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.
This paper reviews the development of xylem vessels in ring-porous dicots and the corresponding leaf phenology. Also included are our original observations on the time-course of vessel element growth, secondary wall deposition, and end wall perforation in the deciduous hardwood Kalopanax septemlobus. Different patterns of xylem growth and phenology serve different strategies of the species for adaptation to seasonal climates. Trees with ring-porous xylem form wide earlywood vessels (EWV) in spring and narrow latewood vessels in summer. The wide EWV become embolized or blocked with tyloses by the end of the growing season while the narrow vessels may remain functional for many years. The co-occurrence of wide and narrow vessels provides both efficiency and safety of the water transport as well as a potentially longer growing season. It has for a long time been assumed that EWV in ring-porous hardwoods are formed in early spring before bud burst in order to supply sap to growing leaves and shoots.
However, the full time-course of development of EWV elements from initiation of growth until maturation for water transport has not been adequately studied until recently. Our observations clarify a crucial relationship between leaf maturation and the maturation of earlywood vessels for sap transport. Accumulated new evidence shows that EWV in branches and upper stem parts develop earlier than EWV lower in the stem. The first EWV elements are fully expanded with differentiated secondary walls by the time of bud burst. In lower stem parts, perforations in vessel end walls are formed after bud burst and before the new leaves have achieved full size. Therefore, the current-year EWV network becomes functional for water transport only by the time when the first new leaves are mature.
A resin-casting method with subsequent scanning electron microscopy (SEM) was used to examine the three-dimensional (3-D) shapes of cells and the cell walls of cambium and differentiating xylem. Glutaraldehyde- fixed and dehydrated specimens were embedded in polystyrene and then organic material was removed by digestion with acidic solutions or enzymes. The acidic solutions used for treatment were sulphuric acid and a mixture of acetic acid and hydrogen peroxide and the enzymes used for treatment were pectinase and cellulase, with a final treatment with sodium hypochlorite. Both methods could be used for studies of the differentiation of cambial cells; however, digestion with enzymes allowed better preservation of the 3-D organisation of the tissue. Negative replicas of inner surfaces of cell walls of differentiating vessel elements revealed the sequential stages of the development of bordered pits and perforation plates. Future bordered pits at the early stages of the differentiation of cell walls were demarcated by the accumulation of organic material between adjacent pit membranes. Subsequent deposition of cell wall material resulted in formation of pit cavities and the rims of perforation plates.
Identification of ancient charcoal fragments is a valuable tool in reconstructing past environments and determining natural and anthropogenic disturbances, and for understanding past cultures and societies. Although in Europe such studies are fairly straightforward, utilising charcoal records from the tropics is more complicated due to the species-richness of the natural vegetation. Comprehensive databases have greatly aided identification but often identification of charcoalified woods from the tropics relies on minute anatomical features that can be difficult to observe due to preservation or lack of abundance.
This article illustrates the relative potential of four imaging techniques and discusses how they can provide optimal visualisation of charcoal anatomy, such that specific difficulties encountered during charcoal examination can be evaluated and fine anatomical characters can be observed enabling high-level identification of charcoal (and wood) taxa. Specifically reflected Light Microscopy is often used to quickly group large numbers of charcoal fragments into charcoal types. Scanning Electron Microscopy and High-Throughput X-ray Computed Tomography are employed to observe fine anatomical detail. More recently X-ray Computed Tomography at very high resolution has proved successful for imaging hidden or ‘veiled’ anatomical features that cannot be detected on exposed surfaces but need three-dimensional volumetric imaging.
Xylem anatomy is fundamental in studies of the evolution of terrestrial plants, tree ecophysiology, forestry, and wood science. Traditional xylem anatomical studies by light microscopy utilize wood sections. However, the procedures are laborious, and high-quality histological sections have been particularly challenging to achieve from recalcitrant wood species and dry wood material. Modern microscopy offers opportunities for speeding up the xylem anatomical preparations. In this regard, the merits of using a sanded surface for wood anatomical research have been largely overlooked. Sanding of wood surfaces is practiced in dendrochronology and wood identification studies exclusively for the investigation of macro features, such as tree rings, wood porosity, or parenchyma patterns. We conducted microscopic level investigations of sanded surfaces of difficult-to-section high-density woods such as Dalbergia and Quercus species by reflected white light and epifluorescence microscopy. Reflected white light or combinations of reflected light and fluorescence could clearly show xylem micro-features in sanded wood surfaces. The resolution of cell types after sanding with 1000-grit was similar to the resolution obtained by transmitted light microscopy in histological slides. The advantages of sanded wood surfaces compared to traditional wood sections can be summarized as cost- and time-effective sample preparation, large sample area, intact cell walls and tissue structure, preservation of chemical content and extractives, and even focus of the field of view. A simple procedure of wood sanding instead of microscopic slides can be used for xylem microscopy and automatic image analysis of xylem structure.
Limited investigations have been carried out on the physiological and growth responses of bark to wounding, even though wound periderms play crucial roles in tree defenses. To understand the mechanisms of wound periderm formation, we studied the growth responses and structural changes of wounded bark of three Cryptomeria japonica individuals. We observed the developmental time frame and morphology of wound periderms around mechanically induced wounds in summer. The wound responses included discoloration, lignification, and suberization in tissues present at the time of wounding, followed by wound periderm formation and secondary metabolite deposition. The trees had developed wound periderms approximately 4 weeks after wounding. The wound periderms were within 3 mm in the axial directions and within 1 mm in the lateral directions from the wound surfaces. The distinct patterns of wound periderm formation in the axial and lateral regions resulted from the arrangement and anatomical features of the cells adjacent to the wounds. The wound phellem cells were tangentially narrower and axially shorter in the side and upper/lower regions, respectively, of the wounds. Therefore, the cell division frequencies in the planes parallel to the wound surface may be greater than those in the other directions. Wound reactions in bark might initially be triggered by microenvironmental changes, such as the spread of desiccation, which depends directly on the morphology of phloem cell complexes.