Creation of New Nitrogen-Fixing Cyanobacterial Associations

Abstract

CREATION OF NEW NITROGEN-FIXING CYANOBACTERIAL ASSOCIATIONS Amar N. Rai and Birgitta Bergman Amar N. Rai (corresponding author; e-mail: rai_amar@ hotmail.com), Biochemistry Department, North-Eastern Hill University, Shillong 793022, India; Birgitta Bergman, Department ofBotany,Stockholm University, SE-10691 Stockholm, Sweden. INTRODUCTION Creation of N2-fixing crop plants is the ultimate goal of N2 fixation research, which aims to de crease our dependence on chemical N fertilisers for food production. The ability to use N2 as an N source is restricted to some prokaryotes only. Des pite the fact that N2 makes up the bulk of the earth's atmosphere, plants remain dependent on an exogenous supply of combined N for their growth and development. Nitrogen-fixing cyanobacteria, particularly Nostoc, form symbioses with plants ranging from algae to angiospenms and provide fixed N to the host (Rai et al. 2000). Nitrogen fixing cyanobacteria also occur epiphytically (phyl losphere and/or rhizosphere) on plants growing in aquatic and high-humidity environments (Rai 1990; Freiberg 1999; Whitton and Potts 2000). There are no natural symbioses between cyanobacteria or Frankia and crop plants, and rhi zobia associate only with legume crops. Although transfer and integration of nifgenes into crop plants is the long-term objective, a promising alternative is to create artificial N2-fixing symbioses or associ ations. Considering the low level of integration achieved during the natural evolution of cyanobac terial-plant symbioses, the creation of an obligate and self-perpetuating N2-fixing symbiosis is a tall order. In fact, none has been created as yet. The creation of facultative associations involving cyanobacteria located extracellularly on or in the plant seems more promising as an alternative. With their ability to colonise awide range of plants and plant tissues or organs, their capacity for aerobic N2 fixation, and their flexible modes of C and N nutrition, diazotrophic cyanobacteria are a more likely candidate than rhizobia to form productive associations with cereals. Nostoc is the most com mon cyanobiont in natural symbioses and, there fore, the ideal choice. Unicellular forms, by contrast, are rarely found as N2-fixing partners in natural symbioses. Attempts to create artificial sym bioses using unicellular forms, as well as attempts to create intracellular symbioses, have not been successful (see Rai 1990; Rai et al. 2000). How ever, co-cultivation of plant seedlings (e.g. from wheat, corn, sugar beet, rice and marine man groves) and N2-fixing cyanobacteria (particularly Nostoc) in liquid and sand cultures, can lead to colonisation of the roots and support seedling growth (see Toledo et al. 1995; Rai et al. 2000; Whitton and Potts 2000). PROTOPLASTS, CELLS, TISSUES AND REGENERATES Early attempts during the 1970s to introduce cyanobacteria i(cyanelles, Gloeocapsa, Anabaena and Nostoc) intomaize, barley, carrot and tobacco proto plasts failed to produce viable regenerates. How ever, Gusev and co-workers at theMoscow State University successfully established N2-fixing associ ations involving cyanobacteria (Nostoc, Anabaena, Chlorogloeopsis) and cells, tissues, plant regenerates or cuttings from carrot, calli from tobacco and mesophyll cells from alfalfa (see Rai 1990; Rai et al. 2000). Such associations have been successfuUy maintained and subcultured for several years. In plant regenerates and cuttings, cyanobacteria oc cupy the stem and leaf surfaces aswell as intemal tissues (stomatal pores, xylem vessels, stem cortex, area under the leaf cuticle). There ismoderate to high heterocyst frequency and N2 fixation, both of which increase with decreasing combined N in the medium. The cells of the cyanobacterium Nostoc in alfalfa regenerates show characteristics similar to those in natural symbioses. These include high heterocyst frequency and N2 fixation, thinner cell walls and heterocyst differentiation in the presence of combined N. More recent observations of these associations (Baulina et al. 2000; Gorelova et al. 2000) indicate that some cells of Nostoc and Anabaena species in artificial associations with higher plants lack a visible peptidoglycan layer and occasionally an outer membrane. It has been sug gested that plants produce active agents that diffuse through the medium and influence hormogonia formation and taxis, heterocyst differentiation, ni trogenase activity, the activity of peptidoglycan metabolsm enzymes and the balance between growth and cell division in the cyanobacterium. The chemical nature of these active agents has not been clarified. Associations between cyanobacteria and...