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

Plant material and FLIP constructs Homozygous Arabidopsis lines (T 3 ) expressing FLIPglu-2μΔ13 (line 1), FLIPglu-600μΔ13 (line 2) or FLIPglu-3.2mΔ13 (line 3) were used for the glucose flux analyses ( Deuschle et al. , 2006 ). To generate lines expressing the sucrose sensor, the FLIPsuc-90μΔ1 insert was excised from pRSETB ( Lager et al. , 2006 ) using Bam HI /Hin dIII and cloned into the Xho I /Bam HI sites of pRT101 ( Töpfer et al. , 1987 ). The Xho I overhang of pRT101 and the Hin dIII overhang from FLIPsuc-90μΔ1 were made compatible to each other by partially filling in using DNA polymerase I (Klenow fragment). The CaMV 35S-Sensor-Terminator cassette was then excised from pRT101 using Hin dIII and cloned into pPZP312 ( Hajdukiewicz et al. , 1994 ). The presence of intact inserts was verified by DNA sequencing. Binary plasmids were introduced into Agrobacterium strain GV3101 and used for transformation of homozygous Arabidopsis rdr6 lines ( Peragine et al. , 2004 ) (a generous gift from Scott Poethig, University of Pennsylvania) using the flower-dip method ( Clough and Bent, 1998 ).

Plant growth conditions

For imaging, plants were germinated in full nutrient (FN) medium (1 m m KH 2 PO 4 , 1 m m MgSO 4 , 0.25 m m K 2 SO 4 , 0.25 m m CaCl 2 , 2 m m NH 4 NO 3 , 0.1 m m Na-Fe-EDTA, 50 μ m KCl, 30 μ m H 3 BO 3 , 5 μ m MnSO 4 , 1 μ m ZnSO 4 , 1 μ m CuSO 4 , 0.7 μ m NaMoO 4 , pH 5.8, adjusted with KOH) ( Loqué et al. , 2005 ) with 1-2% sucrose, buffered with 20 m m MES and solidified with 0.7% agar in a growth chamber with 16 h light at 190 μmol m -2 sec -1 , 50% humidity, 22 °C for 7 days. Before imaging, the plants were transferred to the same medium lacking sucrose for 6 h to 5 days depending on the affinity of the sensor expressed in the plants. Where indicated, plants were imaged in potassium phosphate buffer or in ARB medium (2 m m MES, 2 m m CaSO 4 , 0.5 m m KH 2 PO 4 and 0.5 m m MgSO 4 , pH 5.8) ( Ehrhardt et al. , 1992 ). For pH experiments, the pH of FN medium was adjusted to 5.8, 6.8 or 7.8 using 10 m KOH. In vivo imaging Roots of intact seedlings were immobilized on cover slips (24 × 50 mm, VWR, http://www.vwr.com ) using medical adhesive (stock number 7730, Hollister, http://www.hollister.com ). For screening the sensor response, five or six plants were mounted onto cover slips, and a chamber was created using polymer clay (Sculpey, http://www.sculpey.com ) and filled with FN medium. The plants were then imaged during perfusion of a near-saturating concentration of sugar to determine the maximal response. Slides with plants that showed a response were then mounted on a P-1 or RC-26G perfusion chamber (Warner Instruments, http://www.warneronline.com ) mounted on a stage adapter for a Leica SA-20L3P inverted microscope (Leica, http://www.leica.com ). The volume of the chambers was 0.4-0.7 ml. Ratio imaging was performed on an inverted fluorescence microscope (DM IRE2, Leica) using a QuantEM digital camera and a 20× oil objective (HC PL APO 20x/0.7IMM CORR, Leica). Dual emission intensities were simultaneously recorded using a DualView with a dual CFP/YFP-ET filter set (ET470/24m; ET535/3, Chroma, http://www.chroma.com ) and Slidebook software (Intelligent Imaging Innovations Inc., http://www.intelligent-imaging.com ). Excitation (filter ET430/24x, Chroma) was provided by a Lambda DG4 light source (Sutter Instruments, http://www.sutter.com ). Images were acquired within the linear detection range of the camera, and exposure times varied between 400 and 600 msec depending on the expression level, with binning 2 and an Electron Multiplying gain of 300. Fluorescence intensities for eCFP and eYFP were typically in the range of 2000-8000 and 6000-14 000, respectively. Regions outside the root were used for background subtraction (background value 1000-1300; 300 msec exposure time). Perfusions were performed with FN medium buffered with 20 m m MES pH 5.8 at 3 ml min -1 . The baselines throughout were corrected using second- or third-order polynomial fits of the ratios measured in the absence of glucose. The obtained function describes the baseline aberration (photobleaching) as a function of time during the perfusion. To correct for this effect, the difference between the ratio at the beginning of the experiment, r (0), and the baseline aberration, f ( t ), were calculated at each time point of the measurement and added to the value of the measured ratio at the respective time point, r ( t ), such that r corr ( t ) = r ( t ) + r (0)) f ( t ). Accumulation and elimination rates for glucose and sucrose were calculated by determining Δratio/time at various external sugar concentrations (for a detailed description of the analysis, see Okumoto et al. , 2008 ). The data fitted well to Michaelis-Menten kinetics. However, it would be more accurate to use a model that incorporates all contributing fluxes (e.g. Fehr et al. , 2005 ).

