Novel application of 2-[18F]fluoro-2-deoxy-d-glucose to study plant defenses
Introduction
Plants respond to stresses, including attack by pests, with extensive reprogramming of primary and secondary metabolism [1]. Because they are modular organisms, these responses vary within plants among tissues and organs in space and time [2]. Many, if not all responses to stress, are systemic (occurring in most or all tissues). Since responses begin at the site of damage (locally) and spread systemically [3], the ability to respond effectively requires integrating individual modules within the plant that may even compete for resources [4]. Transport of metabolites and signals among tissues is frequently constrained by vascular architecture [5].
The long-distance transport of primary metabolites [6], hormones associated with growth and defense [7], [8], and proteins and their RNA fragments [9] have all been shown to play a role in systemic defense induction. Sugars transported from source to sink tissues provide the necessary resources for growth of young tissues and have been shown to serve as building blocks for the production of carbon-based defenses in developing plant foliage [10].
Oxylipins, especially the jasmonates (JAs), are important signals integrating plant responses to the environment, especially those elicited by tissue damage, pathogen infection and herbivory [8], [11]. JAs play roles in diverse processes including embryogenesis, seed germination, flowering, pollen development, drought stress and leaf senescence [12]. They also elicit transcriptional changes in numerous genes that impact whole-plant resource allocation of both nitrogen and carbon containing compounds [13]. In Arabidopsis thaliana, JAs such as methyl JA (MeJA) have been shown to be essential for the transcriptional activation of pathways producing several classes of secondary metabolites, including glucosinolates, phenylpropanoids, anthocyanins and isoprenoids [14]. We know much less about their role in regulating resource allocation among tissues and to secondary metabolism in response to stressors.
The short-lived, positron-emitting radioisotopes carbon-11 (t1/2 20.4 min) [11], [15] and nitrogen-13 (t1/2 9.97 min) [16], [17], [18] are useful for studying long-distance transport of resources between tissues over relatively short time intervals. They also have been used to study resource reallocation after insect attack [8], [19], [20], [21] and to assess changes in metabolic partitioning into key defense pathways [11], [22], [23].
The radioisotope 18F (t1/2 = 110 min) also has been extremely useful for radiolabeling molecules of interest for use in life sciences [24], but has been little used in plant biology. 2-[18F]fluoro-2-deoxy-d-glucose (18FDG) is a radioactive surrogate of glucose used to image sugar metabolism in the human brain and locate metabolically active tissues such as cancer cells using positron emission tomography. 18FDG's high specific activity and short half-life offer a unique opportunity to plant scientists to administer tracer well below biologically active levels and probe, with high sensitivity, sugar transport and metabolism simultaneously. Only one report has demonstrated uptake and movement of 18FDG in plants [25], and there are no studies of its incorporation into plant metabolism. The utility of this tool in physiological, biochemical and ecological contexts depends on the degree to which it functions normally as glucose in the plant.
We used 18FDG as a radiotracer glucose surrogate to examine its transport and incorporation into phenolic metabolism in Arabidopsis in response to wounding and MeJA application. Plant phenolic compounds are a diverse group of secondary metabolites, characterized by hydroxylated aromatic rings, and can range from simple acids like gallic acid to more complex flavonoids, condensed tannins (proanthocyanins) and lignins. Many plant phenolics such as chlorogenic acid, caffeic acid and rutin have been shown to have roles in plant defense against pathogens and insects [26]. In this study, we were particularly interested in whether plants would use 18FDG in anthocynanin biosynthesis, as these compounds typically require glucose in glycoside structures (Fig. 1).
Further, we examined normal patterns of 18FDG distribution in both vegetative and reproductive plants and determined whether the combined effect of wounding and exogenous MeJA treatment induces whole-plant changes in resource allocation and partitioning of 18FDG into secondary metabolite biosynthesis. Our results indicate that 18FDG is a powerful tool in plant science.
