospective microsymbionts and the cereal host will eventually lead to effective nitrogen-fixing associations. In the attempt to achieve this goal, continued study of the endophytes occurring naturally in sugar cane and other grasses (e.g. Azoarcus sp.) should be of assistance. P.M. Reddy1, J.K. Ladha1, R.B. So1, R.J. Hernandez1, M.C. Ramos1, O.R. Angeles1, F.B. Dazzo and F.J. de Bruijn2,3 1 International Rice Research Institute, P.O. Box 933, Manila 1099, Philippines, 2 Department of Microbiology and 3DOE Plant research Laboratory, Michigan State University, East Lansing, MI 48824, USA. Legume-rhizobial interactions culminate in the formation of structures known as nodules. In this specialized niche, rhizobia are insulated from microbial competition and fix nitrogen which becomes directly available to the legume plant. It has been a long-standing goal in the field of biological nitrogen fixation to extend the nitrogen-fxing symbiosis to non-nodulated cereal plants, such as rice. To achieve this goal, extensive knowledge of the legume-rhizobia symbioses should help in formulating strategies for developing potential rice-rhizobia symbioses or endophytic interactions. As a first step to assess opportunities for developing a rice-rhizobia symbiosis, we evaluated certain aspects of rice-rhizobia. Our studies indicate that: a) Rice root exudates do not activate the expression of nodulation genes such as nodY of Bradyrhizobium japonicum USDA110, nodA of R. leguminosarum bv. Trifolii, or nodSU of Rhizobium. Sp. NGR234; b) Neither viable wild-type rhizobia, nor purified chitolipooligosaccharide (CLOS) Nod factors elicit root hair deformation or true nodule formation in rice; c) Rhizobia-produced indole-3-acetic acid, but neither trans-zeatin nor CLOS Nod factors, seem to promote the formation of thick, short lateral roots in rice; d) Rhizobia develop neither the symbiont-specific pattern of root hair attachment nor extensive cellulose microfibril production on the rice root epidermis; e) A primary mode of rhizobial invasion of rice roots is through cracks in the epidermis and fissures created during emergence of lateral roots; f) This infection process is nod-gene independent, nonspecific, and does not involve the formation of infection threads; g) endophytic colonization observed so far is restricted to intercellular spaces or within host cells undergoing lysis; h) The cortical sclerenchymatous layer containing tightly packed, thick walled fibers appears to be a significant barrier that restricts rhizobial invasion into deeper layers of the root cortex. Therefore, we conclude that the molecular and cell biology of the Rhizobium-rice association differs in many respects from the biology underlying the development of root nodules in the Rhizobium-legum symbiosis. Y.G. Yanni1, R.Y. Rizk1, V. Corich2, A. Squartini2, K. Ninke3,4, S. Philip-Hollingsworth3, G. Orgambide3, F. de Bruijn3,4,5, J. Stoltzfus5, D. Buckley3,4, T.M. Schmidt3,4, P.F. Mateos6, J.K. Ladha7 and F.B. Dazzo3,4 1 Sakha Agricultural Research Station, Kafr el-Sheikh, 33717 A.R. Egypt; 2 Dipt. Di Biotecnologie Agrarie, Universita Degli Studi di Padova, Padova, Italy; 3 Dept. of Microbiology, 4 Center for Microbial Ecology and 5 Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, U.S.A.; 6 Dept. de Microbiologia y Genetica, Universidad de Salamanca, Salamanca, Spain and 7 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. For over 7 centuries, production of rice (Oryza sativa L.) in Egypt has benefited from rotation with Egyptian berseem clover (Trifolium alexandrinum). The nitrogen supplied by this rotation replaces 25-33% of the recommended rate of fertilizer-N application for rice production. This benefit to the rice cannot be explained solely by an increased availability of fixed N through mineralization of N-rich clover crop residues. Since rice normally supports a diverse microbial community of internal root colonists, we have examined the possibility that the clover symbiont, Rhizobium leguminosarum bv. Trifolii colonizes rice roots endophytically in fields where these crops are rotated, and it so, whether this novel plant-microbe association benefits rice growth. MPN plant infection studies were performed on macerates of surface-sterilized rice