Article Figures & Data
Figures
- Fig. 1.
POR mRNA and POR protein amounts in etiolated Arabidopsis POR-antisense seedlings. A, PORA andPORB mRNA levels in transgenic PAA and PBA lines. Gel blots were prepared with 5 μg of total RNA from 4-d-old seedlings and were probed for the mRNAs indicated at the left. ACT mRNA provides a control for the sample loading. Numbers at the bottom represent the ACT-corrected amounts of the relevant mRNAs from transgenic seedlings, relative to the wild-type (Wt) control. B, Total POR protein levels. A gel blot was prepared using 5 μg of total protein from extracts of 4-d-old seedlings that, independent of genotype, contained similar amounts of total protein on a per seedling basis. Numbers at the bottom represent the amounts of immunoreactive POR protein detected relative to the wild-type control.
- Fig. 2.
Etioplast ultrastructure in POR-antisense and POR-overexpressing seedlings. A, Wild type. B, Line PBO-10 (strong PORB overexpression). C, Line PAA-28 (weak POR antisense) with PLB. D, Line PAA-28 without PLB. E, Line PBA-24 (strong POR antisense) with PLB. F, Line PBA-24 without PLB. Membrane ultrastructure of cotyledon plastids from 4-d-old seedlings was examined by transmission electron microscopy. Magnifications were 20,000× for A and B; 25,000× for C and D;, 15,000× for E; and 12,000× for F. Bars in A through F = 1 μm.
- Fig. 3.
Fluorescence emission spectra of Pchl(ide) pigment forms present in situ in etiolated POR-antisense and POR-overexpressing seedlings. A, Wild type. B, Line PAA-28 (weak POR antisense). C, Line PBA-24 (strong POR antisense). D, Line PBO-10 (strong PORB overexpression). In situ low temperature fluorescence spectra of unilluminated (solid curves) and flash-illuminated (dashed curves) cotyledons of 4-d-old seedlings. Emission bands observed with an excitation wavelength of 440 nm include non-photoactive Pchl(ide)-F632, photoactive Pchlide-F655 arising from the aggregated Pchlide:NADPH:POR ternary complex, and, after flash treatment applied at −20°C, Chlide-F690. The broad pre-flash fluorescence emissions centered at about 693 nm in PBA-24 cotyledons (C) and roughly 720 nm in PBO-10 cotyledons (D) represent vibrational sublevels of Pchl(ide)-F632 and Pchlide-F655, respectively (Böddi et al., 1992).
- Fig. 4.
Quantitative determination of Pchl(ide) pigment forms present in etiolated seedlings. A, Total Pchl(ide). B, Photoactive Pchlide. C, Ratio of photoactive Pchlide-to-non-photoactive Pchl(ide). Room temperature fluorescence emission measurements of the acetone-extracted total pigments from unilluminated and flash-illuminated cotyledons of 4-d-old wild-type, POR-antisense, and POR-overexpressing seedlings collected at an excitation wavelength of 435 nm. Total Pchl(ide) was calculated from the pre-flash fluorescence emission band at 634 nm. Photoactive Pchlide-F655 was determined based on the post-flash emission at 672 nm that results from its quantitative POR-mediated reduction to Chlide. Non-photoactive Pchl(ide)-F632 was set equal to the difference between these two quantities. Pigment amounts are given on a per cotyledon pair basis. Seedling genotypes are given from left to right in the order of increasing total POR content (see Fig. 5).
- Fig. 5.
Quantitative correlation analysis of the amounts of total POR protein and photoactive Pchlide in etiolated seedlings. The ratio of total POR protein in seedlings-to-photoactive Pchlide in cotyledons calculated for 4-d-old seedlings of each transgenic genotype relative to the wild type. A best-fit trendline through the data points was calculated and its correlation coefficient determined.
- Fig. 6.
Fluorescence excitation spectra from the Soret region of Pchl(ide) pigment forms present in situ in etiolated seedlings. A, Photoactive Pchlide-F655. B, Non-photoactive Pchl(ide)-F632. In situ low temperature fluorescence spectra normalized at their respective maxima from cotyledons of 4-d-old wild-type, POR-antisense, and POR-overexpressing seedlings. Seedling genotypes are given and the wavelengths positions of the maxima and a shoulder are indicated.
- Fig. 7.
