Cell-wall antigens in mesophyll cells and mesophyll-derived protoplasts of sugar beet: possible implication in protoplast recalcitrance?

We have investigated the possible relation between plant cell-wall constituents and the recalcitrance of the cell to regenerate organs and whole plants in vitro. A temporal and spatial expression of several carbohydrate epitopes was observed both within leaf tissue used for protoplast isolation and within new walls reformed by recalcitrant mesophyll protoplasts of sugar beet ( Beta vulgaris L.); these include four pectic epitopes, one xyloglucan (rhamnogalacturonan I) epitope, two carbohydrate motifs of arabinogalactan proteins (AGPs) and callose. The walls of mesophyll cells and newly formed walls of protoplasts were similar with respect to the presence of large amounts of pectins recognized by JIM7 antibodies, the scarcity of JIM5-pectins and the complete absence of LM5-responding pectin molecules. Their main differences were the significantly higher accumulation of LM6-recognizing pectins and the very conspicuous greater accumulation of AGPs and callose in walls deposited by protoplasts than in those synthesized by donor cells.     

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.     

Distribution of pectin and arabinogalactan protein epitopes during organogenesis from androgenic callus of wheat

The distribution of several arabinogalactan protein and pectic epitopes were studied during organogenesis in androgenic callus of wheat. In cell wall of mature and degenerating parenchyma cells, the arabinogalactan epitopes JIM4, JIM14, JIM16 or LM2 were expressed differently according to the cells location. LM2 was observed also in meristematic cells of regenerated shoot buds and leaves. Anti-pectin JIM7 labelled the wall of meristematic cells but fluorescence was strongest in outer walls of surface cells of callus and shoot buds coated by extracellular matrix surface network (ECMSN). During leaves growth the ECMSN disappeared, and JIM7 fluorescence decreased. JIM5 epitope was abundant in the cell walls lining the intercellular spaces of callus parenchyma and in tricellular junctions within regenerated buds and leaves.     

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.     

The differences in cell wall composition in leaves and regenerating protoplasts of Beta vulgaris and Nicotiana tabacum

Cell wall composition in leaves and regenerating leaf-derived protoplasts was studied in Beta vulgaris L. and Nicotiana tabacum L. Several oligosaccharides that characterize arabinogalactan proteins (AGPs) and pectins were visualized in situ by a series of immunocytochemical reactions. The most conspicuous difference between the two species involved the expression of AGP epitopes that bind LM2 and MAC207 in only sugar beet cells; both epitopes being characterized by the presence of glucuronic acid (GlcA). Moreover, B. vulgaris leaves and protoplast-derived cells showed lower content of pectin side-chains bearing (1→4)-β-D-galactose residues as compared to N. tabacum.     

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.     

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.     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Arabinogalactan proteins in embryogenic and non-embryogenic callus cultures of Euphorbia pulcherrima

The arabinogalactan protein (AGP) fractions of embryogenic and non-embryogenic callus lines of Euphorbia pulcherrima Willd. ex. Klotzsch were analysed over a cultivation period of 9 weeks using the β -glucosyl Yariv reagent and an anti-AGP antibody (LM2). The amount of AGPs detected with the Yariv reagent increased in embryogenic cultures during the development of somatic embryos. The embryogenic and non-embryogenic callus contained different sets of AGPs characterized with the Yariv reagent and the LM2 monoclonal antibody. AGPs recognized by LM2 are localized primarily in the protodermal cells of globular somatic embryos. The development of somatic embryos of E. pulcherrima appears to be associated with the presence of particular AGPs.     

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.     

Immunohistochemical analysis of cell wall hydroxyproline-rich glycoproteins in the roots of resistant and susceptible wax gourd cultivars in response to Fusarium oxysporum f. sp. Benincasae infection and fusaric acid treatment

Hydroxyproline-rich glycoproteins (HRGPs) play a defensive role in host–pathogen interactions. However, specific roles of individual HRGPs in plant defense against pathogen are poorly understood. Changes in extracellular distribution and abundance of individual cell wall HRGPs were investigated on root sections of two wax gourd (Benincasa hispida Cogn.) cultivars (Fusarium wilt resistant and susceptible, respectively), which were analyzed by immunolabelling with 20 monoclonal antibodies recognizing different epitopes of extensins and arabinogalactan proteins (AGPs) after being inoculated with Fusarium oxysporum f. sp. Benincasae or treated with fusaric acid (FA). These analyses revealed the following: (1) The levels of JIM11 and JIM20 interacting extensins were higher in the resistant cultivar. Either treatment caused a dramatic decrease in signal in both cultivars, but some new signal appeared in the rhizodermis. (2) The AGPs or rhamnogalacturonan containing CCRCM7-epitope were enhanced in the resistant cultivar, but not in the susceptible one by either treatment. (3) Either treatment caused a slight increase in the levels of the AGPs recognized by LM2 and JIM16, but there were no differences between two cultivars. (4) The MAC204 signal nearly disappeared after FA treatment, but this was not the case with pathogen attack. (5) The LM14 signal slightly decreased after both treatments in both cultivars, but a less decrease was observed with the resistant cultivar. These results indicate that the CCRCM7 epitope likely contributed to the resistance of wax gourd to this pathogen, and JIM11 and JIM20 interacting extensins as well as LM2, LM14, MAC204 and JIM16 interacting AGPs were involved in the host–pathogen interaction.     

