The sub-cellular localisation of the potato ( Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2
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ORIGINAL ARTICLE
The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2 Stefania Pasare & Kathryn Wright & Raymond Campbell & Wayne Morris & Laurence Ducreux & Sean Chapman & Peter Bramley & Paul Fraser & Alison Roberts & Mark Taylor
Received: 15 April 2013 / Accepted: 10 June 2013 # Springer-Verlag Wien 2013
Abstract Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by overexpressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the
Handling Editor: Jaideep Mathur S. Pasare : K. Wright : R. Campbell : W. Morris : L. Ducreux : S. Chapman : A. Roberts : M. Taylor (*) Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK e-mail: [email protected] S. Pasare : P. Bramley : P. Fraser School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, Surrey TW20 OEX, UK
location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers. Keywords Amyloplast . Carotenoid . Localisation . Plastid . Potato . Tuber Abbreviations CrtRb β-Carotene hydroxylase DiOC6 3,3′-Dihexyloxacarbocyanine iodide GFP Green fluorescent protein HPLC High-performance liquid chromatography PG Plastoglobuli PSY PHYTOENE SYNTHASE RFP Red fluorescent protein
Introduction Carotenoids are one of the most diverse classes of natural compounds, with over 600 types known to occur in nature (Fraser and Bramley 2004). Plant carotenoids are derived from isopentenyl diphosphate and are generally composed of a C40 isoprenoid skeleton with or without epoxy, hydroxyl and keto groups. Hydrocarbon carotenoids are known as carotenes, whilst oxygenated carotenoids are known as xanthophylls (Britton 1995). Carotenoids play fundamental roles in human nutrition as ant
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