JAGGER localization and function are dependent on GPI anchor addition.

KEY MESSAGE: GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule-polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.

Cisto gigante de segundo arco branquial em adulto: Relato de caso e revisão de literatura / Giant cyst of the second branchial arch in an adult: case report and literature review

Introdução: Os arcos branquiais são os precursores embriológicos da face, pescoço e faringe. As anomalias dos arcos branquiais são a segunda lesão congênita mais comum de cabeça e pescoço em crianças. Entre essas anomalias, estão os cistos de arcos branquiais (BCC), que surgem devido a uma incorreta obliteração das fendas branquiais, ainda no período embrionário. Os BCC podem ser assintomáticos, apenas percebidos incidentalmente, e não se manifestar até a idade adulta. Resultados: Anomalias do segundo arco branquial devem ser consideradas como um dos possíveis diagnósticos diferenciais de massas cervicais, especialmente as que se manifestam como um abaulamento em região lateral do pescoço. Os BCC são formações de revestimento epitelial, sem aberturas externas. Após seu diagnóstico, o tratamento é cirúrgico, usualmente por meio de uma incisão cervical transversa e cuidadosa dissecação das estruturas, com o objetivo de extirpar toda a lesão. Conclusão: O método descrito, de excisão da lesão, por meio de incisão transversa em região cervical, dissecção tecidual por planos e ressecção de massa cística, é uma opção para o tratamento dessa deformidade, com adequado resultado estético e boa reprodutibilidade.

Introduction: The branchial arches are the embryological precursors of the face, neck, and pharynx. Branchial arch anomalies are the second most common congenital head and neck lesions in children. Among these anomalies are branchial arch cysts (BCC), which arise due to incorrect obliteration of the branchial slits, still in the embryonic period. BCCs may be asymptomatic, only noticed incidentally, and not manifest until adulthood. Results: Anomalies of the second branchial arch should be considered as one of the possible differential diagnoses of neck masses, especially those that manifest as a bulge in the lateral region of the neck. BCCs are epithelial lining formations without external openings. After diagnosis, treatment is surgical, usually through a transverse cervical incision and careful dissection of the structures, with the aim of extirpating the entire lesion. Conclusion: The method described of excision of the lesion through a transverse incision in the cervical region, tissue dissection in planes, and resection of the cystic mass is an option for the treatment of this deformity, with adequate aesthetic results and good reproducibility.

AGPs as molecular determinants of reproductive development.

BACKGROUND AND AIMS: Morphogenesis occurs through accurate interaction between essential players to generate highly specialized plant organs. Fruit structure and function are triggered by a neat transcriptional control involving distinct regulator genes encoding transcription factors (TFs) or signalling proteins, such as the C2H2/C2HC zinc-finger NO TRANSMITTING TRACT (NTT) or the MADS-box protein SEEDSTICK (STK), which are important in setting plant reproductive competence, feasibly by affecting cell wall polysaccharide and lipid distribution. Arabinogalactan proteins (AGPs) are major components of the cell wall and are thought to be involved in the reproductive process as important players in specific stages of development. The detection of AGPs epitopes in reproductive tissues of NTT and other fruit development-related TFs, such as MADS-box proteins including SHATTERPROOF1 (SHP1), SHP2 and STK, was the focus of this study. METHODS: We used fluorescence microscopy to perform immunolocalization analyses on stk and ntt single mutants, on the ntt stk double mutant and on the stk shp1 shp2 triple mutant using specific anti-AGP monoclonal antibodies. In these mutants, the expression levels of selected AGP genes were also measured by quantitative real-time PCR and compared with the respective expression in wild-type (WT) plants. KEY RESULTS: The present immunolocalization study collects information on the distribution patterns of specific AGPs in Arabidopsis female reproductive tissues, complemented by the quantification of AGP expression levels, comparing WT, stk and ntt single mutants, the ntt stk double mutant and the stk shp1 shp2 triple mutant. CONCLUSIONS: These findings reveal distinct AGP distribution patterns in different developmental mutants related to the female reproductive unit in Arabidopsis. The value of the immunofluorescence labelling technique is highlighted in this study as an invaluable tool to dissect the remodelling nature of the cell wall in developmental processes.

New insights on the expression patterns of specific Arabinogalactan proteins in reproductive tissues of Arabidopsis thaliana.

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high proportion of carbohydrates, widely distributed in the plant kingdom and ubiquitously present in land plants. AGPs have long been suggested to play important roles in plant reproduction and there is already evidence that specific glycoproteins are essential for male and female gametophyte development, pollen tube growth and guidance, and successful fertilization. However, the functions of many of these proteins have yet to be uncovered, mainly due to the difficulty to study individual AGPs. In this work, we generated molecular tools to analyze the expression patterns of a subgroup of individual AGPs in different Arabidopsis tissues, focusing on reproductive processes. This study focused on six AGPs: four classical AGPs (AGP7, AGP25, AGP26, AGP27), one AG peptide (AGP24) and one chimeric AGP (AGP31). These AGPs were first selected based on their predicted expression patterns along the reproductive tissues from available RNA-seq data. Promoter analysis using ß-glucuronidase fusions and qPCR in different Arabidopsis tissues allowed to confirm these predictions. AGP7 was mainly expressed in female reproductive tissues, more precisely in the style, funiculus, and integuments near the micropyle region. AGP25 was found to be expressed in the style, septum and ovules with higher expression in the chalaza and funiculus tissues. AGP26 was present in the ovules and pistil valves. AGP27 was expressed in the transmitting tissue, septum and funiculus during seed development. AGP24 was expressed in pollen grains, in mature embryo sacs, with highest expression at the chalazal pole and in the micropyle. AGP31 was expressed in the mature embryo sac with highest expression at the chalaza and, occasionally, in the micropyle. For all these AGPs a co-expression analysis was performed providing new hints on its possible functions. This work confirmed the detection in Arabidopsis male and female tissues of six AGPs never studied before regarding the reproductive process. These results provide novel evidence on the possible involvement of specific AGPs in plant reproduction, as strong candidates to participate in pollen-pistil interactions in an active way, which is significant for this field of study.

