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.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that 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 evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Localisation pattern of homogalacturonan and arabinogalactan proteins in developing ovules of the gymnosperm plant Larix decidua Mill

We have identified and characterised the temporal and spatial distribution of the homogalacturonan (HG) and arabinogalactan proteins (AGP) epitopes that are recognised by the antibodies JIM5, JIM7, LM2, JIM4, JIM8 and JIM13 during ovule differentiation in Larix decidua Mill. The results obtained clearly show differences in the pattern of localisation of specific HG epitopes between generative and somatic cells of the ovule. Immunocytochemical studies revealed that the presence of low-esterified HG is characteristic only of the wall of megasporocyte and megaspores. In maturing female gametophytes, highly esterified HG was the main form present, and the central vacuole of free nuclear gametophytes was particularly rich in this category of HG. This pool will probably be used in cell wall building during cellularisation. The selective labelling obtained with AGP antibodies indicates that some AGPs can be used as markers for gametophytic and sporophytic cells differentiation. Our results demonstrated that the AGPs recognised by JIM4 may constitute molecules determining changes in ovule cell development programs. Just after the end of meiosis, the signal detected with JIM4 labelling appeared only in functional and degenerating megaspores. This suggests that the antigens bound by JIM4 are involved in the initiation of female gametogenesis in L. decidua. Moreover, the analysis of AGPs distribution showed that differentiation of the nucellus cells occurs in the very young ovule stage before megasporogenesis. Throughout the period of ovule development, the pattern of localisation of the studied AGPs was different both in tapetum cells surrounding the gametophyte and in nucellus cells. Changes in the distribution of AGPs were also observed in the nucellus of the mature ovule, and they could represent an indicator of tissue arrangement to interact with the growing pollen tube. The possible role of AGPs in fertilisation is also discussed.     

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39–4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39–4 gene, JIM13 and JIM14 epitopes found specifically in 2–4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.     

Localization of arabinogalactan proteins in anther,pollen, and pollen tube of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Summary. Western blot analysis indicated the presence of two epitopes recognized by the anti-arabinogalactan protein antibodies JIM13 and LM2 and the absence of the JIM4 epitope in mature tobacco anthers. Immunoenzyme localization of arabinogalactan proteins (AGPs) with JIM13 showed that AGPs accumulate mainly at the early stages of anther development. AGP content and distribution were also investigated at the ultrastructural level in pollen tubes grown in vivo and in vitro. Abundant AGPs were present in the transmitting tissue of styles, and the AGP content of the extracellular matrix changed during pollen tube growth. In pollen tubes, immunogold particles were mainly distributed in the cell wall and cytoplasm, especially around the peripheral region of the generative-cell wall. β-D-Glucosyl Yariv reagent, which specifically binds to AGPs, caused slow growth of pollen tubes and reduced immunogold labeling of AGPs with JIM13 in vitro. These data suggest that AGPs participate in male gametogenesis and pollen tube growth and may be important surface molecules in generative and sperm cells. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Immunolocalization of arabinogalactan proteins inAmaranthus hypochondriacus L. ovules

Summary Amaranthus hypochondriacus ovules are of the crassinucellate type, having several layers of nucellus cells between the micropyle and the embryo sac through which pollen tubes have to penetrate. The ultrastructural features of the micropylar nucellus cells appear to reflect cells with high metabolic activity. With the monoclonal antibodies MAC207 and JIM8 (against arabinogalactan proteins) we have shown that the presence of the two epitopes was different in the gametophytic tissues and embryo sac. The young embryo and suspensor cells are reactive only to Mab JIM8. The selective presence and localization of these two epitopes was also demonstrated in the micropylar nucellus cells. The expression of these arabinogalactan proteins in this ovule seems to be closely aligned with the pathway of the pollen tube, possibly providing directional guides for tube growth inside the ovule.     

Immunogold localization of arabinogalactan proteins,unesterified and esterified pectins in pollen grains and pollen tubes ofNicotiana tabacum L.

Summary The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the cell wall of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.Abbreviations AGP arabinogalactan protein - BSA bovine serum albumin - GA glutaraldehyde - MAb monoclonal antibody - NGS normal goat serum - PFA paraformaldehyde     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.     

Arabinogalactan proteins 6 and 11 are required for stamen and pollen function in Arabidopsis

Successful male reproductive function in plants is dependent on the correct development and functioning of stamens and pollen. AGP6 and AGP11 are two homologous Arabidopsis genes encoding cell wall-associated arabinogalactan glycoproteins (AGPs). Both genes were found to be specifically expressed in stamens, pollen grains and pollen tubes, suggesting that these genes may play a role in male organ development and function. RNAi lines with reduced AGP6 and AGP11 expression were generated. These, together with lines harboring point mutations in the coding region of AGP6, were used to show that loss of function in AGP6 and AGP11 led to reduced fertility, at least partly as a result of inhibition of pollen tube growth. Our results also suggest that AGP6 and AGP11 play an additional role in the release of pollen grains from the mature anther. Thus, our study demonstrates the involvement of specific AGPs in pollen tube growth and stamen function.     

