Some of the most important changes that occur in plants during sexual reproduction involve the transition from a sporophytic to a gametophytic type of development. In this paper, these changes were evaluated for Arabidopsis thaliana. The results obtained clearly show differences in the pattern of distribution of specific arabinogalactan protein (AGP) sugar epitopes, during anther and ovule development. AGPs are hydroxyproline-rich glycoproteins that are massively glycosylated and ubiquitous in plants. The molecular mechanism of action of AGPs is still unknown, mainly due to the difficulties posed by the complex saccharide chains. However, the complex structure of the sugar fraction of AGPs makes them a potential source of signalling molecules. The selective labelling obtained with AGP mAbs JIM8, JIM13, MAC207, and LM2, during Arabidopsis pollen and pistil development, suggests that some AGPs can work as markers for gametophytic cell differentiation. Specific labelling of the first gametophytic cells in the pistil, the strong labelling of the secretory cells of the embryo sac, the synergid cells, and the labelling of the integument micropylar cells, apparently outlining the pollen tube pathway into its final target, the embryo sac, have all been shown. In the anthers, the specific labelling of gametophytic cells, and of the male gametes that travel along the pollen tube, may indicate AGP epitopes acting as signals for the pollen tube to reach its final destiny. The specific labelling of cells destined to go into programmed cell death is also discussed.

Arabinogalactan proteins (AGPs) are structurally complex plasma membrane and cell wall proteoglycans that are implicated in diverse developmental processes, including plant sexual reproduction. Male gametogenesis (pollen grain development) is fundamental to plant sexual reproduction. The role of two abundant, pollen-specific AGPs, AGP6, and AGP11, have been investigated here. The pollen specificity of these proteoglycans suggested that they are integral to pollen biogenesis and their strong sequence homology indicated a potential for overlapping function. Indeed, single gene transposon insertion knockouts for both AGPs showed no discernible phenotype. However, in plants homozygous for one of the insertions and heterozygous for the other, in homozygous double mutants, and in RNAi and amiRNA transgenic plants that were down-regulated for both genes, many pollen grains failed to develop normally, leading to their collapse. The microscopic observations of these aborted pollen grains showed a condensed cytoplasm, membrane blebbing and the presence of small lytic vacuoles. Later in development, the generative cells that arise from mitotic divisions were not seen to go into the second mitosis. Anther wall development, the establishment of the endothecium thickenings, the opening of the stomium, and the deposition of the pollen coat were all normal in the knockout and knockdown lines. Our data provide strong evidence that these two proteoglycans have overlapping and important functions in gametophytic pollen grain development.

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Key message: AGP update: plant reproduction. Arabinogalactan proteins (AGPs) are a large family of hydroxyproline-rich proteins, heavily glycosylated, ubiquitous in land plants, including basal angiosperms and also in many algae. They have been shown to serve as important molecules in several steps of the reproductive process in plants. Due to their special characteristics, such as high sugar content and their means of association with the membrane, they are often perceived as likely candidates for many different aspects of the reproductive process such as signalling molecules, cell identity determinants, morphogens, nutrient sources and support for pollen tube growth, among others. Nevertheless, the study of these proteins pose many difficulties when it comes to studying them individually. Most of the work done involved the use of the β-glucosyl Yariv reagent and antibodies that recognize the carbohydrate epitopes only. Recently, new approaches have been used to study AGPs largely based in the remarkable growing volume of microarray data made available. Either using older techniques or the most recent ones, a clearer picture is emerging for the functions and mode of action of these molecules in the plant reproductive processes. Here, we present an overview about the most important studies made in this area, focusing on the latest advances and the possibilities for future studies in the field.

