Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem. Page: 2 of 5
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Antisense BvSUTI
Sense BvSUT1
Fig. 1. Companion cell-specific localization of BvSUTI mRNA. In sections of mature sugar beet leaf hybridized with antisense BvSUTI cRNA probe, staining was
localized to cytoplasmically dense companion cells proximal to the sieve elements, whereas no cells were stained in sections hybridized with the sense probe.
The veins shown are directly embedded in the mesophyll and lack supporting parenchyma, indicating they are minor veins involved in phloem loading.tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution
(Sigma) for 15 min. A diffuse band between 43 and 49 kDa was
detected by serum directed against the C-terminal peptide.
Specificity of this band was demonstrated by performing immu-
noblots with 1:500 dilution of antiserum that had been precom-
peted with 5 sg/ml BSA-conjugated C-terminal peptide for 8 h
at 40C. Recognition of the 43-49 kDa band was completely
abolished, but other nonspecific bands remained.
RNA Isolation and Gel Blot Analysis. RNA was isolated by using the
RNAeasy kit according to the manufacturer's directions (Qia-
gen, Valencia, CA). Total RNA (15 g) from each sample was
fractionated on a 1.0% agarose-formaldehyde gel transferred
to a Hybond N+ (Amersham Pharmacia) nylon membrane.
BvSUTI (GenBank accession no. U64967) and EFlA (Gen-
Bank accession no. AF030517) [a_32P]-dCTP probes were
made by using the Megaprime DNA Labeling System (Amer-
sham Pharmacia). Hybridizations were performed in Ultrahyb
solution (Ambion, Austin, TX) for 18 h at 420C. Blots were
washed twice in 420C 5X SSC/0.1% wt/vol SDS for 15 min,
twice in 420C 0.1X SSC/0.1% wt/vol SDS for 30 min, and
exposed to x-ray film. Band intensities were measured by using
a digital densitometer.
Isolation of Nuclei. Frozen leaf tissue was crushed under liquid
nitrogen, added to grind solution (2.5% wt/vol Ficoll 400/5.1%
wt/vol Dextran T-40/25 mM Tris-HCl, pH 8.5/2.5% vol/vol
Triton X-100/4.6 mM MgCl2/0.44 M sucrose/0.007% 2-
mercaptoethanol/0.2 g/liter spermine), and homogenized by
using a Brinkmann Polytron at setting 1 for 45 s. The homog-
enate was filtered through 6 layers of rinsed, chilled cheese-
cloth and centrifuged at 40C for 8 min at 6,000 rpm in a Sorvall
SS-34 rotor. The pellet was suspended in grind solution (minus
spermine) and centrifuged. This step was repeated once.Pellets were suspended in ice-cold wash buffer (50 mM
Tris-HCl, pH 8.5/5 mM MgCl2/20% vol/vol glycerol) and
centrifuged as described. Finally, pellets were suspended in 500
M1 ice-cold storage buffer (50 mM Tris-HCl, pH 8.5/5 mM
MgCl2/50% vol/vol glycerol), flash-frozen, and stored at
-80C. An aliquot from each isolation was counted in a
hemacytometer.
Nuclear Run-On Analysis. Run-on transcription was performed as
described in Liu (9). Transcription reactions containing [a-32P]-
UTP-labeled mRNA were extracted in phenol/chloroform/
isoamyl alcohol (25:24:1, pH 4.6) followed by chloroform and the
RNA was ethanol-precipitated overnight at -20C. RNA pellets
were suspended in diethyl pyrocarbonate-treated water. RNA
samples (1 X 106 cpm/ml) were incubated with membranes
containing 5 g spots of BvSUT1,ArabidopsisACT2 (GenBank
accession no. U41998), rice EFlA, and pBluescript-KS (Strat-
agene) in 2 ml Ultrahyb solution at 420C for 24 h. Blots were
washed twice in 420C 2X SSC, 0.1% wt/vol SDS for 5 min and
twice for 20 min in 420C 0.1X SSC, 0.1% wt/vol SDS, and
exposed to x-ray film for 1 to 2 days. Signal intensities were
determined by using a digital densitometer. To determine
relative transcription rates, BvSUTI signal was expressed as the
ratio between its signal intensity and the intensity of the control
gene EFlA. These values were then compared between blots to
derive relative BvSUTI transcription.
Results
Sucrose transporters are expressed not only in the phloem, but also
in other cell types and tissues where they fulfill diverse physiological
roles (10, 11). In sugar beet leaves, BvSUTI mRNA expression
corresponds to the onset of photosynthetic maturity, supporting a
role in photoassimilate transport (12). However, the prospect that
this transporter might also be found in other leaf cell typesPNAS I August 6, 2002 1 vol. 99 1 no. 16 1 10877
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Vaughn et al.
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Vaughn, Matt; Harrington, Greg & Bush, Daniel R. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem., article, August 6, 2002; United States. (https://digital.library.unt.edu/ark:/67531/metadc788915/m1/2/: accessed April 25, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.