Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

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     

Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination

In lily, adhesion of the pollen tube to the transmitting-tract epidermal cells (TTEs) is purported to facilitate the effective movement of the tube cell to the ovary. In this study, we examine the components of the extracellular matrices (ECMs) of the lily pollen tubes and TTEs that may be involved in this adhesion event. Several monoclonal antibodies to plant cell wall components such as esterified pectins, unesterified pectins, and arabinogalactan-proteins (AGPs) were used to localize these molecules in the lily pollen tube and style at both light microscope (LM) and transmission electron microscope (TEM) levels. In addition, (-d-Glc)₃ Yariv reagent which binds to AGPs was used to detect AGPs in the pollen tube and style. At the LM level, unesterified pectins were localized to the entire wall in in-vivo- and in-vitro-grown pollen tubes as well as to the surface of the stylar TTEs. Esterified pectins occurred at the tube tip region (with some differences in extent in in-vivo versus in-vitro tubes) and were evenly distributed in the entire style. At the TEM level, esterified pectins were detected inside pollen tube cell vesicles and unesterified pectins were localized to the pollen tube wall. The in-vivo pollen tubes adhere to each other and can be separated by pectinase treatment. At the LM level, AGP localization occurred in the tube tip of both in-vivo- and in-vitro-grown pollen tubes and, in the case of one AGP probe, on the surface of the TTEs. Another AGP probe localized to every cell of the style except the surface of the TTE. At the TEM level, AGPs were mainly found on the plasma membrane and vesicle membranes of in-vivo-grown pollen tubes as well as on the TTE surface, with some localization to the adhesion zone between pollen tubes and style. (-d-Glc)₃ Yariv reagent bound to the in-vitro-grown pollen tube tip and significantly reduced the growth of both in-vitro- and in-vivo-grown pollen tubes. This led to abnormal expansion of the tube tip and random deposition of callose. These effects could be overcome by removal of (-d-Glc)₃ Yariv reagent which resulted in new tube tip growth zones emerging from the flanks of the arrested tube tip. The possible roles of pectins and AGPs in adhesion during pollination and pollen tube growth are discussed.Abbreviations AGP arabinogalactan-protein - ECM extracellular matrix - Glc glucose - MAbs monoclonal antibodies - LM light microscope - Man mannose - TEM transmission electron microscope - TTE transmitting tract epidermal cell The authors thank Michael Georgiady for assistance with the preparation of material for the TEM immunolocalization, Diana Dang for her help with the pectinase experiment, and Kathleen Eckard for assistance in all aspects of this study. The MAbs were the generous gifts of Dr. J.P. Knox. G.Y. Jauh thanks Dr. E.A. Nothnagel for assistance in making the Yariv reagent and for the gift of the control (-d-Man)₃ Yariv reagent. This work is in partial fulfilment of the dissertation requirements for a PhD degree in Botany and Plant Sciences for G.Y. Jauh at the University of California, Riverside. This work was supported by National Science Foundation grant 91-18554 and an R.E.U. grant to E.M.L.     

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     

Loss of arabinogalactan-proteins from the plasma membrane of NaCl-adapted tobacco cells

Cultured cells of tobacco (Nicotiana tabacum L.) adapted to 428 mM NaCl exhibited a reduced rate of cell enlargement, which is probably due to decreased cell-wall extensibility. Arabinogalactan-protein (AGP) has been implicated as a cell-wall-loosening factor (Schopfer 1990). Levels of plasma membrane and extracellular AGPs that react with Yariv reagent were measured and compared between NaCl-adapted and unadapted tobacco cells. Unadapted cells contained a very high level of AGPs on the plasma membrane, which amounted to 0.16 g·g^–1 membrane protein. In contrast, AGPs were virtually undetectable on the plasma membrane of NaCl-adapted cells. Accumulation of AGPs was also decreased in culture media of NaCl-adapted cells. These data support the hypothesis that AGPs participate in cell expansion. Possible mechanisms of the proposed cell-expansion role of AGPs are discussed.Abbreviations AGP arabinogalactan-protein - S₀, S₂₅ cells un-adapted, NaCl-adapted tobacco cells This work was supported in part by a Mcknight Foundation fellowship to J.K.Z. This is journal paper No. 13,569 of Purdue University Agricultural Experimental Station. The authors thank Dr. Eugene A. Nothnagel for the Yariv reagent gift and for helpful discussion. The authors also thank Glenda McClatchey for excellent technical assistance.     

