Ultrastructural study of archaeological Vitis vinifera L. seeds using rapid-freeze fixation and substitution.

The ultrastructure of Vitis vinifera seeds from different archaeological sites was studied. Preservation status differed between sites. Preliminary investigations of grape seeds from Poggio Bacherina (Chianciano Terme, Siena) and Miranduolo (Chiusdino, Siena) showed collapsed or charred tegument, making this material suitable for morphometric studies only. Rapid-freeze fixation and substitution of grape seeds from Shahr-I Sokhta in Iran and via De' Castellani in Florence revealed well preserved tegument suitable for chemical and cytochemical analysis. Energy dispersive X-ray microanalysis was used to determine chemical composition. Cytochemical analysis based on fluorescent staining with DAPI suggested the presence of cytoplasm residues.

Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold.

In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes--positively or negatively charged nanogold--were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasma-membrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tubes.

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes.

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation.