Artificially-induced organelles are optimal targets for optical trapping experiments in living cells.

Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads.

The bifactorial endosperm box of gamma-zein gene: characterisation and function of the Pb3 and GZM cis-acting elements.

The proximal region of the gamma-zein promoter (gamma Z) has a functional bifactorial prolamin box element containing two cis-acting elements, a prolamin-box motif (Pb3) and a GCN4-like motif (GZM). By particle bombardment of maize endosperms with 5' deletions and internal deletions of gamma Z fused to the GUS gene, we have shown that a 135 bp region containing the bifactorial element is involved in the transcriptional activation of the gamma Z promoter. However, the 135 bp region was unable to activate the gamma Z promoter in the absence of a 84 bp downstream sequence. Using in vivo footprinting and gel mobility shift assays with 15 DAP endosperm nuclear extracts, we have demonstrated the presence of trans-acting factors that interact with Pb3 and GZM target sites. Base-substitution mutations within Pb3 and GZM decreased transcription activity of the gamma Z promoter suggesting a co-ordinated function between the two cis-acting elements. Two additional cis-motifs upstream of the bifactorial prolamin element have been identified: a motif with high homology to the AACA elements of rice glutelin genes and an AZM motif containing an ACGT core which binds nuclear proteins other than the Opaque 2 (O2).

Expression of a gene encoding a ribosomal p40 protein and identification of an active promoter site.

The promoter of a gene encoding a ribosome-associated protein of 40 kDa from Arabidopsis thaliana (A-p40) was sequenced and the expression of the gene studied. A-p40 was expressed in the same organs and with the same variations as the eukaryotic elongation factor 1 alpha (eEF1A), another gene coding for a protein involved in translation Arabidopsis plants transformed with a beta-glucuronidase (GUS) gene driven by the A-p40 promoter confirm that A-p40 is expressed in actively dividing and growing cells. eEF1A promoter-GUS fusions have the same pattern of expression. Comparison of cis-acting elements from A-p40 and eEF1A revealed some common elements. A-p40 promoter deletions and transient gene expression in transfected Arabidopsis protopasts allowed the identification of trap40, a cis-acting element regulating gene expression. Gel retardation experiments indicate that eEF1A and A-p40 are regulated by different cis-acting elements. The role of such elements is discussed.

CD of proline-rich polypeptides: application to the study of the repetitive domain of maize glutelin-2.

An overview of CD of proline-rich peptides is reported. First, structural characteristics, theoretical CD studies, and the biological relevance of polyproline II structure in such peptides are discussed. Second, a CD study of peptides belonging to the repetitive domain of maize glutelin-2, H-(Val-His-Leu-Pro-Pro-Pro)n-OH (n = 3, 5, 8), is described. This series of peptides displayed the CD features of polyproline II structure in water (5 degrees C, pH 5). Moreover, it was shown that the addition of increasing amounts of the polyanionic molecule heparin forced a displacement of the conformational equilibrium of those peptides toward higher proportions of the polyproline II structure. In contrast, when the temperature is raised such a structure gradually disappears, leading to more disordered conformations.

RNA binding characteristics of a 16 kDa glycine-rich protein from maize.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.

Accumulation of cell wall hydroxyproline-rich glycoprotein mRNA is an early event in maize embryo cell differentiation.

The accumulation of the mRNA coding for a hydroxyproline-rich glycoprotein (HRGP), an abundant component of the wall from the cells of vegetative tissues, has been observed in maize embryo by in situ hybridization. The HRGP mRNA accumulates in the embryo axis and not in the scutellum and preferentially in dividing and provascular cells. The histone H4 mRNA is distributed in similar tissues but is restricted to defined groups of cells, indicating that these two gene products have a different steady-state level of accumulation during the cell cycle. The HRGP mRNA appears to be a useful marker for early formation of the vascular systems. The mRNA accumulation correlates in space and time with cells having a low content of cellulose in their walls, suggesting that the mRNA is produced in the early stages of cell wall formation before complete deposition of cellulose.

Differential Protein Accumulation in Banana Fruit during Ripening.

Banana (Musa acuminata, cv Dwarf Cavendish) proteins were extracted from pulp tissue at different stages of ripening and analyzed by two-dimensional electrophoresis. The results provide evidence of differential protein accumulation during ripening. Two sets of polypeptides have been detected that increase substantially in ripe fruit. These polypeptides were characterized as glycoproteins by western blotting and concanavalin A binding assays. Antibodies againts tomato polygalacturonase cross-react with one of these sets of proteins.

Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.

