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     

Arabinogalactan proteins: actors or spectators during abiotic and biotic stress in plants?

Arabinogalactan proteins (AGPs) are a family of hydroxyproline-rich glycoproteins (HRGP) ubiquitous in the plant kingdom. They are probably one of the most heterogeneous and complex families of macromolecules, making them able to perform different and multiple functions. Located at the plasma membrane–cell wall interface, AGPs are involved in several processes, from plant growth and development to reproduction. An additional function of AGPs in response to biotic and abiotic stress has been suggested by several studies. The purpose of this review is to summarize critically and analytically the available knowledge on the effects of abiotic stress (low and high temperatures, drought, flooding, anoxia and metal deficiency/toxicity) and biotic stress (bacteria, fungi, nematodes and viruses) on AGPs. A deeper understanding of the role of AGPs during these conditions can be an important tool for understanding AGP biology and for the possible development of efficient breeding strategies.     

Synthesis of 3,6-branched arabinogalactan-type tetra- and hexasaccharides for characterization of monoclonal antibodies

Synthesis of tetra- and hexasaccharides built up from a β-(1→6)-linked galactopyranosyl backbone with arabinofuranosyl side chains at position 3 and with a 3-aminopropyl spacer related to arabinogalactans is described. These oligosaccharides were prepared for investigation of monoclonal antibodies raised against arabinogalactan proteins (AGPs) from pressed juice of Echinacea purpurea.     

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [¹⁴C]Gal from UDP-[¹⁴C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)₇ and anhydrous hydrogen fluoride-deglycosylated d(AO)₅₁. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [¹⁴C]Gal to (AO)₇, and the resulting product co-eluted with (AO)₇ by reverse-phase HPLC. Acid hydrolysis of the [¹⁴C]Gal-(AO)₇ product released ¹⁴C-radiolabel as Gal only. Base hydrolysis of the [¹⁴C]Gal-(AO)₇ product released a ¹⁴C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg²⁺ or Mn²⁺ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.     

Cadmium induces the expression of specific stress proteins in sea urchin embryos

Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a simple though significant model system to test how specific stress can simultaneously affect development and protein expression. Here, we used Paracentrotus lividus sea urchin embryos to study the effects of time-dependent continuous exposure to subacute/sublethal cadmium concentrations. We found that, between 15 and 24 h of exposure, the synthesis of a specific set of stress proteins (90, 72-70, 56, 28, and 25 kDa) was induced, with an increase in the rate of synthesis of 72-70 kDa (hsps), 56 kDa (hsp), and 25 kDa, which was dependent on the lengths of treatment. Recovery experiments in which cadmium was removed showed that while stress proteins continued to be synthesized, embryo development was resumed only after short lengths of exposure.     

Localization of arabinogalactan-proteins in different stages of embryos and their role in cotyledon formation of <Emphasis Type=Italic>Nicotiana tabacum</Emphasis> L.

Arabinogalactan proteins (AGPs) have been implicated in plant development including sexual plant reproduction. In this paper, the expression of AGPs and the effects of β-glucosyl Yariv reagent (βGlcY, which binds arabinogalactan proteins) in embryo development and cotyledon formation were investigated. Immunofluorescence assay displayed that the expression of AGPs labeled with antibody JIM13 was developmentally regulated. In early stages, AGPs were evenly distributed in the whole embryo, except for a short polar expression in the basal suspensor cell. In the globular stage of embryo, AGPs were condensed in the embryo proper (EP), apex of the EP, and at the juncture of the EP and suspensor. In heart-shaped embryo, APGs were only present at the juncture of the EP and suspensor. Immunogold labeling assay showed that the strong expression of AGPs at the juncture of the EP and suspensor was localized in the cell wall. Provision of βGlcY to the in vitro ovule culture medium caused delayed growth of embryos, cotyledon defect and abnormal venation pattern. Consequently, βGlcY induced the death of defective seedlings with the characteristics of deformed or irregular single cotyledon. Our results suggested that AGPs play functional roles in embryo development, cotyledon formation and seedling morphology establishment in Nicotiana tabacum L.     

Expression analyses of AtAGP17 and AtAGP19, two lysine-rich arabinogalactan proteins, in Arabidopsis

AtAGP17 and AtAGP19 are members of the lysine-rich arabinogalactan protein (AGP) subfamily in Arabidopsis. Detailed anatomical analysis of promoter activity of the AtAGP19 gene was carried out using transgenic Arabidopsis plants expressing a P(AtAGP19):GUS fusion. AtAGP19 promoter activity was tissue-specific and associated with vascular bundles, particularly differentiating xylem elements. Peptide-specific antibodies were raised against the Lys-rich regions of AtAGP17 and AtAGP19 and used to study the organ-specific expression patterns of these two AGPs. AtAGP17 and AtAGP19 were most abundant in roots and flowers, moderately abundant in stems, seedlings and siliques and virtually absent in leaves. Antibodies specific for AtAGP17 and AtAGP19, as reported here, represent valuable tools for understanding the biology of these two AGPs.     

Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies)

The morphology of somatic embryos of Norway spruce ( Picea abies ) varies among different cell lines, from less developed somatic embryos with small embryonic regions (group B) to well developed embryos with large embryonic regions (group A). Only well developed somatic embryos will undergo a maturation process after a treatment with ABA and develop into mature somatic embryos, which is required for plant regeneration. We have previously shown that the presence of specific extracellular proteins can be correlated with the morphology of the somatic embryos. In the present study we show that extracellular proteins concentrated from group A cell lines can stimulate group B embryos to develop further and that seed extract can stably convert B embryos into A embryos. The arabinogalactan protein (AGP) fraction of the extracellular proteins and of the seed extract was shown to be an active component for stimulating B embryos to develop further. Furthermore, the amount and type of extracellular AGPs, as detected with β-glucosyl Yariv reagent and monoclonal antibodies, varied among different types of tissues and cell lines. The data show that development of somatic embryos in Norway spruce is associated with particular extracellular AGPs, which have a regulatory function.     

Hypocotyl growth of Pinus pinaster seedlings. Changes in the molecular weight distribution of hemicellulosic polysaccharides

Lorences, E. P., Suárez, L. and Zarra, I. 1987. Hypocotyl growth of Pinus pinaster seedlings. Changes in the molecular weight distribution of hemicellulosic polysaccharides.
The changes in the molecular weight distribution of water-soluble hemicelluloses and xyloglucan during hypocotyl growth of intact seedlings of Pinus pinaster Aiton were investigated. The mass-average molecular weight of total polysaccharides of the hemicellulose fraction soluble in 4% KOH dramatically increased during hypocotyl growth while xyloglucan slightly decreased. These phenomena were due to an increase in the degree of polymerization of an arabinogalactan and a slight depolymer-ization in the xyloglucan present in this fraction. In the hemicellulose fraction soluble in 24% KOH, xyloglucan increased its degree of polymerization from day 7 to 10 after which it decreased slightly. The xyloglucan of the hemicellulose fraction soluble in 4% KOH may thus be involved in cell wall loosening which makes cell wall expansion possible during hypocotyl growth.     

Syntheses of beta-(1-->6)-branched beta-(1-->3)-linked d-galactans that exist in the rhizomes of Atractylodes lancea DC

Effective syntheses of galactose hepta-, octa-, nona-, and decasaccharides that exist in the rhizomes of Atractylodes lancea DC were achieved with 2,3,4,6-tetra-O-benzoyl-alpha-d-galactopyranosyl trichloroacetimidate (1), 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-d-galactopyranoside (2), 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-d-galactopyranosyl trichloroacetimidate (5), 4-methoxyphenyl 6-O-acetyl-2,4-di-O-benzoyl-beta-d-galactopyranoside (22), and 4-methoxyphenyl 2,4,6-tri-O-benzoyl-beta-d-galactopyranoside (26) as the key synthons. Coupling of 2 with 1, followed by oxidative cleavage of 1-OMP and subsequent trichloroacetimidate formation gave the beta-(1-->6)-linked disaccharide donor 4. Condensation of 2 with 5 and subsequent selective deacetylation by methanolysis produced the beta-(1-->6)-linked disaccharide acceptor 7. Reaction of 7 with 4, oxidative cleavage of 1-OMP, and trichloroacetimidate formation produced the tetrasaccharide donor 9. The penta- (15), the hexa- (17), and the heptasaccharide donor 19 were synthesized similarly. Meanwhile, treatment of 1 with 22 yielded beta-(1-->3)-linked disaccharide 23 and alpha-(1-->3)-linked disaccharide 25. Oxidative cleavage of 1-OMp of 23 followed by trichloroacetimidate formation produced the disaccharide donor 24. Coupling of 26 with 24, again, gave beta-linked 27 and alpha-linked 29. Selective 6-O-deacetylation of 27 afforded the trisaccharide acceptor 28. TMSOTf-promoted condensation 28 of with the tetra- (9), penta- (15), hexa-(17), and heptasaccharide donor 19, followed by deprotection, gave the target compounds.     

Divergent selection for in vitro developmental capacity of preimplantation mouse embryos

Summary Replicated divergent selection was conducted for two generations in ICR mice for in vitro developmental capacity (IVDC; percentage of fertilized one-cell zygotes developing to blastocysts in vitro per female donor). Realized heritabilities based on high and low selection were 0.03±0.08 and –0.11±0.09 in replicate 1, and 0.10±0.11 and 0.08±0.10 in replicate 2. No differences were detected between selection lines (P>0.2) or replicates (P>0.1). Estimate of heritability in the base population based on 332 daughter-dam pairs was 0.14±0.18. These results indicate that additive genetic variance contributes little to the phenotypic variance in this trait. Considerable phenotypic variation in IVDC was observed (mean=49.3; SD=31.0), with a range of IVDC from 0%–100%. Utilization of donor female as a blocking factor is suggested for designs of experiments with preimplantation embryos to increase precision and power of statistical analyses.     

枣合子胚和体细胞胚发育过程的观察与比较

本实验对临猗梨枣、壶瓶枣、晋矮1号等13个品种的枣胚的发育过程进行了观察,并诱导晋矮1号成熟胚的愈伤组织通过体细胞胚发生途径形成再生植株。结果表明:体细胞胚产生于愈伤组织的表层细胞或内部细胞。在鱼雷胚期已有导管的分化,子叶期的维管组织呈“Y”形。枣合子胚及体细胞胚的发育均经历了原胚、球形胚、心形胚、鱼雷胚和子叶胚五个时期。大多数品种的枣胚从球形胚期或心形胚期即开始败育,只有极少数品种可发育到成熟胚,而且合子胚形成的能力、胚败育时发育的程度等均存在着大的品种间差异,同一品种甚至同一子房内胚的发育进程也不同步。