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tRNA is the source of low-level trans-zeatin production in Methylobacterium spp
Robbin L Koenig1, Roy O Morris, Joe C Polacco
1Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Abstract:
Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.
Insights
Pink-pigmented facultatively methylotrophic bacteria (PPFMs) produce trans-zeatin, a plant hormone, from tRNA turnover, not de novo synthesis. This discovery impacts understanding of plant-microbe interactions and bacterial signaling pathways.
Area of Science:
- Microbiology
- Plant Science
- Biochemistry
Background:
- Pink-pigmented facultatively methylotrophic bacteria (PPFMs), or Methylobacterium spp., are common plant leaf surface inhabitants.
- Previous research suggests a dialogue between PPFMs and plants, hinting at bacterial signaling molecules.
- Cytokinins are plant hormones known to regulate growth and development.
Purpose of the Study:
- To investigate cytokinin production in PPFMs.
- To determine the origin and synthesis pathway of zeatin in Methylobacterium spp.
- To assess the role of bacterial cytokinin production in plant-microbe interactions.
Main Methods:
- Isolation and purification of trans-zeatin from PPFM culture filtrates using immunoaffinity and HPLC.
- Identification of trans-zeatin using HPLC and radioimmunoassay with specific monoclonal antibodies.
- Genetic manipulation of the miaA gene in Methylobacterium extorquens to create cytokinin-null mutants.
- Confirmation of mutations using PCR and analysis of zeatin secretion.
- Soybean seed germination assays with wild-type and mutant bacteria.
Main Results:
- Trans-zeatin and smaller amounts of trans-zeatin riboside were successfully isolated from PPFM culture filtrates.
- Zeatin was also detected in tRNA hydrolysates, suggesting tRNA turnover as the source.
- Mutating the miaA gene abolished zeatin production in tRNA and its secretion into the medium.
- Cytokinin-null mutants retained the ability to stimulate soybean seed germination, similar to wild-type bacteria.
Conclusions:
- PPFMs secrete trans-zeatin, primarily derived from tRNA turnover, not de novo synthesis.
- The miaA gene is crucial for the production of zeatin from tRNA in Methylobacterium.
- While cytokinin production is a characteristic of PPFMs, it is not the sole factor responsible for stimulating seed germination.