Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an α zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.     

Scanning electron microscopy in nematode-induced giant transfer cells.

A study of giant cells induced by the root-knot nematode, Meloidogyne incognita, in roots of Impatiens balsamina was made by scanning electron microscopy. The cytoplasmic contents of giant cells were removed by a procedure based on KOH digestion, to reveal inner wall structure. Wall ingrowths typical of transfer cells are present in giant cells from six days onwards after induction. They develop on walls adjacent to vascular tissues, and their distribution and development was examined. Pit fields contianing plasmodesmata become elaborated in walls between giant cells, but pit fields are lost between giant cells and cells outside them. The distribution of plasmodesmata in pit fields suggests that de novo formation of plasmodesmata occurs in walls between giant cells. Various aspects of giant cell formation and function are discussed and wall ingrowth development is compared in giant cells and normal transfer cells.     

Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs

Summary. The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47–50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.Received January 3, 2003; accepted February 7, 2003; published online August 26, 2003     

Purification, sequencing and functions of calreticulin from maize

The most abundant proteins in the lumen of the endoplasmic reticulum(ER) are thought to be molecular chaperones, some of which mightalso be involved in calcium storage and release. We have purifiedcalreticulin from maize by ion exchange and reverse-phase chromatography.Identity with plant and animal calreticulins was confirmed byN-terminal amino acid sequencing and it was shown to bind calciumwith a calcium overlay technique. An antiserum raised to thepurified protein was used to screen an expression library andthe full coding sequence for maize calreticulin was determinedfrom the clones selected. The sequence shows 96% identity tobarley calreticulin and 55% identity to animal calreticulins.The three major functional regions are conserved, as are targetingand retention features. When visualized by indirect immunofluorescencemicroscopy, calreticulin was found to be confined to the ERand nuclear envelope of maize root cells. It was distributedthroughout the ER compartment and we found no evidence of calreticulin-enriched areas of ER, such as might be associated with specializedcalcium storage domains. Increasing or decreasing extracellularcalcium did not induce measurable changes in calreticulin levels.In addition, maize calreticulin, as well as other recognizedchaperones, was shown to bind to denatured protein and couldbe eluted specifically by nucleoside trisphosphates. Key words: Endoplasmic reticulum, calcium-binding protein, immunofluorescence, targeting, Zea mays L     

Plasmodesmata in onion (Alliurn cepa L.) roots: a study enabled by improved fixation and embedding techniques

Summary Onion (Allium cepa L. cv. Ebeneezer) roots from vermiculite culture were examined with transmission electron microscopy to detect the plasmodesmata in all tissues. In young root regions, plasmodesmata linked all living cells together in all directions. In old zones, the plasmodesmatal connections of the endodermis to its neighbor tissues were not interrupted by later suberin lamella and cellulosic wall deposition. Moreover, plasmodesmata in the fully mature endodermis usually exhibited a large central cavity. In the exodermis, however, upon deposition of suberin lamellae in long cells, all plasmodesmata that initially linked them to their adjacent cells were severed. Afterwards, the long cells lost the capability of forming wound pit callose and their protoplasts began to degenerate. The mature exodermal layer was symplastically bridged to its neighbors only by the short (passage) cells that lacked suberin lamellae. Compared to the long cells, the short cells not only had thicker cytoplasm surrounding their central vacuoles but also a higher density of mitochondria and rough endoplasmic reticulum, consistent with an active involvement in the transport processes of the root. The above results were obtained by an improved, extended transmission electron microscopy procedure devised to analyze plasmodesmata in cells with suberin lamellae. By prefixing root tissues in glutaraldehyde and acrolein, all cells were well preserved. Postfixation was carried out in osmium tetroxide at a low concentration (0.5%). Following dehydration in acetone and transfer to propylene oxide, infiltration with Spurr's resin was accomplished by incubating samples in the accelerator-free mixture for 4 days, then infiltrating samples in the accelerator-amended mixture for additional 4 days.Abbreviations IE immature exodermis - ME mature exodermis - TBO toluidine blue O - TEM transmission electron microscopy     

Rapid detection of plasmodesmata in purified cell walls

Summary Plasmodesmata are complex channels within the plant cell wall, which create plasma membrane and symplastic continuity between neighbouring cells. To detect plasmodesmata in cell wall preparations fromNicotiana cle elandii, we have used 3,3-dihexyl-oxacarbocyanine iodide (DiOC₆), a cationic amphiphilic fluorescent probe, widely employed for general studies of membrane structure and dynamics. Punctate fluorescent staining was readily seen in pit fields, small depressions within the cell wall known to be rich in plasmodesmata. Scanning electron microscopy was used to demonstrate that the punctate staining corresponded to plasmodesmata. Treatment of cell wall fragments with chloroform-methanol to remove lipids did not alter the staining of plasmodesmata. In contrast, pronase E-sodium dodecyl sulfate treatment completely abolished staining, indicating that the DiOC₆ labelling of plasmodesmata may be protein rather than lipid specific. Although not membrane mediated, DiOC₆ staining of plasmodesmata is a simple, rapid, and specific tool for the detection of plasmodesmata in isolated cell walls and will prove useful for studies of plasmodesmal location, structure, and composition.     

