ULTRASTRUCTURAL DYNAMICS OF PHOTOSYNTHETIC THYLAKOID MEMBRANES IN VASCULAR PLANTS

Vaclav Svoboda

doi:10.7273/000007478

Sessile photoautotrophic organisms, such as vascular plants, developed multiple-level adaptations for natural light energy fluctuations to sustain optimal energy conversion. The adaptations begin in chloroplasts on the thylakoid membrane, where the photosynthetic electron transport chain occurs. Plant thylakoid is a three-dimensional continuous membrane network hosting all protein complexes involved in photosynthetic light reactions and forming stacked grana of multiple tightly appressed thylakoid membrane discs interconnected by unstacked stroma lamellae. The two thylakoid domains host different sets of protein complexes and thus are involved in different parts of electron transport. The thylakoid is a highly dynamic network and undergoes dramatic short- and long-term light-induced adaptations. Because of the resolution needed to measure the fine ultrastructural features of thylakoids, the traditional method of choice has been transmission electron microscopy (TEM). We optimized reproducible TEM fixation for quantitative image analysis for the model plant Arabidopsis thaliana. We developed an automated Python-based image analysis pipeline to extract detailed Grana stack parameters quantitatively. The ImageJ Fiji-based semi-automated pipeline was designed to quantitatively analyze the entire thylakoid membrane and whole chloroplast parameters. These approaches were used to get insight into light-dependent ultrastructural adaptations and underlying mechanisms. We complemented the structural TEM information with a thorough biochemical analysis of thylakoids and thylakoid fractions to better understand the light-dependent structure-function relationship. To determine stoichiometric details of the main thylakoid photosynthetic protein complexes, light-harvesting complex II (LHCII), photosystem II (PSII), photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase, different quantitative spectroscopic and biochemical methods were optimized. We found a light-induced change in the thylakoid ultrastructure that consisted of a dramatic increase of stacked thylakoid doublets, a recently discovered membrane region having characteristics of both stacked and unstacked thylakoid regions that were previously suggested to be formed by specific LHCII thylakoid phosphorylation. Our experiments under moderate highlight confirmed that this light adaptation is controlled by thylakoid phosphorylation. The particular control, however, seems to be more complex since our experimental conditions exclude significant involvement of LHCII phosphorylation as the sole mechanism. Instead, our data show that light-dependent phosphorylation of PSII by STN8 kinase is involved in the thylakoid ultrastructural response under high-energy quenching conditions.

ULTRASTRUCTURAL DYNAMICS OF PHOTOSYNTHETIC THYLAKOID MEMBRANES IN VASCULAR PLANTS

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