Section snippets
Plant material
Arabidopsis Col-0 plants were grown in individual pots in potting soil (Pro-Mix; Premier Horticulture Inc., Quakertown, PA, USA) supplemented with 1.8 kg of Osmocote slow-release fertilizer (The Scotts Company, Marysville, OH, USA) per cubic meter of soil. Plants were grown under metal halide lamps at 24°C and 62% relative humidity with a 12/12-h 180-μmol m− 2 s− 1 photoperiod. Vegetative (rosette only) and reproductive plants (rosette with bolted stem) used in experiments were 4 and 5 weeks
18FDG distribution among aboveground tissues
Three hours after the petiole of leaf seven was labeled with 18FDG, radioactivity was most concentrated in the petiole of that leaf and in the younger and older leaves most directly above and below it in the phyllotaxy (Fig. 2, Fig. 3). This pattern of tracer movement is consistent with previously described vascular connections between the labeled leaf and others in its orthostichy [29], [30] and occurred in both vegetative and reproductive plants (Fig. 2, Fig. 3). Sectoriality was also
Discussion
Our results illustrate how the use of short-lived tracers like 18FDG provide another tool for exploring rapid resource dynamics and factors controlling resource partitioning into secondary metabolic pathways in plants. Earlier work demonstrated that 18FDG can be loaded into phloem and transported between source and sink tissues [25], but our results suggest that this tracer travels as an intact 18FDG sugar molecule to distal tissues where it is incorporated into secondary metabolites. While it
Conclusions
Our conclusions may differ from those of investigators finding export away from elicited tissues for several reasons. With one exception, studies finding elicited increases in transport to roots commonly employ 11C as the tracer, while others have used 13C or 18FDG. It is possible that molecules moving in different directions in the plant differentially acquire these tracers, thus highlighting the need to identify the materials actually transported in future studies. The direction and extent of
Acknowledgments
We thank M. Kasel for methods related to 18FDG desalination, as well as other members of the Schultz-Appel lab for insightful comments that improved this manuscript. This research was supported by the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-98CH10886 to R.A.F.; DOE DE-SC0002040 Research Projects for Interrogations of Biological Systems: Training for the Development of Novel Radiotracer (University of Missouri, Chemistry Department),
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2015, Trends in Plant ScienceCitation Excerpt :For administration of this tracer, it is necessary to damage the plant surface (Figure 2), after which it is transported and incorporated into secondary metabolites. Because intact 18FDG is transported, questions arise whether it really mimics actual sugar transport [45], and this might explain why 18FDG has not yet found wide application in plant research [46]. Phloem vessels are known to be notoriously difficult to investigate because they operate under high pressures [47].
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2014, Applied Radiation and IsotopesCitation Excerpt :IAA labeled with long-lived radioisotopes, such as [3H]IAA (T1/2=12.32 y) and [14C]IAA (T1/2=5730±40 y), has been used in botany research since the middle of last century (Bendaña et al., 1965; Hertel et al., 1969; Hicks et al., 1989; Jones et al., 1984; PerkinElmer, 2012; Sabnis et al., 1969; Zhu and Davies, 1997). Utilizing short-lived radioisotopes, carbon-11 (T1/2=20.4 min) and fluorine-18 (T1/2=109.7 min), for in vivo carbon flow kinetic measurements only became possible when the Positron Emission Tomography (PET) technology became widely available for medical research with human and animal subjects (Sweet, 1951, 1953), as well as other biological systems (Agtuca et al., 2013; Babst et al., 2012, 2013; Ferrieri et al., 2012; Kinsella et al., 2012; Minchin and Thorpe, 1989; Pritchard et al., 2004; Thorpe et al., 2007; Weisenberger et al., 2011, 2012). The non-destructive nature of PET imaging coupled with the high specific activity of the radiotracer make it possible to perform longitudinal physiology and metabolism research in a single plant throughout its life cycle (Babst et al., 2012; Ferrieri et al., 2005, 2013; Fowler and Wolf, 1981; Minchin and Thorpe, 1989).
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2014, Nuclear Medicine and BiologyCitation Excerpt :Arabidopsis plants photosynthesize sucrose as photoassimilate and it is the main sugar component translocated in phloem. It had been reported that 18FDG persist as an intact sugar molecule during its translocation to other plant parts [19]. This is contradictory to the literature that only sugars such as sucrose, raffinose and sugar alcohol such as galactinol have been known to be translocated in Arabidopsis thaliana phloem [42,43].