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

Localization of an arabinogalactan protein epitope and the effects of Yariv phenylglycoside during zygotic embryo development of Arabidopsis thaliana

Summary. Arabinogalactan proteins (AGPs) are a class of highly glycosylated proteins widely distributed in higher plants and thought to be involved in plant growth and development. In the present paper, Western blotting with the monoclonal antibodies JIM4, JIM13, and LM2 showed that JIM13 reacted best with total protein extracts from flowers and siliques of Arabidopsis thaliana. This monoclonal antibody was therefore used as a probe to localize the AGP epitope in zygotic embryos at different developmental stages. Immunofluorescent labeling with JIM13 showed that AGPs were mainly distributed in the embryo proper and the top 1 to 2 cells and basal part of suspensors. The results of immunogold labeling confirmed the JIM13 epitope distribution in the different cells of the suspensor. AGP immunofluorescence was also observed at the shoot apex meristem during transition from the globular to the heart embryo stage, but this gradually disappeared after the torpedo stage. After (β-D-Glc)₃ Yariv phenylglycoside (βGlcY), a synthetic reagent that specifically binds to AGPs, was added to A. thaliana ovule culture medium, the survival rate and frequency of development of ovules at the zygote stage decreased in a concentration-dependent manner, with complete inhibition at 100 μM. The frequency of embryo differentiation from the globular stage to heart or later stages also decreased sharply. When βGlcY was removed 24 h after inoculation, the inhibitory effects were reversible in a concentration-dependent and time-dependent manner. The results show that βGlcY can inhibit embryo development and differentiation in A. thaliana, and the inhibitory effects are concentration dependent and reversible, indicating that AGPs are involved in embryo differentiation and shoot meristem formation. The possible roles of AGPs in A. thaliana zygotic embryo development are also discussed. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Immunolocalization of Arabinogalactan Proteins and Pectins in Floral Buds of Cucumber （Cucumis sativus L.） During Sex Determination

Arabinogalactan proteins (AGPs) and pectins were detected in the floral buds of cucumber(Cucumis sativus L.) during its sex determination using the following monoclonal antibodies: MAC 207(recognizes AGP epitopes); JIM 8 (recognizes a subset ofAGP epitopes); and JIM 5 and JIM 7 (epitopes of pectins esterified to various degrees). In the stem apex meristem (SAM) of the cucumber, epitopes of MAC 207, JIM 7, and JIM 5 were localized in the cells from second to third peripheral layers when the sex organ primodium began to differentiate; epitopes of MAC 207 and JIM 5 were also detected in the ragged edge cells. A very dense labeling signal with MAC 207 was observed in the carpel and pistil primodium. The AGP epitopes recognized by JIM 8 were localized in the anther of the male flower and the anther-like portion of the stagnant stamen of the female flower. This suggests that the AGPs and pectins in the SAM of the cucumber are closely associated with the differentiation of the SAM, from meristematic cells to floral primodium. The subset of AGPs recognized by JIM 8 may play an important role in stamen formation.     

Arabinogalactan proteins and the extracellular matrix surface network during peach palm somatic embryogenesis

Somatic embryogenesis has been described in peach palm as a reliable method for its in vitro multiplication and conservation. In this study, we evaluated the possible role of arabinogalactan proteins (AGPs) during this morphogenetic pathway. The presence of Yariv reagent, a synthesized chemical antibody that specifically binds AGP molecules, affected somatic embryos and callus development rate, but no effect was observed on fresh weight increment. This substance also had profound effects on embryo morphology: somatic embryos presented loose cells in the protoderm and no signs of polarization could be observed. To better evaluate the role of AGPs, analyses of specific monoclonal antibodies (MAbs) against different AGP epitopes revealed a specific pattern of distribution for each epitope. MAb JIM13 had differential expression and showed intense signal on the embryogenic sector and some immediately adjacent layers. MAb JIM7 against pectin recognized cell walls and a specific layer over the developing somatic embryo, as well as over the shoot meristem region of mature somatic embryos. This corresponds to an extracellular matrix surface network (ECMSN) associated with the development of somatic embryos and closely related to the expression of MAb JIM13. Scanning electron microscopy confirmed the presence of an ECMSN covering a specific group of cells and ultra-structural analyses revealed that the ECMSN had lipophilic substances.     

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet

Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts--these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.     

Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation

Two polymorphic forms of an extracellular arabinogalactan protein (AGP1 and AGP2), obtained from the conditioned media of two carrot suspension-cultured cell lines, have been identified in terms of binding of the anti-plasma membrane antibodies JIM4 and MAC207. AGP1 and AGP2 have been used as immunogens to generate further anti-AGP monoclonal antibodies. JIM14 identified an epitope carried by AGP2 and also by glycoproteins of low molecular weight localized to the plant cell wall. In addition, further antibodies (JIM13 and JIM15) identified carbohydrate epitopes of the AGPs that also occur on plasma membrane glycoproteins and are expressed by patterns of cells that reflect cell position at the carrot root apex. Indirect immunofluorescence microscopy indicated that JIM13 recognized the surface of cells forming the epidermis and cells marking the region and axis of the future xylem. JIM15 recognized a pattern of cells directly complementary to the JIM13 pattern. The panel of anti-AGP monoclonal antibodies now available indicates groups of cells within the root meristem that may reflect an early pre-pattern of the tissues of the mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex.     

Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside

Arabinogalactan proteins (AGPs) are abundant complex macromolecules involved in both reproductive and vegetative plant growth. They are secreted at pollen tube tips in Lilium longiflorum. Here, we report the effect of the (beta-D-glucosyl)3 Yariv phenylglycoside, known to interact with AGPs, on pollen tube extension in several plant species. In Annona cherimola the Yariv reagent clearly inhibited pollen tube extension within 1-2 h of treatment, as demonstrated previously for L. longiflorum, but had no effect on Lycopersicon pimpinellifolium, Aquilegia eximia, and Nicotiana tabacum. With the monoclonal antibody JIM13 we also examined these same species for evidence that they secreted AGPs at their pollen tube tips. Only A. cherimola showed evidence of AGPs at the pollen tube tip as does lily. The Yariv reagent causes arrest of tube growth in both A. cherimola and lily, but its removal from the medium allows regeneration of new tip growth in both species. We show that the site of the new emerging tip in lily can be predicted by localization of AGP secretion. Labeling with JIM13 appeared on the flanks of the arrested tip 1 h after removal of the Yariv reagent from the growth medium. After 4 h, many of the Yariv reagent-treated pollen tubes had regenerated new pollen tubes with the tips brightly labeled by JIM13 and with a collar of AGPs left at the emergence site. During this recovery, esterified pectins colocalized with AGPs. Secretion at the site of the new tip may be important in the initial polarization event that occurs on the flanks of the arrested tube tip and results in a new pollen tube.     

Immunolocalization of Arabinogalactan Proteins and Pectins in Floral Buds of Cucumber (Cucumis sativus L.) During Sex Determination

Arabinogalactan proteins (AGPs) and pectins were detected in the floral buds of cucumber (Cucumis sativus L.) during its sex determination using the following monoclonal antibodies: MAC 207 (recognizes AGP epitopes); JIM 8 (recognizes a subset ofAGP epitopes); and JIM 5 and JIM 7 (epitopes of pectins esterified to various degrees). In the stem apex meristem (SAM) of the cucumber, epitopes of MAC 207, JIM 7, and JIM 5 were localized in the cells from second to third peripheral layers when the sex organ primodium began to differentiate; epitopes of MAC 207 and JIM 5 were also detected in the ragged edge cells. A very dense labeling signal with MAC 207 was observed in the carpel and pistil primodium. The AGP epitopes recognized by JIM 8 were localized in the anther of the male flower and the anther-like portion of the stagnant stamen of the female flower. This suggests that the AGPs and pectins in the SAM of the cucumber are closely associated with the differentiation of the SAM, from meristematic cells to floral primodium. The subset of AGPs recognized by JIM 8 may play an important role in stamen formation.     

Cell-specific expression of two arabinogalactan protein epitopes recognized by monoclonal antobodies JIM8 and JIM13 in maize roots

Summary The cell-specific expression of two arabinogalactan protein (AGP) epitopes recognized by monoclonal antibodies JIM8 and JIM13 is reported in maize roots. Employing immunofluorescence and immunogold electron microscopy, the JIM8 antibody was shown to label exclusively protophloem sieve elements, while the JIM13 antibody labelled sieve elements very strongly and adjacent pericycle and companion cells, as well as sloughing root cap cells less strongly. Since the labelling of sieve elements with JIM8 antibody was specific and did not spread to other cell types during root development, it is concluded that this AGP epitope can serve as a specific marker of these specialized cells within the maize root. In the case of the AGP epitope recognized by JIM13 antibody, part of the immunofluorescence label was also found to be associated with cytoplasmic strands in the pericycle and sloughing root cap cells. Immunogold-labelling of sieve elements revealed the association of both AGP epitopes (JIM8 and JIM13) with cortical sieve element reticulum and plasma membranes. Labelling of sieve element reticulum was prominent at its domains of adhesion to the plasma membrane, P-type plastids, and mitochondria. Based on our subcellular studies, we propose a new function of AGP epitopes in endomembrane recognition and adhesion within the sieve elements of maize roots.Abbreviations AGP arabinogalactan protein - SER sieve element reticulum