Transgene integration,organization and interaction in plants

It has been appreciated for many years that the structure of a transgene locus can have a major influence on the level and stability of transgene expression. Until recently, however, it has been common practice to discard plant lines with poor or unstable expression levels in favor of those with practical uses. In the last few years, an increasing number of experiments have been carried out with the primary aim of characterizing transgene loci and studying the fundamental links between locus structure and expression. Cereals have been at the forefront of this research because molecular, genetic and cytogenetic analysis can be carried out in parallel to examine transgene loci in detail. This review discusses what is known about the structure and organization of transgene loci in cereals, both at the molecular and cytogenetic levels. In the latter case, important links are beginning to be revealed between higher order locus organization, nuclear architecture, chromatin structure and transgene expression.     

The formation,function and fate of protein storage compartments in seeds

Seed storage proteins (SSPs) have been studied for more than 250 years because of their nutritional value and their impact on the use of grain in food processing. More recently, the use of seeds for the production of recombinant proteins has rekindled interest in the behavior of SSPs and the question how they are able to accumulate as stable storage reserves. Seed cells produce vast amounts of SSPs with different subcellular destinations creating an enormous logistic challenge for the endomembrane system. Seed cells contain several different storage organelles including the complex and dynamic protein storage vacuoles (PSVs) and other protein bodies (PBs) derived from the endoplasmic reticulum (ER). Storage proteins destined for the PSV may pass through or bypass the Golgi, using different vesicles that follow different routes through the cell. In addition, trafficking may depend on the plant species, tissue and developmental stage, showing that the endomembrane system is capable of massive reorganization. Some SSPs contain sorting signals or interact with membranes or with other proteins en route in order to reach their destination. The ability of SSPs to form aggregates is particularly important in the formation or ER-derived PBs, a mechanism that occurs naturally in response to overloading with proteins that cannot be transported and that can be used to induce artificial storage bodies in vegetative tissues. In this review, we summarize recent findings that provide insight into the formation, function, and fate of storage organelles and describe tools that can be used to study them.     

Plant bioreactors - the taste of sweet success

Thaumatins are intensely sweet proteins (3000 times sweeter than the same weight of sucrose) that are found in the arils of the tropical perennial plant Thaumatococcus daniellii Benth and are produced commercially by aqueous extraction from the fruits. The proteins are widely used as sweeteners and flavor enhancers in the food industry, and the European Food Safety Association (EFSA) has recently confirmed that their use as feed additive (1 to 5 mg/kg complete feed) is safe for all animal species. Given the large market for sweeteners and flavor enhancers, thaumatins could become increasingly important in the food and feed additives sector. In this issue of Biotechnology Journal, a study examines the production of thaumatin in tobacco hairy root cultures.     

Plant-based production of biopharmaceuticals

Plants are now gaining widespread acceptance as a general platform for the large-scale production of recombinant proteins. The first plant-derived recombinant pharmaceutical proteins are reaching the final stages of clinical evaluation, and many more are in the development pipeline. Over the past two years, there have been some notable technological advances in this flourishing area of applied biotechnology, as shown by the continuing commercial development of novel plant-based expression platforms. There has also been significant success in tackling some of the limitations of plant bioreactors, such as low yields and inconsistent product quality, that have limited the approval of plant-derived pharmaceuticals.     

The Quest to Understand the Basis and Mechanisms that Control Expression of Introduced Transgenes in Crop Plants

We discuss mechanisms and factors that influence levels and stability of expressed heterologous proteins in crop plants. We have seen substantial progress in this field over the past two decades in model experimental organisms such as Arabidopsis and tobacco. There is no question such studies have resulted in furthering our understanding of key processes in the plant cell and the elaboration of sophisticated models to explain underlying mechanisms that might influence the fate, levels and stability of expression of recombinant heterologous proteins in plants. However, very often, such information is not applicable outside these laboratory experimental models. In order to generate a knowledge basis that can be used to achieve high levels and stability of heterologous proteins in relevant crop plants it is imperative to perform such studies on the target crops. With this in mind, we discuss key elements of the process at the DNA, RNA and protein levels. We believe it is essential to discuss recombinant protein production in crops in a holistic manner in order to develop a comprehensive knowledge base that will in turn serve plant biotechnology applications well.Key Words: transgene expression, protein trafficking, silencing mechanisms, chromatin remodeling, crop plants     

Expression and Immunolocalisation of the Snowdrop Lectin,GNA in Transgenic Rice Plants

Transgenic rice (Oryza sativa L.) plants generated through particle bombardment expressed high levels of an insecticidal protein (the snowdrop lectin, GNA) directed against sap-sucking insects. Engineered plants expressed GNA either constitutively or in a tissue specific manner, depending on the nature of the promoter used to drive expression of the gene. We used specific antibodies raised against GNA to localize its expression in phloem tissue in plants engineered with the rice sucrose synthase promoter driving GNA expression. We report here molecular, biochemical and immunological analyses for fifteen independently-derived transformants out of more than 200 plants we generated.     

Practical considerations for pharmaceutical antibody production in different crop systems

The potential of plant cells to produce functional recombinantantibodies has been demonstrated in a number of different plant systems. Wepresent a comparative study of a well-defined target protein, a single chain Fvantibody, in different transgenic crop species and cultured tissues. The effectof different regulatory elements and signals for subcellular targeting areconsidered. Practical considerations for the choice of a particular cropsystem,such as yield, storage, distribution and containment properties are discussed.