Immunolocalization of LM2 arabinogalactan protein epitope associated with endomembranes of plant cells

Summary Arabinogalactan proteins (AGPs) are proteoglycans detected in high amounts at plant cell surfaces; however, details of their subcellular localization are largely unknown. Immunolocalization studies with the anti-AGP monoclonal antibody LM2 have indicated that this AGP epitope is associated with secretory compartments such as endoplasmic reticulum and Golgi apparatus within plant cells actively producing and secreting AGPs. The LM2 epitope contains a -linked glucuronic acid residue and occurs in the polysaccharide moiety of AGPs. We have localized this AGP epitope also to the tonoplast and to cytoplasmic strands. Endomembrane association of AGPs was confirmed with two other monoclonal antibodies, JIM13 and MAC207, both reacting with carbohydrate AGP epitopes containing GlcpA-(13)-D-GalpA-(12)-L-Rha residues. Immunocytochemistry is supported by biochemical analysis which shows that LM2 reacts with the microsomal fraction and also with low-molecular-weight material of the detergent phase after Triton X-114 phase separation prepared from maize roots. Our results indicate that some AGP epitopes are closely associated with endomembranes.Abbreviations AGP arabinogalactan protein - ER endoplasmic reticulum - GlcA glucuronic acid Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

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.     

Immunolocalization of arabinogalactan proteins (AGPs) in reproductive structures of an early-divergent angiosperm,Trithuria (Hydatellaceae)

Background and Aims Trithuria

is the sole genus of Hydatellaceae, a family of the early-divergent angiosperm lineage Nymphaeales (water-lilies). In this study different arabinogalactan protein (AGP) epitopes in T. submersa were evaluated in order to understand the diversity of these proteins and their functions in flowering plants.

Methods

Immunolabelling of different AGPs and pectin epitopes in reproductive structures of T. submersa at the stage of early seed development was achieved by immunofluorescence of specific antibodies.

Key Results

AGPs in Trithuria pistil tissues could be important as structural proteins and also as possible signalling molecules. Intense labelling was obtained with anti-AGP antibodies both in the anthers and in the intine wall, the latter associated with pollen tube emergence.

Conclusions

AGPs could play a significant role in Trithuria reproduction, due to their specific presence in the pollen tube pathway. The results agree with labellings obtained for Arabidopsis and confirms the importance of AGPs in angiosperm reproductive structures as essential structural components and probably important signalling molecules.     

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.Abbreviations DAA days after anthesis - IgG immunoglobulin G - M_r apparent molecular mass - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis     

Mature Amaranthus hypochondriacus seeds contain non-processed 11S precursors

Amaranth is a dicotyledonous plant whose major seed storage proteins are globulins and glutelins. An unique feature of amaranth seeds is the presence of a fraction named albumin-2, that is extractable with water only after an exhaustive extraction of globulins and albumin-1. In this work, we tested the hypothesis that albumin-2 fraction could be constituted by a non-processed 11S globulin (proglobulin). To this end, the gene encoding the amaranth 11S subunit was cloned and expressed in Escherichia coli. Subsequently, the recombinant proglobulin and albumin-2 purified from seeds were treated with a sunflower vacuolar processing enzyme (VPE). A 55 kDa component of albumin-2 was specifically cleaved into 38 and 17-15 kDa polypeptides, as a consequence of this endoproteolytic cleavage a change of the oligomeric state from trimeric to hexameric was observed. Amaranth 11S globulin fraction was not modified under these proteolysis conditions. Using VPE-specific antibodies, it was shown that amaranth expresses a 57 kDa VPE, and that both developing and mature amaranth seeds have VPE activity, although the increase of this activity during amaranth seed development is higher than that observed for sunflower seeds. These results confirm the presence of unprocessed 11S precursors in mature amaranth seeds; this phenomenon cannot, however, be attributed to low VPE activity during developing of amaranth seeds.     

Subcellular distribution of arabinogalactan proteins in pollen grains and tubes as revealed with a monoclonal antibody raised against stylar arabinogalactan proteins

Summary Monoclonal antibody PCBC3, raised against stylar extracts fromNicotians, alata flowers, was deduced from enzyme-linked immunosorbent assays and inhibition of immuno-gold labelling on tissue sections to bind specifically to carbohydrate epitopes on arabinogalactan proteins (AGPs) but not to other arabinose-containing cell wall polysaccharides. When pollen grains ofN. tabacum were hydrated in fixative, PCBC3 bound to vesicles in the vicinity of the endoplasmic reticulum but, when grains were hydrated for 20 min in culture medium before fixation, binding was restricted to the plasma membrane. The generative-cell plasma membrane was also labelled in grains ofLycopersicon peruvianum. In pollen tubes ofN. tabacum grown in liquid culture, the AGPs detected by PCBC3 were located in several regions, including the plasma membrane, tubular-vesicular structures (plasmalemmasomes) at and under the plasma membrane, and multilamellar bodies within vacuoles, features generally associated with endocytosis. Labelling was not evident in secretory vesicles or the plasma membrane at the pollen-tube tip. The AGPs detected with PCBC3 were also present in pollen-tube walls, near the interface between the inner, callosic layer and the outer, fibrillar, pectic layer. Pollen tubes ofN. tabacum grown in medium lacking added CuSO₄ produce a wall with an abnormally thickened fibrillar layer, and this layer was uniformly labelled with PCBC3. The disposition of wall AGPs thus changes in pollen tubes of different morphologies.Abbreviations AGP arabinogalactan protein - -L-Araf -L-arabinofuranose - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - PBS phosphate-buffered saline     

