Global spectral–kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley

This study presents a novel measurement, and simulation, of the time–resolved room temperature chlorophyll a fluorescence emission spectra from leaves of the barley wild–type and chlorophyll– b –deficient chlorina ( clo ) f2 and f104 mutants. The primary data were collected with a streak–camera–based picosecond–pulsed fluorometer that simultaneously records the spectral distribution and time dependence of the fluorescence decay. A new global spectral–kinetic analysis programme method, termed the double convolution integral (DCI) method, was developed to convolve the exciting laser pulse shape with a multimodal–distributed decay profile function that is again convolved with the spectral emission band amplitude functions. We report several key results obtained by the simultaneous spectral–kinetic acquisition and DCI methods. First, under conditions of dark–level fluorescence, when photosystem II (PS II) photochemistry is at a maximum at room temperature, both the clo f2 and clo f104 mutants exhibit very similar PS II spectral–decay contours as the wild–type ( wt ), with the main band centred around 685 nm. Second, dark–level fluorescence is strongly influenced beyond 700 nm by broad emission bands from PS I, and its associated antennae proteins, which exhibit much more rapid decay kinetics and strong integrated amplitudes. In particular a 705–720 nm band is present in all three samples, with a 710nm band predominating in the clo f2 leaves. When the PS II photochemistry becomes inhibited, maximizing the fluorescence yield, both the clo f104 mutant and the wt exhibit lifetime increases for their major distribution modes from the minimal 250–500 ps range to the maximal 1500–2500 ps range for both the 685 nm and 740 nm bands. The clo f2 mutant, however, exhibits several unique spectral–kinetic properties, attributed to its unique PS I antennae and thylakoid structure, indicating changes in both PS II fluorescence reabsorption and PS II to PS I energy transfer pathways compared to the wt and clo f104 . Photoprotective energy dissipation mediated by the xanthophyll cycle pigments and the PsbS protein was uninhibited in the clo f104 mutant but, as commonly reported in the literature, significantly inhibited in the clo f2 ; the inhibited energy dissipation is partly attributed to its thylakoid structure and PS II to PS I energy transfer properties. It is concluded that it is imperative with steady–state fluorometers, especially for in vivo studies of PS II efficiency or photoprotective energy dissipation, to quantify the influence of the PS I spectral emission.