roots inoculated on T. alexandrinum as the legume trap host. The results indicated that the root interior of rice grown in fields rotated with clover in the Nile Delta contained ~106 clover-nodulating rhizobial endophytes g-1 fresh weight of root. Plant tests plus microscopical, cultural, biochemical, and molecular structure studies indicated that the numerically dominant isolates of clover-nodulating rice endophytes represent 3-4 authentic strains of R. leguminosarum bv. Trifolii that were Nod+ Fix+ on berweem clover. Pure cultures of selected strains were able to colonize the interior of rice roots grown under gnotobiotic conditions. These rice endophytes were reisolated from surface-sterilized roots and shown by molecular methods to be the same as the original inoculant strains, thus verifying koch's postulates. Two endophytic strains of R. leguminosarum bv. Trifolii significantly increased shoot and root growth of rice in growth chamber experiments, and grain yield plus agronomic fertilizer N-use efficiency of Giza-175 hybrid rice in a field inoculation experiment conducted in the Nile Delta. Thus, field where rice has been grown in rotation with clover since antiquity contain Fix+ strains of R. leguminosarum bv. trifolii that naturally colonize the rice root interior, and these true rhizobial endophytes have the potential to promote rice growth and productivity under laboratory and field conditions. Interactions of rhizobia with rice and wheat G. Webster1,2, C. Gough2, J. Vasse2, C.A. Batchelor1, K.J. O'Callaghan1, S.L. Kothari1, .R. Davey1, J. Dénarié2 and E.C. Cocking1 1 Plant Genetic Manipulation Group, Department of life Science, University of Nottingham, Nottingham NG7 2RD, U.K. and 2 Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, INRA-CNRS, BP27, 31326 Castanet-Tolosan, France. Recently, evidence has been obtained that naturally occurring rhizobia, isolated from the nodules of non-legume Parasponia species and from some tropical legumes, are able to enter the roots of rice, wheat and maize at emerging lateral roots by crack entry. We have now investigated whether Azorhizobium caulinodans strains ORS571, which induces root and stem nodules on the tropical legume Sesbania rostrata as a result of crack entry invasion of emerging lateral roots, might also enter rice and wheat by a similar route. Following inoculation with ORS571 carrying a lacZ reporter gene, azorhizobia were observed microscopically within the cracks associated with emerging lateral roots of rice and wheat. A high proportion of inoculated rice and wheat plants had colonized lateral root cracks. The flavanone naringenin at 10-4 and 10-5 M stimulated significantly the colonization of lateral root cracks and also intercellular colonization of wheat roots. Naringenin does not appear to be acting as a carbon source and may act as a signal molecule for intercellular colonization of rice and wheat by ORS571 by a mechanism whhich is nod gene-independent, unlike nodule formation in Sesbania rostrata. The opportunity now arises to compare and to contrast the ability of Azorhizobium caulinodans with that of other rhizobia, such as Parasponia rhizobia, to intercellularly colonize the roots of non-legume crops. C. Gough1, Jacques Vasse1, C. Galera1, G. Webster1, E. Cocking2 and J. Dénarié1 1 Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, INRA-CNRS, BP27, 31326 Castanet-Tolosan, France and 2 Plant Genetic Manipulation Group, Department of Life Science, University of Nottingham, Nottingham NG7 2RD, U.K. When interactions between diazotrophic bacteria and non-legume plants are studied within the context of trying to extend biological nitrogen fixation to non-legume crops, an important first step is to establish reproducible internal colonization at high frequency of these plants. Using Azorhizobium caulinodans ORS571 (which induces stem and root nodules on the tropical legume Sesbania rostrata), tagged with a constitutively expressed lacZ reporter gene, we have studied the possibilities of internal colonization of the rot system of the model dicot Arabidopsis thaliana. ORS571 was found to be able to enter A. thalianai roots after first colonizing lateral root cracks (LRCs), at the points of emergence of lateral roots. Cytological