The isopentenyltransferase (ipt) gene associated with cytokinin biosynthesis in plants was cloned from a tumor-inducing plasmid carried by Agrobacterium tumefaciens and placed under the control of promoters of differing activities, the cauliflower mosaic virus 35S promoter and the nopaline synthase promoter. These promoter-gene constructs were introduced into wounded Nicotana stems, leafpieces, and cucumber seedlings by A. tumefaciens infection. Shoots were observed in the infection site on all responding genotypes of Nicotana plants infected with the 35S promoter construct (35S-ipt), whereas only 41% responded similarly to infection with the unmodified gene. Furthermore, shoots were observed 19 days after infection with the 35S-ipt gene but not until 28 to 45 days with the unaltered ipt gene. Shoots were more numerous (>40) on galls incited by 35S-ipt and were up to 6 times taller than shoots induced by the native gene. On Cucumis (cucumber), shoots were observed only on galls incited by the 35S-ipt construct. These galls were on the average 7.5 times larger than those incited by the nopaline synthase promoter construct (NOS-4pt) or the unmodified ipt gene. Zeatin and zeatinriboside concentrations averaged 23 times greater in the 35Spt transformed shoots than in ones transformed with the native ipt gene. These results suggest that a more active promoter on the ipt gene can enhance or change the morphogenic potential of transformed plant cells by increasing their endogenous cytokinin levels.Patternsiof differentiation in plants can be affected by several phytohormones including two major classes, the cytokinins and auxins. The relative levels of cytokinins and auxins in plants appear to be critically important. When cultured in vitro, cells of most plants require exogenous cytokinin and auxin in the medium for cell division to occur, presumably because of low endogenous pools of these phytohormones. Moreover, regeneration of shoots from cultured cells can be achieved with many plant species by increasing the cytokinin-to-auxin ratio in the medium (1, 2). However, failure of some species, among them important crop species, to respond in a morphogenic fashion to such treatment may reflect problems in hormone uptake, compartmentalization, or metabolism. The availability of phytohormone-specifying genes with plant regulatory sequences from Agrobacterium tumefaciens provides an alternate genetic approach to the study of morphogenesis through the in vivo manipulation of hormone ratios (3).A widely studied method of gene transfer to dicotyledonous plants is based on the tumor-inducing (Ti) plasmid of A.

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorgh-um was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, a-keto-ymethylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.Incorporation of "4C from added methionine-'4C into ethylene was curtailed by rhizobitoxine to about the same extent as was ethylene production. These results suggest that rhizobitoxine interferes with ethylene biosynthesis by blocking the conversion of methionine to ethylene and not indirectly by inhibiting the biosynthesis of methionine. Ethylene production by Pemicillium digitatum, a fungus which produces ethylene via pathways not utilizing methionine as a precursor, was not affected by rhizobitoxine.Two model systems for the generation of ethylene in plant tissues have been described by Lieberman and co-workers, one utilizing methionine as a substrate (8), and the other utilizing linolenate (1 1). In addition, methionine can serve as a precursor of ethylene in plant tissues (2, 7). To help assess the physiological importance of methionine as an ethylene precursor, a specific inhibitor of methionine biosynthesis was sought. Rhizobitoxine appeared to offer that potential.Rhizobitoxine is a phytotoxin produced by certain strains of the soybean root nodule bacterium Rhizobium japonicum (15).It inhibits greening of new leaf tissue of many plants and causes the main visual symptom of the disease in soybean known as rihizobial-induced chlorosis (14). The precise structure of rhizobitoxine remains to be elucidated; however, it is known to be a basic sulfur-containing amino acid which yields an ether derivative of homoserine upon desulfurization (13). Rhizobitoxine inhibits the growth of Salmonella typhimurium by inhibiting /3-cystathionase, an enzyme in the methionine biosynthetic pathway (12). It also irreversibly inactivates /Bcystathionase isolated from spinach leaves (4); however, the physiological effect of this lesion on the biosynthesis of methionine in spinach has yet to be assessed. We report here that rhizobitoxine inhibits ethylene biosynthesis in sorghum seedlings and in senescent apple tissue by the unexpected mechanism of blocking the conversion of methionine to ethylene. Hegari were surface-sterilized by wetting with ethanol and then immersing in an aqueous solution of 0.2% HgCl2 + 1% HCl for 2 min. After rinsing well, the seeds were germinated on moist filter paper in a Petri dish at 27 C in the dark. Two days after imbibition, the seedlings were transplanted to 50-ml Erlenmeyer flasks constructed with a side arm to collect CO2. Six seedlings per flask (about 300 mg fresh wt) were supported on a nylon mesh screen held 1.0 cm above the flask bottom...