CORTICAL MICROTUBULE INVOLVEMENT IN BORDERED PIT FORMATION IN SECONDARY XYLEM VESSEL ELEMENTS OF AESCULUS-HIPPOCASTANUM L (HIPPOCASTANACEAE) - A CORRELATIVE STUDY USING ELECTRON-MICROSCOPY AND INDIRECT IMMUNOFLUORESCENCE MICROSCOPY

A correlative study, using indirect immunofluorescence microscopy (IIF ) of anti-alpha-tubulin stained sections and transmission electron mic roscopy (TEM), gave details of the involvement of cortical microtubule s (CMTs) in the development of bordered pits in secondary xylem vessel elements of Aesculus hippocastanum L. In addition, aspects of wall cy tochemistry were studied during this process using the Thiery (PATAg) test, immunolocalization with the monoctonal antibodies JIM5 and JIM7, and a range of other cytochemical procedures. IIF showed that the alt ernately-arranged pits are pre-figured as perforations within a reticu lum of randomly-oriented CMTs before any secondary wall thickening is obvious. Each incipient pit border is subsequently delimited by a circ le of CMTs whose diameter decreases as deposition of secondary wall ta kes place around the perforation. These IIF observations are corrobora ted by a parallel TEM study. During the period of bordered pit formati on, the secondary walls of the cell are lignifying. At maturity, howev er, the pit membrane is unlignified and continues to stain strongly wi th the monoclonal antibody JIM45, a marker of primary, ''juvenile'' wa ll. The results are discussed in terms of the relationship of the CMT cytoskeleton with development of bordered pits.