Figure 6.

 Mutations in the Auxin Binding Site Stabilize SKP2A and DPB and Affect Cell Division.

(A) Five-day-old PER8:SKP2A-GUS, PER8:SKP2Amut2-GUS, and the control PER8:GFP-GUS transgenic seedlings were transferred to MS plates containing 15 μM estradiol to induce the expression of the proteins for 16 h. After induction, these seedlings were stained for GUS activity (tp = 0) or incubated during 5 h in a MS medium (5 h MS) or MS containing 5 × 10⁻⁷ M 2,4-D (5 h+Aux) and then stained for GUS activity. Bar = 500 μm.

(B) Higher magnification of PER8:SKP2A-GUS, PER8:SKP2Amut2-GUS, and the control PER8:GFP-GUS root tip shown in (A). Bar = 500 μm.

(C) Expression of the SKP2A-GUS and SKP2Amut2-GUS transgenes analyzed by RT-PCR using primers from SKP2A and GUS coding regions. As a control, the expression of the ACTIN1 (ACT) gene was analyzed.

(D) Immunoblotting analyses of the E2FC or MYC-DPB levels into the skp2a/MYC-DPB plants. Total protein was extracted from wild-type, MYC-DPB^OE, skp2a/MYC-DPB^OE, or skp2a/MYC-DPB^OE seedlings that overexpress HA-SKP2A, HA-SKP2Amut1 (two independent lines), or HA-SKP2mut2 (two independent lines) and immunoblotted (I-blot) with anti-E2FC or anti-MYC. LC is the loading control corresponding to the Ponceau-stained blot. Ø indicates skp2a/MYC-DPB^OE plants that overexpress the HA tag alone. The expression levels of both E2FC and MYC-DPB genes were analyzed by RT-PCR. As a control, the ACTIN gene levels were analyzed.

(E) Root meristem cell number of control MYC-GFP, MYC-SKP2A^OE, or MYC-SKP2Amut2^OE plants grown in MS medium. For monitoring root meristem growth, cortex meristematic cells were counted at the indicated days. Error bars represent se (n = 15). Asterisks indicate statistically significant difference compared with wild-type meristems as determined by Student´s t test (*P < 0.02 and **P < 0.001, respectively).

[See online article for color version of this figure.]