Characteristics of self-incompatibility in Schlumbergera truncata and S. x buckleyi (Cactaceae)

The influence of self-incompatibility (SI) on fruit set, seed set, and pollen tube growth was investigated in Schlumbergera truncata (Haworth) Moran and S.xbuckleyi (T. Moore) Tjaden. Four Schlumbergera clones were crossed in a complete diallel to verify the presence of SI. Fruit did not set when the clones were selfed or when two of the clones were crossed reciprocally, but all other outcrosses yielded fruit which contained 100–200 seeds each. Compatible outcrosses were characterized by large numbers of pollen tubes in the style and ovary cavity at 72 h after pollination. When pistils were selfed or incompatibly crossed, pollen tubes were inhibited in the upper third of the style and few pollen tubes reached the base of the style by 72 h after pollination. Schlumbergera exhibits several characteristics often associated with sporophytic SI systems (tricellular pollen and dry stigmas with elongate papillae), together with those commonly observed in gametophytic SI systems (stylar inhibition of incompatible pollen tubes and absence of reciprocal differences in outcrosses).Publication no. 3174 of the Massachusetts Agricultural Experiment Station     

Arabinogalactan-Proteins of the Female Sexual Tissue of Nicotiana alata: I. Changes during Flower Development and Pollination

Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed-electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination.     

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.     

Induction of homozygous Spathiphyllum wallisii genotypes through gynogenesis

In Spathiphyllum wallisii the production of doubled haploids was attempted. Different combinations of growth regulators were tested, as well as different cultivars. The use of TDZ (0.25–1 M) in ovary cultures of Spathiphyllum was required. On the contrary, cytokinins were not crucial during ovule culture; in fact, the use of a too high TDZ concentration induced diploid parthenogenesis in ovules of `Alfa'. The use of the imidazole fungicides IMA, PRO or TRI during ovary culture was not critical, though they enhanced the swelling of ovules during ovary culture, making the isolation of the ovules easier. The ovule cultures yielded different plantlets. Flow cytometry and AFLP-patterns showed that two doubled haploid genotypes could be obtained from `Stefanie'. These plants showed a normal phenotype. We concluded that the induction of homozygous Spathiphyllum through gynogenesis is possible and is genotype dependent.     

Pollen-tube growth and fertilization mode in<Emphasis Type="Italic"> Gymnostoma</Emphasis> (Casuarinaceae): their characteristics and evolution

A unique mode of fertilization called chalazogamy, whereby the pollen tube passes through the chalaza instead of the micropyle, is known in several species of derived genera in Casuarinaceae. In this paper we report the occurrence of chalazogamy in Gymnostoma (G. poissonianum), the most primitive genus in the family. We also show that the pollen tube grows discontinuously from the stigma to ovules in about 3 months. At the time of pollination, the ovules have not yet formed in the ovary, and require a long time to develop. The pollen tube(s) lie in a zigzag line and are branched in the upper region of the style, and their growth is arrested there until the ovary develops further. Studies of the relevant literature further revealed discontinuous pollen-tube growth in relation to a prolonged period between pollination and fertilization, as well as chalazogamy, in Betulaceae, Juglandaceae and/or Fagaceae that are closely related to Casuarinaceae. This feature may have derived early in the evolution of Fagales.     

Different kinds of male flowers in the dioecious plant Asparagus of officinalis L.

Summary In the dioecious plant Asparagus officinalis L. the female plants bear flowers that are all strictly of the same type, with well-developed pistils and collapsed and consistently sterile rudiments of anthers, while male plants, on the contrary, show a great variety of vestigial female organs, from small, rudimentary ovaries with no style and stigma, up to pistils provided with a rather long style that is often enlarged in a stigma. In our investigations, we used homozygous male and female doubled haploid plants obtained from in vitro anther culture, the all-male F₁ progeny and male individuals from subsequent backcrosses. The results showed that: (1) the character length of the style is genetically inherited and involves at least two genes, the influence of the environment being quite negligible; (2) in male pistils provided with style and stigmatic papillae, the pollination and growth of the pollen tubes up to the ovules do actually occur as a rule, the only barrier to fertilization being the absence of normal embryo sacs inside the ovules; (3) the character length of the style is a very reliable marker of the trend towards hermaphroditism in Asparagus, since a correlation always exist between length of the style, size of the ovary, tendency to self-pollination, vascularization and rate of development of the ovules inside the male ovaries. On the whole, most of our observations, together with the high inbreeding depression observed when occasional andromonoecious plants are selfed, are consistent with the hypothesis of the origin of dioecy in Asparagus from hermaphroditism via the gynodioecy pathway.     

