Luís Gustavo Pereira

Molecular Cloning and Characterization of a Vacuolar Class III Peroxidase Involved in the Metabolism of Anticancer Alkaloids in Catharanthus roseus

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to α-3′,4′-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in α-3′,4′-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

Pollen grain development is compromised in Arabidopsis agp6 agp11 null mutants

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.

Expression of arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana.

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.

Arabinogalactan proteins: rising attention from plant biologists

AbstractKey messageAGP 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.

Early germination of Arabidopsis pollen in a double null mutant for the arabinogalactan protein genes AGP6 and AGP11.

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.

Expression-based and co-localization detection of arabinogalactan protein 6 and arabinogalactan protein 11 interactors in Arabidopsis pollen and pollen tubes

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.

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the posibility that AGPs in the sperm cells are probably involved in the double fertilization process.

Arabinogalactan Proteins in Arabidopsis thaliana Pollen Development

Among the genes, or gene families that conform to the transition from a sporophytic type of development to a gametophytic program are the arabinogalactan protein (AGP) genes. AGPs are a class of plant proteoglycans, virtually present in all plant cells and in all plant species, from Algae to Angiosperms. They are predominantly located at the periphery of cells, i.e. on the plasma membrane and in the apoplast. Such ubiquitous presence insinuates that AGPs are vital components of the plant cell. Indeed, many studies have implicated AGPs in important biological phenomena, such as cell expansion, cell division, cell death, seed germination, pollen tube growth and guidance, resistance to infection, etc. (Seifert & Roberts, 2007). Evidences implicating AGPs in sexual reproduction have been obtained in our group, for several plant species (Coimbra & Duarte, 2003; Coimbra & Salema 1997; Coimbra et al., 2005) but how these molecules exert their function or how they interact with other cell components is yet to be defined. Only then the fragmentary knowledge that we have today about the function of these proteins may become pieces of a puzzle.

Growth Media Induces Variation in Cell Wall Associated Gene Expression in Arabidopsis thaliana Pollen Tube

The influence of three different pollen germination media on the transcript profile of Arabidopsis pollen tubes has been assessed by real-time PCR on a selection of cell wall related genes, and by a statistical analysis of microarray Arabidopsis pollen tube data sets. The qPCR assays have shown remarkable differences on the transcript levels of specific genes depending upon the formulation of the germination medium used. With the aid of principal component analysis performed on existing microarray data, a subset of genes has been identified that is more prone to produce diverging transcript levels. A functional classification of those genes showed that the clusters with higher number of members were those for hydrolase activity (based in molecular function) and for cell wall (based in cellular component). Taken together, these results may indicate that the nutrient composition of the pollen germination media influences pollen tube metabolism and that caution must be taken when interpreting transcriptomic data of pollen tubes.

Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes

Following studies on the transcriptome of pollen tubes of an agp6 agp11 Arabidopsis double null mutant, together with the knowledge that arabinogalactan protein (AGP) 6 is important for male gametogenesis and pollen germination, we sought to know whether AGP6 could be present in the vegetative cell wall or in the generative cell wall or in both. To that end, Arabidopsis plants were transformed with AGP6 gene sequence fused with Sirius fluorescent protein. Fluorescent blue light emission could be detected in the vegetative cell wall only. This result supports the model by which AGP6 and other pollen AGPs are probably important signaling molecules at the pollen tube apex.