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Auxin-Dependent Cell Expansion Mediated by Overexpressed Auxin-Binding Protein 1

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Science  06 Nov 1998:
Vol. 282, Issue 5391, pp. 1114-1117
DOI: 10.1126/science.282.5391.1114

Abstract

To test the hypothesis that auxin-binding protein 1 (ABP1) is a receptor controlling auxin-mediated plant cell expansion,ABP1 complementary DNAs were expressed in a controllable fashion in tobacco plants and constitutively in maize cell lines. Induction of Arabidopsis ABP1 expression in tobacco leaf strips resulted in an increased capacity for auxin-mediated cell expansion, whereas induction of ABP1 in intact plants resulted in leaves with a normal morphology, but larger cells. Similarly, constitutive expression of maize ABP1 in maize cell lines conferred on them the capacity to respond to auxin by increasing cell size. These results support a role of ABP1 as an auxin receptor controlling plant growth.

Auxins are plant growth hormones that cause rapid increases in plant cell wall extensibility, alter ion flux at the plasma membrane, and cause specific changes in gene expression (1, 2). A receptor mediating these effects has not been unequivocally identified, although ABP1 is a leading candidate for at least the plasma membrane and wall effects (1). ABP1 has been carefully characterized with regard to its auxin-binding properties (3). Moreover, it has been shown to fit the criteria of a receptor mediating auxin-induced cell expansion, on the basis of its expected tissue distribution, its plasma membrane location, and on results from experiments designed to antagonize or mimic auxin action using antibodies to ABP1 and an ABP1 peptide mimetic (4, 5). These results, while consistent with ABP1 receptor function, are nonetheless indirect. Therefore, we took a molecular genetic approach by examining the effect of inducible overexpression of ABP1 in plants and constitutive expression in cell lines.

Tobacco plants expressing the tetracycline repressor were transformed with the full-length, Arabidopsis ABP1 cDNA placed under the control of a tetracycline promoter (6). The inducible expression of ABP1 in these plants is shown in Fig. 1A. One transformant designated MJ10B was used to further characterize the induction kinetics and dose dependence of induction (Fig. 1, B and C). Steady-state transcript is detectable in uninduced transformants and is shown to greatly increase within 6 hours after application of anhydrotetracycline (AhTet). Arabidopsis ABP1 protein (7) is detected by immunoblot analysis in uninduced MJ10B transformants (homozygous for ABP transgene) and increases significantly during the 48 hours after induction (Fig. 1D).

Figure 1

Anhydrotetracycline-inducible expression of ABP1 in tobacco. (A) Northern blot analysis of Arabidopsis ABP1 transcript level in total RNA (15 μg) isolated from all leaves of 40-day-old (3 to 4 leaf stage) control plants (R7, expressing the tetracycline repressor) and four independently transformed tobacco plants (MJ10B, MJ10D, MJ10Z, MJ10Y) expressing both the tetracycline repressor and the tet-inducible Arabidopsis ABP1 construct. Plants were hydroponically fed 12 μg/ml of the nontoxic tetracycline analog AhTet via the roots for 48 hours (+) or left untreated (–) on the lab bench under 10/14 light/dark cycles at 25°C. (B) Northern blot analysis of the Arabidopsis ABP1 transcript level in total RNA from R7 and MJ10B tobacco plants after the indicated number of hours in the presence of 12 μg/ml of AhTet. (C) Northern blot analysis of the Arabidopsis ABP1 transcript level in total RNA from R7 and MJ10B tobacco plants after 48 hours treatment with AhTet at the indicated concentrations (micrograms per milliliter). (D) Protein immunoblot analysis of ArabidopsisABP1 in crude microsomal fractions from R7 and MJ10B tobacco plants after the indicated hours of treatment with 4 μg/ml of AhTet. MJ10B plants are homozygous for the Arabidopsis ABP1 transgene. Polyclonal antisera directed against recombinant ArabidopsisABP1 were used. Note that this serum does not recognize tobacco ABP1 (lane R7, 48 hours). Band “b” represents the ArabidopsisABP1. The nonspecific recognition of band “a” shows equal loading between the samples.

Having established the transgene induction kinetics and expression characteristics, we began our analysis of the physiological effect of ectopic expression of ABP1 in leaves, because previous work found that ABP1 mediates auxin-induced hyperpolarization in mesophyll protoplasts. The capacity of leaf cells to respond to auxin is developmentally acquired (8). Cells in the tips of young leaves respond to auxin by ethylene-independent expansion, whereas cells in the base of the lamina do not acquire this capacity until much later as the basal lamina expands. We therefore chose to examine the effect of controlled expression of ABP1 in cells of the basal region of the lamina at a time point when they are not normally auxin responsive. Interveinal lamina tissue taken from the basal region of ABP1 transformants exhibited AhTet-inducible epinastic growth (reported as leaf curvature) that is significantly higher than the basal growth of control tissues from R7 leaves (Fig. 2). This inducible growth is strictly dependent on the presence of auxin, as expected for any receptor mediating the ligand response. The structurally similar nonauxin, 2-naphthaleneacetic acid (2-NAA), was ineffective, indicating structural specificity is required for inducible growth. The increased auxin-regulated growth in ABP1 transgenic plants without induction with AhTet (Fig. 2A; MJ10B and MJ10Y, gray bars) is most likely due to leaky basal expression of ABP1 (Fig. 1D, lane 0). The consistency of the effect of ABP1 overexpression is seen in Fig. 2B, which shows AhTet-inducible growth in an additional five independent transformants.

Figure 2

AhTet-inducible, auxin-dependent, epinastic growth of leaf tissue. (A) Strips of interveinal leaf tissue taken from the lamina base of primary ABP1 transformants (MJ series) were floated on solutions containing 4 μg/ml AhTet plus 10 μM 1-NAA (solid bars), AhTet alone (open bars), NAA alone (gray bars), or buffer (striped bars). Epinastic growth (Degree curvature) was measured as described (8). R7 tobacco plants express only the tetracycline repressor (6). MJ10B and MJ10Y plants are two transformants homozygous for both the tetracycline repressor and the ABP1 transgene. Several different primary ABP1transformants (hemizygous) indicated by letter, as well as the R7 control, are shown in (B), where growth is expressed as the difference between degree curvature in the presence of AhTet plus 1-NAA versus 1-NAA alone, the difference between the solid and gray bars in (A). (C) Strips of interveinal leaf tissue were taken from the indicated position on the leaf designated tip, mid, and base. (D) Leaf strips removed at the indicated positions from leaves of MJ10B plants homozygous for the ABP1transgene (open bars) and from R7 control (solid bars) plants were treated with 4 μg/ml AhTet plus 10 μM NAA. (E) Cross sections of R7 and MJ10B leaf strips were prepared from the midsection after growth shown in (D) was recorded.

To determine if overexpression of ABP1 alters the spatial pattern of auxin-regulated growth in young leaves described above, we measured growth of the leaf at different positions within lamina of control and ABP1-overexpressing leaves (Fig. 2C). Auxininduced growth of the control leaf occurs in the tip only, whereas AhTet-treated MJ10B leaves exhibit growth at all three positions of the young leaf (Fig. 2D). This effect was dependent on induction of ABP1 expression with AhTet. Cross sections of the strips show that this increase in growth is due to increased cell expansion, most notably by the palisade mesophyll cells (Fig. 2E).

Plants transformed with the ABP1 transgene did not have an altered phenotype when treated with AhTet (Fig. 3, A and B). Furthermore, the rate of expansion of leaves on plants treated with AhTet did not differ from the control R7, AhTet-treated plants. However, despite normal morphology, cells from mature leaves of MJ10B plants treated with AhTet are larger. Plants were fed 4 μg/ml AhTet via roots for a period greater than a plastochron, then protoplasts of interveinal cells located in the midsection of the youngest, fully expanded leaves were prepared and analyzed (9). The distribution of protoplast volumes of cells from control plants and MJ10B plants not treated with AhTet were similar. In contrast, the protoplast volume of cells in AhTet-treated MJ10B plants was greater (Fig. 3), evident both in a shift toward the largest class of protoplasts and the average protoplast volume. These results indicate that ABP1 mediates auxin-dependent cell expansion in the intact plant. Furthermore, these results are consistent with the intriguing observation that the basic unit of morphology is not the cell, because leaves having the same morphology and size can be produced by fewer but larger cells (10).

Figure 3

Protoplast volumes of cells of leaves from control and tobacco plants inducibly expressing theArabidopsis ABP1 transgene. (A) MJ10B plants grown in the absence of AhTet; (B) MJ10B plants fed 4 μg/ml AhTet via the roots for 13 days. Duplicate interveinal tissue disks were punched from the youngest, fully expanded leaves at the positions indicated [inset to (C)] and digested to completion. (C) The protoplast volumes were determined and grouped into size classes. The smallest class of protoplasts (class “a”) had a diameter range of 16 to 19 μm. Each subsequent class, indicated by letters “b” through “n”, increase by 3-μm increments, ending at the largest class (“n”), which has a diameter range of 56 to 59 μm. (D) The average volumes of protoplasts (in cubic micrometers) were calculated for the four treatments (n = sample size). Symbols correspond to data in (C).

