Anne Kearns

Bio: Anne Kearns is an academic researcher from Oxford Brookes University. The author has contributed to research in topics: Fusion protein & Green fluorescent protein. The author has an hindex of 4, co-authored 5 publications receiving 1346 citations.

Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.

Abstract: Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.

Plant cell culture

Abstract: Plant cell culture has found wide applications ranging from studies on basic plant biochemistry and molecular biology to mass propagation and genetic engineering of crop species. This chapter talks about basic requirements for common procedures in plant cell culture. Successful plant cell culture requires extremely close attention to proper sterile technique and temperature control. Some laboratories use containment hoods when introducing microbes into plant cell cultures, such as during genetic transformation or inoculation with plant pathogens. Continuous light is sometimes detrimental to whole-plant growth and is rarely used in plant cell cultures. Many methods have been used to estimate the growth of plant cell cultures both directly and indirectly. The method chosen will depend on the requirements for maintaining the sterility of the culture and the speed and accuracy of the measurement. Assays in which the stain (such as fluorescein diacetate [FDA] or triphenyltetrazolium chloride [TTC]) is meant to be taken up only by living cells are known to sometimes give false-positive results with cells that no longer divide. One application of plant cell cultures is the study of plant secondary-metabolite biosynthesis and the commercial production of such valuable natural products.

BY-2 cells: culture and transformation for live cell imaging.

Abstract: Tobacco Bright Yellow-2 (BY-2) suspension cells are a widely used biological material for studying plant cell morphology and physiology. These cells are easy to transform and maintain in culture and tolerate transformation with fluorescent proteins such as the green fluorescent protein and its derivatives. These, by the addition of plant or mammalian targeting sequences, can be directed to specific subcellular locations for the study of cell dynamics in vivo. This unit describes the production of BY-2 cell stable transformants via an Agrobacterium based method to permit the visualisation of cellular components in vivo by epifluorescence or confocal microscopy.

PP7, a gene encoding a novel protein Ser/Thr phosphatase, is expressed primarily in a subset of guard cells in Arabidopsis thaliana

Abstract: Recently, we have identified a cDNA encoding a novel protein Ser/Thr phosphatase (EC 3.1.3.16) from Arabidopsis thaliana (L.) Heynh. (ecotype Columbia), designated as PP7, which is only distantly related to the known phosphatases of the PPP family. We examined PP7 expression in A. thaliana using Northern blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridisation. Although the PP7 transcript could be detected in all organs analysed by the first two methods, whole-mount in situ hybridisation revealed specific expression in a subset of stomata in A. thaliana seedlings. Possible implications of this expression pattern are discussed.

Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.

Abstract: Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.

Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.

Abstract: Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Additionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus-induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions currently used by the research community. In addition to addressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer.

Abstract: The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H(2)O(2) or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mm cytosolic glutathione buffer would contain only 25 nm oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.

Plant cell cultures an alternative and efficient source for the production of biologically important secondary metabolites

Abstract: Plant cell culture systems represent a potential renewable source of valuable medici- nal compounds, flavors, fragrances, and colorants, which cannot be produced by microbial cells or chemical synthesis. Biotechnological applications of plant cell cultures presents the most up- dated reviews on current techniques in plant culture in the field. The evolving commercial im- portance of the secondary metabolites has in recent years resulted in a great interest, in secon- dary metabolism, and particularly in the possibility to alter the production of bioactive plant me- tabolites by means of cell culture technology. The principle advantage of this technology is that it may provide continuous, reliable source of plant pharmaceuticals and could be used for the large-scale culture of plant cells from which these metabolites can be extracted. In addition to its importance in the discovery of new medicines, plant cell culture technology plays an even more significant role in solving world hunger by developing agricultural crops that provide both higher yield and more resistance to pathogens and adverse environmental and climatic conditions. This paper describes the callus and suspension culture methods that we have established in our labo- ratory for the production of bioactive secondary metabolites from medicinal plants.