Proteins produced by barley microspores and their derived androgenic structures promote in vitro zygotic maize embryo formation

Maize zygotes formed in planta were isolated and co-cultured with barley microspores. Evidence suggests that culture with microspores and/or conditioned media in barley promoted zygotic embryo formation. In order to characterise active substances present in the conditioned media, we collected medium at various times after the initiation of culture. We showed that proteins appeared over time and that their quantity increased during the course of the culture. Some proteins were glycosylated as revealed by the ConA peroxidase test. The use of the antigen -glucosyl Yariv reagent has shown that arabinogalactan-proteins (AGPs) were present. In addition, conditioned medium samples were analysed for their oligosaccharide content. New oligosaccharides appear in the course of the culture but they do not seem to affect development. We discuss in details the results in the context of understanding cell-cell interactions between embryo and nurse cells and the possible parallel with that occurs in ovulo between endosperm and embryo.     

Purification and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Petunia hybrida

We report isolation and N-terminal amino acid sequencing of three style glycoproteins, which segregate with three S (self-incompatibility) alleles of Petunia hybrida. The S-glycoproteins were expressed mainly in the upper part of the pistil and showed an increasing concentration during flower development. The glycoproteins were purified by a combination of ConA-Sepharose and cation exchange fast protein liquid chromatography. The amount of S-glycoproteins recovered from style extracts varied from 0.5 to 1.6 g per style, which was 40–60% of the amount recovered by a simplified analytical method. N-terminal amino acid sequences of S₁-, S₂- and S₃-glycoprotein showed homology within the fifteen amino terminal residues. These amino acid sequences were compared with the previously published sequences of S-glycoproteins from Nicotiana alata and Lycopersicon peruvianum.     

Analysis of the predetermining effect of a sex realizer by ovary transplantations in the monogenic flyChrysomya rufifacies

Summary In the heterozygousF/f female-producing females of the strictly monogenic blowflyChrysomya rufifacies the gene product of the dominant or epistatic female sex realizerF which causes sexpredetermination is thought to be synthesized either by cells of the germ line (oocytes, nurse cells or oogonia) or by somatic cells and then transferred into the oocytes. To determine the possible site of synthesis, reciprocal transplantations were made of prepupal ovaries between female-producing (thelygenic; t) and male-producing (arrhenogenic; a) females ofChrysomya rufifacies. In another series of experiments prepupal host females of the wild t-type and a-type were each provided with one additional ovary either from a0type (f/f) or a t-type (F/f) prepupa (neither were distinguishable by their phenotypes). In all these experiments the donor females were marked by the recessive sex-linked mutation white (w/w); white eyes, white Malpighian tubules). In a considerable number of cases the implanted ovaries were in contact with the host's own oviduct and grew normally, but the rate of hatched adults was somewhat reduced. Crosses between such host females andw/w males (f/f) produced female or male offspring with white eyes from the eggs of the implantedw/w ovary, as well as flies with wild-type eyes (+/w) which had developed in the host's own ovaries. In all cases so far examined, the genetically thelygenic (or arrhenogenic) host females with an additional ovary implanted from an arrhenogenic (thelygenic) donor, produced progeny of both sexes: sons (daughters) from the eggs of the donor's ovary and daugthers (sons) from the eggs of the host's own gonads.These results demonstrate that the ovaries of the t-females ofChrysomya rufifacies at least from the early prepupal stage, are autonomous for the product of theF gene. Transplantations of the premordial germ cells (pole cells) are planned to find out whether the predeterminingF gene product is synthesized before the prepupal stage, by somatic cells outside the ovary or by somatic (follicle) cells of the ovary itself.Dedicated to Professor Dr. Hans Bauer with gratitude in commemoration of his 75th birthday     

