Simultaneous analysis of shape and internal structure of a curved Hibiscus cannabinus pulvinus: X-ray microtomography and semi-automated quantification.

In the Malvaceae family, dynamic solar tracking by leaves is actuated by the deformation of the pulvinus, a thickened region at the leaf blade-petiole junction. While the internal structure is believed to play a crucial role in this process, experimental verification has been challenging due to technical limitations. To address this gap, we developed a semi-automated workflow, which integrates data analysis and image processing to simultaneously analyze the shape and internal structure of a Malvaceae pulvinus using X-ray microtomography. Firstly, we found that kenaf (Hibiscus cannabinus L.), a Malvaceae species with curved pulvini, exhibited solar-tracking leaf movement and selected it as a model system. We employed diffusible iodine-based contrast-enhanced computed tomography to visualize the internal structure of the kenaf pulvinus. Analysis of the pulvini's shape revealed variations in pulvinus morphology, yet plausible prediction of the centerline was accomplished using polar polynomial regression. Upon slicing the pulvini perpendicular to the centerline, we observed distinct gray value gradients along the proximo-distal and adaxial-abaxial axes, challenging threshold-based tissue segmentation. This workflow successfully generated three modified 3D images and derived quantitative parameters. Using these quantitative parameters, we conducted network analysis and found the linkage between the size-normalized cortex cross-sectional area and curvature. Polynomial least absolute shrinkage and selection operator (LASSO) regression revealed the relationship between the size-normalized cortex cross-sectional area and curvature commonly in all three tested samples. This workflow enables simultaneous analysis of the shape and internal structure, significantly improving the reproducibility of Malvaceae leaf pulvinus characterization.