studies showed that after LRC colonization, bacteria moved into the intercellular space between the cortical and endodermal cell layers of roots. In our experimental conditions, this LRC and intercellular colonization are reproducible and occur at high frequency, although the level of colonization at each site is low. The flavonoids naringenin and daidzein, at low concentrations, were found to significantly stimulate (at the p=0.01 level) the frequency of LRC and intercellular colonization of A. thaliana roots by A. caulinodans. The role in colonization of the structural nodABC genes, as well as the regulatory gene nodD, was studied and it was found that both colonization and flavonoid stimulation of colonization are nod gene-independent. These systems should now enable the various genetic and physiological factors which are limiting both for rhizobial colonization and for endophytic nitrogen fixation in non-legumes, to be investigated. In particular, the use of A. thalianai, whch has many advantages over other plants for molecular genetic studies, to study interactions between diazotrophic bacteria and non-legume dicots, should provide the means of identifying and understanding the mechanisms by which plant genes are involved in these interactions. B.G. Rolfe1, M.a. Djordjevic1, J.J. Weinman1, U. Mathesius1, C. Pittock1, E. Gärtner1, K.M. Ride1, Z. Dong2, M. McCully2 and J. McIver1 1 PMI Group, RSBS, ANU, Canberra, ACT 0200, Australia and 2Biology Department, Carlton University, Ottawa, Canada Root morphology is both genetically programmed and environmentally determined. We have begun an analysis into the components of root development by: (a) constructing a range of transgenic clover plants to assess some of the genetic programs involved as both roots and nodules are initiated and develop. These transgenic plants report on auxin activity, flavonoid synthesis and chitinase expression and suggest a role for flavonois as regulators of auxin levels; and (b) determining in cereals the effect of both added auxin and specific microorganisms on the initiation and development of modified root outgrowths and lateral roots. Appropriate combinations of auxin, the nitrogen fixing Acetobacter diazotrophicus, and rice variety did give rise to some plants which grew slowly for over 12 months in a nitrogen-free medium. R. Colnaghi1, A. Green, Luhong He2, P. Rudnick and C. Kennedy3 Department of Plant Pathology, College of Agriculture, P.O. Box 210036, The University of Arizona, Tucson, AZ 85721, USA. Current addresses: 1Departimento di Scienze Molecolari Agroalimentari, University of Milano, Italy and 2111 Koshland Hall, Department of Plant Biology, University of California at Berkeley, Berkeley, CA 94720, USA. Strategies considered and studied for achieving ammonium excretion in nitrogen fixing bacteria include 1) inhibition of ammonium assimilation and 2) interference with the mechanisms by which ammonium inhibits either nitrogenase synthesis or activity. These aspects of nitrogen fixation have been best studied in diazotrophic proteobacteria and Cyanobacteria and those of the former are reviewed in this paper. Ammonium assimilation by glutamine synthetase (GS) can be diminished or prevented by treatment of bacteria with chemicals that inhibit GS activity and in some diazotrophs, such treatment results in excretion of up to 15mM ammonium into liquid growth medium. Also, mutants with altered GS activity, isolated by selection for resistance to GS inhibitors, often excrete ammonium. In Proteobacteria, ammonium inhibits nitrogenase activity and/or synthesis, the latter by preventing activity or expression of nifA, a transcriptional activator required for expression of other nif genes. In Azotobacter vinelandii, ammonium inhibits nifA activity but not its synthesis; NifL mediates this effect by interacting directly with nifA causing its inactivation. In nifL insertion mutants, NifA is constitutively active and up go 10 mM ammonium is excreted during nitrogen fixation. GlnD insertion/deletion mutations are unable to be stably maintained in A. vinelandii wild type but are stable and viable in a mutant that produces constitutively active GS (cannot be adenylylated). This confirms the hypothesis that GlnD is required for activity