Rapid Metabolism of Glucose Detected with FRET Glucose Nanosensors in Epidermal Cells and Intact Roots of Arabidopsis RNA-Silencing Mutants

Abstract

Genetically encoded glucose nanosensors have been used to measure steady state glucose levels in mammalian cytosol, nuclei, and endoplasmic reticulum. Unfortunately, the same nanosensors in Arabidopsis thaliana transformants manifested transgene silencing and undetectable fluorescence resonance energy transfer changes. Expressing nanosensors in sgs3 and rdr6 transgene silencing mutants eliminated silencing and resulted in high fluorescence levels. To measure glucose changes over a wide range (nanomolar to millimolar), nanosensors with higher signal-to-noise ratios were expressed in these mutants. Perfusion of leaf epidermis with glucose led to concentration-dependent ratio changes for nanosensors with in vitro K d values of 600 mM (FLIPglu-600mD13) and 3.2 mM (FLIPglu-3.2mD13), but one with 170 nM K d showed no response. In intact roots, FLIPglu-3.2mD13 gave no response, whereas FLIPglu-600mD13, FLIPglu-2mD13, and FLIPglu-170nD13 all responded to glucose. These results demonstrate that cytosolic steady state glucose levels depend on external supply in both leaves and roots, but under the conditions tested they are lower in root versus epidermal and guard cells. Without photosynthesis and external supply, cytosolic glucose can decrease to <90 nM in root cells. Thus, observed gradients are steeper than expected, and steady state levels do not appear subject to tight homeostatic control. Nanosensor-expressing plants can be used to assess glucose flux differences between cells, invertase-mediated sucrose hydrolysis in vivo, delivery of assimilates to roots, and glucose flux in mutants affected in sugar transport, metabolism, and signaling.

Alterations in Cytosolic Glucose-Phosphate Metabolism Affect Structural Features and Biochemical Properties of Starch-Related Heteroglycans

“…Although the exact reason for this remains unclear in this study, this observation is not without precedent in plant primary metabolism. The commonly ascribed view that cytosolic Glc levels are vanishingly small was recently augmented by both nonaqueous fractionation studies within the potato tuber (Farre et al, 2001) and the use of metabolite-sensing technology in Arabidopsis roots (Deuschle et al, 2006). Moreover, increasing evidence is building, suggesting an important role for metabolite channeling within primary metabolism, including the pathway of glycolysis (Giege et al, 2003;Holtgrawe et al, 2005;Graham et al, 2007).…”

Salicylic Acid Biosynthesis and Metabolism

“…An engineered Acinetobacter biosensor that produces SA-induced bioluminescence has provided important spatial and temporal data on apoplastic SA in tobacco (Figure 4; Huang et al, 2006); unfortunately, it does not work well with Arabidopsis. Synthetic SA-responsive promoters coupled to reporter proteins similar to those used in auxin research (Benková, et al, 2003), SA FRET nanosensors such as those developed to detect a range of glucose concentrations (Deuschle et al, 2006), and other SA reporters/sensors using emerging technologies (e.g. riboswitch sensor for B12 (Fowler et al, 2010)) would be invaluable components of an SA tool box.…”

Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular Resolution

“…Sensors with a single fluorescent protein report ligand-dependent changes in conformation as changes in fluorescence intensity, whereas sensors with two fluorescent proteins can yield changes in fluorescence resonance energy transfer (FRET), which can be quantified through ratiometric imaging. FRET-based sensors have been used in live plants to assess a variety of analytes, including Glc, maltose, Suc, Gln, calcium, zinc, and pH (Deuschle et al, 2006;Chaudhuri et al, 2008Chaudhuri et al, , 2011Kaper et al, 2008;Rincón-Zachary et al, 2010;Adams et al, 2012;Gjetting et al, 2012Gjetting et al, , 2013Krebs et al, 2012). Gu et al (2006) engineered a FRET-based Pi sensor named fluorescence indicator protein for inorganic phosphate (FLIPPi) that consists of a cyanobacterial inorganic phosphate binding protein (PiBP) fused to enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP) and showed the use of one of these sensors for monitoring cytosolic Pi in cultured animal cells.…”