阿拉伯半乳糖蛋白在被子植物有性生殖中的作用

阿拉伯半乳糖蛋白(arabinogalactan-proteins,AGPs)是一类主要分布在细胞表面和胞外基质中的糖蛋白.它们在植物的雄性器官(花粉、花粉管、精细胞)、雌性器官(柱头、花柱、子房)和胚胎(合子胚和体细胞胚)等组织和细胞中均有大量的表达.大量研究表明AGPs在被子植物有性生殖过程中起着非常重要的作用,既可能参与花粉管粘附、营养、传导或提供信号的作用,也可能参与受精过程中配子识别和受精后胚胎的发育与分化等过程.该文就其分子结构、特性以及在植物有性生殖过程中各种器官和组织内的表达和功能研究进展做了较为全面的概述.     

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     

Extracellular matrix formation by amebocytes during epithelial regeneration in the pearl oysterPinctada fucata

Summary To identify the cells which produce the extracellular matrix during bivalve wound healing, we observed epithelial regeneration inPinctada fucata and evaluated the ability of amebocytes to produce the matrix in vitro. Between days 1 and 3 after an ovary was implanted with abiotic material (a shell ball) via an incision, agranular amebocytes formed a sheath, consisting of 10–20 cell layers, between the implant and incised ovarian tissue. Extracellular matrix was deposited in the spaces between the amebocytes in the sheath. At the incised follicle, gonadal epithelial cells were attached to the newly formed matrix. When a mantle allograft (2 mm square) was implanted with abiotic material to bring them into close contact, epithelial cells emigrated from the allograft along the surface of the abiotic material where they attached to the newly formed matrix at the sheath of amebocytes. In vitro, agranular amebocytes formed a matrix composed of fibrils with a diameter of 20 nm during a 6-day culture period. Pepsin-digested extract of the cell layer forming the matrix gave protein bands with electrophoretic mobilities identical to - and -sized components of a collagen purified from this animal. The matrix exhibited immunoreaction to antiserum raised against the collagen and was stained by alcian bluc. Thus, the agranular amebocyte apparently has the ability to produce an extracellular matrix containing collagen and possibly proteoglycan(s).     

Polyamines in grapevine microcuttings cultivated in vitro. Effects of amines and inhibitors of polyamine biosynthesis on polyamine levels and microcutting growth and development

The main free amines identified during growth and development of grapevine microcuttings of rootstock 41 B, (Vitis vinifera cv. Chasselas × Vitis berlandieri) cultivated in vitro were agmatine, putrescine, spermidine, spermine, diaminopropane and tyramine (an aromatic amine). Amine composition differed according to tissue, with diaminopropane the major polyamine in the apical parts, internodes and leaves. Putrescine predominated in the roots. There was also a decreasing general polyamine and specific tyramine gradient along the stem from the top to the bottom. Conjugated amines were only found in roots. The application of exogenous amines (agmatine, putrescine, spermidine, tyramine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these amines can be growth limiting. Diaminopropane (the product of oxidation of spermidine or spermine by polyamine oxydases) strongly inhibited microcutting growth and development. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), led to inhibition of microcutting development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition indicating that polyamines are involved in regulating the growth and development of grapevine microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis from ornithine decarboxylase (ODC), had no effect on microcutting development and growth. We propose that ADC regulates putrescine biosynthesis during microcutting development.     

Wide hybridization in Brassica

Summary Crosses were made to obtain interspecific hybrids between B. fruticulosa (wild species , 2n = 16) × B. campestris (cultivar , 2n = 20). Although many pollen grains germinated and their tubes entered the style, only about 30% of the ovules received pollen tubes. Fertilized ovules aborted at various stages of development. A few hybrid seeds resulted from hand pollinations in the field, and they showed poor germination and seedling establishment. The in vitro culture of ovaries, ovules, and seeds increased the frequency of obtaining hybrid seeds and plants: the most effective method was ovary culture followed by ovule culture. The hybrid nature of the plants was confirmed through morphological, cytological, and electrophoretic studies. A meiotic analysis of F₁ hybrids (2n = 18) showed that they had 0–5 bivalents and were completely pollen sterile. Electrophoretic analysis of leaf esterases and acid phosphatases of F₁ hybrids revealed bands derived from each parent. Induced amphidiploids of F₁ hybrids contained mostly bivalents, and had about 50% fertile pollen.     

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.