To elucidate the role of protein conformation at the air/water interface, we measured the interfacial dilatational rheology of bovine serum albumin (BSA) and β-casein adsorbed over long time periods using a modified dynamic pendant-drop tensiometer. Companion long-time dynamic surface pressure and ellipsometry measurements are also reported. BSA has well-characterized structural isomers (conformers) whose structural transitions depend on solution pH. It is, therefore, possible to establish a connection between protein structure and interfacial rheology without the ambiguity of comparing different proteins. We also studied β-casein to verify the conclusions obtained from BSA. Adsorbed BSA and β-casein protein layers are primarily elastic with the dilatational elastic modulus arising from two contributions: (1) conformational rearrangement following adsorption that leads to formation of an interconnected, samplespanning interfacial protein network (i.e., an interfacial gel), and (2) the intrinsic structural stability of the individual protein units within the network. The latter component is most important to the interfacial dilatational modulus and explains why adsorbed layers of rigid, globular proteins are more elastic than those of flexible, random-coil proteins. We identify a new surface elasticity relaxation mechanism at the interface due to interprotein conformational rearrangement that is enhanced by electrostatic screening.

The expression of "classical" arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana was characterized by RT-PCR. Transcripts of Agp6 and Agp11 were consistently found to be more abundant in pollen tubes and seem to be pollen-specific. Transcripts of other AGP genes were also detected in pollen but in lesser amounts and in a non-specific fashion. Two reference genes, ubiquitin-conjugating enzyme 9 and tubulin beta-4 chain, were evaluated and selected for gene expression studies in pollen. Expression characterization was complemented with immunolocalization studies using monoclonal antibodies specific to several glycosidic epitopes of AGPs. These studies were performed on in vitro germinated pollen tubes with the antibodies MAC207 and LM2. MAC207 produced labelling at the tip of the pollen tube, while LM2 produced a ring-like fluorescence around the emerging region of the tube, suggesting a role of AGPs during Arabidopsis pollen tube germination. To our knowledge, this is the first report establishing the presence of AGPs on Arabidopsis pollen tubes.

The pollen specificity of the Arabidopsis arabinogalactan protein (AGP) genes AGP6 and AGP11 suggests that they are integral to pollen biogenesis, and their high percent of sequence similarity may indicate a potential for overlapping function. Arabidopsis agp6 agp11 double null mutants have been studied in our laboratory, and in the present work, we characterize the germination and growth of its pollen. When compared to wild type, mutant agp6 agp11 pollen displayed reduced germination and elongation, both in vivo and in vitro, and precocious germination inside the anthers, provided that sufficient moisture was available. This characteristic was not observed in wild type plants, even in water content conditions which for the mutant were sufficient for pollen germination. Therefore, an additional distinctive phenotypic trait of arabinogalactan proteins AGP6 and AGP11 may be to avert untimely germination of pollen. Such AGPs may control germination through water uptake, suggesting an important biological function of this gene family in pollen.

BackgroundArabinogalactan proteins (AGPs) are cell wall proteoglycans that have been shown to be important for pollen development. An Arabidopsis double null mutant for two pollen-specific AGPs (agp6 agp11) showed reduced pollen tube growth and compromised response to germination cues in vivo. A microarray experiment was performed on agp6 agp11 pollen tubes to search for genetic interactions in the context of pollen tube growth. A yeast two-hybrid experiment for AGP6 and AGP11 was also designed.ResultsThe lack of two specific AGPs induced a meaningful shift in the gene expression profile. In fact, a high number of genes showed altered expression levels, strengthening the case that AGP6 and AGP11 are involved in complex phenomena. The expression levels of calcium- and signaling-related genes were found to be altered, supporting the known roles of the respective proteins in pollen tube growth. Although the precise nature of the proposed interactions needs further investigation, the putative involvement of AGPs in signaling cascades through calmodulin and protein degradation via ubiquitin was indicated. The expression of stress-, as well as signaling- related, genes was also changed; a correlation that may result from the recognized similarities between signaling pathways in both defense and pollen tube growth.The results of yeast two-hybrid experiments lent further support to these signaling pathways and revealed putative AGP6 and AGP11 interactors implicated in recycling of cell membrane components via endocytosis, through clathrin-mediated endosomes and multivesicular bodies.ConclusionsThe data presented suggest the involvement of AGP6 and AGP11 in multiple signaling pathways, in particular those involved in developmental processes such as endocytosis-mediated plasma membrane remodeling during Arabidopsis pollen development. This highlights the importance of endosomal trafficking pathways which are rapidly emerging as fundamental regulators of the wall physiology.