Cell and chloroplast anatomical features are poorly estimated from 2D cross‐sections

Abstract

Summary Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF‐SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under‐ and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF‐SEM has strong potential to contribute to improved understanding of leaf form and function.

Recent developments in mesophyll conductance in C3, C4, and crassulacean acid metabolism plants

“…The few publications comparing 2‐D versus 3‐D leaf structure suggest that 2‐D projections maybe underestimating key traits. For example, wheat and chickpea using geometric models with 2‐D micrographs underestimated mesophyll chloroplast volume by c. 61% compared with 3‐D analysis from serial block‐face scanning electron microscopy (Harwood et al , ). Furthermore, 3‐D reconstruction of X‐ray microcomputed tomography (microCT) of leaves from 23 species with diverse leaf anatomy demonstrated were compared with four commonly reported 2‐D methods (Theroux‐Rancourt et al , ).…”

“…Additionally, these models can theoretically account for variation in CA activity throughout the leaf and how it potentially facilitates both CO 2 and HCO À 3 diffusion between compartments (Tholen and Zhu, 2011;Ho et al, 2016) or the interaction of chloroplast and mitochondria orientation on re-fixation of CO 2 released from F and R L Berghuijs et al, 2017). Therefore, combining this modelling with the advancement in 3-D visualization such as X-ray microscopy and serial blockface scanning electron microscopy will potentially provide better mechanistic descriptions for individual anatomical and biochemical traits affecting g m Harwood et al, 2019).…”

Maximum CO2diffusion inside leaves is limited by the scaling of cell size and genome size

“…For the liquid phase conductance, gliq, we approximated cells as capsule shaped, as capsules give the best approximation of cell shape 60,61 . The capsule height was two times its diameter.…”

Shape matters: the pitfalls of analyzing mesophyll anatomy

“…Manual serial sectioning of mesophyll tissue for electron microscopy is very labor‐intensive (Zellnig et al , ), allowing only small parts of the mesophyll cells to be examined. Harwood et al show that the block‐face scanning technique, where the sectioning is automated, makes it now less labor‐demanding to acquire larger volumes of the leaf in three dimensions.…”

“…If the object of interest is a single cell or organelle, it becomes even more difficult to obtain unbiased estimates of volume and surface area. A number of approaches, discussed in Harwood et al , have been used to quantify cell or organelle volume and surface area by using exact or approximate estimates of volumes and surface areas for spheres, cylinders, capsules, or ellipsoids. These approaches not only rely on questionable assumptions on the cell shape as highlighted by Harwood et al , but they cannot be applied on single sections because they usually underestimate the true dimensions of the structure under investigation (see geometrical example in Box 1).…”