Auxin-dependent cell division and cell elongation. 1-Naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid activate different pathways

Abstract

During exponential phase, the tobacco (Nicotiana tabacum) cell line cv Virginia Bright Italia-0 divides axially to produce linear cell files of distinct polarity. This axial division is controlled by exogenous auxin. We used exponential tobacco cv Virginia Bright Italia-0 cells to dissect early auxin signaling, with cell division and cell elongation as physiological markers. Experiments with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated that these 2 auxin species affect cell division and cell elongation differentially; NAA stimulates cell elongation at concentrations that are much lower than those required to stimulate cell division. In contrast, 2,4-D promotes cell division but not cell elongation. Pertussis toxin, a blocker of heterotrimeric G-proteins, inhibits the stimulation of cell division by 2,4-D but does not affect cell elongation. Aluminum tetrafluoride, an activator of the G-proteins, can induce cell division at NAA concentrations that are not permissive for division and even in the absence of any exogenous auxin. The data are discussed in a model where the two different auxins activate two different pathways for the control of cell division and cell elongation.

Figure 1.

Response of the tobacco cell line VBI-0 to different concentrations of exogenous auxins. Cells were inoculated into fresh medium supplied with different concentrations of NAA and 2,4-D in a fixed ratio (1:1 w/w, respectively) and scored after 6 d of cultivation. A, Average number of cells per file. Each experiment was repeated at least 4 times, counting more than 10³ cell files/sample every time. The dashed line indicates the basal level of cell division observed in auxin-free medium. B, Morphological effects. The images are confocal sections of cells stained with rhodamin-G6-chloride, a fluorescent membrane dye. Size bars correspond to 50 μm. C, Average ratio of cell length to width as measure of cell elongation. More than 300 individual cell files from 3 independent experiments were viewed by confocal microscopy and images recorded for the central section of the cell.

Figure 2.

Differential effects of NAA and 2,4-D on division and elongation in VBI-0. Cells were cultivated either with both NAA and 2,4-D at 1:1 w/w ratio (auxins 10 μm) or with either 10 μm NAA or 10 μm 2,4-D alone. A, Average number of cells per file. Each experiment was repeated 8 times, counting more than 10³ cell files/sample every time. B, Morphological effects. C, Average ratio of cell length to width. For details refer to the legend to Figure 1.

Figure 3.

Dose response of cell division (A) and cell elongation (B) over different concentrations of either 2,4-D or NAA. Each data point represents the mean from 3 independent experiments comprising more than 10³ cell files/sample. The dashed line indicates the basal level of basal cell division in the absence of exogenous auxins. For comparison, the dose response curve for combined addition of NAA and 2,4-D in a 1:1 w/w ratio is shown as well.

Figure 4.

Response of auxin-dependent cell division and cell elongation to PTX and AlF₄⁻. Cells were cultivated for 5 d either in standard conditions (1:1 w/w ratio of NAA and 2,4-D for a total auxins concentration of 10 μm) or alternatively in 10 μm 2,4-D or NAA alone. Every 24 h, either 10 ng/mL PTX or 100 μm AlF₄⁻ was added. The average number of cells per file obtained from 3 independent experimental series comprising more than 10³ cell files/sample (A) and the average ratio cell length to width from 3 independent experiments with more than 300 individual cell files/sample (C) were measured. Representative images showing the morphological effects of the treatments are shown in B. For details refer to the legend of Figure 1.

Figure 5.

Expression of tobacco ABP1 genes in the VBI-0 cell line under different hormonal treatments. Total RNA from VBI-0 cells cultured in standard conditions (auxin 10 μm) or in medium supplied with 10 or 50 μm NAA or 2,4-D alone were used as template in a OneStep RT-PCR to detect the presence of transcript specific for the tobacco ABP1 genes NtERabp1, NtERabp2, and the transcription factor mybB as internal standard for loading of lanes. The amplificates were separated in 2% (w/v) agarose gel.

Figure 6.

Model of early auxin signaling to cell division and cell elongation in the tobacco cell line VBI-0. A receptor with high affinity for NAA (presumably ABP1) triggers cell elongation, a second unidentified receptor with high affinity for 2,4-D (Rx) activates cell division through a trimeric G-protein. Both pathways are connected by a negative crosstalk (dashed lines 1 and 2). DP stays for a generic docking protein activated by G-proteins.