of GS, an essential enzyme in A. vinelandii. In addition, the stable glnD mutants are Nif-, supporting also the previous conclusion that GlnD is involved in mediating NifL/NifA interaction. Mechanisms of inhibition of synthesis or activity of NifA by ammonium in other diazotrophs are discussed and compared. Genetics of Azospirillum brasilense with respect to ammonium transport, sugar uptake, and chemotaxis A. Van Dommelen, E. Van Bastelaere, V. Keijers and J. Vanderlyden F.A. Janssens Laboatory of Genetics, Willem de Croylaan 42, B-3001 Heverlee, Belgium This paper describes molecular aspects of Azospirillum-plant root association with respect to nitrogen flux and carbon utilization. In the first part, biochemical and genetic data are reported on the transport of ammonium and methylammonium in A. brasilense cells. Amonium excreting A. basilense mutants reported so far appear to result from alterations in genes encoding for enzymes involved in ammonium assimilation. Solid genetic evidence is given on the occurrence of a postulated ammonium transporter in A. brasilense. In the second part, biochemical and genetic evidence is likewise given for the occurrence of a high-affinity uptake system for D-galactose in A. brasilense. A sugar-binding protein that is part of this uptake system is required for chemotaxis of A. brasilense towards particular sugars, including D-galactose. Chitin recognition in rice and legumes G. Stacey1 and N. Shibuya2 1 Center for Legume Research, Department of Microbiology and Department of Ecology and Evolutionary Biology, The University of Tennessee, Knoxville, TN 37996-0845, USA and 2Department of Biotechnology, National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan This review focuses on a comparison of plant reception of chitin oligosaccharides by legumes and rice. Chitin oligosaccharides (dp=6-8) released from fungal pathogens induce plant defense reactions in rice, while lipo-chitin oligosaccharides (dp=4-5) induce the development of a new plant organ, the nodule, in legumes during infection by rhizobia. The former situation is pathogenic and the latter situation beneficial to the plant. However, these two systems do share some common features. We hypothesize that rice and legumes, as well as other plants, may possess members of an evolutionarily conserved family of chitin binding proteins. These proteins may play an important role in chitin reception and subsequent signal transduction. However, data support the idea that legumes may possess a second chitin binding receptor that shows a greater specificity for the lipo-chitin nodulation signals . The presence of this second receptor may be one of the key factors that distinguishes plants capable of nodulation by rhizobia (e.g., soybean) from those that cannot be nodulated (e.g., rice). The role of phytohormones in plant-microbe symbioses A.M. Hirsch1,2, Y. Fang1, S. Asad1,4 and Y. Kapulnik3 1Department of Molecular; Cell and Development Biology, 2Molecular Biology Institute, University of California, Los Angeles, CA 90095-1606, USA and 3Institute of Field and Garden Crops ARO, The Volcani Center, Bet Dagan 50-250, Israel. 4Present address: National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Plant hormones, especially auxin, cytokinin, and ethylene, have long been implicated in nodule development. In addition, plant hormones have been shown to have increased concentrations in mycorrhizal associations. We show that the early nodulin (ENOD) genes can be used as indicators for the status of endogenous hormones in symbiotic root tissues. Transcripts for ENOD2 and ENOD40 genes are shown to accumulate in uninoculated, cytokinin-treated alfalfa roots, even in roots of the non-nodulating alfalfa mutant MN1008, which is unresponsive to Rhizobium meliloti inoculation and to Nod factor treatment. Transcripts for these ENOD genes also accumulate in mycorrhizal roots of alfalfa. A model describing the involvement of cytokinin and auxin in stimulating cell divisions in the inner cortex which leads to nodule formation is presented. S.M. Swensen1 and B.C. Mullin2 1 Department of Biology, Ithaca College, Ithaca, NY 14850, USA and 2The Department of Botany and the Center for Legume Research, The University of Tennessee, Knoxville, TN 