Flash-kinetics as a complementary analytical tool in PAM fluorimetry.

A new measuring system based on the already existing Multi-Color-PAM Fluorimeter (Schreiber et al. in Photosynth Res 113:127-144, 2012) was developed that in addition to standard PAM measurements enables pump-and-probe flash measurements and allows simultaneous measurements of the changes in chlorophyll fluorescence yield (F) during application of saturating flashes (ST). A high-power Chip-on-Board LED array provides ST flashes with close to rectangular profiles at wide ranges of widths (0.5 µs to 5 ms), intensities (1.3 mmol to 1.3 mol 440 nm quanta m-2 s-1) and highly flexible repetition times. Using a dedicated rising-edge profile correction, sub-µs time resolution is obtained for assessment of initial fluorescence and rise kinetics. At maximal to moderate flash intensities the flash-kinetics (changes of F during course of ST, STK) are strongly affected by 'High Intensity Quenching' (HIQ), consisting of Car-triplet quenching, TQ, and donor-side-dependent quenching, DQ. The contribution of TQ is estimated by application of a second ST after 20 µs dark-time. Upon application of flash trains (ST sequences with defined repetition times) typical period-4 oscillations in dark fluorescence yield (F0) and ST-induced fluorescence yield, FmST, are obtained which can be measured in vivo both with suspensions and from the surface of leaves. Examples of application with dilute suspensions of Chlorella and an intact dandelion leaf are presented. It is shown that weak far-red light (730-740 nm) advances the S-state distribution of the water-splitting system by one step, resulting in substantial lowering of FmST and also of the I1-level in the polyphasic rise of fluorescence yield induced by a multiple-turnover flash (MT). Based on comparative measurements of STK and the polyphasic rise kinetics with the same Chlorella sample, it is concluded that the generally observed lower values of maximal fluorescence yields using ST-protocols compared to MT-protocols are due to a higher extent of HIQ (mainly DQ) and the contribution of variable PSI fluorescence to FmST.

Light-induced changes of far-red excited chlorophyll fluorescence: further evidence for variable fluorescence of photosystem I in vivo.

Recently, the long-standing paradigm of variable chlorophyll (Chl) fluorescence (Fv) in vivo originating exclusively from PSII was challenged, based on measurements with green algae and cyanobacteria (Schreiber and Klughammer 2021, PRES 149, 213-231). Fv(I) was identified by comparing light-induced changes of Fv > 700 nm and Fv < 710 nm. The Fv(I) induced by strong light was about 1.5 × larger in Fv > 700 nm compared to Fv < 710 nm. In the present communication, concentrating on the model green alga Chlorella vulgaris, this work is extended by comparing the light-induced changes of long-wavelength fluorescence (> 765 nm) that is excited by either far-red light (720 nm, mostly absorbed in PSI) or visible light (540 nm, absorbed by PSI and PSII). Polyphasic rise curves of Fv induced by saturating 540 nm light are measured, which after normalization of the initial O-I1 rises, assumed to reflect Fv(II), display a 2 × higher I2-P transient with 720 nm excitation (720ex) compared with 540ex. Analysis of the Fo(I) contributions to Fo(720ex) and Fo(540ex) reveals that also Fo(I)720ex is 2 × higher than Fo(I)540ex, which supports the notion that the whole I2-P transient is due to Fv(I). The twofold increase of the excitation ratio of F(I)/F(II) from 680 to 720 nm is much smaller than the eight-tenfold increase of PSI/PSII known from action spectra. It is suggested that the measured F > 765 nm is not representative for the bulk chlorophyll of PSI, but rather reflects a small fraction of far-red absorbing chlorophyll forms ("red Chls") with particular properties. Based on the same approach (comparison of polyphasic rise curves measured with 720ex and 540ex), the existence of Fv(I) is confirmed in a variety of other photosynthetic organisms (cyanobacteria, moss, fern, higher plant leaves).

Evidence for variable chlorophyll fluorescence of photosystem I in vivo.

Room temperature fluorescence in vivo and its light-induced changes are dominated by chlorophyll a fluorescence excited in photosystem II, F(II), peaking around 685 nm. Photosystem I fluorescence, F(I), peaking around 730 nm, so far has been assumed to be constant in vivo. Here, we present evidence for significant contributions of F(I) to variable fluorescence in the green unicellular alga Chlorella vulgaris, the cyanobacterium Synechococcus leopoliensis and a light-green ivy leaf. A Multi-Color-PAM fluorometer was applied for measurements of the polyphasic fluorescence rise (O-I1-I2-P) induced by strong 440 nm light in a dilute suspension of Chlorella, with detection alternating between emission above 700 nm (F > 700) and below 710 nm (F < 710). By averaging 10 curves each of the F > 700 and F < 710 recordings even small differences could be reliably evaluated. After equalizing the amplitudes of the O-I1 phase, which constitutes a specific F(II) response, the O-I1-I2 parts of the two recordings were close to identical, whereas the I2-P phase was larger in F > 700 than in F < 710 by a factor of 1.42. In analogous measurements with Synechococcus carried out in the dark state 2 using strong 625 nm actinic light, after O-I1 equalization the I2-P phase in F > 700 exceeded that in F < 710 even by a factor of 1.99. In measurements with Chlorella, the I2-P phase and with it the apparent variable fluorescence of PS I, Fv(I), were suppressed by moderate actinic background light and by the plastoquinone antagonist DBMIB. Analogous measurements with leaves are rendered problematic by unavoidable light intensity gradients and the resulting heterogenic origins of F > 700 and F < 710. However, a light-green young ivy leaf gave qualitatively similar results as those obtained with the suspensions, thus strongly suggesting the existence of Fv(I) also in leaves.

Rapidly reversible chlorophyll fluorescence quenching induced by pulses of supersaturating light in vivo.

The saturation pulse method provides a means to distinguish between photochemical and non-photochemical quenching, based on the assumption that the former is suppressed by a saturating pulse of light (SP) and that the latter is not affected by the SP. Various types of non-photochemical quenching have been distinguished by their rates of dark relaxation in the time ranges of seconds, minutes, and hours. Here we report on a special type of non-photochemical quenching, which is rapidly induced by a pulse of high-intensity light, when PS II reaction centers are closed, and rapidly relaxes again after the pulse. This high-intensity quenching, HIQ, can be quantified by pulse-amplitude-modulation (PAM) fluorimetry (MULTI-COLOR-PAM, high sensitivity combined with high time resolution) via the quasi-instantaneous post-pulse fluorescence increase that precedes recovery of photochemical quenching in the 100-400-µs range. The HIQ amplitude increases linearly with the effective rate of quantum absorption by photosystem II, reaching about 8% of maximal fluorescence yield. It is not affected by DCMU, is stimulated by anoxic conditions, and is suppressed by energy-dependent non-photochemical quenching (NPQ). The HIQ amplitude is close to proportional to the square of maximal fluorescence yield, Fm', induced by an SP and varied by NPQ. These properties are in line with the working hypothesis of HIQ being caused by the annihilation of singlet excited chlorophyll a by triplet excited carotenoid. Significant underestimation of maximal fluorescence yield and photosystem II quantum yield in dark-acclimated samples can be avoided by use of moderate SP intensities. In physiologically healthy illuminated samples, NPQ prevents significant lowering of effective photosystem II quantum yield by HIQ, if excessive SP intensities are avoided.