Louis Berrios

Date of Award

Summer 2021

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Berten Ely

Abstract

Bacteria play an integral role in regulating plant growth and development. However, many of the mechanisms encompassing bacteria-plant interactions are poorly understood and thus require detailed assessments (see CHAPTER 1). To this end, I coupled bacterial (Caulobacter sp.) and plant model organisms (Arabidopsis) to determine 1) the degree to which select bacteria can enhance the growth and development of plants, and 2) what functions these bacteria possess that enable them to aid plant development. Employing bacterial isolation techniques, monoculture inoculum-based plant growth assays, biochemical assays, comparative genomics, functional genetics, and real-time quantitative PCR (RT-qPCR), I determined that 1) Caulobacter-Arabidopsis interactions vary from mutualistic to parasitic; 2) common biosynthates are not required for many beneficial Caulobacter-Arabidopsis interactions; 3) redox-related genes and bacterial cell curvature facilitate Caulobacter-Arabidopsis interactions, and 4) bacterial concentration and bacterial induced pH reductions contribute to Caulobacter-mediated seed germination inhibition.

Collecting and processing soil and root samples from South Carolina and Florida, I uncovered two novel Caulobacter strains that can enhance the biomass of Arabidopsis. To contextualize these findings, I tested the ability of previously obtained stock cultures of Caulobacter strains (collected from both aquatic and soil environments) to also enhance plant growth. As a result, I determined that 1) plant growth enhancement is not a conserved feature in the Caulobacter genus, and 2) isolation source did not correlate with plant-growth-promoting (PGP) factors (i.e., not all soil-derived strains enhanced plant growth and not all aquatic-derived strains failed to enhance plant growth). Using established biochemical tests as proxies for plant-growth-promotion factors, I determined that (among the 11 Caulobacter strains that I assayed) Caulobacter strains do not use these common PGP factors to enhance plant growth. Employing a comparative genomics approach, I determined that each of the PGP Caulobacter strains that I assayed harbors a unique set of genes (cyo operon) with predicted functions in betalain biosynthesis—a ROS scavenging metabolite—in its genome. Since ROS molecules are critical for plant growth and development, I hypothesized that these genes may be involved in the ability of PGP Caulobacter strains to enhance the growth and development of Arabidopsis (see CHAPTER 2).

To determine whether the cyo operon genes are necessary for Caulobacter-mediated plant growth enhancement, I disabled the function of one of the subunits (cyoB) using homologous recombination in two different PGP Caulobacter species and assessed the potential of the resultant mutant strains to enhance plant growth relative to their parental strain. As a result, I determined that a functional cyo operon facilitates Caulobacter-mediated growth enhancement of Arabidopsis since the mutant strains were unable to enhance plant growth relative to their parental strains. Interestingly, using RT-qPCR, I determined that one PGP Caulobacter strain expresses the cyoB gene (and additional genes with predicted betalain biosynthesis functions; see CHAPTER 3) significantly more than other strains and subsequently hinders the germination rate of Arabidopsis seeds. I also constructed a flux balance analysis (FBA) to gauge the relative metabolic activity between Caulobacter strains since a large portion (~80%) of variation in seed germination inhibition was explained by the culturing media type (media used for bacterial-seed plating assays). To this end, the FBA and subsequent pH measurements suggested that increased H+ ion excretion likely contributes to Caulobacter-mediated seed germination inhibition, although abundant bacterial growth also contributes to the observed inhibition. Moreover, I hypothesized that bacterial cell shape would facilitate plant growth since previous reports have shown that Caulobacter cell shape impacts niche habitancy, and I showed that Caulobacter cell curvature is required for this bacterium to enhance the growth of Arabidopsis. Therefore, I established a genetic framework to investigate the mechanisms that undergird Caulobacter-Arabidopsis interactions. Taken together, I fused two reliable genetic models (Caulobacter and Arabidopsis) to generate a working model for bacteria-plant interactions. Leveraging the high-quality genomic database for Caulobacter strains, I discovered genetic factors that facilitate the ability of select Caulobacter strains to enhance the growth of Arabidopsis plants.

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© 2021, Louis Berrios

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