Thylakoid membrane appression in the giant chloroplast of Selaginella martensii Spring: A lycophyte challenges grana paradigms in shade-adapted species

In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid system among plant species is mainly determined by the adaptation to the growth light regime, according to a paradigm stating that shade-tolerant species are featured by a high membrane extension with an enhanced number of thylakoid layers per granum. In this study, the thylakoid system was analysed in Selaginella martensii Spring, a shade-adapted rainforest species belonging to lycophytes, a diminutive plant lineage, sister clade of all other vascular plants (euphyllophytes, including ferns and seed plants). The species is characterized by giant cup-shaped chloroplasts in the upper epidermis and, quantitatively less important, disk-shaped chloroplasts in the mesophyll and lower epidermis. The study aimed at the quantitative assessment of the thylakoid appression exploiting a combination of complementary methods, including electron microscopy, selective thylakoid solubilisation, electron paramagnetic resonance, and simultaneous analysis of fast chlorophyll a fluorescence and P700 redox state. With a chlorophyll a/b ratio of 2.6 and PSI/PSII ratio of 0.31, the plant confirmed two typical hallmarks of shade-adaptation. The morphometric analysis of electron micrographs revealed a 33% fraction of non-appressed thylakoid domains. However, contrasting with the structural paradigm of thylakoid shade-adaptation in angiosperms, S. martensii privileges the increase in the granum diameter in place of the increase in the number of layers building the granum. The very wide grana diameter, 727 nm on average, largely overcame the threshold of 500 nm currently hypothesized to allow an effective diffusion of long-range electron carriers. The fraction of non-appressed membranes based on the selective solubilisation of thylakoids with digitonin was 26%, lower than the morphometric determination, indicating the presence of non-appressed domains inaccessible to the detergent, most probably because of the high three-dimensional complexity of the thylakoid system in S. martensii. Particularly, strong irregularity of grana stacks is determined by assembling thylakoid layers of variable width that tend to slide apart from each other as the number of stacked layers increases.