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The effects of high concentrations of Hg (2+) (10 (-2) M and 10 (-3) M) were investigated on the ultrastructure and on the light-induced transformation of isolated prolamellar bodies (PLBs) of dark-grown wheat leaves. Our earlier work on wheat leaf homogenates ( , Plant Biology 6, 358 - 368) showed that, depending on the concentration, Hg (2+) reacts with protochlorophyllide, NADPH and the NADPH : protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme and induces disaggregation of the macrodomain structure of this latter. Spectroscopic analyses confirmed that 15 min incubation with 10 (-2) M Hg (2+) at 4 degrees Celsius completely inhibited the activity of POR also in isolated PLBs. Ultrastructural investigations revealed the loosening of the PLB structure in the Hg (2+)-treated sample, i.e., intensive vesicle formation on the surface of the PLB membranes. The hexagonal geometry of the inner lattice was not disturbed, however, the unit cell size significantly increased. The disruption of the PLB membranes upon irradiation was studied after 40 min incubation with 10 (-3) M Hg (2+) at 4 degrees Celsius and a subsequent irradiation for 40 min at 20 degrees Celsius. Equimolar concentrations (10 (-3) M) of NADPH and Hg (2+) were added to the samples 10 min prior or after the addition of Hg (2+). Our results suggest that Hg (2+) accelerates the disruption of the PLB membranes and that NADPH can only partially prevent this process. These membrane transformations were similar to those observed in the initial steps of the Shibata shift of control samples.

The molecular background of Hg (2+)-induced inhibition of protochlorophyllide (Pchlide) photoreduction was investigated in homogenates of dark-grown wheat leaves. Our earlier work showed that 15 min incubation with 10 (-2) M Hg (2+) completely inhibits the activity of NADPH : Pchlide oxidoreductase ( ). Detailed analysis of spectra recorded at 10 K indicated the appearance of emission bands at 638 and 650 nm, which are characteristic for NADP (+)-Pchlide complexes. Fluorescence emission spectra recorded with different excitation wavelengths, fluorescence lifetime measurements and the analysis of acetone extractions revealed that Hg (2+) can also react directly with Pchlide, resulting in protopheophorbide formation. At 10 (-3) M Hg (2+), the phototransformation was complete but the blue shift of the chlorophyllide emission band speeded up remarkably. This indicates oxidation of the NADPH molecules that have a structural role in keeping together the etioplast inner membrane components. We suggest a complex model for the Hg (2+) effect: depending on concentration it can react with any components of the NADPH : Pchlide oxidoreductase macrodomains.

Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide(650-656) form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3-0.8 ns and a slow component in the range 5.1-7.1 ns were present in all samples, but the values varied, depending on species. The long-wavelength Pchlide(650-656) form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short-wavelength Pchlide(628-633) form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein-bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.