A hydroxyproline-rich glycoprotein (HRGP) component of the maize cell wall was shown to be present in different organs of the plant by extraction of cell wall proteins and detection by Western blotting and immunocytochemistry. Antibodies raised against the protein or against synthetic peptides designed from the protein sequence immunoprecipitated a proline-rich polypeptide which was synthesized in-vitro from poly(A) (+) RNA extracted from different tissues of the plant and from the complete in-vitro-transcribed mRNA. A very low amount of the protein was found in immature embryos. In particular, the protein could not be detected in the scutellum either by Western blotting or by immunocytochemistry. In agreement with this finding, HRGP mRNA was barely detected in the scutellum, in contrast to its accumulation in the embryo axis. Our results indicate the existence of a unique cell wall structure in embryonic tissues from maize as well as a tissuespecific component of the control of maize HRGP gene expression, distinct to others already described such as cell division.

Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation.

The spatial pattern of expression for a maize gene encoding a hydroxyproline-rich glycoprotein (HRGP) was determined by in situ hybridization. During normal development of roots and leaves, the expression of the gene was transient and particularly high in regions initiating vascular elements and associated sclerenchyma. Its expression was also associated with the differentiation of vascular elements in a variety of other tissues. The gene encoded an HRGP that had been extracted from the cell walls of maize suspension culture cells and several other embryonic and post-embryonic tissues. The gene was present in one or two copies in different varieties of maize and in the related monocots teosinte and sorghum. A single gene was cloned from maize using a previously characterized HRGP cDNA clone [Stiefel et al. (1988). Plant Mol. Biol. 11, 483-493]. In addition to the coding sequences for the HRGP and an N-terminal signal sequence, the gene contained a single intron in the nontranslated 3' end.

Expression of genes for cell-wall proteins in dividing and wounded tissues ofZea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.

Storage-protein hydrolysis and protein-body breakdown in germinatedZea mays L. seeds.

Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein 1. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.

The use of two-dimensional gel electrophoresis in the analysis of organ-specific maize proteins.

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means.

Molecular cloning of cDNAs encoding a putative cell wall protein from Zea mays and immunological identification of related polypeptides.

Copy DNAs corresponding to a highly repetitive, proline-rich protein from maize have been cloned by differential screening of a coleoptile cDNA library. The deduced amino acid sequence contains a single repetitive element of carrot extensin (Ser-Pro-Pro-Pro-Pro). The related mRNAs have a defined distribution in tissues of the plant and are accumulated mainly in the coleoptile node and root tip. A peptide that corresponds to one of the repetitive elements of the protein has been synthesized and antisera have been obtained in rabbits. These antibodies react against crude preparations of coleoptile cell wall and against polypeptides extracted following the protocols described for the extraction of extensin. From these data it is concluded that the cDNAs correspond to a family of cell wall glycoproteins from maize.

In maize, glutelin-2 and low molecular weight zeins are synthesized by membrane-bound polyribosomes and translocated into microsomal membranes.

Experiments to establish the site of biosynthesis and the possible translocation into microsomes of glutelins-2 (28 kD G2) and low molecular weight zeins (10, 14, 15 kD Z2) have been carried out. Free and membrane-bound polyribosomes as well as microsomal membranes were isolated from immature endosperms of W64A Zea mays L. In vitro translation studies were carried out in the presence and in the absence of membranes using [(35)S]-methionine or [(35)S]-cysteine as precursors. Cell-free translation products were characterized by electrophoretic mobility, solubility and antigenic properties. The results obtained indicate that 28 kD G2 and low molecular weight zeins are primarily synthesized on membrane-bound polysomes. From experiments using proteinase K as a probe, we also conclude that these proteins are translocated into microsomes where they accumulate. The translocated and pre-28 kD G2 proteins do not present changes in the apparent molecular weight. However we show that there are differences in their isoelectric points, a fact that indicates the existence of 28 kD G2 processing.

Subcellular localization of glutelin-2 in maize (Zea mays L.) endosperm.

Accumulation of the 28 KD protein of the glutelin-(G2) fraction was followed in developing maize endosperm, using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peak integration of scanned gels. 28 KD glutelin-2 could already be observed from 15 days after pollination and its accumulates reached a plateau during the second half of the development period. The process of biosynthesis of 28 KD glutelin-2 and zeins occurs in a parallel way. Subcellular fractions obtained from linear sucrose gradient centrifugation of developing maize endosperms were analyzed by SDS-PAGE and immunoblotting using a serum reacting against glutelin-2 and 14 KD Z2. Glutelin-2 was found to be present in the protein bodies when subcellular fractionation was carried out without dithiothreitol (DTT). The presence of a reducing agent causes the elution of glutelin-2 from protein bodies. Immunocytochemical labelling using the protein A-colloidal gold technique in protein bodies incubated with anti-G2 IgG revealed that G2 is located mainly in the periphery of protein bodies. These results are interpreted as indicating a structural role for glutelins in protein bodies.