Article Figures & Data
Figures
- Figure 1.
Analysis of AtPGP1 Expression in Wild-Type and Transgenic AS and OE Plants.
(A) Autoradiography of an RNA gel blot probed with a double-stranded 32P-labeled probe. Each lane contains 8 μg of total RNA. The expected lengths of AtPGP1 and antisense transcripts are 4.5 and 1.9 kb, respectively. The lengths in kilobases of marker fragments are indicated at right. WT, wild type.
(B) Ethidium bromide–stained gel before blotting.
(C) and (D) Gel blot analysis of membrane proteins extracted from OE, AS, and wild-type plants. Ten and 100 μg of microsomal proteins were loaded per lane on the gels corresponding to the blots shown in (C) and (D), respectively. The lane labeled AS2/OE3 in (D) contained 100 μg of AS2 and 2 μg of OE3 microsomal proteins. The blots were probed with an AtPGP1-specific rabbit antiserum and processed with the ECL chemiluminescent detection system. The sizes of the marker proteins are given at right in kilodaltons.
(E) Relative intensities of the upper band (corresponding to AtPGP1) to the lower band (serving as an internal standard) in corresponding lanes in (D). The histogram represents the percentage values of this ratio relative to the wild type.
- Figure 2.
Analysis of the Hypocotyl Growth Rate of Wild-Type and Transgenic Plants.
The plants were grown under long-day conditions (18-hr photoperiod at 24°C) under white light at a fluence rate of 75 μmol m−2 sec−1. Each data point represents the average length of 10 to 25 plants. For the wild type (WT), 20 to 35 hypocotyls were measured per day. The error bars indicate the standard error of the mean.
- Figure 3.
Number and Length of Hypocotyl Cells in 7-Day-Old Wild-Type, AS, and OE Plants Grown at a Light Fluence Rate of 52 μmol m−2 sec−1.
(A) Scanning electron microscopy of hypocotyls.
(B) Number of epidermal cells per file in hypocotyls. Eight hypocotyls from each of two AS lines, two OE lines, and the wild type were scored. The error bars indicate the standard error of the mean. WT, wild type.
- Figure 4.
Influence of Light Quality on Hypocotyl Length in Wild-Type, AS, and OE Plants.
(A) Hypocotyl lengths of etiolated seedlings. The data represent the averages of three independent measurements conducted after 5, 7, and 8 days of growth in complete darkness. Hypocotyls already had reached maximal length after 5 days. A total of 142 AS and 190 OE seedlings of all transgenic lines and 102 wild-type seedlings were scored. The differences between OE, AS, and wild-type plants are statistically not significant at the 95% confidence level.
(B) to (D) Seedlings of two AS lines, two OE lines, and the wild type were grown in light of different qualities, and hypocotyls were measured at two different time points that yielded identical results, thus ensuring full elongation. In (B), 40 seedlings per line were measured after 19 days of growth in red light at a fluence rate of 25 μmol m−2 sec−1. In (C), 20 seedlings per line were scored after 14 days in blue light at a fluence rate of 15 μmol m−2 sec−1. In (D), 18-day-old seedlings were grown under far-red light at a fluence rate of 2 μmol m−2 sec−1. Twenty seedlings per line were scored.
Error bars indicate the standard error of the mean. WT, wild type.
- Figure 5.
Influence of Light Quantity on Hypocotyl Length in Wild-Type, AS, and OE Plants.
Seedlings were grown under white light at different light fluence rates, and hypocotyls were measured at two different time points to ensure full elongation. The star on the vertical axis indicates the average hypocotyl length of etiolated seedlings (taken from the data presented in Figure 4). Data points represent measurements of 30 to 40 seedlings of the wild type (WT) and each of three AS and two OE lines, respectively. Error bars indicate the standard error of the mean.
- Figure 6.
Root Length of Wild-Type, AS, and OE Seedlings.
Seedlings were grown on soil under white light at a fluence rate of 48 μmol m−2 sec−1. Roots of two AS lines (30 seedlings), two OE lines (55 seedlings), and the wild type (WT; 19 seedlings) were scored after 12 days. Roots of OE plants were significantly different from those of AS and wild-type plants, whereas the difference between AS and wild-type roots was not statistically significant at the 95% confidence level. Error bars indicate the standard error of the mean.
- Figure 7.
Localization of AtPGP1 Gene Expression and of AtPGP1 Protein.
(A) to (D) Histochemical localization of GUS activity in transgenic plants harboring an AtPGP1–GUS reporter gene. In (A) and (B), 7-day-old seedlings were photographed with two different background colors. In (C) and (D), the shoot and root apex, respectively, of a 5-day-old seedling are shown. In (A) and (B), bars = 1 mm; in (C) and (D), bars = 100 μm.
(E) to (N) Detection of AtPGP1-specific RNA by in situ hybridization in wild-type seedlings. In (E) to (L), longitudinal sections through seeds 12 hr after transfer from vernalization to growth conditions ([E] and [F]) and through seedlings 12 hr ([G] and [H]), 24 hr ([I] and [J]), and 5 days ([K] and [L]) after germination were hybridized with a digoxigenin-labeled AtPGP1-specific antisense RNA probe ([E], [G], and [I]) or with a sense RNA probe as a control ([F], [H], and [J]). In (M), a whole mount of a root of a 5-day-old seedling was hybridized with a digoxigenin-labeled antisense RNA probe. In (N), a section similar to the one shown in (M) was hybridized with a sense RNA probe as a control. Arrows indicate the shoot apex. Bars = 100 μm.
(O) to (R) Immunocytochemical localization of c-Myc epitope–tagged AtPGP1 in transgenic plants. False-color confocal laser scanning microscopy was performed with leaf sections probed with the c-Myc–specific monoclonal antibody 9E10. Detection was achieved with DTAF-conjugated secondary antibody (shown in green). The autofluorescence of chloroplasts is shown in red, whereas the purple color in (Q) and (R) indicates calcofluor white staining of cell walls. (O) and (Q) are transverse sections through cotyledon of a transgenic seedling carrying an ectopically expressed gene encoding c-Myc epitope–tagged AtPGP1. The arrow in (O) indicates the cuticle. (P) and (R) are control sections of wild-type seedlings treated exactly as the sections shown in (O) and (Q). In (O) and (P), bars = 20 μm; in (Q) and (R), bars = 10 μm.
co, cotyledon; hy, hypocotyl.
- Figure 8.
Protein Gel Blot Analysis of Membrane Vesicles Fractionated on Density Gradients.
(A) Membrane vesicles of wild-type Arabidopsis plants and transgenic plants ectopically expressing c-Myc epitope–tagged AtPGP1 were separated simultaneously on continuous sucrose density gradients. Gradients were fractionated, and appropriate aliquots of equal volume were separated on SDS–polyacrylamide gels and blotted. Shown are blots of wild-type samples of a single gradient probed with the AtPGP1-specific polyclonal antiserum or specific antisera to the tonoplast protein VM23, the endoplasmic reticulum protein BiP, and the plasmalemma protein PIP. The blot shown at bottom was prepared with samples derived from transgenic plants ectopically synthesizing c-Myc epitope–tagged AtPGP1 and was probed with the c-Myc epitope–specific monoclonal antibody 9E10. This gradient was run in conjunction with the one containing the wild-type samples, and the distribution of the marker proteins was identical in both gradients (data not shown). Numbers at left indicate the sizes in kilodaltons of marker proteins.
(B) Sucrose concentration of gradient fractions as measured with a refractometer.
