Arabinogalactan proteins and pectin distribution during female gametogenesis in Quercus suber L.

BACKGROUND AND AIMS: Quercus suber L. (cork oak) is one of the most important monoecious tree species in semi-arid regions of Southern Europe, with a high ecological value and economic potential. However, as a result of its long reproductive cycle, complex reproductive biology and recalcitrant seeds, conventional breeding is demanding. In its complex reproductive biology, little is known about the most important changes that occur during female gametogenesis. Arabinogalactan proteins (AGPs) and pectins are the main components of plant cell walls and have been reported to perform common functions in cell differentiation and organogenesis of reproductive plant structures. AGPs have been shown to serve as important molecules in several steps of the reproductive process in plants, working as signalling molecules, associated with the sporophyte-gametophyte transition, and pectins have been implicated in pollen-pistil interactions before double fertilization. In this study, the distribution of AGP and pectin epitopes was assessed during female gametogenesis. METHODS: Immunofluorescence labelling of female flower cells was performed with a set of monoclonal antibodies (mAbs) directed to the carbohydrate moiety of AGPs (JIM8 and JIM13) and pectic homogalacturonans (HGs) (mAbs JIM5 and JIM7). KEY RESULTS: The selective labelling obtained with AGP and pectin mAbs JIM8, JIM13, JIM5 and JIM7 during Q. suber female gametogenesis shows that AGPs and pectic HG can work as markers for mapping gametophytic cell differentiation in this species. Pectic HG showed different distribution patterns, depending on their levels of methyl esterification. Methyl-esterified HGs showed a uniform distribution in the overall female flower cells before fertilization and a more specific pattern after fertilization. A low methyl-ester pectin distribution pattern during the different developmental stages appears to be related to the pathway that pollen tubes follow to reach the embryo sac. AGPs showed a more sparse distribution in early stages of development, but specific labelling is shown in the synergids and their filiform apparatus. CONCLUSIONS: The labelling obtained with anti-AGP and anti-pectin mAbs in Q. suber female flower cells showed a dynamic distribution of AGPs and pectic HGs, which may render these molecules useful molecular markers during female gametogenesis. Changes occurring during development will be determined in order to help describe cork oak ovule structural properties before and after fertilization, providing new insight to better understand Q. suber female gametogenesis.

Evaluation of the presence of arabinogalactan proteins and pectins during Quercus suber male gametogenesis.

BACKGROUND AND AIMS: Quercus suber (cork oak) is a dominant tree of the Fagaceae in forests of the south-west Iberian Peninsula. It is monoecious with a long progamic phase that provides a comprehensive system for comparative studies in development and sexual reproduction. In this study the distribution of arabinogalactan protein (AGPs) and pectin epitopes in anthers of Q. suber was assessed to map these hydroxyproline-rich glycoproteins and the galacturonate-rich acidic polysaccharides during pollen development. Methods Immunolocalization in male flowers was performed with a set of monoclonal antibodies directed against the carbohydrate moiety that recognizes AGPs and pectins. To identify AGP genes involved in cork oak male flower development, a search was conducted for annotated AGP genes in the available transcriptome data of the Cork Oak EST Consortium database (www.corkoakdb.org). KEY RESULTS: Ubiquitous labelling in all cell types was obtained with anti-homogalacturan antibodies for methyl-esterified pectins. In contrast, the antibody that labelled non-methyl-esterified homogalacturans had a preferential presence in microsporocyte cells walls at the beginning of pollen development. Intense labelling was obtained with anti-AGP antibodies both in the tapetum and in the intine wall near the pollen apertures and later in the generative cell wall and vegetative cell. Evaluation of the putative AGPs highly expressed in the male gametophyte was achieved by quantitative RT-PCR analysis in male and female cork oak flowers. CONCLUSIONS: Four putative AGP genes were identified that are preferentially expressed in the male flower compared with the female flower. The putative Arabidopsis thaliana orthologues of these genes are associated with preferential expression in pollen, suggesting that the AGPs probably play a significant role in cork oak reproduction.

Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana reproductive tissues.

Arabinogalactan proteins (AGPs) are heavily glycosylated proteins existing in all members of the plant kingdom and are differentially distributed through distinctive developmental stages. Here, we showed the individual distributions of specific Arabidopsis AGPs: AGP1, AGP9, AGP12, AGP15, and AGP23, throughout reproductive tissues and indicated their possible roles in several reproductive processes. AGP genes specifically expressed in female tissues were identified using available microarray data. This selection was confirmed by promoter analysis using multiple green fluorescent protein fusions to a nuclear localization signal, ß-glucuronidase fusions, and in situ hybridization as approaches to confirm the expression patterns of the AGPs. Promoter analysis allowed the detection of a specific and differential presence of these proteins along the pathway followed by the pollen tube during its journey to reach the egg and the central cell inside the embryo sac. AGP1 was expressed in the stigma, style, transmitting tract, and the chalazal and funiculus tissues of the ovules. AGP9 was present along the vasculature of the reproductive tissues and AGP12 was expressed in the stigmatic cells, chalazal and funiculus cells of the ovules, and in the septum. AGP15 was expressed in all pistil tissues, except in the transmitting tract, while AGP23 was specific to the pollen grain and pollen tube. The expression pattern of these AGPs provides new evidence for the detection of a subset of specific AGPs involved in plant reproductive processes, being of significance for this field of study. AGPs are prominent candidates for male-female communication during reproduction.

In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH).

The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins' distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.

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.

Immunolocalization of AGPs and Pectins in Quercus suber Gametophytic Structures.

The arabinogalactan proteins (AGPs) are highly glycosylated proteins, ubiquitous in plants that have been linked to numerous aspects of sexual reproduction in several plant species, including the monoecious tree species Quercus suber. AGPs are found in cell membranes and cell walls of all types of tissues, including reproductive cells and organs. Pectins are cell wall components that also have been shown to change in composition and quantity during the maturations of the male and female gametophyte in cork oak. These findings were only possible to reveal, due to the histological study of AGP and pectins epitopes by immunolabeling. The immunofluorescence microscopy technique uses antibodies linked to fluorophores and relies on the specificity of the antibody binding to its antigen, labeling the epitope with a fluorescent dye.In the method presented here, we explore the immunolocalization technique performed in male and female flowers of Quercus suber, using London Resin (LR-White) as the embedding medium, after vacuum fixation with formaldehyde/glutaraldehyde. An extensive description of all the aspects of this technique is provided, from the plant material developmental stages selection to the critical analysis of results performed, continuously supported by troubleshooting recommendations.

"Love Is Strong, and You're so Sweet": JAGGER Is Essential for Persistent Synergid Degeneration and Polytubey Block in Arabidopsis thaliana.

Successful double fertilization and subsequent seed development in flowering plants requires the delivery of two sperm cells, transported by a pollen tube, into the embryo sac of an ovule. The embryo sac cells tightly control synergid cell death, and as a result the polyspermy block. Arabinogalactan proteins are highly glycosylated proteins thought to be involved in several steps of the reproductive process. We show that JAGGER, Arabinogalactan Protein 4, is an important molecule necessary to prevent the growth of multiple pollen tubes into one embryo sac in Arabidopsis thaliana. In jagger, an AGP4 knockout mutant, the pistils show impaired pollen tube blockage as a consequence of the survival of the persistent synergid. JAGGER seems to be involved in the signaling pathway that leads to a blockage of pollen tube attraction. Our results shed light on the mechanism responsible for preventing polyspermy in Arabidopsis and for safeguarding successful fertilization of all ovules in one pistil, ensuring seed set and the next generation.

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.