Exploring the Potential Applications of the Noninvasive Reporter Gene RUBY in Plant Genetic Transformation

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Abstract

Betalains can be conveniently observed and quantified and, accordingly, have the potential as naked-eye visual screening reporters during plant transformation. RUBY is a new reporter system that uses “2A” peptides to fuse three key genes, CYP76AD1, DODA, and glucosyl transferase, for betalain biosynthesis, and has been successfully used for transformation of rice, Arabidopsis, and cotton, but its potential applications in the genetic transformation of various other plant species remain to be verified. In this study, RUBY was transferred into the hairy roots of Plukenetia volubilis and Nicotiana benthamiana, and was transferred into Arabidopsis by the floral-dip method. The expression levels of CYP76AD1, DODA, and glucosyl transferase were detected by RT−PCR and RT−qPCR, the relationship between the expression level of RUBY and red coloration was analyzed, and the genetic stability of RUBY in transgenic Arabidopsis was studied. The results showed that the expression of RUBY could reconstruct the betalain biosynthesis pathway in the hairy roots of P. volubilis, N. benthamiana, and Arabidopsis plants, indicating that it has the potential for versatile use across species. As a reporter, betalain did not affect callus induction, plant regeneration, development, or fertility. However, when used in plant transformation for observation and visual screening, it needed to accumulate to a certain extent to show red coloration, and it was positively correlated with gene expression. In general, RUBY is a convenient reporter for plant transformation, and has no obvious side effects during plant growth and development. However, the potential application of RUBY for visual screening is highly determined by the expression level, and further improvement is needed.

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Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana

Steven J Clough, Andrew Bent, Kazuhiro Oiwa (1999)

The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.

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GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

R A Jefferson, T. Kavanagh, M W Bevan (1987)

We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

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Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids.

Yoshikazu Tanaka, Nobuhiro Sasaki, Akemi Ohmiya (2008)

Plant compounds that are perceived by humans to have color are generally referred to as 'pigments'. Their varied structures and colors have long fascinated chemists and biologists, who have examined their chemical and physical properties, their mode of synthesis, and their physiological and ecological roles. Plant pigments also have a long history of use by humans. The major classes of plant pigments, with the exception of the chlorophylls, are reviewed here. Anthocyanins, a class of flavonoids derived ultimately from phenylalanine, are water-soluble, synthesized in the cytosol, and localized in vacuoles. They provide a wide range of colors ranging from orange/red to violet/blue. In addition to various modifications to their structures, their specific color also depends on co-pigments, metal ions and pH. They are widely distributed in the plant kingdom. The lipid-soluble, yellow-to-red carotenoids, a subclass of terpenoids, are also distributed ubiquitously in plants. They are synthesized in chloroplasts and are essential to the integrity of the photosynthetic apparatus. Betalains, also conferring yellow-to-red colors, are nitrogen-containing water-soluble compounds derived from tyrosine that are found only in a limited number of plant lineages. In contrast to anthocyanins and carotenoids, the biosynthetic pathway of betalains is only partially understood. All three classes of pigments act as visible signals to attract insects, birds and animals for pollination and seed dispersal. They also protect plants from damage caused by UV and visible light.