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

Abstract

Granum is a basic structural unit of the thylakoid membrane network of plant chloroplasts. It is composed of multiple flattened membranes forming a stacked arrangement of a cylindrical shape. Grana membranes are composed of lipids and tightly packed pigment-protein complexes whose primary role is the catalysis of photosynthetic light reactions. These membranes are highly dynamic structures capable of adapting to changing environmental conditions by fine-tuning photochemical efficiency, manifested by the structural reorganization of grana stacks. Due to a nanometer length scale of the structural granum features, the application of high-resolution electron microscopic techniques is essential for a detailed analysis of the granum architecture. This mini-review overviews recent approaches to quantitative grana structure analyses from electron microscopy data, highlighting the basic manual measurements and semi-automated workflows. We outline and define structural parameters used by different authors, for instance, granum height and diameter, thylakoid thickness, end-membrane length, Stacking Repeat Distance, and Granum Lateral Irregularity. This article also presents insights into efficient and effective measurements of grana stacks visualized on 2D micrographs. The information on how to correctly interpret obtained data, taking into account the 3D nature of grana stacks projected onto 2D space of electron micrograph, is also given. Grana ultrastructural observations reveal key features of this intriguing membrane arrangement, broadening our knowledge of the thylakoid network’s remarkable plasticity.

Thylakoid ultrastructural variations in chlorophyll-deficient wheat: aberrations or structural acclimation?

“…The basic ultrastructural parameters were measured with Fiji software ( https://imagej.net/software/fiji ; Abramoff et al 2004 ), using electron micrographs recorded at a magnification of 13.500 × on areas of 90 μm 2 . The morphometric parameters descriptive of the grana structure were calculated according to Mazur et al ( 2021 ) and included: the granum height ( h ), established by measuring the distance between the granum end-membranes; the number of thylakoid layers per granum ( N ); the stacking repeat distance (SRD), which represents the average thylakoid thickness obtained dividing h by N ; the average granum diameter ( d ), which is the mean of all layers forming a granum stack; the granum area in cross section ( Area ); GLI, which is the coefficient of variation (the ratio of the standard deviation to the mean) of layers diameters within the granum; GSI, which is calculated by comparing the granum cross-sectional area and the rectangle area with the same perimeter and height as the granum cross section [(rectangle area– granum area)/rectangle area]. In case GSI was negative, the granum was considered regular and the zero value was assigned.…”

“…In land plants and their closest algal relatives, the thylakoid system is a unique membrane network with a bipartite architecture, which consists of appressed grana stacks and unstacked stromal lamellae (Solymosi 2012 ; Solymosi and Keresztes 2012 ). Typically, in angiosperms, a granum is formed by 5–25 thylakoid layers with diameters between 300 and 550 nm, and includes core, margins, and end membranes (Mustárdy and Garab 2003 ; Kirchhoff 2019 ; Mazur et al 2021 ). The grana–intergrana organization is the result of photosynthetic complexes segregation between thylakoid domains, which ensures the highest possible packing density of the grana membranes (Garab 2016 ).…”

“…The thylakoid system, with its special architecture, is the theater where these complex events occur to allow photosynthesis, while preserving the biochemical machinery responsible for light-harvesting and energy conservation. The intimate relation between thylakoid structure and function is not fully understood and is a hot topic in plant cell biology, with methodological contributions deriving from electron microscopy, biochemistry and biophysics (Kirchhoff 2018 , 2019 ; Staehelin and Paolillo 2020 ; Mazur et al 2021 ).…”

“…Different studies have already recognized the components of the thylakoid membrane as possible regulators of grana architecture and size (for review, Mazur et al 2021 ). A classical approach is the structural grana analysis in mutants characterized by an altered composition of the photosynthetic membranes.…”

“…The Granum Lateral Irregularity (GLI) quantifies the irregularity of a granum stack with respect to the uniformity of the thylakoid disk diameters (Kowalewska et al 2016 ). The Granum Cross-Sectional Irregularity (GSI) evaluates the shifting of granum membranes in the lateral plane (Mazur et al 2021 ).…”