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.     

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-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv.

The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of arabinogalactan-protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation.     

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.     

Immunodetection of pectin and arabinogalactan protein epitopes during pollen exine formation of Beta vulgaris L.

Summary. We present the results of ultrastructural and immunocytochemical studies of sugar beet microsporocytes during the developmental phase that begins with the first meiotic metaphase and ends with the formation of young tetrads. The most prominent feature noted during this period of microsporogenesis was the presence of numerous cisternae of endoplasmic reticulum which frequently lie perpendicular to the surface of the plasma membrane and eventually fuse to it. Microscopic observations have been combined with the detection of several carbohydrate epitopes representing pectins and arabinogalactan proteins in the primexine and incipient exine. Pectin domains that possess both low and highly methylesterified epitopes, as well as pectin side chains enriched in (1→4)-β-D-galactose residues, are deposited in this young microspore wall. The epitopes of arabinogalactan protein that bind to JIM13, JIM8, and LM2 antibodies are localised within the callose wall surrounding posttelophase tetrads. The possibility of endoplasmic-reticulum involvement in the synthesis, transport, or metabolism of several microspore wall compounds is discussed. Correspondence and reprints: Institute of Plant Breeding and Acclimatization, Powstańców Wielkopolskich 10, 85-090 Bydgoszcz, Poland.     

Variation in the Degree of Pectin Methylesterification during the Development of Baccharis dracunculifolia Kidney-Shaped Gall

Insect galls may be study models to test the distribution of pectins and arabinogalactan-proteins (AGPs) and their related functions during plant cell cycles. These molecules are herein histochemically and immunocitochemically investigated in the kidney-shaped gall induced by Baccharopelma dracunculifoliae (Psyllidae) on leaves of Baccharis dracunculifolia DC. (Asteraceae) on developmental basis. The homogalacturonans (HGAs) (labeled by JIM5) and the arabinans (labeled by LM6) were detected either in non-galled leaves or in young galls, and indicated stiffening of epidermal cell walls, which is an important step for cell redifferentiation. The labeling of HGAs by JIM7 changed from young to senescent stage, with an increase in the rigidity of cell walls, which is important for the acquaintance of the final gall shape and for the mechanical opening of the gall. The variation on the degree of HGAs during gall development indicated differential PMEs activity during gall development. The epitopes recognized by LM2 (AGP glycan) and LM5 (1–4-β-D-galactans) had poor alterations from non-galled leaves towards gall maturation and senescence. Moreover, the dynamics of pectin and AGPs on two comparable mature kidney-shaped galls on B. dracunculifolia and on B. reticularia revealed specific peculiarities. Our results indicate that similar gall morphotypes in cogeneric host species may present distinct cell responses in the subcelular level, and also corroborate the functions proposed in literature for HGAs.     

Isolation of de-exined pollen and cytological studies of the pollen intines of Pinus bungeana Zucc. Ex Endl. and Picea wilsonii Mast

To study the cytological and biochemical characteristics of intine, pollen deprived of exine, or de-exined pollen, was isolated from the gymnosperms Pinus bungeana and Picea wilsonii. The factors influencing the isolation rate were examined. Cellulose, callose, pectin, and arabinogalactan proteins (AGPs) were localized in this material using fluorescent probes, and components of the isolated intine were further analyzed by Fourier transform infrared (FTIR) microspectroscopy. The isolation protocol was repeatable and reliable. Cellulose was found to be evenly distributed on the surface of the intine, as indicated by strong calcofluor White ST (CW) fluorescence, and aniline blue staining revealed that callose was present on the intine of P. bungeana but not on that of P. wilsonii. Immunolabeling revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present on the pollen intine, as well as esterified pectin recognized by the monoclonal antibody JIM7, and AGPs recognized by the LM2 antibody. Two lectin binding sites, the concanavalin agglutinin (Con A) and soybean agglutinin (SBA) binding sites, were present on the intine surface, but no wheat germ agglutinin (WGA) binding sites were detectable. These results were confirmed by FTIR analysis.     

Spatio-temporal distribution and methyl-esterification of pectic epitopes provide evidence of developmental regulation of pectins during somatic embryogenesis in Arabidopsis thaliana

The aim of the present study was to describe the occurrence of three pectic epitopes, recognized by JIM7, LM19, and LM5 antibodies, during somatic (SE) and zygotic (ZE) embryogenesis in Arabidopsis thaliana. The epitopes recognized by JIM7 and LM19 antibodies showed different distributions during SE stages. Moreover, in the early stages of somatic embryo development, a cytoplasmic occurrence of LM19 epitope was detected. Distribution of a pectic epitope recognized by LM5 antibody corresponded to a vascular system differentiation pattern. Occurrence of LM5 epitope was the same in both zygotic and somatic embryos and often restricted to newly synthesized walls of two adjacent cells. These data suggest that both low and high methyl-esterified pectins (recognized by LM19 and JIM7 antibodies, respectively) are developmentally regulated during SE stages and (1→4)-β-D-galactan epitope (recognized by LM5 antibody) may play a role in cell cytokinesis.