Increases in binding protein (BiP) accompany changes in protein body morphology in three high-lysine mutants of maize

Summary A maize 75 kDa protein recently has been identified as a plant homolog of the mammalian binding protein (BiP). To better understand the function of BiP in protein body formation in maize endosperm, immunomicroscopy studies were conducted on three maize endosperm mutants, floury-2, Mucronate, and Defective endosperm-B 30, in which the level of BiP is highly elevated. Our results showed that protein body morphology in all three mutants was altered. In addition, BiP was localized in both the ER and peripheral regions of the abnormal protein bodies. The degree to which protein body morphology differed from normal was positively correlated with increased amounts of BiP. In addition, the accumulation of BiP in abnormal protein bodies increased with protein body maturation. In the three endosperm mutants, the arrangement of zeins within protein bodies had been perturbed, yet none of the specific zein subclasses exhibited the staining pattern found for BiP. The association of BiP with abnormal packaging of proteins in protein bodies may reflect a biological function to mediate protein folding and assembly in maize endosperm.Abbreviations BiP binding protein - BSA bovine serum albumin - De*-B 30 Defective endosperm B 30 - DAP day after pollination - ER endoplasmic reticulum - fl 2 floury-2 - hsp 70 70 kDa heat shock protein - Mc Mucronate - TBST 20mM Tris-HCl, pH8.2 at 20°C, 500mM NaCl, 0.3% Tween 20 - TBST-B TBST with 1% (w/v) BSA     

Involvement of JIM13- and JIM8-Responsive Carbohydrate Epitopes in Early Stages of Cell Wall Formation

Beta vulgaris L.). The spatial and temporal expression of both antigens was studied in suspension cells used as the source-tissue for protoplast isolation, in suspension- and mesophyll-derived protoplasts, and in cells which developed from both types of protoplast. Immunofluorescence and immunocytochemical-electron microscopic methods revealed that labeling was present in the cell walls of most suspension cells and also in the incipients of cell walls synthesized around the protoplasts. This signal became much more intense as rebuilding of the cell wall progressed during culture. Relatively weaker labeling was observed in the cytoplasm, where it was frequently associated with the vacuolar compartment. Signal intensity varied between individual cells of the same population and in successive stages of development, but was always stronger with JIM13 than with JIM8. The role of JIM13-responsive epitope in the development of suspension-derived protoplasts was further studied by its ability to bind antibody added to cultures of different ages. Both JIM8- and JIM13-responsive epitopes were widespread in sugar beet cells of different origin and stage of cell wall synthesis. These epitopes may play an important role in cell wall formation and growth under in vitro conditions. Received 17 August 1998/ Accepted in revised form 13 January 1999     

Distribution of the extended family of protein kinase C isoenzymes in fetal organs of mice: an immunohistochemical study

Using isoenzyme-specific antisera, we have studied the distribution of protein kinase C isoforms in fetal mouse organs at the developmental age of 17 days. Two different sets of antibodies, produced by different manufacturers, were employed in this study. The specificity of the antisera was tested by immunoblotting experiments using whole fetal mouse extracts. Immunohistochemistry was carried out by means of an alkaline phosphatase-conjugated secondary antibody. Analysis of fetal mouse longitudinal cryostat sections stained with the antibodies demonstrated a distinct distribution of protein kinase C isoforms in the tissues. Protein kinase C- and C-I were present in all tissues examined, whereas the C-II isoform was absent in the lung and the liver. Protein kinase C- was identified in brain, spinal ganglia, and adrenal gland. The C- isoenzyme was abundantly expressed in spinal ganglia and in the smooth muscle cells of the bronchial wall. Antisera to C- and C- isoforms heavily stained liver, kidney, and spinal ganglia, whereas the C- isozyme was mainly detected in brain, stomach and kidney. Thus, protein kinase C-, C-I, C-II, C-, C- and C- were the isoforms present in many of the organs investigated. The two sets of antibodies gave slightly different results that might be ascribed to the different epitopes recognized by the antisera. One set of antisera was employed to investigate the distribution of the isoforms in selected organs from an earlier developmental age (15 days) and from adult animals. Both qualitative and quantitative differences were seen in comparison with the same organs from a 17-day fetus.     

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.     

Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells

Members of the Pinaceae family have complex chemical defense strategies. Conifer defenses associated with specialized cell types of the bark involve constitutive and inducible accumulation of phenolic compounds in polyphenolic phloem parenchyma cells and oleoresin terpenoids in resin ducts. These defenses can protect trees against insect herbivory and fungal colonization. The phytohormone ethylene has been shown to induce the same anatomical and cellular defense responses that occur following insect feeding, mechanical wounding, or fungal inoculation in Douglas fir (Pseudotsuga menziesii) stems (Hudgins and Franceschi in Plant Physiol 135:2134–2149, 2004). However, very little is known about the genes involved in ethylene formation in conifer defense or about the temporal and spatial patterns of their protein expression. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the final step in ethylene biosynthesis. We cloned full-length and near full-length ACO cDNAs from three conifer species, Sitka spruce (Picea sitchensis), white spruce (P. glauca), and Douglas fir, each with high similarity to Arabidopsis thaliana ACO proteins. Using an Arabidopsis anti-ACO antibody we determined that ACO is constitutively expressed in Douglas fir stem tissues and is up-regulated by mechanical wounding, consistent with the wound-induced increase of ethylene levels. Immunolocalization showed cytosolic ACO is predominantly present in specialized cell types of the wound-induced bark, specifically in epithelial cells of terpenoid-producing cortical resin ducts, in polyphenolic phloem parenchyma cells, and in ray parenchyma cells.J.W. Hudgins and Steven G. Ralph contributed equally to this work.     

