Cytokinin Production by Bradyrhizobium japonicum

Abstract

Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobhum Japoncum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Meff.), was grown in defined medium.Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found.Total cytokinin activity In the B. Japoncum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. Japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.Cytokinin production in several plant-associated microbes has been well characterized (13,14,23,33). These include the gall-forming phytopathogenic bacteria, Agrobacterium tumefaciens and Pseudomonas savastanoi, and the causative agent of witches broom, Corynebacterium fascians. In all of these organisms, the disease symptoms are correlated with cytokinin production (20,22,23). Almost thirty years ago, Arora et al. (3) suggested that the action of rhizobia in nodule formation might likely involve the supply or activation of several different growth factors. Despite considerable evidence for the production of cytokinins by Rhizobium and for the involvement of cytokinins in legume nodulation, neither the nature of cytokinin production by Rhizobium nor the role of these cytokinins in the Rhizobium-legume symbiosis has been determined. 'A portion of this research was presented in preliminary form at the annual meeting of the American Society of Plant Physiologists, St. Louis, 1987. There are reports that at least some strains of Rhizobium produce cytokinins in culture, though they have not been quantitated or rigorously identified. Three cytokinin-active compounds were detected in the culture filtrate ofRhizobium leguminosarum, one of which cochromatographed with i6Ade2 (9). Phillips and Torrey (27) also reported cytokininlike...

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

“…5), and 17 Ϯ 7 ng/mg of tRNA was recovered from Arabidopsis PPFM isolates (data not shown). These values are much lower than the 200 ng/mg reported for B. japonicum (40), but the recovery procedures differed; using our procedures, we recovered less than one-half of the previously reported levels of trans-zeatin riboside from B. japonicum tRNA (data not shown). No significant signal corresponding to the cis isomer was obtained, as shown by the lack of active fractions in an RIA for the cis isomer (Fig.…”

“…Because of the difference in stereoisom-erism between the two paths of cytokinin biosynthesis, it has been assumed that tRNA turnover makes a minor contribution, at best, to the plant cytokinin pool. However, trans-hydroxylated zeatin has been found in the tRNAs and culture filtrates of Bradyrhizobium japonicum (40) and the rhizosphere bacterium Azotobacter vinlandii (1,43). The trans isomer in the tRNA of Agrobacterium that was described by Chapman and Morris (6) was later shown by workers in the same laboratory to be the cis isomer (32).…”

A simple model based on known plant defence reactions is sufficient to explain most aspects of nodulation

“…An additional level of complexity is added by the perturbation of hormone levels in nodules, which can be in response to. or even mediated by, the microsymbiont (Sturtevant and Taller, 1989), and which can also affect the expression of plant defence response genes (Robinette and Matthysse. 1990;Dominov et al, 1992).…”

“…Furthermore, Maxwell et al (1993) reported that isoflavonoid biosynthesis is higher than, and uncoupled from, nod-on synthesis in Medicago nodules, implying that either nod-offs (or, at a later stage, phytoalexins) are being produced. An additional level of complexity is added by the perturbation of hormone levels in nodules, which can be in response to, or even mediated by, the microsymbiont (Sturtevant and Taller, 1989), and which can also affect the expression of plant defence response genes (Robinette and Matthysse, 1990;Dominov et al, 1992).…”

Signals in Root Nodule Organogenesis and Endocytosis of Rhizobium.

“…A/-Acetylglutamic acid induces nodulelike primordia on clover (Philip-Hollingsworth et al, 1991), whereas diglycosyldiacylglyceride causes uniform cell division in the cortex, leading to a thick and short root phenotype (F. Dazzo, personal communication). Rhizobia are also known to secrete cytokinins (Sturtevant and Taller, 1989). These compounds apparently have distinct receptors and use separate signal transduction pathways that may converge to elaborate their responses via a common route.…”