A nitrogénkötő szimbiózis génjeinek evolúciós és funkcionális vizsgálata Medicago truncatula modellnövényen

Abstract

Soil nutrient availability is a major limiting factor in plant growth and productivity at many areas under agricultural cultivation. An evolutionary answer to this demand is the symbiotic association of plants with soil microbes that provide valuable macronutrients to the host for a share of the sugar compounds of photosynthesis, as an exchange. An ancient type of coexistence is the arbuscular mycorrhiza symbiosis formed with fungi that provide mainly phosphates to the plants. The majority of land plant species are able to form mycorrhiza symbiosis. It is hypothesized that this capability was present already in the common ancestor of all land species, hence those recent species that do not form mycorrhiza, most probably lost this ability during their evolution. In addition, much later in evolution, another form of symbiotic co-existence appeared between certain nitrogen fixing soil bacteria and a group of closely related plant species. During nitrogen fixing symbiosis a new organ, the root nodule is formed, where the symbiotic bacteria reside and fix atmospheric nitrogen, which is than converted to organic molecules that can be used by the plant. Analyzing the plant genes needed for these symbioses revealed that some of them are indispensable for both types of co-existence. These are the elements of the so-called common symbiotic pathway. It is hypothesized that the phylogenetically much younger nitrogen fixing symbiotic system was developed on the molecular grounds of the more ancient mycorrhiza symbiosis, recruiting elements from the already existing signaling pathways. We aim to understand this process through the evolutionary and functional analyses of these genes and gene products. For our evolutionary analysis we chose 16 M. truncatula genes coding for proteins with diverse functions during nitrogen fixing symbiosis. Most of them are specific for nitrogen fixing symbiosis, but some elements are part of the common symbiotic pathway. Additionally, we chose 12 sequences with no proven symbiotic function, to use them as controls. The selected symbiotic and control M. truncatula sequences were used as queries while searching for their homologs in the available genome sequences of ten plant species from different groups of the angiosperm phylogenetic tree. Two other legume species, that are able to form nitrogen fixing root nodule symbiosis, were picked, and other non-nodulating dicot and also monocot species were selected for further analysis. All these species were capable of mycorrhiza symbiosis with the one exception of A. thaliana that is incapable of either the mycorrhiza or the nitrogen fixing symbiosis. After the BLAST searches with the M. truncatula amino acid sequences a single homologous sequence from each plant genome - that showed the highest similarity to the respective reference sequence - was chosen to work with. These homologous sequences were considered as putative orthologs. Reciprocal BLAST searches were used to identify and filter out the false ortholog hits. The general model plant A. thaliana revealed the highest number of missing orthologous sequences. It is unable to form even mycorrhiza symbiosis, most probably lost this ability during its evolution, and supposedly, in connection with this, also lost the vast majority of its symbiosis related genes. Those orthologous sequences that are still present in the A. thaliana genome may have an important role in other, non-symbiotic function. First we compared the collected most similar sequences in their length on the amino acid level to see whether some of them went through larger changes in their length during their evolution that could possibly affect their domain composition. Considerable length differences could only be detected for orthologous sequences for some symbiotic proteins. More than 20% difference in length compared to the respective reference M. truncatula sequence were shown for three A. thaliana paralogs, and the monocot DMI2 orthologous sequences. We know from the literature that DMI2 orthologs developed by extracellular domain acquisition during plant evolution. Among these, the ones with longer sequences were able to complement both the defective mycorrhiza and nitrogen fixing symbiosis phenotype of the respective L. japonicus mutants, while the shorter monocot orthologous sequence could only restore mycorrhiza symbiosis in trans-complementation experiments. Moreover, the two monocot NSP2 homologs and the Z. mays ERN1 sequence showed 10-15% difference in length compared to the respective reference sequence, but no extra domains in these proteins could be detected by InterPro database analysis. Also it is known from the literature that the extended length O. sativa NSP2 ortholog with extended length was able to fully complement the L. japonicus nsp2 mutants...