Plant nuclear envelope proteins

Compared to research in the animal field, the plant NE has been clearly under-investigated. The available data so far indicate similarities as well as striking differences that raise interesting questions about the function and evolution of the NE in different kingdoms. Despite a seemingly similar structure and organization of the NE, many of the proteins that are integral components of the animal NE appear to lack homologues in plant cells. The sequencing of the Arabidopsis genome has not led to the identification of homologues of animal NE components, but has indicated that the plant NE must have a distinct protein composition different from that found in metazoan cells. Besides providing a selective barrier between the nucleoplasm and the cytoplasm, the plant NE functions as a scaffold for chromatin but the scaffolding components are not identical to those found in animal cells. The NE comprises an MTOC in higher plant cells, a striking difference to the organization of microtubule nucleation in other eukaryotic cells. Nuclear pores are present in the plant NE, but identifiable orthologues of most animal and yeast nucleoporins are presently lacking. The transport pathway through the nuclear pores via the action of karyopherins and the Ran cycle is conserved in plant cells. Interestingly, RanGAP is sequestered to the NE in plant cells and animal cells, yet the targeting domains and mechanisms of attachment are different between the two kingdoms. At present, only a few proteins localized at the plant NE have been identified molecularly. Future research will have to expand the list of known protein components involved in building a functional plant NE.     

Geminivirus-based shuttle vectors capable of replication in Escherichia coli and monocotyledonous plant cells

Shuttle vectors have been constructed that are able to replicate in either Escherichia coli or plant cells. They contain the ColE1 origin of replication and parts of the wheat dwarf virus genome, a geminivirus infecting a variety of species of monocotyledonous plants. Such plasmids are able to replicate in E. coli and wheat cells. The plasmids can be rescued in E. coli and show no changes during their passage through plant cells. Such an E. coli/plant cell shuttle vector system could be used for the amplification of foreign genes in plant cells, for studies on DNA rearrangement or the isolation of plant transposons.     

Membrane transport in the endocytic pathway: Animal versus plant cells

Summary The endocytic pathway is a well established process in animal cells, but it is not well understood in plant cells. At the morphological level, all the compartments involved in endocytosis in animal cells seem to have counterparts in plant cells, and the organization of the pathway appears to share some striking similarities. Several Rab homologues have been found in plant cells, including homologues of Rab5, Rab7, and Rab11, markers of endocytic compartments in animal cells. Coat proteins are also present in plant cells, including clathrin, adaptins, and ADP ribosylation factor proteins. However, endocytic compartments in plant cells also exhibit specific features both in organization and function. The molecular composition of these compartments remains to be established, and future work will be necessary to identify the key regulators of endocytic trafficking in plant cells.Abbreviations EE early endosome - LE late endosome - ECV-MVB endosomal carrier vesicle-multivesicular body - PCR partially coated reticulum - MPR mannose 6-phosphate receptor - TGN trans-Golgi network     

Advances in selectable marker genes for plant transformation

Plant transformation systems for creating transgenics require separate process for introducing cloned DNA into living plant cells. Identification or selection of those cells that have integrated DNA into appropriate plant genome is a vital step to regenerate fully developed plants from the transformed cells. Selectable marker genes are pivotal for the development of plant transformation technologies because marker genes allow researchers to identify or isolate the cells that are expressing the cloned DNA, to monitor and select the transformed progeny. As only a very small portion of cells are transformed in most experiments, the chances of recovering transgenic lines without selection are usually low. Since the selectable marker gene is expected to function in a range of cell types it is usually constructed as a chimeric gene using regulatory sequences that ensure constitutive expression throughout the plant. Advent of recombinant DNA technology and progress in plant molecular biology had led to a desire to introduce several genes into single transgenic plant line, necessitating the development of various types of selectable markers. This review article describes the developments made in the recent past on plant transformation systems using different selection methods adding a note on their importance as marker genes in transgenic crop plants.     

Can plant oncogenes inhibit programmed cell death? The rolB oncogene reduces apoptosis-like symptoms in transformed plant cells

The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.     

Suspension-cultured plant cells as a platform for obtaining recombinant proteins

Production of recombinant proteins in suspension cultures of genetically modified plant cells is a promising and rapidly developing area of plant biotechnology. In the present review article, advantages related to using plant systems for expression of recombinant proteins are considered. Here, the main focus is covering the literature on optimization of cultivation conditions of suspension-cultured plant cells to obtain a maximal yield of target proteins. In particular, certain examples of successful use of such cells to produce pharmaceuticals were described.     

Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity

A Ti plasmid mutant was constructed in which all the on-cogenic functions of the T-DNA have been deleted and replaced by pBR322. This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T-DNA into infected plant cells. Moreover, integration and expression of this minimal T-DNA in plant cells does not interfere with normal plant cell differentiation. A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T-region upon a single recombination event through the pBR322 region of pGV3850 producing a co-integrate useful for the transformation of plant cells. Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.     

Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells.

