Confocal microscopy of thylakoid autofluorescence in relation to origin of grana and phylogeny in the green algae

Confocal microscopy was used to examine heterogeneity of chlorophyll fluorescence in chloroplasts of selected green algae, in the light of evidence that the technique reveals the distribution of photosystem II (PSII). Three levels of complexity were seen: (1)uniform fluorescence (Codium) or intergrading zones of bright and less bright fluorescence in genera known from electron microscopy to have irregular areas of thylakoid appression (e.g. Chlamydomonas - in which Bertos and Gibbs (J. Phycol., 34, 1009, 1998) have found absence of segregation of phoiosystem I (PSI) and PSII, Ulothrix, Stigeoclonium, Draparnaldia); (2) a pattern of 1-2 μm patches of fluorescence on a less bright uniform background, in taxa where more organized thylakoid stacking (but not grana sensu higher plants) is seen by electron microscopy (Ulva, Oedogonium); and (3) Discrete 0.5-2 μm spots of fluorescence in a relatively fluorescence-free background, closely resembling higher plant grana (Cladophorophyceae, Zygnematales, Coleochaete, Nitella). Further investigation of these states may illuminate the evolution of higher plant thylakoid systems, where PSII is segregated into grana, and may provide clues concerning the adaptive advantages of grana. Association of putative PSII fluorescence with pyrenoids was found in most taxa, although fluorescence of trans-pyrenoid thylakoids was seen in one case only. The association prevails whether or not there is a starch sheath around the pyrenoids, and is persistent in pyrenoids isolated from Spirogyra. We speculate that this fluorescence may represent a subset of PSII that functions with thylakoid carbonic anhydrase to provide locally high concentrations of CO₂ to the Rubisco in the pyrenoid core, as predicted by Raven (Plant, Cell and Environment, 20, 147, 1997).