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.

We investigated the effects of 1 and 10 mg L(-1) AgNPs on germinating Triticum aestivum L. seedlings. The exposure to 10 mg L(-1) AgNPs adversely affected the seedling growth and induced morphological modifications in root tip cells. TEM analysis suggests that the observed effects were due primarily to the release of Ag ions from AgNPs. To gain an increased understanding of the molecular response to AgNP exposure, we analyzed the genomic and proteomic changes induced by AgNPs in wheat seedlings. At the DNA level, we applied the AFLP technique and we found that both treatments did not induce any significant DNA polymorphisms. 2DE profiling of roots and shoots treated with 10 mg L(-1) of AgNPs revealed an altered expression of several proteins mainly involved in primary metabolism and cell defense.

Silver nanoparticles (AgNPs) are widely used in commercial products, and there are growing concerns about their impact on the environment. Information about the molecular interaction of AgNPs with plants is lacking. To increase our understanding of the mechanisms involved in plant responses to AgNPs and to differentiate between particle specific and ionic silver effects we determined the morphological and proteomic changes induced in Eruca sativa (commonly called rocket) in response to AgNPs or AgNO3. Seedlings were treated for 5 days with different concentrations of AgNPs or AgNO3. A similar increase in root elongation was observed when seedlings were exposed to 10 mg Ag L1 of either PVP-AgNPs or AgNO3. At this concentration we performed electron microscopy investigations and 2-dimensional electrophoresis (2DE) proteomic profiling. The low level of overlap of differentially expressed proteins indicates that AgNPs and AgNO3 cause different plant responses. Both Ag treatments cause changes in proteins involved in the redox regulation and in the sulfur metabolism. These responses could play an important role to maintain cellular homeostasis. Only the AgNP exposure cause the alteration of some proteins related to the endoplasmic reticulum and vacuole indicating these two organelles as targets of the AgNPs action. These data add further evidences that the effects of AgNPs are not simply due to the release of Ag ions.

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.

Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell membrane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching (FRAP) experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM.