Dynamic Thylakoid Stacking Is Regulated by LHCII Phosphorylation but Not Its interaction with PSI

Abstract

Grana stacking in plant chloroplast thylakoid membranes dynamically responds to the light environment. These dynamics have been linked to regulation of the relative antenna sizes of PSI and PSII (state transitions), the PSII repair cycle, and the regulation of photosynthetic electron transfer. Here, we used 3D structured illumination microscopy, a subdiffraction-resolution fluorescence imaging technique, to investigate the light-intensity dependence, kinetics, reversibility, and regulation of dynamic thylakoid stacking in spinach (Spinacia oleracea) and Arabidopsis (Arabidopsis thaliana). Low-intensity white light (150 mmol photons m 22 s 21) behaved similarly to light preferentially exciting PSII (660 nm), causing a reduction in grana diameter and an increased number of grana per chloroplast. By contrast, high-intensity white light (1000 mmol photons m 22 s 21), darkness, and light preferentially exciting PSI (730 nm) reversed these changes. These dynamics occurred with a half-time of 7 to 8 min and were accompanied by state transitions. Consistent with this, the dynamics were dependent on STN7 (light-harvesting complex II [LHCII] kinase) and TAP38 (LHCII phosphatase), which are required for state transitions but were unaffected by the absence of STN8 (PSII kinase) or PSII core phosphatase (PSII phosphatase). Unlike state transitions, however, thylakoid stacking dynamics did not rely on the presence of the LHCI and PSI subunit L phospho-LHCII binding sites on PSI. Since oligomerization of thylakoid curvature protein (CURT1A) was unaffected by the absence of STN7 or TAP38, we conclude that the primary determinant of dynamic thylakoid stacking is LHCII phosphorylation.

Modelling the role of LHCII-LHCII, PSII-LHCII and PSI-LHCII interactions in state transitions

“…2B,D). In both cases, only LHCII was allowed to move between the grana and stromal lamellae, consistent with the majority of the biochemical data (31, 40, 42). The grana radius was set to 190 nm and 170 nm for SI and SII respectively as observed in SIM images of spinach chloroplasts (23).…”

“…While the LHCII phosphorylation is clearly established as the mechanistic basis of state transitions it remains unclear precisely how the thylakoid macro-organization changes to bring about the relative change in the PSI/ PSII antenna size. STN7-dependent phosphorylation of LHCII causes a reduction in grana diameter and the number of membrane layers per stack while simultaneously increasing the number of grana per chloroplast (23, 31). Puthiyaveetil et al .…”

“…The net result is that increasing the negative surface charge density on LHCII by phosphorylation is sufficient to weaken the lateral and stacking interactions that sustain grana organization (13, 14). These changes bring about a reduction in grana dimeter and effectively increase the contact area between the grana and stromal lamellae, which may facilitate the exchange of LHCII between the domains (23, 31, 32). The changes in thylakoid stacking were also found to weaken the connectivity between PSII reaction centres, again contributing to the relative shift in antenna size (33).…”

“…Given the unanswered questions that remain on the precise mechanism of state transitions new approaches are needed to clarify how the altered balance of forces upon LHCII phosphorylation affect the thylakoid macro-organization. The structural model of the thylakoid membrane at the individual protein complex level presented here was made possible by recent breakthroughs in elucidating thylakoid organization and dynamics using in atomic force microscopy (AFM) (7, 23, 43) and structured illumination microscopy (SIM) (31, 44), and publication of high-resolution structures of the key protein complexes involved in the light reactions (45–50). Recently developed algorithms, which made the calculation of whether two or more particles spatially overlap tractable, were also essential (see methods).…”

Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley due to Reduced Plastid Translation

“…Large grana have been further described in mutants with impaired starch formation (Hausler et al, 2009). Recent studies point to phosphorylation levels of PSII and LHCII and/or the degree of oligomerization of the thylakoid curvature protein family as regulators of the dynamic changes in thylakoid stacking (Armbruster et al, 2013; Pietrzykowska et al, 2014; Puthiyaveetil et al, 2017; Wood et al, 2018; Wood et al, 2019). HvCMF3 encodes a chloroplast-localized CMF protein.…”