3-D reconstruction of rice leaf tissue for proper estimation of surface area of mesophyll cells and chloroplasts facing intercellular airspaces from 2-D section images

Abstract

Background and aims The surface area of mesophyll cells (Smes) and chloroplasts (Sc) facing the intercellular airspace (IAS) are important parameters for estimating photosynthetic activity from leaf anatomy. Although Smes and Sc are estimated based on the shape assumption of mesophyll cells (MCs), it is questionable if the assumption is correct for rice MCs with concave-convex surfaces. Therefore, in this study, we establish a reconstruction method for the 3-D representation of the IAS in rice leaf tissue to calculate the actual Smes and Sc with 3-D images and to determine the correct shape assumption for the estimation of Smes and Sc based on 2-D section images. Methods We used serial section light microscopy to reconstruct 3-D representations of the IAS, MCs and chloroplasts in rice leaf tissue. Actual Smes and Sc values obtained from the 3-D representation were compared with those estimated from the 2-D images to find the correct shape-specific assumption (oblate or prolate spheroid) in different orientations (longitudinal and transverse sections) using the same leaf sample. Key results The 3-D representation method revealed that volumes of the IAS and MCs accounted for 30 and 70 % of rice leaf tissue excluding epidermis, respectively, and the volume of chloroplasts accounted for 44 % of MCs. The shape-specific assumption on the sectioning orientation affected the estimation of Smes and Sc using 2-D section images with discrepancies of 10-38 %. Conclusions The 3-D representation of rice leaf tissue was successfully reconstructed using serial section light microscopy and suggested that estimation of Smes and Sc of the rice leaf is more accurate using longitudinal sections with MCs assumed as oblate spheroids than using transverse sections with MCs as prolate spheroids.

Plant phenotyping relevance

イネ葉組織の3D再構成法を開発し、2D画像による葉肉細胞・葉緑体の表面積推定と比較検証しており、植物形質の取得・推定法が中心である。

Code and data availability

The paper's supplementary materials include 3-D reconstruction videos of the IAS and chloroplast regions facing the IAS (Videos S1 and S2) and a table of shape assumptions used for curvature correction factors (Table S1), all directly reproducing this paper's phenotyping measurements. These are hosted online via the Oy