37996, USA Current taxonomic schemes place plants that can participate in root nodule symbioses among disparate groups of angiosperms. According to the classification scheme of A. Cronquist, which is based primarily on the analysis of morphological characters, host plants of rhizobial symbionts are placed in subclasses Rosidae and Hamamelidae, and those of Frankia are distributed among subclasses Rosidae, Hamamelidae, magnoliidae and dilleniidae. This broad phylogenetic distribution of nodulated plants has endangered the notion that nitrogen fixing endosymbionts, particularly those of actinorhizal plants, can interact with a very broad range of unrelated host plant genotypes. New angiosperm phyloenies based on DNA sequence comparisons reveal a markedly different relationship among nodulated plants and indicate that they form a more coherent group than has previously been thought. Molecular data support a single origin of the predisposition for root nodule symbiosis and at the same time support the occurrence of multiple origins of symbiosis within this group. Nif gene transfer and expression in chloroplasts: Prospects and problems R. Dixon1, Q. Cheng1, G.-F. Shen2, A. Day3 and M. Dowson-Day4 1 Nitrogen Fixation Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK, 2Biotechnology Research Center, Chinese Academy of Agricultural Sciences, Beijing 10081, China, 3School of Biological Sciences, University of Manchester, Oxford road, Manchester M13 9PT, UK and 4School of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requiring the coordinated and regulated expression of 16 nif genes in an appropriate cellular location. We suggest that plastids may provide a favourable environment for nif gene expression provided that the nitrogenase enzyme can be protected from oxygen damage. Using the non-heterocystous cyanobacteria as a model, we argue that photsynthesis could be temporally separated from nitrogen fixation in chloroplasts by restricting nitrogenase synthesis to the dark period. We report preliminary data on the introduction and expression of one of nitrogenase components, the Fe protein, in transgenic tobacco and Chlamydomonas reinhardtii. Finally we discuss potential avenues for further research in this area and the prospects for achieving the ultimate goal of expressing active nitrogenase in cereal crops such as rice. S. Shantharam1 and A.K. Mattoo2 1 Biotechnology Evaluations, USDA/APHIS, BSS,Riverside, MD 20737-1237, USA and 2The USDA Vegetable Laboratory, ARS, BARC-W, Bldg. 010A, Beltsville, MD 20705-2350, USA Biological nitrogen fixation (BNF) involves a highly specialized and intricately evolved interactions between soil micoorganisms and higher plants for harnessing the atmospheric elemental nitrogen (N). This process has been researched for almost a century for efficient N input into plants. The basic mechanism and biochemical steps involved in BNF have been unraveled. It has become abundantly clear that the host plant (legumes) dominates in regulating the BNF process. Environmental factors as well influence this process. Perturbation or any manipulation of the interactions between the bacteria and the legumes seems to offset the critical balance, usually to the detriment of N fixation efficiency. Not much success has been obtained in either enhancing BNF in legumes or transferring important BNF traits to non-nitrogen fixing organisms. An appraisal is given for the lack of success in making the BNF process a popular and efficient agronomic practice. Alternative physiological approaches are presented for improving mobilization, redistribution and utilization of stored N reserves within the host plant. |
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Introduction: Assessing opportunities for nitrogen fixation in rice Isolationof endophytic diazotrophic bacteria from wetland rice Azoarcus spp. and their interactions with grass roots Interactions of rhizobia with rice and wheat Genetics of Azospirillum brasilense with respect to ammonium transport, sugar uptake, and chemotaxis Chitin recognition in rice and legumes The role of phytohormones in plant-microbe symbioses Nif gene transfer and expression in chloroplasts: Prospects and problems |
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