Cyanobacteria in symbiosis with plants : Protein patterns and regulatory mechanisms

University dissertation from Stockholm : Botaniska institutionen

Abstract: Cyanobacteria belonging to the heterocystous genus Nostoc, capable of oxygenic photosynthesis and nitrogen fixation via the enzyme nitrogenase, may form symbiotic associations with plants. In these associations most of the N2 fixed by the symbiotic cyanobacteria (cyanobiont) is transferred to the host, which in turn supplies the cyanobiont with fixed carbon. By using a proteomic approach adapted for investigating unsequenced organisms, adaptations of the cyanobiont to symbiotic conditions within the angiosperm Gunnera, for which each generation needs to be newly infected, and the water fern Azolla, which is in permanent association with its cyanobiont, were investigated.Despite morphological and physiological modifications of the cyanobionts, many basic functions appear to be intact in symbiosis compared to when free-living, as indicated by similar protein levels. Some differences were identified however, and in the view of parallel studies on photoautotrophic and heterotrophic growth of free-living cyanobacteria, these indicated that cellular functions were focused on N2 fixation and the associated heterocyst specific metabolism, and also reflected a mainly heterotrophic growth. Stress responses were induced in both cyanobionts, while surface adaptations mainly in that of Gunnera, possibly a reflection of its intracellular location in combination with the microaerobic and dark conditions inside the Gunnera glands. The heterocyst envelope was reduced, which may be involved in ammonia release. The level of nitrogenase was considerably higher in the Azolla cyanobiont, potentially reflecting a co-evolution with its host plant. The results also indicate that the Azolla cyanobiont may be classified as a new genus. Probably induced by oxygen, some nitrogenase in the Azolla cyanobiont carried a post-translational modification, located within a specific peptide corresponding to the part of nitrogenase that is ADP-ribosylated in certain other N2-fixing bacteria. However, the modification, with a mass of 300-400 Da, was not identified.The regulation behind heterocyst differentiation, N2-fixation and N-assimilation in symbiosis was also investigated. The mechanisms involving the regulatory proteins NtcA and HetR appear to be intact in symbiosis but distinctly upregulated, generating the higher heterocyst frequencies observed. This upregulation may be induced by a high C:N ratio in symbiosis or a plant effector molecule. These results also indicate that glutamine synthetase levels are reduced in symbiosis by a separate, but unknown mechanism. A sugar uptake regulator located near the hrm hormogonium repressing operon may be involved in carbohydrate uptake in the Gunnera symbiosis. Expression of isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase and a possible redox regulation of certain enzymes may be involved in regulation of metabolic pathways in symbiotic as well as in free-living cyanobacteria. Potential host-induced mechanisms responsible for cyanobiont adaptations, other than the environment offered in symbiosis, remain to be identified.

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