Formation of long-wavelength chlorophyllide (Chlide695) is required for the assembly of Photosystem II in etiolated barley leaves

Chlorophyll(ide) spectroscopic properties and Photosystem II assembly, monitored by 77 K variable fluorescence, were studied in etiolated barley leaves as a function of the extent of protochlorophyllide photoreduction by a single millisecond light flash of different intensities. Variable fluorescence, measured 2 hours after the flash, was only detected when the extent of phototransformation was higher than a threshold value of 0.4. Its development paralleled the formation of a chlorophyll emission component at 685 nm, which itself derived from long-wavelength chlorophyllide with an emission maximum at 695 nm. At low flash intensities, short-wavelength chlorophyllide forms preferentially accumulated and no Photosystem II fluorescence was detected after 2 hours. Chlorophyllide esterification was independent of the extent of phototransformation. These results suggested that the formation of long-wavelength chlorophyllide was essential for further assembly of Photosystem II. This interpretation was strengthened by the observed inhibition of both long-wavelength chlorophyllide formation and of variable fluorescence development in leaves treated with 6-aminolevulinic acid or in untreated leaves subjected to repeated flashes of low intensity.