Acknowledgements

This research was supported by CIDA/(Havana U./Carleton U. Project). Technical assistance of M. Diez Cabezas and V. Garcia is gratefully acknowledged.

BIOLOGICAL NITROGEN FIXATION IN SUGARCANE: A GENOME PERSPECTIVE

E.M. Nogueira1, F. Vinagre1, H.P. Masuda1, C. Vargas1, V.L.M. de Padua1,

F.R. da Silva2, R.V. dos Santos2, J.I. Baldani3, P.C.G. Ferreira1, A.S. Hemerly1

!Lab. Biología Molecular, Depto. Bioquímica Médica, ICB, UFRJ, 21941-590,

Rio de Janeiro, RJ 2CBMEG, UNICAMP, Campinas, SP, Brazil 3EMBRAPA/Agrobiologia, Seropedica, RJ, Brazil

1. Introduction

Distinct endophytic diazotrophic bacteria have been isolated from sugarcane organs, including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans. In this unique type of association, the bacteria live in the intercellular spaces and vascular tissues of most plant organs without causing disease symptoms (Baldani et al. 2000). Besides fixing nitrogen, the endophytic diazotrophs produce plant growth-regulating hormones, such as auxin (Fuentes-Ramirez et al. 1993). It is still not clear which mechanisms are involved in the establishment of this particular type of interaction and which kinds of molecules are mediating signaling between plant and bacteria. To address these questions, we have investigated gene expression profiles in sugarcane plants colonized by the endophytic diazotrophs G. diazotrophicus and Herbaspirillum spp., using the SUCEST (Sugar Cane EST Sequencing Project) database.

2. Material and Methods

For the annotation, the database (http://sucest.led.ic.unicamp.br) containing 81,223 clusters was searched. Transcriptional profiles were constructed by electronic northern. Nineteen cDNA libraries that represent distinct tissues/organs of sugarcane plants and the two that represent in vitro growing plants co-cultivated for seven days with G. diazotrophicus and H. rubrisubalbicans, named AD1 and HR1 respectively, were used in our analysis. EST representation (frequency in AD1 or HR1 library/frequency in the second best represented library of non-infected tissues) > 2 was classified as Preferential. ESTs represented only in AD1 and/or HR1 cDNA libraries were classified as Exclusive.

3. Results and Discussion

Using the EST database of SUCEST, genes encoding proteins that might function in processes involved in the association with the endophytic diazotrophs, such as nitrogen metabolism, plant growth, plant-microbe signaling and early nodulin homologs, were annotated and their expression profile was analyzed by electronic northern. The selected dataset comprised 1827 ESTs. In all the processes investigated, various ESTs preferentially or exclusively expressed in the AD1 and /or HR1 cDNA libraries were identified. An inventory of sugarcane genes, whose expression was modulated by the association, was generated. These preliminary data suggest that the plant might be actively involved in the interaction, responding to distinct processes during the association.

4. References

Baldani J et al. (2000) In Pedrosa F et al. (eds), Nitrogen Fixation: From Molecules to Crop Productivity, pp. 397-400, Kluwer Academic Publishers, Dordrecht, The Netherlands Fuentes-Ramirez LE et al. (1993) Plant Soil 154,145-150

5. Acknowledgement

Supported by FAPERJ (in collaboration with ONSA - FAPESP), FINEP, CNPq, CAPES.

AZOSPIRILLUM DOEBEREINERAE AND HERBASPIRILL UM FRISINGENSE: TWO NEW DIAZOTROPHIC SPECIES FROM C4-FIBER PLANTS

B. Eckert1, G. Kirchhof1, O.B. Weber2, V.M. Reis2, F. Olivares2, M. Stoffels1, M. Schloter1,

IJ. Baldani2, A. Hartmann1

'GSF-Institute of Soil Ecology, D-86764 Neuherberg/Munich, Germany

2EMBRAPA, CNPAB, Seropedica, CEP 23851-970, Rio de Janeiro, Brazil

The C4-plants Miscanthus sinensis, Spartina pectinata and Pennisetum purpureum are becoming increasingly important as industrial crops for alternative agricultural production of renewable resources for energy and fiber as well as charcoal production in Europe and Brazil on set-aside land. Since it is known that these plants have only a low requirement for additional nitrogen fertilization, the presence of nitrogen-fixing bacteria associated with these plants was investigated. Using washed pieces of roots and stems of M. sinensis cv. Giganteus, Miscanthus sacchariflorus, and Spartina pectinata growing in Freising, Bavaria, Germany as well as Pennisetum purpureum cvs. grown in Brazil, nitrogen-fixing bacteria were isolated using NFb- and JNFb-semisolid media. Pure cultures of diazotrophs were characterized using a polyphasic approach (Eckert et al. 2001; Kirchhof et al. 2001). Besides known diazotrophic bacteria like Herbaspirillum seropedicae and Azospirillum lipoferum two new bacterial species were found.

Azospirillum doebereinerae sp. nov.: These bacteria are closely related to A. lipoferum and A. brasilense with 96.6 and 95.9% 16S rDNA sequence similarity. Two 16S rDNA targeting diagnostic oligonucleotide probes were developed to rapidly identify A. doebereinerae with fluorescence in situ hybridization. The bacteria differ from A. lipoferum by their inability to use N-acetylglucosamine and D-ribose and their ability to grow without supplemented biotin and from A. brasilense by their growth with D-mannitol and D-sorbitol. The new bacterial species was named in honor of Dr. Johanna Dobereiner for her great achievements.

Herbaspirillum frisingense sp. nov.: These bacteria form a homogenous group within the Herbaspirillum genus with only 1-34% similarity to H. seropedicae and H. rubrisubalbicans in DNA-DNA hybridization. The 16S rDNA similarity is 98.5-99.1%. Species-specific oligonucleotide probes were designed for diagnostic fluorescence in situ hybridization. On the basis of utilization of adipate (-), N-acetyl-D-glucosamine (+), meso-erythritol (-), L-rhamnose (-) and meso-inositol (-), H. frisingense can be distinguished from the other Herbaspirillum spp. Two days after inoculation of H. frisingense to axenically grown Miscanthus seedlings, bacteria could be demonstrated invading intracellular spaces in the rhizodermis; seven days after inoculation, they abundantly colonized xylem vessels in the vascular tissue.

References

Eckert B et al. (2001) Int. J. System. Evolut. Microbiol. 51, 17-26 Kirchhof G et al. (2001) Int. J. System. Evolut. Microbiol. 51, 157-168

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