Cortical microtubule involvement in bordered pit formation in secondary xylem vessel elements ofAesculus hippocastanum L. (Hippocastanaceae): A correlative study using electron microscopy and indirect immunofluorescence microscopy

Summary A correlative study, using indirect immunofluorescence microscopy (IIF) of anti--tubulin stained sections and transmission electron microscopy (TEM), gave details of the involvement of cortical microtubules (CMTs) in the development of bordered pits in secondary xylem vessel elements ofAesculus hippocastanum L. In addition, aspects of wall cytochemistry were studied during this process using the Thiéry (PATAg) test, immunolocalization with the monoclonal antibodies JIM5 and JIM7, and a range of other cytochemical procedures. IIF showed that the alternately-arranged pits are pre-figured as perforations within a reticulum of randomly-oriented CMTs before any secondary wall thickening is obvious. Each incipient pit border is subsequently delimited by a circle of CMTs whose diameter decreases as deposition of secondary wall takes place around the perforation. These IIF observations are corroborated by a parallel TEM study. During the period of bordered pit formation, the secondary walls of the cell are lignifying. At maturity, however, the pit membrane is unlignified and continues to stain strongly with the monoclonal antibody JIM5, a marker of primary, juvenile wall. The results are discussed in terms of the relationship of the CMT cytoskeleton with development of bordered pits.Abbrevations BSA bovine serum albumin - CMT(s) cortical micro-tubule(s) - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - IIP indirect immunofluorescence - MAP microtubule-associated protein - mf(s) wall microfibril(s) - MTSB microtubule-stabilising buffer - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PBS Phosphate-buffered saline - PIPES piperazine-N,N-bis-2-ethylsulphonic acid] - SVS secondary vascular system - TEM transmission electron microscopy