Three-Dimensional Imaging and Analysis of Differentiating Secondary Xylem by Confocal Microscopy

Abstract

We examined the three-dimensional (3-D) structure of differentiating xylem in a hardwood tree, Kalopanax pictus, by confocallaser scanning microscopy (CLSM) using relatively thick, hand-cut histological sections. 3-D studies of plant tissues by mechanical serial sectioning with a microtome are very time-con suming. By contrast, the preparation of samples for CLSM is easier and the 3-D structure of intact tissue is preserved during optical sectioning. We obtained extended-focus images of the surface of specimens and these images resembled the stereographic images obtained by scanning electron microscopy. In addition , we observed radial files of cambial derivative cells at various stages of differenti ation and the internal structure along the 'z' axis of specimens on serial optical sections. We analysed the developmental changes in the morphology of cambial derivat ive cells, for example, the 3-D shape and arrangement of cells, the readjustment of the position of cells, and the development of secondary walls, pits and perforation plates. Our results showed that the arrangement of the differentiating xylem cells mirror s that of the cambial cell s. Deviations from the longitudinal orientation of vessel elements were specified by similar patterns of orientation of fusiform and ray cambial cells. The development of vessel elements progressed more rapidly than that of other xylem elements. When secondary walls with bordered pits and perforation plates with membranes were present in vessel elements and their expansion ceased, no secondary wall formation was detected in adjacent ray cells. The delay in secondary wall formation by the ray parenchyma cells, as compared to that by vessel elements, might facilitate the readju stment of the position of cells in the developing xylem tissue that is a consequence of the considerable expan sion of the vessel elements.