Imaging in the presence of inhibitors

CCCP and 2,4-DNP (both Sigma, http://www.sigmaaldrich.com/ ) were prepared as 250 m m stock in 100% DMSO and frozen; nigericin (EMD Biosciences, http://www.emdbiosciences.com ) was prepared as 5 m m stock in 95% ethanol and frozen. Before use, CCCP and 2,4-DNP stocks were diluted to 100 μ m in FN medium (final DMSO concentration 0.04%); nigericin was diluted to 25 μ m . All experiments were repeated at least three times. Controls were performed with solutions containing 0.04% DMSO or 0.5% ethanol, respectively. Tetraethylammonium was prepared as a 100 m m stock solution in H 2 O and was diluted prior to use. Plants were incubated with the indicated inhibitor for 15 min before resuming perfusion with either sugar. 3- O -methylglucose was prepared as a 1 m stock in H 2 O. Estimate of exchange rates for the RC-26G perfusion chamber Exchange rates were determined on the same perfusion set-up as used for the imaging experiments using fluorescent dye Alexa Fluor 430 (Invitrogen, http://www.invitrogen.com/ ) at 3 ml min -1 . After the fluorescence intensity reached saturation, perfusion was switched back to the buffer. The rates were calculated by taking the maximum fluorescence intensity as 100% and determining the time that it takes to reach 85% or 15% fluorescence, i.e. the linear uptake or efflux phases, respectively. The addition and removal rates are the mean of two experiments.

Confocal microscopy

For analysis of Golgi movement, Arabidopsis thaliana seedlings expressing YFP:SYP32 were grown for 6 days on FN medium and root tips were imaged on a spinning-disk confocal microscope as described by Takanaga et al. (2008) . Plants were mounted and treated as for imaging experiments. Images were acquired every 4 sec for 2 min before adding 100 μ m CCCP and for 8 min thereafter. Image processing was performed using Metamorph (Molecular Devices, http://www.moleculardevices.com ) and ImageJ software (W. Rasband, National Institutes of Health, Bethesda, MD). Two images (20 sec apart) were merged after false coloring in red for the first frame and green for the second. YFP:SYP32 wave lines were a generous gift from Niko Geldner and Joanne Chory. More information about these lines can be found at http://www.unil.ch/dbmv/page49637_en.html . The expression of the nanosensors expressed under the control of the CaMV 35S promoter was analyzed by collecting an image series for root tips expressing either FLIPglu-600μΔ13 or FLIPsuc-90μΔ1 as z-stacks using a Leica confocal SP5 microscope equipped with an argon laser (514 nm excitation for YFP) and a 442 diode laser (442 nm excitation for CFP). The emission signal was captured from 525-560 nm for YFP and 460-500 nm for CFP. The pinhole was adjusted to an airy disk size of 0.5, and images were captured at 1.5 μm intervals. In vitro titration of glucose sensor proteins under various pH conditions FLIPglu-2μΔ13 and FLIPglu-600μΔ13 were expressed in E. coli (BL21 DE3 gold) and grown for 3 days at room temperature. Protein was isolated as described previously ( Deuschle et al. , 2005 ). Substrate titration curves and substrate specificity analysis were performed using a monochromator microplate reader (Safire; excitation 433/12 nm; emission 485/12 and 528/12 nm; gain 80, http://www.tecan.com ). In vitro analyses at various pH were performed in MES/Tris buffer; pH was adjusted by mixing 40 m m MES and 40 m m Tris buffers. Proteins were titrated using various glucose concentrations in each of the pH buffers. FRET was approximated as the peak emission intensity ratio at 480 nm (first eCFP peak) and 528 nm (eYFP peak). Measurements were performed on two independent protein extracts.