In silico and expression analyses of fasciclin-like arabinogalactan proteins reveal functional conservation during embryo and seed development.

KEY MESSAGE: The fasciclin-like arabinogalactan proteins organization into four groups is conserved and may be related to specific roles in developmental processes across angiosperms. Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs), which contain fasciclin-like domains in addition to typical AGP domains. FLAs are present across all embryophytes, and despite their low overall sequence similarity, conserved regions that define the fasciclin functional domain (FAS) have been identified, suggesting that the cell adhesion property is also conserved. FLAs in Arabidopsis have been organized into four subgroups according to the number and distribution of functional domains. Recent studies associated FLAs with cell wall-related processes where domain organization seemed to be related to functional roles. In Arabidopsis, FLAs containing a single FAS domain were found to be important for the integrity and elasticity of the plant cell wall matrix, and FLAs with two FAS domains and two AGP domains were found to be involved in maintaining proper cell expansion under salt stress conditions. The main purpose of the present work was to elucidate the expression pattern of selected FLA genes during embryo and seed development using RT-qPCR. AtFLA8 and AtFLA10, two Arabidopsis genes that stood out in previous microarray studies of embryo development, were further examined using promoter-driven gene reporter analyses. We also studied the expression of cork oak FLA genes and found that their expression partially parallels the expression patterns of the putative AtFLA orthologs. We propose that the functional organization of FLAs is conserved and may be related to fundamental aspects of embryogenesis and seed development across angiosperms. Phylogenetic studies were performed, and we show that the same basic four-subgroup organization described for Arabidopsis FLA gene classification is valid for most Arabidopsis FLA orthologs of several plant species, namely poplar, corn and cork oak.

Arabinogalactan proteins: rising attention from plant biologists.

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 ß-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.

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.

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

BACKGROUND: Arabinogalactan 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. RESULTS: The 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. CONCLUSIONS: The 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.

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.

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.

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.

Asymptomatic infection in family contacts of patients with human visceral leishmaniasis in Três Lagoas, Mato Grosso do Sul State, Brazil.

The Brazilian city of Três Lagoas, Mato Grosso do Sul State, has experienced an urban outbreak of visceral leishmaniasis since 2000. In 2002, due to the increase in the number of cases, 46 families with cases of visceral leishmaniasis were studied to verify the prevalence of asymptomatic infection in household contacts. Indirect immunofluorescence and ELISA showed a 36.4% positive infection rate. There were no cases of symptomatic disease among these contacts. There was no statistically significant difference in gender or age. Median age was 21 years, and the 10-19-year age bracket was the most heavily affected (23%). As for family characteristics, no differences were observed in schooling or family income; most families (58.7%) owned their homes, which were built of masonry (97.8%) and had adequate infrastructure. All the families reported what were probably phlebotomine sand flies in the peridomicile. In conclusion, asymptomatic visceral leishmaniasis infection is frequent and occurs in both males and females, regardless of age.

Asymptomatic infection in family contacts of patients with human visceral leishmaniasis in Três Lagoas, Mato Grosso do Sul State, Brazil / Infecção assintomática em contactantes de pacientes com leishmaniose visceral humana em Três Lagoas, Mato Grosso do Sul, Brasil

The Brazilian city of Três Lagoas, Mato Grosso do Sul State, has experienced an urban outbreak of visceral leishmaniasis since 2000. In 2002, due to the increase in the number of cases, 46 families with cases of visceral leishmaniasis were studied to verify the prevalence of asymptomatic infection in household contacts. Indirect immunofluorescence and ELISA showed a 36.4 percent positive infection rate. There were no cases of symptomatic disease among these contacts. There was no statistically significant difference in gender or age. Median age was 21 years, and the 10-19-year age bracket was the most heavily affected (23 percent). As for family characteristics, no differences were observed in schooling or family income; most families (58.7 percent) owned their homes, which were built of masonry (97.8 percent) and had adequate infrastructure. All the families reported what were probably phlebotomine sand flies in the peridomicile. In conclusion, asymptomatic visceral leishmaniasis infection is frequent and occurs in both males and females, regardless of age.

O Município de Três Lagoas, Mato Grosso do Sul, Brasil, foi alvo de uma epidemia de leishmaniose visceral a partir de 2000. Em 2002, devido ao incremento de casos, estudou-se 46 famílias que apresentavam um caso de doença para verificar-se o percentual de positividade de infecção assintomática por leishmaniose visceral em contactantes. Encontrou-se 36,4 por cento de positividade pelos testes sorológicos Reação de Imunofluorescência Indireta e/ou imunoenzimático ELISA, sem diferença estatisticamente significativa quanto ao sexo e faixa etária. A mediana de idade foi de 21 anos, sendo a faixa etária mais acometida de 10 a 19 anos (23 por cento). Quanto às características familiares não observaram-se diferenças quanto ao nível de instrução e renda familiar; a moradia, em sua maioria, era própria (58,7 por cento), em alvenaria (97,8 por cento), com infra-estrutura adequada. Todas as famílias relataram a presença de provável flebotomíneo no peridomicílio. Conclui-se que a infecção assintomática por leishmaniose visceral é freqüente, ocorrendo em homens e mulheres, independente de faixa etária.

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 possibility that AGPs in the sperm cells are probably involved in the double fertilization process.

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 alpha-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 alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction.

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.

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.