Expression of nutritionally well-balanced protein, AmA1, inSaccharomyces cerevisiae

Food yeast.Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins. AmA1, a seed storage albumin fromAmaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further improving food and animal feed additives. In order to find an effective means of expressingAmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter. The recombinantAmA1 genes were then introduced into the yeastSaccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed thatAmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount of AmA1 protein in the recombinant yeast was 1.3–4.3% of the total soluble proteins. The highest expression level was obtained from the hybrid ADH2-GPD promoter.     

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 H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

Summary A heterogeneous distribution of H⁺-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H⁺-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H⁺-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H⁺-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.Abbreviations BSA bovine serum albumine - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - Tris 2-amino-2-hydroxymethyl-1,3-propandiol     

Genetic studies of yield contributing traits in Amaranthus

Summary Genetical studies on grain yield and its contributing traits were made in a six parent complete diallel in the F₁ and F₂ generations of one of the most widely grown grain species of grain amaranths (Amaranthus hypochondriacus L.). Graphical analysis indicated that epistasis exists for 1,000-grain weight in the F₁. Grain weight/panicle, yield/plant and harvest index indicated absence of non-allelic gene interaction. The harvest index in the F₁ and F₂ and grain weight/ panicle, 1,000-grain weight, yield/plant in the F₂ appeared to be controlled by overdominance effects. Higher grain yield appeared to be associated with dominant genes. Both additive and non-additive gene effects were responsible for the genetic variation in the diallel population. However, dominance variance was more important than additive variance in grain yield/ plant and harvest index in the F₁ and F₂. For 1,000-grain weight additive genetic variance was more important in the F₁ and non-additive in F₂. There was overdominance of a consistent nature in the two analyses for harvest index in the F₁ and F₂, grain weight/ panicle, 1,000-grain weight and yield/plant in the F₂ and partial dominance for 1,000-grain weight in the F₁.     

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     

Purification and immunocytochemical localization of kafirins inSorghum bicolor (L. Moench) endosperm

Summary Kafirins are the storage proteins of sorghum and are found in protein bodies in the seed endosperm. They have been classified as -, -, and -kafirins according to differences in molecular weight, solubility, and structure. The kafirins were purified, amino acid composition was determined, and immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. All three groups of kafirins were low in lysine. -Kafirins and -kafirins were relatively high in cysteine, and -kafirins were relatively high in methionine. Transmission electron microscopy showed that protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed primarily of -kafirins with minor portions of - and -kafirins. The dark staining regions, however, are composed primarily of - and -kafirins. Immunoelectron microscopy showed that protein bodies in the peripheral endosperm contain predominantly a-kafirin with minor amounts of - and -kafirin. Central endosperm protein bodies are also predominantly -kafirin, but have a higher proportion of -kafirin and -kafirin than the peripheral endosperm protein bodies.Abbreviations GAR-HRP Goat anti-rabbit horseradish peroxidase - IgG immunoglobulin G - 2-ME 2-mercaptoethanol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffer saline - TBS-T Tris buffer saline with Tween - TBS-T-B Tris buffer saline with Tween and bovine serum albumin - TCA trichloroacetic acid - UV ultraviolet     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

Immunolocalization of abscisic acid by monoclonal antibodies in Lavandula stoechas L. leaves

A monoclonal antibody was used to localize abscisic acid in Lavandula stoechas L. plants treated with 1 M abscisic acid. ABA localization was performed using EDC as the only fixative, which preserves antigenicity and improves the specificity of monoclonal antibodies. A relationship was observed between the effect of abscisic acid on chloroplast ultrastructure and ABA accumulation. Gold particles accumulated on swollen chloroplasts. Our findings provide cytochemical evidence that the chloroplast is a trapping compartment, and yield valuable information on the relation between chloroplast ultrastructure and ABA accumulation.Abbreviations ABA abscisic acid - BSA bovine serum albumin - EDC 1-(3-dimethyl-aminopropyl)-3 ethyl carbodiimide - IgG immunoglobulin G - mAB monoclonal antibodies - pAB polyclonal antibodies     

Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth—a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkalinepH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such asTribolium castaneum andLocusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.     

Immunolocalization and histochemical evidence for the association of two different<Emphasis Type="Italic"> Arabidopsis</Emphasis> annexins with secretion during early seedling growth and development

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following ³H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.     

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.