Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Abstract

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.

Figure 1

Morphology of mature tomato plants. A, Seven-week-old plants: untreated wild type (left), wild type watered with 10 μm BA (center), and untreated dgt (right). B, Leaves from 7-week-old plants, arranged from cotyledon (left) to youngest leaf (right); untreated wild-type (top row), wild type treated with 10 μm BA (middle row), and untreated dgt (bottom row). C, Leaves emerging from first internode of untreated dgt plant (left) and wild-type plant treated with 10 μm BA (right). D, Lateral branches emerging from first internode of a 3-month-old dgt plant.

Figure 2

Dose response curve for auxin-induced elongation of tomato hypocotyl segments in the presence and absence of 100 μm BA. Segment growth was determined after 14 h of incubation in the light (A) or in the dark (B). In addition to receiving BA during the auxin treatment, segments were pre-incubated for 2 h in the presence or absence of the indicated BA concentration. Error bars show ses from at least three independent experiments.

Figure 3

Time course of elongation for wild-type hypocotyl segments incubated in the light. Segments were treated with or without 100 μm IAA in the presence of 100 μm BA after a 2-h pre-incubation with 100 μm BA and measured at the indicated times after auxin addition. Error bars show SEs from three independent experiments.

Figure 4

Elongation kinetics of abraded wild-type tomato hypocotyl segments. Growth rates were monitored with a CCD camera. Arrows indicate addition of 10 μm IAA to the incubation medium at 120 min. A, Elongation rates in bright white light. B, Elongation rates in dim light. Experiments were repeated three or more times, and representative results for each treatment are shown.

Figure 5

H⁺ secretion in abraded wild-type tomato hypocotyl segments. The pH of the unbuffered incubation medium (10 mm KCl and 1 mm CaCl₂) was monitored continuously. A, Continuous pH measurements. After equilibration of the incubation medium to a stable pH, a pH-adjusted aqueous IAA solution (white triangles), or ethanolic FC solution (black triangles, final ethanol concentration 0.1% [v/v]) were added at the indicated times. Additions to the medium indicated in the left column were made from aqueous stocks and were present during the entire experiment. One representative experiment for each treatment is shown. Equilibrium pH before IAA or FC addition is given to the left of each graph. B, Quantitative comparison of IAA-induced changes in the pH of the medium at 0.5 and 2 h after IAA addition. Bars represent averages from two or three experiments. Error bars show SEs.

Figure 6

Influence of auxin, cytokinin, and the dgt mutation on the expression of the LeSAUR gene as determined by RNA gel blots. Hypocotyl segments were harvested and treated as described for elongation assays. The final incubation was for 2 h in Suc/MES (SM) buffer containing the indicated hormones at 100 μm. A representative autoradiograph is shown above the quantification of signals from three independent experiments. Values from densitometer scans of films were expressed as percent of the highest signal in each respective experiment and subsequently averaged. Error bars indicate the se from three independent experiments. The ethidium bromide-stained gel is shown at the bottom.

Figure 7

Influence of cytokinin on auxin-induced ethylene synthesis in wild-type and dgt tomato hypocotyl segments. Accumulated ethylene was measured after a 3-h treatment with the indicated auxin concentrations in the presence and absence of 100 μm BA, after a 2-h pre-incubation with or without BA. Segments were harvested as described for elongation experiments and incubated in the dark. Error bars represent the se from independent experiments (n = 4 for wild type, n = 2 for dgt).

Figure 8

Influence of auxin, cytokinin, and the dgt mutation on the expression of two ACC synthase genes (LE-ACS3 and LE-ACS5) as determined by RNAse protection assays. Hypocotyl segments were harvested and treated as described for ethylene biosynthesis assays. The final incubation was for 2 h in SM buffer containing the indicated hormones at 100 μm. A representative fluorograph (LE-ACS3 and LE-ACS5) is shown above the quantification of signals from three independent experiments. Values from densitometer scans of films were expressed as percent of the highest signal in each respective experiment and subsequently averaged. Error bars indicate the se from three independent experiments.

Figure 9

Hypothetical model for the action of cytokinin and the DGT gene product on auxin responses in tomato hypocotyl segments. Through a DGT-dependent process, auxin stimulates the expression of auxin-inducible genes and H⁺ secretion, leading to auxin-induced ethylene synthesis and elongation. Cytokinin antagonizes a subset of these DGT-dependent auxin responses by inhibiting one branch of the auxin signal transduction pathway.