Successful studies on receptor function in animal cells have used ectopic expression of the receptor in cell lines that otherwise do not express the receptor. In such an approach, the expressed receptor often confers to the cells a hormone-mediated response. Therefore, as a second test of the hypothesis that ABP1 is a functional auxin receptor, we ectopically expressed maize ABP1 in a clonal maize endosperm cell line that does not contain detectable levels of ABP1 (Fig. 4) and does not have a strict requirement for auxin to proliferate. This enabled us to examine the effect of ABP1 on cell expansion without the complication of high endogenous ABP1 levels; by this approach, we have shown that ectopic expression of ABP1 confers on these cells an auxin-dependent response.

Figure 4

ABP1 expression and growth of transformed maize cells. Immunoblot analysis of crude extracts of equal fresh weight amount of maize cells transformed with CaMV35S:ABP1 and CaMV 35S:Bar (Cell lines F652 and F631) compared to a control line transformed with35S:Bar alone (C101). Immunoblots were probed with a polyclonal serum (NC13) directed against the full-length maize ABP1 expressed recombinantly in Escherichia coli (left panel) or monoclonal antibodies (right panel) directed against the purified maize ABP1 [pooled MAC 256, 257, 259, 260; (14)]. S, standards.

Maize cells were cotransformed with a CaMV 35S:maizeABP1 construct and the bar gene (11). Immunoblot analysis using several different antibodies to ABP1 (12) showed that cell lines F652 and F631 overexpressABP1 (Fig. 4), whereas ABP1 in control cells (C101) was not detectable.

F652 and F631 cells grown in the presence of auxin and expressing detectable levels of ABP1 are significantly larger than the C101 control cells of the same age (Table 1). Ploidy differences between F652, F631, and C101 cannot account for the observed size differences (13). The increased cell size of the ABP1-overexpressing lines (designated cell size class “c”) is not neomorphic, because this phenotype is observed in the control cells, albeit at low frequency (Fig. 5A). This means that these clonal endosperm cells are competent to expand to the class “c” cell size, but only do so infrequently. In contrast, F652 and F631 lines are predominantly larger class “c” cells, suggesting that ABP1, through its normal action, shifts the growth capacity of endosperm cells toward the formation of larger cells.

Figure 5

Auxin-dependent phenotype conferred on cells overexpressing ABP1. (A) Frequency of cell size classes “a” (open bars; approximate diameter, 20 μm), “b” (hatched bars; approximate diameter, 30 μm), and “c” (solid bars; approximate diameter, 50 μm) in two control cultures, C101 and C403, and two ABP1-overexpressing lines, F652 and F631. These cells were cultured in the presence of 45 μM 2,4-dichlorophenoxyacetic acid (2,4-D). (B) Cell size class frequency of control (C101) and ABP1-overexpressing (F652) cells cultured 4 weeks in the absence of auxin (noted 0 μM) and in the presence of 2,4-D (90 μM) for C101 and F652 cells. Numbers above the bars indicate the cell sample size.

Table 1

Maize cells overexpressing ABP1 are larger and contain more cell wall material.

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The most important observation supporting auxin receptor function is that expression of ABP1 in maize endosperm cells confers on these cells an auxin-dependent response. Figure 5B illustrates that while auxin had no effect on cell size in the control cell line (cell size classes “b+c”), the large cell size of cells overexpressingABP1 was strongly auxin-dependent. When auxin was removed from the medium, the frequency of cell size class “a” (Fig. 5B) approximated that seen in control lines with or without auxin in the medium. At auxin concentrations twice the normal culture levels, the frequency of cell types “b+c” increased, indicating dose dependency.

Our data demonstrate that an auxin-inducible growth phenotype is conferred on tobacco cells in planta by the AhTet-inducibleABP1 construct, and in cultured maize cells by expression of a 35S:ABP1 construct. This molecular genetic evidence, taken together with the extensive characterization of ABP1 auxin-binding properties, unequivocally demonstrates that ABP1 is an auxin receptor that controls cell expansion. Considering the complexity of auxin action even in a single response such as cell expansion and considering the evidence supporting multiple auxin pathways, it is likely that additional auxin receptors yet to be identified are involved in auxin perception and cell expansion.

  • * To whom correspondence should be addressed. E-mail: alan_jones{at}unc.edu

  • Present address: Department of Biology, University of West Florida, Pensacola, FL 32514, USA.

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