A new approach for the study of the chemical composition of bordered pit membranes: 4Pi and confocal laser scanning microscopy.

UNLABELLED PREMISE OF THE STUDY Coniferous bordered pits are some of the most unique and fascinating microstructures of the lignified cell wall. The pit membrane consists of a margo and a torus region, hence facilitating both xylary water transport and also limiting air intrusion by pit aspiration. Additionally, bordered pits have been reported to play a decisive role in the control of rapid liquid flow via the shrinkage and swelling of pectin. The study of the nanostructural chemical composition of pit membranes has been difficult with common imaging/chemical techniques, which involve drying and/or coating of the samples. • METHODS Using fluorescent tagging and antibodies specific to pectin, and a His-tagged cellulose-binding module that reacts with crystalline cellulose, in combination with confocal laser scanning microscopy (CLSM) and 4Pi microscopy, we generated three-dimensional images of intact pit membranes. • KEY RESULTS With enhanced resolution in the z-direction of the 4Pi microscope, it was possible to distinguish cellulose in the torus and the margo strands of Pinus strobus. The torus was surrounded by pectin, and a pectin ring was found at the margin of the torus. We also found differences in the structure of the pit membrane between aspirated and unaspirated pits, with a displacement of pectin to form a ring-like structure, the collapse of a void in the interior of the torus, and an apparent change in the chemical structure of cellulosic components, during the aspiration process. • CONCLUSIONS The 4Pi microscope is well suited to scanning pit membranes to discover previously undescribed anatomical features in bordered pits and can provide information on chemical composition when used in combination with appropriate probes.

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