Changes in the cellular localization of estrogen receptor alpha in the growing and regressing ovaries of Gallus domesticus during development

In this work, the presence of estrogen receptor alpha (ER-α) was determined in different cell subpopulations in the left growing and right regressing ovaries of Gallus domesticus from 13-day-old chicken embryos to one-month-old chickens by immunohistochemistry. Results revealed positive ER-α immunostaining in both ovaries during development, but the percentage, staining intensity, and cellular distribution of ER-α immunostaining changes according to whether it is the left or right ovary and with the animal’s age. In the left ovary, the ER-α was localized in the nuclei of the germinal epithelium and in germ cells of the ovarian cortex, as well as in the interstitial cells, undifferentiated cells, and epithelial cells of the lacunar channels of the ovarian medulla in all ages. In contrast, in the right ovary from 13-day-old chicken embryos to one-week-old chickens, only the epithelial cells of lacunar channels were ER-α immunoreactive, but in the right ovary of one-month-old chickens both the epithelial cells of lacunar channels and the interstitial cells presented ER-α. These results demonstrate differential expression of ER-α in both chicken ovaries during development in a cell type-specific distribution, suggesting that these differences may be regarded as an important cause in the process of asymmetric ovarian development in the chicken.     

Structure of maize protein bodies and immunocytochemical localization of zeins

Summary Zeins, the seed storage proteins of maize (Zea mays L.), are synthesized by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum in developing endosperm, where they assemble into protein bodies. To better understand the organization of protein bodies and the mechanism by which zeins are assembled, we have used immunolocalization to study their distribution within isolated protein bodies. In sections stained with uranyl acetate and lead citrate, the protein body matrix consists of light- and dark-staining regions with the darker stain predominating at the periphery and the lighter stain in the central region. Immunogold staining of the storage proteins in isolated protein bodies reveals a distinct segregation with -zein localized in the light-staining region and - and -zein localized in the dark-staining regions. However, the relative amounts and distribution of these proteins varies substantially among different protein bodies. These results indicate a more complex internal organization than has been previously observed, and suggest that spatial and/or temporal differences in zein synthesis account for this complexity.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - PB phosphate buffer - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TTBS Tween-20/tris-buffered saline - TBS-T Tris-buffered saline/Tween-20 - TBS-T-B Tris-buffered saline/Tween-20/bovine serum albumin     

Cytochemical immunolocalization of the abundant pistil protein Sk2 in potato (Solanum tuberosum)

S_k2 protein is the most abundant member of the pistil-specific proteins of Solanum tuberosum. S_k2 protein has been localized by use of a polyclonal antibody (anti-S_k2) in the pistils of four clones of Solanum tuberosum. In the stigmas S_k2 protein accumulates to a high level in the cytoplasm of the internal secretory cells underlying the papillae one day prior to anthesis. In styles, the intercellular matrix of the transmitting tissue cells is intensely labelled by anti-S_k2. S_k2 protein is present in all four clones and shows the same labelling pattern. The possible role of the S_k2 protein in pollen tube growth is discussed.     

Dynein heavy chain-related polypeptides are associated with organelles in pollen tubes of Nicotiana tabacum

 It is known that pollen tubes contain two high molecular weight polypeptides which share some biochemical and immunological properties with dynein heavy chains. This paper reports data on the subcellular localization of the two dynein heavy chain-related polypeptides during pollen tube growth. Immunofluoresence studies using a purified antibody (Dy-1) raised against a synthetic peptide reproducting the P-loop conserved sequence of dynein heavy chains showed spot-like structures, with a characteristic distribution pattern that depended on the tube length. Biochemical evidence confirmed the presence of dynein heavy chain-related bands in the pollen tube membrane fraction. The association of proteins carrying dynein heavy chain-related polypeptides to cell membranes was affected by detergent (Triton×100), whereas other stripping agents, like NaCl and Na₂CO₃, did not significantly influence the interaction of dynein heavy chain-related doublet with their cytoplasmic targets. These data suggest that dynein heavy chain-related polypeptides associate with membranous organelles within the vegetative cell of Nicotiana tabacum pollen tubes, implying their involvement in the cytoplasmic distribution of these organelles. Received: 22 May 1997 / Revision accepted: 11 November 1997