Applications for the DUAL-KLAS-NIR
The DUAL-KLAS-NIR is unique in providing online deconvoluted signals of the in vivo redox states of P700, PC and Fd in real time. This opens up a whole new research field with applications that may not have come to our minds yet. In particular, for the first time simultaneous kinetic information on the complex interplay between reactions at the PS I donor and acceptor sides can be obtained, including information on cyclic electron transport around PS I.
Some examples of applications, which have been described in Klughammer and Schreiber, Photosynth Res 128 (2016) 195-214, Schreiber and Klughammer, Plant Cell Physiol 57 (2016) 1454-1467 and Schreiber Photosynth Res 134 (2017) 343-360, are given here.
The effective quantum yield of photosystem I. In all older measurements the P700 signal contained always contributions of Fd and variable amounts of PC, depending on the method used to reduce this contribution. Panel C of the figure illustrates the light intensity dependence of the effective PSI quantum yield Y(I), the effective quantum yield of PSII Y(II) and the effective quantum yield of PSII corrected for PSI fluorescence Y(II)corr of Brassica napus.
The effective antenna size of photosystem I. On a dark-to-light transition, first oxidized PC starts to accumulate and only with some delay oxidized P700. The initial slope of the clean PC signal can now be used as a measure for the effective antenna size of PS I. The second figure zooms in on the initial absorbance changes and shows the large difference in the initial slope of PC (red) and P700 (blue). For a dark-acclimated leaf, with inactive photosystem I acceptor side, the initial slope of Fd-reduction can also be used for this purpose. The figure illustrates the delay in the oxidation kinetics of P700 relative to those of PC for a barley leaf, which has a relatively high PC/P700 ratio.
The DUAL-KLAS-NIR has a window to visualize the simultaneous changes in the P700 and PC redox states relative to each other. This allows an estimation of the apparent equilibrium constant between PC and P700. The figure gives an example of a P700 versus PC redox plot.
During a saturation pulse given to a well dark-adapted leaf, the Fd-pool becomes reduced and in a subsequent period of darkness slowly re-oxidizes again. When the electron flow on the acceptor side of PSI has become activated the re-oxidation kinetics become much faster. Following activation of the acceptor side of PSI, the dark-inactivation can be followed by monitoring the rate at which Fd becomes re-oxidized following a probe pulse. These kinetics can be fit with an exponential fit routine. Panel A gives examples of the Fd reoxidation kinetics fit with an exponential fit routine and Panel B shows the dark-inactivation kinetics of the acceptor side of PSI for a Hedera helix leaf.
The maximal NIR transmittance changes of PC, P700 and Fd are proportional to the leaf/sample content of these compounds and the ratios of the extinction coefficients of PC, P700 and Fd are constant. This makes it possible to probe the PC/P700 and Fd/P700 ratios and thereby the relative PC and Fd pool sizes in different species or under different conditions (e.g. sun/shade, stressed/non-stressed) on a routine basis with the DUAL-KLAS-NIR. It is observed that high PC/P700 ratios correlate with high ETR values. The figure illustrates that sun and shade leaves of, in this case, Hedera helix have considerably different PC and Fd contents relative to P700.