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.

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.

Structural changes of mesophyll cells in the rice leaf tissue in response to salinity stress based on the three-dimensional analysis.

Rice leaf blades have intricate-shaped mesophyll cells (MCs) with a large volume of chloroplasts enhancing gas exchange between stroma and intercellular airspace (IAS). Since the rice MCs do not form palisade or spongy tissue cells and are considered monotypic cells, the structural analysis of MCs in the middle part of the leaf tissue has been done, neglecting the various shapes of MCs can be observed on the cross-section of rice leaves. Moreover, the middle MC layer is sandwiched between the upper and lower layers and is more restricted in its demand for light and CO2 entering from the outside. Therefore, the different layers of MCs may differ in their sensitivity to salt stress that causes structural changes in cells. This study aims to elucidate the intra- and extra-cellular structures of MC in different layers of leaf tissue and determine how salinity affects the MC structure in each layer. The mesophyll tissue was divided into adaxial, middle and abaxial layers, and eight MCs and chloroplast regions were selected from each layer and reconstructed into three-dimensional (3D) representations. The whole leaf anatomical and physiological parameters were measured to find the effects of salinity stress on the MC structures. As a result, the 3D analysis of rice leaf tissue revealed the different structures of MCs with greater diversity in the adaxial and abaxial layers than in the middle layer. Salinity stress reduced the size and height of the MCs and coverage of the chloroplast on the cytoplasm periphery of the adaxial and abaxial layers, as well as the chloroplast size of adaxial MCs. Overall, these results reveal the variation of rice MC in leaf tissue and suggest the higher sensitivity to salt stress in the adaxial mesophyll among the layers, which may partly account for the decrease in photosynthetic capacity.