The fine structure of the cells that perceive gravity in the root tip of maize

Summary Within the root cap, in maize, the cells believed to be responsible for the perception all possess large well-developed amyloplasts. They also have normal mitochondria and Golgi bodies, normal rough-surfaced ER with a very striking pattern of distribution, few free ribosomes, walls with an abnormal reticulate encrusting material, irregularly distributed plasmodesmata and an as yet unidentified fine quadruple membranous system. All of these features are discussed in relation to the role of the cells in perception.     

Proteins reacting with cadherin and catenin antibodies are present in maize showing tissue-, domain-, and development-specific associations with endoplasmic-reticulum membranes and actin microfilaments in root cells

Summary With heterologous antibodies raised against animal N-cadherin, -catenin, and -catenin, we have visualized their reactive proteins within cells of maize root apices. Embedding using Steedman's wax allowed us to accomplish tissue-specific analysis which revealed that cells of epidermis, endodermis/pericycle, and outer stele tissues, all of which are tightly associated to each other, are especially enriched with presumed plant homologues of N-cadherin and both catenins. In the root epidermis, trichoblasts initiating root hairs showed prominent accumulations of cadherin-like antigens at outgrowing domains where they co-localize with actin. Close associations of cadherin-like proteins with F-actin were detected in parenchymatic cells of the stele, also at the immunogold electron microscopy level. A possible role of these interesting proteins in membrane-membrane interactions is indicated by their prominent accumulations at endoplasmic-reticulum-enriched pit-field-based plant cell adhesion domains in plasmolyzing cells of maize root apices exposed to mannitol. Intriguingly, these unique adhesion domains of plasmolyzing cells are enriched with endoplasmic-reticulum-resident calreticulin. Cadherin-like, but not catenin-like, proteins were abundant also within the nucleoplasm.Abbreviations AGPs arabinogalactan proteins - EM electron microscopy - ER endoplasmic reticulum - MFs microfilaments - SB stabilizing buffer     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

Monitoring endoplasmic reticulum-to-Golgi traffic of a plant calreticulin by protein glycosylation analysis

Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.     

Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention

Using pulse-chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post-endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan-related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs.     

Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges

Plant root hair formation is initiated when specialized elongating root epidermis cells (trichoblasts) assemble distinct domains at the plasma membrane/cell wall cell periphery complexes facing the root surface. These localities show accumulation of expansin and progressively transform into tip-growing root hair apices. Experimentation showed that trichoblasts made devoid of microtubules (MTs) were unaffected in root hair formation, whereas those depleted of F-actin by the G-actin sequestering agent latrunculin B had their root hair formation blocked after the bulge formation stage. In accordance with this, MTs are naturally depleted from early outgrowing bulges in which dense F-actin meshworks accumulate. These F-actin caps remain associated with tips of emerging and growing root hairs. Constitutive expression of the GFP-mouse talin fusion protein in transgenic Arabidopsis, which visualizes all classes of F-actin in a noninvasive mode, allowed in vivo confirmation of the presence of distinct F-actin meshworks within outgrowing bulges and at tips of young root hairs. Profilin accumulates, at both the protein and the mRNA levels, within F-actin-enriched bulges and at tips of emerging hairs. ER-based calreticulin and HDEL proteins also accumulate within outgrowing bulges and remain enriched at tips of emerging hairs. All this suggests that installation of the actin-based tip growth machinery takes place only after expansin-associated bulge formation and requires assembly of profilin-supported dynamic F-actin meshworks.     

The quality control of MHC class I peptide loading

The assembly of major histocompatibility complex (MHC) class I molecules is one of the more widely studied examples of protein folding in the endoplasmic reticulum (ER). It is also one of the most unusual cases of glycoprotein quality control involving the thiol oxidoreductase ERp57 and the lectin-like chaperones calnexin and calreticulin. The multistep assembly of MHC class I heavy chain with beta(2)-microglobulin and peptide is facilitated by these ER-resident proteins and further tailored by the involvement of a peptide transporter, aminopeptidases, and the chaperone-like molecule tapasin. Here we summarize recent progress in understanding the roles of these general and class I-specific ER proteins in facilitating the optimal assembly of MHC class I molecules with high affinity peptides for antigen presentation.     

Immunocytochemical localisation of auxin-binding proteins in coleoptiles and embryos ofZea mays L.

