Plant growth promotion and HM tolerance induced by CR30: As reflected by the values of a variety of plant parameters, enhanced growth was observed in CR30-inoculated plants, compared to non-inoculated control plants, mainly in plants exposed to low HM concentrations. A genomic analysis of CR30 was carried out to identify genes related with its well-known plant growth promotion traits. Relevantly, genes involved in plant hormone regulation, production of siderophores and phosphate solubilization were identified. In vitro tests were used to phenotypically confirm the ability of CR30 to produce these compounds, as well as a whole set of degrading enzymes related with plant-bacteria interactions. The isolation of the strain from nodules confirmed plant tissue colonization by CR30, showing its remarkable capacity to colonize internal tissues of P. sativum and then become a plant growth-promoting endophyte.
Greenhouse experiment: One of the major effects observed in HM-exposed P. sativum plants, even after a short period of exposure, was the reduction in the number of nodules developed at plant roots. Interestingly, this effect was reduced in CR30-inoculated plants. Pertaining to other growth parameters (number of leaves, chlorophyll intensity), no significant differences were detected between treatments. However, in HM-exposed plants, the presence of CR30 did result in an enhanced root and shoot development. The evolution of P. sativum plants was measured every week. At the end of the experiment, the observed differences between HM-treated plants and their respective controls were accentuated. Moreover, plant development was significantly influenced by the presence of CR30, which led to a considerable reduction of HM-induced adverse effects on plant growth.
HM concentrations in plant tissues: Heavy metal accumulation was detected in pea plants after ten days of irrigation with a solution containing several HMs. CR30-inoculated plants presented lower HM concentrations than non-inoculated plants, being this effect more pronounced in roots than in shoots. Accumulation values depended on the specific HM under analysis, with some of them being translocated to aerial plant parts while others were not. The analysis of shoot, root and nodule samples indicated that HM accumulation was dependent of the presence of CR30.
Transcriptomic analysis: In both the presence and absence of HMs, the transcriptomic profiles of CR30-inoculated plants showed an important number of differentially expressed genes (DEGs), with respect to non-inoculated control plants. Some of these DEGs are related to organelle organization and chromatin assembly. In addition, genes related to metabolic processes, including nitrogen metabolism, were identified. Other noteworthy genes that presented a differential expression had a gene ontology of response to stimuli, stress, defence, and binding. In CR30-inoculated HM-exposed plants, the number of DEGs compared to HM-exposed non-inoculated controls, was different, showing an increasing level of expression of the genes in the presence of CR30.
