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Unraveling the molecular dialogue in microbial-assisted plant growth in the presence of heavy metals

Periodic Reporting for period 1 - BACTEPEA (Unraveling the molecular dialogue in microbial-assisted plant growth in the presence of heavy metals)

Reporting period: 2020-05-01 to 2022-04-30

Agriculture is currently confronting an increasing human population and limitations of soil use due to, among other reasons, pollution levels above food safety threshold values. Some agricultural practices increase the heavy metal (HM) content of agricultural soils, representing an important threat for human health and ecosystems. The use of microorganisms (bacteria, fungi) as plant growth promoters has been increasingly studied for a number of years, but it has only recently been proposed to improve plant HM tolerance. Regrettably, plant-microorganism-pollutant interactions are still poorly understood, and the molecular underlying mechanisms are mostly unknown. The abovementioned challenges for agricultural production require the study of these mechanisms to better promote a more efficient and sustainable agriculture.

Deciphering the molecular interactions between plants and microorganisms under HM stress is necessary to provide new pathways for improved soil management. This project addresses a crucial objective in food security, i.e. the development of sustainable agricultural practices to control potentially adverse effects of HM exposure on plant health and growth.

Objectives of the project:

- To demonstrate the plant growth promotion capacity of the actinobacterial strain Micromonospora cremea CR30T on a highly valuable crop (Pisum sativum) in the presence and absence of HMs

- To identify genes implicated in P. sativum response to the presence of M. cremea CR30T

- To identify genes implicated in P. sativum response to HMs

- To identify genes implicated in P. sativum response to HMs in the presence of M. cremea CR30T

- To determine HM accumulation in P. sativum tissues in the presence and absence of M. cremea CR30T.
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.
In BACTEPEA, we have confirmed the plant growth promotion capacity of the rhizospheric actinobacterial strain, Micromonospora cremea CR30T, as well as its remarkable capacity to colonize the internal tissues of Pisum sativum, thus becoming an endophyte. In addition, we have confirmed the presence of genes, and their corresponding physiological function (at the phenotype level), involved in IAA (auxin) production, ACC-deaminase activity, phosphatase activity, and siderophore production, all of them implicated in plant development. Furthermore, high tolerance to HMs and their accumulation in plant internal tissues have been confirmed, and a number of genes implicated in these processes have been identified in the CR30 genome. Both traits, plant promotion and HM tolerance, were used to determine the capacity of CR30 to protect plant development under HM stress, finding out that plant development was influenced by CR30, whose presence considerably reduced HM-induced adverse effects on plant performance. Similarly, the presence of CR30 altered metal translocation to upper plant tissues. This finding opens up the possibility of using CR30 for bacteria-assisted phytoextraction or phytostabilization strategies depending on the specific HM under consideration.

According to our transcriptomics results, several genes appear to be specifically expressed in the presence of CR30, being probably implicated in the root penetration process performed by this actinobacterial strain when inoculated to P. sativum plants. This finding will be the focus of future projects to better understand the different steps needed for CR30 colonization. This information could be of great interest to enhance plant colonization by other bacterial species. Finally, an important number of genes responded to HM exposure (including genes encoding HM transport and binding proteins), which will also be the focus of futures studies with potential interest for the field of metal phytoremediation.
Protection of pea plants against HM by CR30