David W Galbraith
Work Summary
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
Abstract:
We present a characterization of transgenic Arabidopsis thaliana (L.) Heynh. plants expressing a chimeric gene comprising the Green Fluorescent Protein (GFP) and β-glucuronidase (GUS) coding sequences, fused to an efficient nuclear localization signal (NLS). The transgenic plants accumulate the fusion protein in their nuclei, and this provides a novel phenotype, that of green-fluorescent nuclei. The fluorescent nuclei are readily observed using conventional epifluorescence and laser scanning confocal microscopy. We describe the use of this phenotype for in vivo studies of nuclear shape and movement, cell division, and for analysis of the transcriptional activities of constitutive and tissue-specific promoters. We propose that the phenotype of fluorescent nuclei will prove particularly valuable in histological, physiological and developmental studies of higher plants that require the facile observation of nuclei within living cells and in the absence of fixation or external staining.
Abstract:
We have identified a callus associated protein (CAP) as a new molecular marker for proliferative growth and early cellular differentiation in higher plants. Antiserum directed against the Sorghum callus associated protein (CAP) was employed to isolate a cDNA clone (CAP-C2) from an expression library prepared from mRNA from actively dividing cell suspension cultures of Nicotiana tabacum L. cv. xanthi. The derived amino acid sequence contained structural features of eukaryotic transcriptional factors, including three putative zinc fingers, three activator domains, and a nuclear localization signal. A fusion protein formed between part of the CAP-C2 polypeptide and Escherichia coli beta-glucuronidase specifically accumulated within the nuclei of transfected protoplasts, suggesting a nuclear localization for the CAP protein. Increased levels of expression of CAP in vivo were observed in actively proliferating tobacco tissues, whereas expression in vitro was induced when mature, differentiated explants proliferated into callus or cell suspensions. A 6.2 kb unstable mRNA and two low-abundance nuclear proteins (p66 and p68) containing epitopes encoded by CAP-C2 accumulated in plant tissues undergoing proliferative growth but were absent in mature, differentiated tissues. The corresponding genes were also present in Nicotiana sylvestris and Petunia hybrida © 1994 Tissue Culture Association.
PMID: 2455722;PMCID: PMC2115190;Abstract:
Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.