Relative efficiency of the excitation of photoactive Pchlide-F655 by 465 nm light in etiolated seedlings. Calculation of the ratio of the in situ low temperature fluorescence emissions at 655 nm in cotyledons of 4-d-old wild-type and transgenic seedlings upon excitation at 465 or 440 nm. Seedling genotypes are arranged left to right as POR-antisense, wild type, and PORA- or PORB-overexpressing. Error bars indicate the standard deviations.
- Fig. 8.
Quantitative determination of the distribution of DV- and MV-Pchl(ide) within the non-photoactive and photoactive pigment fractions of etiolated seedlings. Fluorimetric analysis of the total Pchl(ide) organically extracted from cotyledons of 4-d-old etiolated wild-type, POR-antisense, and POR-overexpressing seedlings. A, Room temperature fluorescence excitation spectra of the Pchl(ide) emission band at 634 nm in acetone-extracted total pigments from unilluminated (solid curves) and flash-illuminated (dashed curves) cotyledons of wild-type seedlings. The spectra were normalized at their respective maxima (434 nm), but have been slightly vertically offset to facilitate their presentation. B, Room temperature fluorescence excitation spectra of the Pchl(ide) emission band at 634 nm in acetone-extracted total pigments from etiolated cotyledons of wild-type, POR-antisense, and POR-overexpressing seedlings. The spectra were normalized at their respective maxima. Seedlings genotypes are given and wavelength positions of the maxima are indicated. C, Low temperature fluorescence excitation spectra of the Pchl(ide) emission band at 625 nm in acetone-extracted total pigments transferred to diethyl ether. The spectra were collected from extracts of etiolated cotyledons of wild-type, POR-antisense, and POR-overexpressing seedlings and were normalized at their respective maxima. Seedling genotypes are given and the wavelength positions of the emission maxima and a shoulder are indicated. D, Determination of the relative amounts of DV- and MV-Pchl(ide) within the photoactive and non-photoactive pigment fractions of the total Pchl(ide) extracted from etiolated cotyledons of wild-type and transgenic seedlings. Pigment amounts are given relative to the wild type for which total Pchl(ide) has been defined as 100%. The respective DV-to-MV pigment ratios for the total Pchl(ide), photoactive Pchlide, and non-photoactive Pchl(ide) fractions of seedlings of each genotype are given below. Seedling genotypes are arranged left to right as POR antisense, wild type, and PORA or PORB overexpressing.
- Fig. 9.
Fluorescence excitation spectra from the red region of photoactive Pchlide-F655 present in situ in etiolated seedlings. A, In situ low temperature excitation spectra of the fluorescence emission at 730 nm, which corresponds to a vibrational sublevel of Pchlide-F655 (Böddi et al., 1992), were collected from cotyledons of 4-d-old wild-type and transgenic seedlings. Spectra were normalized at their respective maxima. B, Gaussian deconvolution of the wild-type excitation spectrum (inset, the difference between the experimental spectrum and the sum of the G628, G640, and G652 gaussian components). C, Relative amplitudes of the G628 and G640 gaussian components (the amplitude of G652 has been defined as 1 for each genotype). Error bars indicate the standard deviations. Seedling genotypes are arranged left to right as POR antisense, wild type, and PORA or PORB overexpressing.
- Fig. 10.
Estimation of the ratio of non-photoactive Pchl(ide)-to-photoactive Pchlide molecules within an energy transfer unit. The ratio of non-photoactive Pchl(ide)-to-photoactive Pchlide obtained from pigment quantitations was plotted against the ratio of the in situ low temperature fluorescence emission bands at 632 and 655 nm for cotyledons of 4-d-old transgenic and wild-type seedlings. A best-fit trendline through the data points was calculated and its correlation coefficient was determined. Error bars indicate thesds.
- Fig. 11.
Photoactive Pchlide reduction kinetics upon illumination of etiolated seedlings. A, Time courses of the in situ Chlide fluorescence increase in cotyledons of 4-d-old wild-type and transgenic seedlings exposed to 650 nm light at room temperature. B, Photoactive Pchlide reduction halftimes determined from the rate of the Chlide fluorescence increase. Error bars indicate the standard deviations. Seedling genotypes are arranged left to right as POR antisense, wild type, and PORA- or PORB-overexpressing.
