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.