Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome

Abstract

SummaryApplications of chloroplast engineering in agriculture and biotechnology will depend critically on success in extending the crop range of chloroplast transformation, and on the feasibility of expressing transgenes in edible organs (such as tubers and fruits), which often are not green and thus are much less active in chloroplast gene expression. We have improved a recently developed chloroplast-transformation system for tomato plants and applied it to engineering one of the central metabolic pathways in fruits: carotenoid biosynthesis. We report that plastid expression of a bacterial lycopene b-cyclase gene results in herbicide resistance and triggers conversion of lycopene, the main storage carotenoid of tomatoes, to b-carotene, resulting in fourfold enhanced pro-vitamin A content of the fruits. Our results demonstrate the feasibility of engineering nutritionally important biochemical pathways in non-green plastids by transformation of the chloroplast genome.

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

“…Five micrograms of total DNA were digested with SphI, separated on a 1 % (w/v) agarose gel and transferred to a Hybond XL membrane (GE Healthcare). A 550 bp PCR product was generated by amplification of a portion of the psaB coding region (Wurbs et al 2007) and was used as RFLP probe to verify plastid transformation and assess the homoplasmic state of the transformants. Probes were labelled with a[ 32 P] dCTP by random priming using the Multiprime DNA labelling system (GE Healthcare).…”

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

“…In transplastomic tomato plants, the fusion protein P24-Nef from HIV accumulated up to 40% TSP in leaves, but significantly less in green and ripe fruits (2.5% and nil, respectively) (Zhou et al 2008). The relative expression level of transgenes in tomato fruit chromoplasts, however, was higher with other proteins (Ruf et al 2001) and in any case sufficient to induce significant metabolic changes (Wurbs et al 2007;Apel and Bock 2009). Very low levels of mRNA and GFP protein were detected in roots of transplastomic Nicotiana benthamiana plants (less than 2% of that in the leaves), albeit expression levels up to 40-75% of those in leaf chloroplasts could be achieved in petal leucoplasts of the same plants or in chromoplasts of carrot taproots (Kumar et al 2004a;Davarpanah et al 2009).…”

“…A full-length plastid rrn operon promoter, with both PEP and NEP consensus sequences, along with the ribosome-binding site of the phage T7 gene 10 leader and the 3 0 -UTR of the plastid gene encoding the ribosomal protein S16 were recently used to facilitate transgene expression in carrot chromoplasts (Kumar et al 2004a). A similar approach was adopted by Wurbs et al (2007), who used the atpI promoter containing both PEP and NEP signals, and the rps16 3 0 -UTR, to express carotenoidrelated genes in tomato fruits. Nevertheless, the short rrn promoter, containing only the sequences for PEP recognition, was successfully used to express transgenes in fruit chromoplasts of transplastomic tomato, and in petal leucoplasts and root amyloplasts of tobacco and Nicotiana benthamiana (Khan and Maliga 1999;Ruf et al 2001;Apel and Bock 2009;Davarpanah et al 2009).…”

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons

“…S5). Regeneration medium and selection conditions for plastid transformation of green-fruited tomato varieties were as described previously for red-fruited varieties (46,48). The plastid transformation efficiencies expressed as number of confirmed transplastomic lines (pSyHPT + pTop2) per number of selection plates (Petri dishes) with bombarded leaf pieces were five pSyHPT + three pTop2 events per 19 plates for IPA-6, nine pSyHPT + eight pTop2 events per 469 plates for Dorothy's Green, and three pSyHPT + 10 pTop2 events per 674 plates for Green Pineapple.…”

“…Young leaves from aseptically grown tobacco and tomato plants were bombarded with plasmid-coated 0.6-μm gold particles using a helium-driven biolistic gun (PDS1000He; BioRad) with the Hepta Adapter setup. Primary spectinomycinresistant lines were selected on plant-regeneration medium containing 500 mg/L spectinomycin (19,46,48).…”