Abstract

The growth of plants in a natural environment is strongly influenced by their ability to overcome the restrictions imposed by their immediate surroundings, such as limited nutrient availability in the soil, the potential attack of pathogens, and stressful conditions like drought, salinity, or the presence of toxic pollutants. Plants are able to establish diverse kinds of mutually beneficial interactions with soil bacteria that colonize the rhizosphere and, in many cases, the internal tissues of the plant host. It has been demonstrated that interaction with such bacteria allows plants to cope with environmentally challenging conditions, and improves the rate and extent of their growth, even in the absence of environmental stress. These plant growth-promoting bacteria (PGPB) are ubiquitously present in natural environments, and their ability to colonize and stimulate the growth of different plant hosts has been reported in a variety of cases. Proteobacterial species of the Burkholderia and Azospirillum genera are among the most versatile PGPB found so far, being able to colonize several plant species, and to use diverse strategies for growth promotion, including the ability to modify plant hormone levels, thus increasing growth and/or down-regulating stress, the expression of antagonistic functions towards pathogens, and the capacity to perform nitrogen fixation, even in the roots of non-nodulating plants. A well-studied example of a versatile growth promoter is Burkholderia phytofirmans PsJN, a bacterium with outstanding abilities for colonization and growth promotion of different plant hosts, including potato, tomato, grape and the model plant Arabidopsis thaliana. On the other hand, Azospirillum brasilense Sp245 is capable of extra-nodular nitrogen fixation, among other growth stimulation abilities. Taken together, these two bacteria possess most of the traits described as relevant for plant growth promotion to date. It is widely recognized that, in order to promote plant growth efficiently, bacteria must interact closely with the root surface and, in most cases, colonize the internal tissues therein. However, despite the intensive research focused on plant growth promotion, little is known about the possible signalling compounds or carbon sources in root secretions that are responsible for driving bacterial colonization, and stimulating the expression of beneficial functions. Regarding this point, it has been determined that a significant portion of the total carbon exuded by plant roots is constituted by phenolic compounds, including lignin-phenolics and flavonoids, some of which are known to inhibit the growth of pathogens, while some stimulate the onset of the specific Rhizobia-legume symbiosis. However, the effect of phenolic compounds on widely generalized mutualistic interactions, as the ones described above, is largely unknown. Intriguingly, the ability to catabolize and respond to certain phenolic compounds is broadly distributed in PGPB, and well represented in Burkholderia and Azospirillum strains. The general goal of this proposal is to assess the influence of relevant plant-derived phenolic compounds on the ability of beneficial proteobacteria to colonize and promote growth of plants, using A. thaliana as a model, based on the hypothesis that such compounds are key determinants of the ability of the bacteria to establish a mutualistic interaction with the plant host, acting as inducers of bacterial colonization and/or growth promotion functions. To fulfil this goal, the effect of specific plant phenolics on colonization and growth promotion of A. thaliana by B. phytofirmans and A. brasilense will be assessed. Once individual phenolic compounds participating in these processes are identified, their effect on the expression of specific bacterial genes encoding colonization and promotion functions will be analyzed. The promoters of genes shown to be controlled by plant phenolics will be fused to the uidA (GUS) reporter gene, to assess their expression when bacteria colonize the plant. To confirm the role of induced genes on colonization and growth promotion, recombinational inactivation will be performed, and the interaction of the resulting mutant strains with A. thaliana will be evaluated. Finally, to further validate the previous results, colonization and growth promotion by either strain will be studied in Arabidopsis mutant lines displaying an altered endogenous production of phenolic compounds. This research proposal is expected to contribute to a new area of groundbreaking research, which receives considerable attention worldwide, and is not currently explored by other researchers in our country. The systematic approach described, combining the study of phenotypic responses, gene expression analyses and gene inactivation, will allow the identification of essential elements involved in plant colonization and growth promotion by mutualistic bacteria, the molecular bases of which are poorly understood. Finally, conclusions of this project are expected to be relevant for the manipulation of the beneficial properties of PGPB, potentially leading to a better management of plant growth in field applications and the enhancement of the resistance of different plants to a variety of environmental stresses by microbial stimulation of fitness.