A Noninvasive Platform for Imaging and Quantifying Oil Storage in Submillimeter Tobacco Seed    

Abstract

While often thought of as a smoking drug, tobacco (Nicotiana spp.) is now considered as a plant of choice for molecular farming and biofuel production. Here, we describe a noninvasive means of deriving both the distribution of lipid and the microtopology of the submillimeter tobacco seed, founded on nuclear magnetic resonance (NMR) technology. Our platform enables counting of seeds inside the intact tobacco capsule to measure seed sizes, to model the seed interior in three dimensions, to quantify the lipid content, and to visualize lipid gradients. Hundreds of seeds can be simultaneously imaged at an isotropic resolution of 25 mm, sufficient to assess each individual seed. The relative contributions of the embryo and the endosperm to both seed size and total lipid content could be assessed. The extension of the platform to a range of wild and cultivated Nicotiana species demonstrated certain evolutionary trends in both seed topology and pattern of lipid storage. The NMR analysis of transgenic tobacco plants with seed-specific ectopic expression of the plastidial phosphoenolpyruvate/phosphate translocator, displayed a trade off between seed size and oil concentration. The NMR-based assay of seed lipid content and topology has a number of potential applications, in particular providing a means to test and optimize transgenic strategies aimed at the manipulation of seed size, seed number, and lipid content in tobacco and other species with submillimeter seeds.

Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture

“…If this transporter is highly abundant and in vivo reversible, the modulation of plastidial PEP levels by PKp can be thought to be propagated across the chloroplast envelope so that the feedback control can take place in the cytosol. Accordingly, the PEP/phosphate translocator has been ascribed an important role in lipid synthesis (Ruuska et al, 2002;Kubis et al, 2004), and its overexpression in tobacco seeds has been demonstrated to promote lipid accumulation (Fuchs et al, 2013).…”

“…Metabolic flux studies with developing seeds have added a quantitative understanding of in vivo pathway usage in oilseeds (O'Grady et al, 2012). To modulate seed oil levels or composition, various attempts have been chosen, mostly relying on introducing specific enzymes (Weselake et al, 2008;Kelly et al, 2013) or metabolite transporters (Fuchs et al, 2013;Kim et al, 2013). A number of seed-specific transcription factors are known to affect oil storage capabilities (Le et al, 2010); their modulated expression as well as the introduction of engineered transcription factors can enable a more global modulation of oilseed metabolism (Century et al, 2008;Gupta et al, 2012;Singh et al, 2013).…”

“…After 10 d of culture, embryos were weighed, freeze dried, and weighed again for the determination of fresh and dry weights. Embryo material was pulverized and used for the quantification of total lipid content (using time-domain NMR as described by Fuchs et al [2013]), fatty acid composition (using gas chromatography as described by Borisjuk et al [2013a]), and total protein content (measured as total nitrogen 3 5.64 using elemental analysis as described by Borisjuk et al [2013a]). This in vitro system has been used in former studies to describe flux distribution and pathway usage in central metabolism, which demonstrated that major aspects of in planta seed metabolism can be mimicked in vitro (Schwender et al, 2003(Schwender et al, , 2004(Schwender et al, , 2006Schwender, 2008).…”

Seed Architecture Shapes Embryo Metabolism in Oilseed Rape

“…However, recent developments in magnetic resonance imaging (MRI; Borisjuk et al, 2012) and mass spectrometry have begun to remove this limitation, thereby allowing for the spatial mapping of storage lipids (Neuberger et al, 2009;Fuchs et al, 2013) and their individual components (Horn et al, 2012). In this article, we designed an array of spatial high-resolution techniques, which allowed us not only to visualize steep gradients in oil deposition within the embryo of oilseed rape but also to define their spatial/ temporal relations to those established for the accumulation of starch and storage proteins, cellular growth, photosynthetic activity, and metabolite pattern.…”

Specialization of Oleosins in Oil Body Dynamics during Seed Development in Arabidopsis Seeds

“…Third harmonic generation microscopy (Débarre et al, 2006) and label-free coherent anti-Stokes Raman scattering microscopy (Paar et al, 2012) allow dyeless observation of LDs but require very specific equipment. Magnetic resonance imaging enables topographic analysis of lipid distribution in cereal grains (Neuberger et al, 2008) and in submillimeter-sized seeds like those of tobacco (Nicotiana tabacum; Fuchs et al, 2013). Nevertheless, the use of fluorescent dyes such as Nile Red (Greenspan and Fowler, 1985), BODIPY (Pagano et al, 1991), or LipidTOX (Invitrogen) associated with confocal microscopy is also a powerful way to monitor LDs in living organisms.…”