Measuring the dynamic response of the thylakoid architecture in plant leaves by electron microscopy

Abstract

The performance of the photosynthesis machinery in plants, including light harvesting, electron transport, and protein repair, is controlled by structural changes in the thylakoid membrane system inside the chloroplasts. In particular, the structure of the stacked grana area of thylakoid membranes is highly dynamic, changing in response to different environmental cues such as light intensity. For example, the aqueous thylakoid lumen enclosed by thylakoid membranes in grana has been documented to swell in the presence of light. However, light‐induced alteration of the stromal gap in the stacked grana (partition gap) and of the unstacked stroma lamellae has not been well characterized. Light‐induced changes in the entire thylakoid membrane system, including the lumen in both stacked and unstacked domains as well as the partition gap, are presented here, and the functional implications are discussed. This structural analysis was made possible by development of a robust semi‐automated image analysis method combined with optimized plant tissue fixation techniques for transmission electron microscopy generating quantitative structural results for the analysis of thylakoid ultrastructure. Significance Statement A methodical pipeline ranging from optimized leaf tissue preparation for electron microscopy to quantitative image analysis was established. This methodical development was employed to study details of light‐induced changes in the plant thylakoid ultrastructure. It was found that the lumen of the entire thylakoid system (stacked and unstacked domains) undergoes light‐induced swelling, whereas adjacent membranes on the stroma side in stacked grana thylakoid approach each other.

Plants cope with fluctuating light by frequency-dependent non-photochemical quenching and cyclic electron transport

“…In contrast, P700 relative oxidation dynamics (Supplementary Materials Fig.SM-6.4) was always lacking this feature and roughly followed the oscillating light dynamics. Considering that PC was proposed to be responsible for long-distance transfer between cyt b 6 f and PSI along the thylakoid membrane, one may expect that the putative light-induced reorganization of the membrane 82,83 might be causing changes in the dynamical equilibrium between PC and P700 oxidation states. This in turn, can be accounting for the different dynamics of PC and P700 (Figs.3 and 4).…”

“…The ratio between the redox of PC and P700 is, thus, influenced by a factor that is changing in the range of minutes. With the trends in the PC dynamics changing at the oscillation phase with Chl-F extremes, we tentatively propose that the PC dynamics, unlike P700 dynamics is modulated by a factor that connects both photosystems, such as photosynthesis control 57,81 and/or systemic properties such as thylakoid topology 82,83 that may influence the diffusion of PC.…”

How to Measure Grana – Ultrastructural Features of Thylakoid Membranes of Plant Chloroplasts

“…The number of thylakoids per granum was also determined (e.g., Brangeon, 1973;Mostowska, 1986). Such basic parameters are also frequently assessed in current microscopy studies using digital micrographs and image analysis software of choice, followed by different approaches in data presentation and appropriate statistical analyses (e.g., Rozak et al, 2002;Anderson et al, 2012;Pietrzykowska et al, 2014;Pribil et al, 2018;Wood et al, 2018;Nicol et al, 2019;Li et al, 2020).…”

“…SRD might also be measured directly through manual or semi-automated segmentation of particular granum elements (membrane, lumen, and partition gap) using high magnifications. Various semi-automated approaches based on pixel gradient and power spectrum analyses were applied by different authors (Kirchhoff et al, 2011;Tsabari et al, 2015;Wood et al, 2018;Li et al, 2020; for details, see Figure 2B). It should be noted that all detailed analyses of granum layers require high-quality images of grana stacks cut parallelly to the vertical granum direction (Figure 2C).…”

Small-Angle X-Ray and Neutron Scattering on Photosynthetic Membranes

“…1-200 nm in near in vivo conditions and is widely applied in structural biology and soft matter sciences. Small-angle scattering complements various microscopic techniques, such as TEM (Menke, 1960;Paolillo and Paolillo, 1970;Staehelin, 1986;Austin and Staehelin, 2011;Armbruster et al, 2013;Heinz et al, 2016;Kowalewska et al, 2016;Wood et al, 2018;Kowalewska et al, 2019;Wood et al, 2019;Li et al, 2020), scanning electron microscopy (Mustárdy and Jánossy, 1979;Armbruster et al, 2013), cryo-EM (Ford et al, 2002;Kirchhoff et al, 2011;Engel et al, 2015), cryoelectron tomography (Shimoni et al, 2005;Austin and Staehelin, 2011;Daum and Kühlbrandt, 2011;Kouřil et al, 2011;Ford and Holzenburg, 2014;Bussi et al, 2019;Rast et al, 2019), atomic force microscopy (Kaftan et al, 2002;Sturgis et al, 2009;Sznee et al, 2011;Grzyb et al, 2013;Onoa et al, 2014), confocal laser scanning microscopy (Kowalewska et al, 2016;Mazur et al, 2019), and live cell imaging (Iwai et al, 2014;Iwai et al, 2016). The first scattering studies on photosynthetic membranes were performed in 1953 by Finean et al (1953) and has continued ever since.…”