tRNA Is the Source of Low-Level trans- Zeatin Production in Methylobacterium spp

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.Although the best-studied plant-bacterium relationships involve pathogenesis or symbiosis, plants interact constantly with phylloplane bacteria and with the bacteria in the soil around their roots (rhizosphere). While it is not widely believed that epi-and endophytic bacteria have overt effects on plants, there is evidence (13) that plants interact with pink-pigmented facultatively methylotrophic bacteria (PPFMs), which are inhabitants of the phylloplane and rhizosphere. PPFMs are gram-negative members of the alpha subclass of the class Proteobacteria that belong to the genus Methylobacterium and are known to inhabit the leaf surfaces of a wide variety of plant species (11). One intriguing aspect of the plant-PPFM relationship is the possibility that PPFMs may provide cytokinins to the plant host or have cytokinin-like effects. Corpe and Basile (12) demonstrated that callus from Streptocarpus prolixus (gracilis) (Cape primrose) regenerated plantlets within 15 days when it was cultured cobiotically with PPFMs. PPFM-free controls exhibited no plant development after 30 days. However, Corpe and Basile did not indicate whether specific phytohormone regimens could mimic the PPFM effects or whether PPFMs produce phytohormones. We demonstrated previously that heat ...

EPS Production, PHB Accumulation and Abiotic Stress Endurance of Plant Growth Promoting Methylobacterium Strains Grown in a High Carbon Concentration

“…Methylobacterium, as plant symbionts, utilize the methanol emitted from plants and in turn, impart beneficial effects on plant growth through one or more mechanisms that include production of phytohormones like indoleacetic acid or cytokinins and vitamins (Basile et al, 1985;Koenig et al, 2002;Trotsenko et al, 2001), synthesis of enzymes such as urease or 1-aminocyclopropane-1-carboxylate deaminase (ACCD) that modulate plant growth (Holland and Polacco, 1992;Madhaiyan et al, 2006Madhaiyan et al, , 2007 and production of siderophores (Idris et al, 2004). Monier and Lindow (2003) reported that on different plant species, large aggregates of bacteria are embedded in extracellular matrix.…”

Impact of Pink Pigmented Facultative Methylotroph and PGRs on Water Status, Photosynthesis, Proline and NR Activity in Tomato under Drought

“…Madhaiyan et al, (2004) observed higher photosynthetic activity in rice cultivar Co-47 that received Methylobacterium and attributed the effect due to enhancement of chlorophyll concentration, maleic acid content and increased number of stomata. Using a variety of biochemical and cytological methods, it has been shown that microbes of the genus Methylobacterium produced cytokinin (Koenig et al, 2002). The "classical" phytohormone auxin is also produced and secreted by different strains of Methylobacterium (Hornschuh et al, 2002).…”

Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis

“…Therefore, it is possible that iP and cZ are produced when modified tRNAs are degraded. In bacteria, modified tRNAs are a source of low-level cytokinin production (31,32); however, there is no experimental evidence for a tRNA-degradation route for cZ production in plants. tRNA IPTs catalyze isopentenylation of certain tRNAs in diverse organisms ranging from bacteria to animals and plants (33), including Arabidopsis (34).…”