Protonophore- and pH-insensitive glucose and sucrose accumulation detected by FRET nanosensors in Arabidopsis root tips

Carnegie Institution for Science, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USA.
The Plant Journal (Impact Factor: 5.97). 09/2008; 56(6):948-62. DOI: 10.1111/j.1365-313X.2008.03652.x
Source: PubMed


Although soil contains only traces of soluble carbohydrates, plant roots take up glucose and sucrose efficiently when supplied in artificial media. Soluble carbohydrates and other small metabolites found in soil are in part products from exudation from plant roots. The molecular nature of the transporters for uptake and exudation is unknown. Here, fluorescence resonance energy transfer (FRET) glucose and sucrose sensors were used to characterize accumulation and elimination of glucose and sucrose in Arabidopsis roots tips. Using an improved image acquisition set-up, FRET responses to perfusion with carbohydrates were detectable in roots within less than 10 sec and over a wide concentration range. Accumulation was fully reversible within 10-180 sec after glucose or sucrose had been withdrawn; elimination may be caused by metabolism and/or efflux. The rate of elimination was unaffected by pre-incubation with high concentrations of glucose, suggesting that elimination is not due to accumulation in a short-term buffer such as the vacuole. Glucose and sucrose accumulation was insensitive to protonophores, was comparable in media differing in potassium levels, and was similar at pH 5.8, 6.8 and 7.8, suggesting that both influx and efflux may be mediated by proton-independent transport systems. High-resolution expression mapping in root tips showed that only a few proton-dependent transport of the STP (Sugar Transport Protein) and SUT/SUC (Sucrose Transporter/Carrier) families are expressed in the external cell layers of root tips. The root expression maps may help to pinpoint candidate genes for uptake and release of carbohydrates from roots.

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Available from: Philip N Benfey, Oct 09, 2015
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    • "When feeding specific sugars to intact plants or excised leaves, it is frequently not clear how fast and evenly the taken up carbohydrates are transported within the tissue and whether a given sugar is converted into related sugar species, which in turn might act as a distinct signal inducing changes in gene expression ([12]. Cells lying on the exterior of the treated plant face a higher sugar concentration than cells on the inside of the plant, leading to gradients in gene expression from cell layer to cell layer. "
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    ABSTRACT: Background: Sugars modulate expression of hundreds of genes in plants. Previous studies on sugar signaling, using intact plants or plant tissues, were hampered by tissue heterogeneity, uneven sugar transport and/or inter-conversions of the applied sugars. This, in turn, could obscure the identity of a specific sugar that acts as a signal affecting expression of given gene in a given tissue or cell-type. Methodology/ Principal Findings: To bypass those biases, we have developed a novel biological system, based on stem-cell-like Arabidopsis suspension culture. The cells were grown in a hormone-free medium and were sustained on xylose as the only carbon source. Using functional genomics we have identified 290 sugar responsive genes, responding rapidly (within 1 h) and specifically to low concentration (1 mM) of glucose, fructose and/or sucrose. For selected genes, the true nature of the signaling sugar molecules and sites of sugar perception were further clarified using non-metabolizable sugar analogues. Using both transgenic and wild-type A. thaliana seedlings, it was shown that the expression of selected sugar-responsive genes was not restricted to a specific tissue or cell type and responded to photoperiod-related changes in sugar availability. This suggested that sugar-responsiveness of genes identified in the cell culture system was not biased toward heterotrophic background and resembled that in whole plants. Conclusions: Altogether, our research strategy, using a combination of cell culture and whole plants, has provided an unequivocal evidence for the identity of sugar-responsive genes and the identity of the sugar signaling molecules, independently from their inter-conversions or use for energy metabolism.
    PLoS ONE 06/2014; in press(6). DOI:10.1371/journal.pone.0100312 · 3.23 Impact Factor
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    • "Transport might also be underestimated by about twofold if transport activity is limiting and non-stereoselective for (+)-ABA (i.e., competition from (−)-ABA limits apparent (+)-ABA import) since roots were treated with (+)-ABA as part of a racemic mix, and ABACUS1 is stereoselective for (+)-ABA (Figure 3F). It is noteworthy that ABA levels accumulated much more slowly compared to glucose (Chaudhuri et al., 2008); saturation of sensor responses was reached only after ∼15 min in a typical experiment compared to less than 1 min for glucose (Grossmann et al., 2011) indicating that the uptake capacity for ABA is significantly lower relative to glucose. In contrast to ABACUS1-2µ, which saturated between 12.5 and 62.5 µM external (+)-ABA, ABACUS1-80µ continued to respond to higher levels of ABA (i.e., 312.5 µM (+)-ABA, Figure 6B). "
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    ABSTRACT: Cytosolic hormone levels must be tightly controlled at the level of influx, efflux, synthesis, degradation and compartmentation. To determine ABA dynamics at the single cell level, FRET sensors (ABACUS) covering a range ∼0.2–800 µM were engineered using structure-guided design and a high-throughput screening platform. When expressed in yeast, ABACUS1 detected concentrative ABA uptake mediated by the AIT1/NRT1.2 transporter. Arabidopsis roots expressing ABACUS1-2µ (Kd∼2 µM) and ABACUS1-80µ (Kd∼80 µM) respond to perfusion with ABA in a concentration-dependent manner. The properties of the observed ABA accumulation in roots appear incompatible with the activity of known ABA transporters (AIT1, ABCG40). ABACUS reveals effects of external ABA on homeostasis, that is, ABA-triggered induction of ABA degradation, modification, or compartmentation. ABACUS can be used to study ABA responses in mutants and quantitatively monitor ABA translocation and regulation, and identify missing components. The sensor screening platform promises to enable rapid fine-tuning of the ABA sensors and engineering of plant and animal hormone sensors to advance our understanding of hormone signaling. DOI:
    eLife Sciences 04/2014; 3(3):e01741. DOI:10.7554/eLife.01741 · 9.32 Impact Factor
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    • "The majority of biosensors that respond to varying levels of carbohydrates such as glucose or sucrose have been developed for mammalian systems, but there are also examples described for the plant field (Chaudhuri et al. 2008, 2011). These biosensors are called " FLIP " sensors and are composed of a recognition element consisting of a bacterial periplasmicbinding protein flanked by CFP and YFP. "
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    ABSTRACT: Genetically encoded biosensors are increasingly used in visualising signalling processes in different organisms. Sensors based on green fluorescent protein technology are providing a great opportunity for using Förster resonance energy transfer (FRET) as a tool that allows for monitoring dynamic processes in living cells. The development of these FRET biosensors requires careful selection of fluorophores, substrates and recognition domains. In this review, we will discuss recent developments, strategies to create and optimise FRET biosensors and applications of FRET-based biosensors for use in the two major eukaryotic kingdoms and elaborate on different methods for FRET detection.
    Protoplasma 12/2013; 251(2). DOI:10.1007/s00709-013-0590-z · 2.65 Impact Factor
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