Approximation of cell-type specific expression at developmental stage resolution

High-resolution genome-wide expression data at the level of each cell type at all developmental stages in the Arabidopsis root are not available, due to the technical limitations regarding the amount of material required for microarray measurements. However, using the large microarray dataset profiling 19 semi-overlapping marker lines and 13 developmental sections published recently ( Brady et al. , 2007 ), a reasonable approximation can be made. Expression of a given gene within a particular tissue or cell type and section was approximated as the mean expression from all marker lines covering that tissue in that section, and scaling that mean by a factor equal to the expression of that gene in that developmental section relative to its mean expression over all longitudinal sections. Thus, expression of gene g within tissue t in developmental section s , denoted G ts , can be approximated as: G t s = ∑ M M g ⋅ C m t s ∑ M C m t s . S g A g where M g is the expression of gene g in marker line m, C mts is a 1/0 indicator variable depending on whether marker m covers tissue t in section s, S g is the expression of gene g in section s , and A g is the mean expression of gene g over all sections. Expression within a region that is not covered by any marker line (e.g. trichoblasts in meristematic zone sections) is reported as 0.

Table

S2 and Figure S6 show the marker line × developmental section, marker line × cell type and developmental section × cell type matrices that were used to approximate expression. Approximating cell type-/developmental stage-specific resolution by this method has some restrictions, such as treating expression within a marker line as constant throughout all developmental stages, the inability to approximate expression in areas not covered by any marker line, and the inability to guarantee that the approximated data exactly reproduce the observed measurements. Methods that are not subject to such restrictions would be very useful for more detailed studies, but, for the purposes of identifying candidate genes, our simplistic approximation is sufficient. The scale was set to a maximum of 5; thus some of the transporters are more highly expressed than shown here (see Table S2 for detailed quantification).

Plant material and FLIP constructs Homozygous Arabidopsis lines (T 3 ) expressing FLIPglu-2μΔ13 (line 1), FLIPglu-600μΔ13 (line 2) or FLIPglu-3.2mΔ13 (line 3) were used for the glucose flux analyses ( Deuschle et al. , 2006 ). To generate lines expressing the sucrose sensor, the FLIPsuc-90μΔ1 insert was excised from pRSETB ( Lager et al. , 2006 ) using Bam HI /Hin dIII and cloned into the Xho I /Bam HI sites of pRT101 ( Töpfer et al. , 1987 ). The Xho I overhang of pRT101 and the Hin dIII overhang from FLIPsuc-90μΔ1 were made compatible to each other by partially filling in using DNA polymerase I (Klenow fragment). The CaMV 35S-Sensor-Terminator cassette was then excised from pRT101 using Hin dIII and cloned into pPZP312 ( Hajdukiewicz et al. , 1994 ). The presence of intact inserts was verified by DNA sequencing. Binary plasmids were introduced into Agrobacterium strain GV3101 and used for transformation of homozygous Arabidopsis rdr6 lines ( Peragine et al. , 2004 ) (a generous gift from Scott Poethig, University of Pennsylvania) using the flower-dip method ( Clough and Bent, 1998 ).

Supplementary Material sm001 Figure S1. Comparison of FRET sensor responses in various regions of the root tip. sm010 Movie S1. Cellular expression of the sucrose nanosensor in root tips of Arabidopsis. sm002 Figure S2. Glucose and sucrose response curves in the absence and presence of various inhibitors. sm003 Figure S3. Effect of CCCP on Golgi movement. sm004 Figure S4. [ 14 C]-glucose uptake into intact seedlings. sm005 Figure S5. Insensitivity of responses to tetraethylammonium. sm006 Figure S6. Marker line coverage. sm007 Table S1 Expression levels for the transporter genes as shown in Figure 9 . sm008 Table S2 Marker line coverage. sm009 Appendix S1. Radioactive glucose uptake in intact seedlings of Arabidopsis thaliana .