Figure 2.
Identification of the SGB1 locus and recapitulation of the SGB1-1D mutant phenotype. (A) The insertion site of a tandem 4X 35S enhancer element in the SGB1-1D/agb-2 mutant plant (T1) was determined by plasmid rescue. The relative steady-state transcript levels of four genes flanking the T-DNA insert (∼10-kb length each side) were estimated by semiquantitative PCR using RNA from 2-wk-old rosette leaves (constant light; 22°C) of agb1-2 single and the SGB1-1D agb-2 double mutants. An elevated steady-state RNA level was found for locus At1g79820, a putative hexose transporter in Arabidopsis. Longer PCR cycling revealed an At1g79820 band in agb1-2. The β-actin 2 gene served as the normalization control. (B) The steady-state At1g79820 RNA levels in plants harboring a transgene driven by the constitutive 35S promoter-driven were measured by RT-PCR in three independent transformed lines in the agb1-2 genotype (lanes 3–5, lines 1→3). Shown for comparison are the steady-state At1g79820 RNA levels in the Col-0 wild-type and agb1-2 genotypes. (C) Recapitulation of the wild-type phenotype in the agb1-2 mutant was performed using 2-d-old seedlings grown in dark. Arabidopsis (agb1-2) was transformed with a 35S CaMV viral promoter driving the expression of the SGB1 cDNA (pHW101). Lines 1→3 mimicked the phenotype of the suppressor sgb1–1D agb1-2 with partially elongated hypocotyl and closed hooks. (D) Quantitation of hypocotyl lengths as a function of d-glucose is shown for wild type (solid diamonds), agb1-2 expressing SGB1 (closed circles), and agb1-2 (open squares). The data are from one experiment repeated twice with the same results. (E) sgb1–1D/− (hemizygous), agb-2/+ (heterozygous) did not display additional phenotypes. (F) Ectopic expression of SGB1 (35S::SGB1) in agb1-2 has increased cell numbers in developing hypocotyls. ♦, Col-0; □, agb1-2; ●, 35S:SGB1,agb1-2. The number and length of epidermal cells of the hypocotyl were determined as described in Materials and Methods. Cell number begins with the basal cell at the root–shoot junction and increases apically toward the hook of the 2-d-old etiolated seedling. The average of the SEM for cell length measurement was ±26 μm.