Summary The auxin-binding protein ABP-1 was localised immunocytochemically in coleoptiles and immature embryos ofZea mays. Two primary polyclonal antibodies raised against ABP-1 and secondary antibodies were either labelled with FITC or 10 nm gold particles for light microscopy, and with 10 nm gold particles for transmission electron microscopy. Light microscopy revealed that ABP-1 was localised in the epidermal cells of etiolated maize coleoptiles, in subepidermal parenchymatic mesophyll cells of the coleoptile and in the companion cells of the vascular bundles. Most labelling was found in the cytoplasm, less in nuclei and vacuoles and cell walls appeared negative. The region of the plasma membrane exhibited prominent labelling. Embryos showed low labelling throughout their tissues just after excision, but after culture for 7 days intensive labelling was found in the epidermis of the scutellum. Quantitative electron microscopy confirmed that ABP-1 was present in the cytoplasm of epidermal, mesophyll, and companion cells of coleoptiles. Gold particles were neither found in cell walls nor in the cuticle. Areas with ER and dictyosomes within epidermal and mesophyll cells of coleoptiles had a denser labelling with gold particles than elsewhere. Labelling at the plasma membrane, being the site where the auxin binds to the ABP, was observed at low levels in all cells examined, which is due to the method applied. Epidermal cells of embryos cultured for 5 days exhibited high levels of gold particles in ER and nuclei, and lower levels in the cytoplasm. The distribution is only partly in accordance with the model in which ABP is thought to cycle through the plant cell from the ER via the Golgi system towards the plasma membrane.Abbreviations ABP-1 auxin-binding protein 1 - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - LM light microscopy - LR Write London resin white - PBS phosphate-buffered saline - PEG polyethylene glycol - TEM transmission electron microscopy     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

ULTRASTRUCTURE OF THE SUBGLANDULAR CELLS FROM THE FOLIAR NECTARIES OF COTTON IN RELATION TO THE DISTRIBUTION OF PLASMODESMATA AND THE SYMPLASTIC TRANSPORT OF NECTAR

Foliar nectaries on the midveins of 7-cm leaves from cotton (Gossypium hirsutum L., cv. Stoneville 213) were examined by light and electron microscopy. The nectaries consist of external multicellular papillae and internal subglandular tissue that extends from the bases of the papillae to the vascular tissue of the midveins. The subglandular tissue is composed of small parenchyma cells; it does not contain sieve elements or xylem vessels. The parenchyma cells are rich in mitochondria, and their walls contain numerous pit fields having a high concentration of plasmodesmata. The absence of vascular tissue and the significance of the pit fields in the subglandular tissue are discussed in relation to symplastic transport of nectar secretions.     

Closure of plasmodesmata in maize (Zea mays) at low temperature: a new mechanism for inhibition of photosynthesis

Background and Aims

Photosynthesis is one of the processes most susceptible to low-temperature inhibition in maize, a tropical C4 crop not yet fully adapted to a temperate climate. C4 photosynthesis relies on symplasmic exchange of large amounts of photosynthetic intermediates between Kranz mesophyll (KMS) and bundle sheath (BS) cells. The aim of this study was to test the hypothesis that the slowing of maize photosynthesis at low temperature is related to ultrastructural changes in the plasmodesmata between KM and BS as well as BS and vascular parenchyma (VP) cells.

Methods

Chilling-tolerant (CT) KW 1074 and chilling-sensitive (CS) CM 109 maize (Zea mays) lines were studied. The effect of moderate chilling (14 °C) on the rate of photosynthesis, photosynthate transport kinetics, and the ultrastructure of plasmodesmata linking the KMS, BS and VP cells were analysed. Additionally, the accumulation of callose and calreticulin was studied by the immunogold method.

Key Results

Chilling inhibited photosynthesis, photosynthate transfer to the phloem and photosynthate export from leaves in both lines. This inhibition was reversible upon cessation of chilling in the CT line but irreversible in the CS line. Simultaneously to physiological changes, chilling induced swelling of the sphincters of plasmodesmata linking KMS and BS cells and a decreased lumen of the cytoplasmic sleeve of plasmodesmata at the BS/VP interface in the CS line but not in the CT line. Accumulation of calreticulin, which occurred near the neck region of the closed plasmodesmata was observed after just 4 h of chilling and over-accumulation of callose at the KMS/BS and BS/VP interfaces occurred after 28 h of chilling.

Conclusions

Stronger chilling sensitivity of the CM 109 maize line compared with the KW 1074 line, shown by decreased photosynthesis and assimilate export from a leaf, is related to changes in the ultrastructure of leaf plasmodesmata at low temperature. The chain of reactions to chilling is likely to include calreticulin action resulting in rapid and efficient closure of the plasmodesmata at both KMS/BS and BS/VP interfaces. Callose deposition in a leaf was a secondary effect of chilling.     

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.