Figure 4.

Transcriptional analysis of cell type-specific IAA7mII and IAA19mII expression. A, A schematic representation of the experiment. Protoplasts derived from the roots of 1-week-old P_WER∷GFP seedlings were transfected with either pMON999_mRFP, expressing only mRFP, or pBeaconRFP, expressing IAA7mII or IAA19mII. After an overnight incubation, protoplast suspensions were treated for 3 h with 5 μm IAA or solvent alone. Dual-labeled protoplasts were isolated by FACS and used for microarray analysis. B, Microscopic examination of protoplasts derived from the roots of 1-week-old P_WER∷GFP seedlings that were transfected with pMON999_mRFP. Bar = 50 μm. C, FACS of the transfected protoplast suspensions. Dot plots are shown depicting the controls used to set up the gates: an untransfected protoplast suspension derived from wild-type roots (blank), an untransfected protoplast suspension derived from P_WER∷GFP roots, and a protoplast suspension derived from wild-type roots transfected with pMON999_mRFP. In addition, a dot plot depicting a protoplast suspension derived from P_WER∷GFP roots transfected with pMON999_mRFP is shown; protoplasts falling within the gate marked “double” were sorted and used for microarray analysis. A total of 100,000 events are displayed in each dot plot. D, Transcriptional analysis of sorted protoplasts. A log-scale heat map and a histogram quantifying the differences in gene expression between the various collected protoplasts are shown. The heat map displays all of the genes that exhibit any significant difference between mock and auxin treatment, between transformation with the different vectors, and by interaction level (see “Materials and Methods”) as measured by microarray analysis; rows represent genes and columns represent treatment and transformation vectors (n = 3). The histogram presents the difference in GH3.5 expression (as measured by microarray) between the various collected protoplasts ± sd (n = 3).