The attachment of virulent strains of Agrobacterium tumefaciens to plant cells is the first step in the bacterial induction of tumors. Binding of A. tumefaciens to carrot tissue culture cells occurred as a two-step process. The initial step was the attachment of the bacteria to the plant cell wall. Living plant cells were not required. Bacterial attachment to heat-killed or glutaraldehyde-fixed carrot cells proceeded with only slightly altered kinetics and unaltered bacterial strain specificity. After the bacteria bound to the carrot cell surface, scanning electron microscopy showed that fibrils developed, surrounded the bacteria, and anchored them to the plant cell surface. These fibrils were synthesized by the bacteria and not by the plant cell since they were also made after the attachment of A. tumefaciens to dead carrot cells and since under some conditions the bacteria synthesized fibrils in the absence of plant cells. Calcofluor staining, acid hydrolysis, enzymatic digestion studies, and infrared spectroscopy showed that the fibrils were composed of cellulose. The formation of these cellulose fibrils occurred during the attachment of virulent strains of A. tumefaciens to plant cells in vitro. The fibrils anchored the bacteria to the plant cell surface and entrapped additional bacteria. The multiplication of entrapped and attached bacteria resulted in the formation of large clusters of bacteria held close to the plant cell wall and plasma membrane by cellulose fibrils. This high concentration of bacteria may facilitate transfer of Ti plasmid deoxyribonucleic acid to the plant cell resulting in the formation of tumors.     

Surface immobilization of plant cells

A novel technique has been developed to immobilize plant cells. The cells are deposited on a surface of manmade fibrous material that provides for strong binding of the plant tissue biomass growing in the submerged culture. The immobilized plant cells remain fully viable. Relatively uniform biomass loadings of up to 20 mg d.w. plant cells/cm(2) support material have been attained. All plant cells from the inoculum suspension became attached within the first 24-48 h depending on the support matrix configuration and hydraulic culture conditions. The advantages and scale-up potential of this technique are discussed and compared to other culturing modes.     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

A Comparative Mechanical Analysis of Plant and Animal Cells Reveals Convergence across Kingdoms

Plant and animals have evolved different strategies for their development. Whether this is linked to major differences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.     

Bacitracin significantly reduces degradation of peptides in plant cell cultures

Production of useful peptides using recombinant plant cells is often limited by the stability of the newly translated peptides in the plant expression system. As an initial step toward producing peptides in transformed plant cell cultures, we examined the stability of exogenously added arginine vasopressin (AVP) peptide in a suspension culture of Nicotiana plumbaginifolia cells. The peptide was lost rapidly from the plant cell culture, but the rate of loss was slowed significantly by the addition of the peptide, bacitracin, at a concentration of 150 mug/mL. At higher concentrations, bacitracin substantially inhibited the growth of the plant cells in culture. (c) 1997 John Wiley & Sons, Inc.     

In vitro activation of chemicals by plants: a comparison of techniques

We have studied the ability of two in vitro plant activation techniques to enhance the mutagenicity of 4-nitro-o-phenylenediamine (NOP) and to activate 2-aminofluorene (2AF). Mutagenic activities of NOP and 2AF were both increased by plant S9 in the Salmonella plate-incorporation and preincubation assays. They were also increased during preincubation with intact plant cells. NOP mutagenic activity was enhanced to a similar extent by plant S9 and by intact plant cells in Salmonella assay, whereas 2AF was activated more extensively by the plant cells than by plant S9. NOP was not enhanced by mammalian hepatic S9 in any assay, whereas 2AF was activated by hepatic S9 under all conditions tested.     

A plant signal sequence enhances the secretion of bacterial ChiA in transgenic tobacco

When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the protein is glycosylated and secreted from the plant cells; however most of the protein remains inside the cells. We tested whether the efficiency of secretion could be improved by replacing the bacterial signal sequence with a plant signal sequence. We found the signal sequence and the first two amino acids of the PR1b protein attached to the ChiA mature protein directs complete glycosylation and secretion of the ChiA from plant cells. Glycosylation of this protein is not required for its efficient secretion from plant cells.     

植物细胞中间纤维角蛋白性质的分析

角蛋白是植物细胞中间纤维的主要成分。应用选择性抽提和生物化学技术,分离纯化了豌豆根尖细胞58-、52 kD、白菜子叶52kD和胡萝卜悬浮细胞64kD角蛋白,测定了它们的氨基酸组成,结果表明上述角蛋白与动物细胞中间纤维角蛋白的氨基酸组成有较大的相似性。比较了动、植物细胞角蛋白的肽谱,结果显示它们之间存在较大的差异,但是植物细胞间角蛋白的肽谱比较一致,这提示它们属于同一蛋白家族,为植物中间纤维及其角蛋白的存在提供了新的论据。     

Evidence for ribosome scanning during translation initiation of mRNAs in transformed plant cells

Summary The recent development of methods for transforming plant cells has permitted testing of the Kozak ribosome scanning hypothesis of translational initiation in plant cells. The experiments described in this paper provide a direct demonstration that an extra translational initiation signal decreases the level of Tn5 neomycin phosphotransferase II enzyme produced in transformed plant cells. Removal of the extra AUG results in an improved chimeric kanamycin resistance gene that expresses a five-fold increase in selectable resistance and assayable enzyme without an increase in stable mRNA levels. This is the first evidence suggesting that the Kozak’s model of ribosome scanning for mammalian translation initiation applies to plant cells.     

细胞培养技术在植物抗性生理研究领域中的应用

本文综述了近10年来有关利用细胞培养技术所进行的抗性生理领域的研究成果,包括在培养过程中植物细胞对外界胁迫的反应、有关利用细胞培养技术研究植物的抗逆性反应,以及植物抗逆性的理论在实际中的应用。大量的实验证明,细胞培养技术在植物抗逆性研究领域具有广阔的应用前景。