Antisense expression of the CK2 alpha-subunit gene in Arabidopsis. Effects on light-regulated gene expression and plant growth.
(9/7901)
The protein kinase CK2 (formerly casein kinase II) is thought to be involved in light-regulated gene expression in plants because of its ability to phosphorylate transcription factors that bind to the promoter regions of light-regulated genes in vitro. To address this possibility in vivo and to learn more about the potential physiological roles of CK2 in plants, we transformed Arabidopsis with an antisense construct of the CK2 alpha-subunit gene and investigated both morphological and molecular phenotypes. Antisense transformants had a smaller adult leaf size and showed increased expression of chs in darkness and of cab and rbcS after red-light treatment. The latter molecular phenotype implied that CK2 might serve as one of several negative and quantitative effectors in light-regulated gene expression. The possible mechanism of CK2 action and its involvement in the phytochrome signal transduction pathway are discussed. (+info)
Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation.
(10/7901)
We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants. (+info)
Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1.
(11/7901)
The utilization of transgenic plants expressing recombinant antigens to be used in the formulation of experimental immunogens has been recently communicated. We report here the development of transgenic plants of alfalfa expressing the structural protein VP1 of foot and mouth disease virus (FMDV). The presence of the transgenes in the plants was confirmed by PCR and their specific transcription was demonstrated by RT-PCR. Mice parenterally immunized using leaf extracts or receiving in their diet freshly harvested leaves from the transgenic plants developed a virus-specific immune response. Animals immunized by either method elicited a specific antibody response to a synthetic peptide representing amino acid residues 135-160 of VP1, to the structural protein VP1, and to intact FMDV particles. Additionally, the immunized mice were protected against experimental challenge with the virus. We believe this is the first report demonstrating the induction of a protective systemic antibody response in animals fed transgenic plants expressing a viral antigen. These results support the feasibility of producing edible vaccines in transgenic forage plants, such as alfalfa, commonly used in the diet of domestic animals even for those antigens for which a systemic immune response is required. (+info)
Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.
(12/7901)
Macromolecular trafficking within the sieve element-companion cell complex, phloem unloading, and post-phloem transport were studied using the jellyfish green fluorescent protein (GFP). The GFP gene was expressed in Arabidopsis and tobacco under the control of the AtSUC2 promoter. In wild-type Arabidopsis plants, this promoter regulates expression of the companion cell-specific AtSUC2 sucrose-H+ symporter gene. Analyses of the AtSUC2 promoter-GFP plants demonstrated that the 27-kD GFP protein can traffic through plasmodesmata from companion cells into sieve elements and migrate within the phloem. With the stream of assimilates, the GFP is partitioned between different sinks, such as petals, root tips, anthers, funiculi, or young rosette leaves. Eventually, the GFP can be unloaded symplastically from the phloem into sink tissues, such as the seed coat, the anther connective tissue, cells of the root tip, and sink leaf mesophyll cells. In all of these tissues, the GFP can traffic cell to cell by symplastic post-phloem transport. The presented data show that plasmodesmata of the sieve element-companion cell complex, as well as plasmodesmata into and within the analyzed sinks, allow trafficking of the 27-kD nonphloem GFP protein. The data also show that the size exclusion limit of plasmodesmata can change during organ development. The results are also discussed in terms of the phloem mobility of assimilates and of small, low molecular weight companion cell proteins. (+info)
The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase.
(13/7901)
The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis. (+info)
The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants.
(14/7901)
Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants. (+info)
Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation.
(15/7901)
Previous analysis of potato spindle tuber viroid (PSTVd) RNA-infected tobacco plants has suggested that an RNA-DNA interaction could trigger de novo methylation of PSTVd transgene sequences. Using the genomic sequencing technique, the methylation pattern associated with the RNA-directed DNA methylation process has been characterized. Three different PSTVd transgene constructs all showed a similar pattern of methylation. Most of the cytosines at symmetrical as well as non-symmetrical positions appeared to be methylated in both DNA strands of the viroid sequences. Heavy methylation was mostly restricted to the viroid cDNA sequences. Flanking DNA regions immediately adjacent to the viroid cDNA displayed a lower but significant level of cytosine methylation. The observation that the heavy methylation was essentially co-extensive with the length of the PSTVd cDNA sequences provided evidence that a direct RNA-DNA interaction can act as a strong and highly specific signal for de novo DNA methylation. These data also confirmed that de novo methylation was not limited to canonical CpG and CpNpG sites, but can also involve all the cytosine residues located in the genomic region where the RNA-DNA interaction takes place. (+info)
N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants.
(16/7901)
Since plants are emerging as an important system for the expression of recombinant glycoproteins, especially those intended for therapeutic purposes, it is important to scrutinize to what extent glycans harbored by mammalian glycoproteins produced in transgenic plants differ from their natural counterpart. We report here the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in a transgenic plant. The structures of the N-linked glycans attached to the heavy chains of the monoclonal antibody Guy's 13 produced in transgenic tobacco plants (plantibody Guy's 13) were identified and compared to those found in the corresponding IgG1 of murine origin. Both N-glycosylation sites located on the heavy chain of the plantibody Guy's 13 are N-glycosylated as in mouse. However, the number of Guy's 13 glycoforms is higher in the plant than in the mammalian expression system. Despite the high structural diversity of the plantibody N-glycans, glycosylation appears to be sufficient for the production of a soluble and biologically active IgG in the plant system. In addition to high-mannose-type N-glycans, 60% of the oligosaccharides N-linked to the plantibody have beta(1, 2)-xylose and alpha(1, 3)-fucose residues linked to the core Man3GlcNAc2. These plant-specific oligosaccharide structures are not a limitation to the use of plantibody Guy's 13 for topical immunotherapy. However, their immunogenicity may raise concerns for systemic applications of plantibodies in human. (+info)