Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA

Abstract

Insertion ofthe transposon Tn5 into the T-region ofthe octopine Ti plasmid ofAgrobacterium tumefiaciens gives rise to crown gall tumors having altered morphology. Three loci within the T-DNA that control tumor morphology have been detected [Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P. & Nester, E. W. (1981). CeU 27, 14-3-1531. They influence tumor size (tnd), production ofroots (tmr), or production of shoots (tins). Cytokinin and auxin levels in such mutant tumors were examined by HPLC/radioimmunoassay and HPLC/fluorescence assay, respectively. Free indoleacetic acid levels (in pmol/g) were: uninfected tobacco stem tissues, 128; wild-type A348 tumors, 295; tin mutant tumors, 307; tmr mutant tumors, 129; and tns mutant tumors, 70. Average trans-ribosylzieatin levels were correspondingly: 0.97, 48, 40, 0.54, and 1,400 pmol/g. transRibosylzeatin/indoleacetic acid ratios were as high as 24 in shootproducing tumors and as low as 0.003 in root-producing tumors. The evidence strongly suggests that tumor phytohormone levels are determined by genes in the T-DNA.It is known from the work ofseveral laboratories that a segment of DNA, the transferred (T) region of a large plasmid (the Ti plasmid) harbored by the inciting bacterium, Agrobacterium tumefaciens, is integrated into the host genome during crown gall tumorigenesis (1). Although seven T-DNA transcripts have been reported (2,3) in octopine' crown gall tumors, no T-DNAcoded gene product has been identified with certainty other than octopine synthase. Other transcripts are presumably determinative in tumor growth and maintenance, but their functions are unknown (4-6). However, crown gall tumors do grow in culture in the absence of added auxin or cytokinin (7), and recent studies (8)(9)(10) indicate that cytokinin levels are increased in many, but not in all,. tumor lines.The morphology of crown gall tumors is determined by at least three factors: (i) the strain ofA. tumefaciens used to incite the tumor, (ii) the plant host, and (iii) the site ofinoculation on the host (11). The work ofSkoog and Miller (12) established that the morphology of normal tobacco tissue in culture is determined by the cytokinin/auxin ratio in the medium. High and low exogenous cytokinin/auxin ratios lead respectively to shoot and root production. It seems very plausible, therefore, that hormone overproduction and imbalance may be general characteristics also of crown gall tumors.Transpositional inactivation of regions of the T-DNA (4-6) has defined several loci that control tumor morphology. We describe here the application of some new (13-14) analytical techniques to the measurement of hormone levels in tumors that have altered morphologies as a result of Tn5 insertion in the T-DNA. The data indicate clearly that endogenous tumor hormone levels are determined by specific loci in the T-DNA that also control tumor morphology. MATERIALS AND METHODSThe strains used in these experiments have been described in detail (4). All mutants were derived from strain...

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.

“…This enzyme catalyzes the condensation of isopentenyl pyrophosphate and adenosine monophosphate giving isopentenyl AMP (iPMP) (McGaw, 1987). In tumorous plant tissue this enzyme may represent the rate-limiting step in cytokinin biosynthesis (Akiyoshi et al, 1983). Furthermore, this enzyme may have a role in nontumorous tissues since a partially purified enzyme with ipt activity has been reported in tobacco cells (Chen and Melitz, 1979).…”

“…Although the mRNA could not be detected without heat induction, the threefold and sevenfold increase in the levels of ZR and ZMP (respectively) in the uninduced transgenic plants indicates that the chimeric gene must be expressed to some extent at control temperatures. Although expression of the maize hsp70 promoter at control temperatures must be nominal, even a low leve1 of constitutive ipt gene expression at control temperatures could account for the accumulation of cytokinins, since expression of ipt mRNA from the promoter normally found in the A. tumefaciens Ti plasmid (a relatively weak promoter) is enough to alter plant morphology (Akiyoshi et al, 1983). Presumably, the uninduced expression from the maize hsp70 gene occurs equally throughout the plant.…”

Cytokinin-to-Auxin Ratios and Morphology of Shoots and Tissues Transformed by a Chimeric Isopentenyl Transferase Gene

“…All of the transformed tissues examined were characterized by high Z + ZR/IAA ratios due mainly, but not entirely, to greatly elevated levels of Z + ZR. To our knowledge, all previous reports of both cytokinin and auxin concentrations were on tissues transformed with wildtype and mutated Ti plasmids carrying the unaltered ipt gene (1,7,15). In those studies, the cytokinin content appeared to be elevated to some extent over levels in normal plants, however, considerable variation was observed.…”

“…Endogenous hormone levels have been reported for normal plant tissues and ones transformed with the T-DNA from wild-type and mutant Ti plasmids of Agrobacterium tumefaciens (1,7,9,11,12,15,20). The T-DNA region encodes genes involved in cytokinin and auxin biosynthesis and imparts the trait of hormone-autonomous growth on transformed cells (16).…”

Structural insight into the reaction mechanism and evolution of cytokinin biosynthesis

“…The Tzs gene is located in the virulence region of nopaline-type Ti-plasmids (16). Tzs stimulates T-DNA transfer efficiency (16,17), whereas Tmr, which is encoded in the T-DNA region, induces tumorigenesis in host plant cells (10,18).…”