Article Figures & Data
Figures
- Fig. 1.
Expression cassettes. 1, The pGED/ARA expression cassette for apoplastic targeting. 2, The pGED/ST::ARA cassette conferring Golgi targeting. Dotted bar, Region encoding theA. aculeatus α-1,5-endo-arabinanase signal sequence. Black bar, Mature A. aculeatus α-1,5-endo-arabinanase. Gray bar, Region encoding the β-galactoside α-2,6-sialyltransferase cytoplasmic domain. Hatched bar, Region encoding the β-galactoside α-2,6-sialyltransferase membrane spanning/signal sequence domain. White bar, Region encoding a truncated β-galactoside α-2,6-sialyltransferase catalytic domain. Pro, Tuber-specific granule-bound starch synthase promoter. Term, Nopaline synthase terminator and polyadenylation site.
- Fig. 2.
Phenotypic appearance of WT and endo-arabinanase expressing potato plants. 1, Transformant harboring the pGED/ARA construct for apoplastic targeting. 2, Transformant harboring the pGED/ST::ARA construct for Golgi targeting. 3, WT potato plant.
- Fig. 3.
Western analysis of protein extracts from pGED/ARA leaves after 24-h induction of the granule-bound starch synthase promoter with Suc and light. 1, Induced pGED/ARA leaves. 2, Noninduced pGED/ARA leaves. 3, Induced WT leaves. 4, Noninduced WT leaves. Blot was probed with an antiserum raised against the A. aculeatus endo-arabinanase.
- Fig. 4.
Immunoblots of fractions collected during separation of organelles from T7.2 and WT tuber tissue. 1, Cytosolic fraction from T7.2. 2, ER fraction from T7.2. 3, Golgi fraction from T7.2. 4, Putative cytosolic fraction from WT. 5, Putative ER fraction from WT. 6, Golgi fraction from WT. A, Western blot probed with mAbs recognizing the Golgi marker RGP1. B, Western blot probed with mAbs recognizing the ER marker CRH. C, Western blot probed with polyclonal antibodies raised against the A. aculeatus endo-arabinanase. The observed molecular masses are in agreement with the 41.5 kD determined for pea RGP1 (Dhugga et al., 1997), the 60 kD determined for the tobacco CRH homolog (Denecke et al., 1995), and the predicted molecular mass of 39 kD for the ST-ARA fusion protein.
- Fig. 5.
Sections of WT (A and C) and ST-ARA-expressing (T7.2, B and D) potato tubers gold-labeled with mAb LM6, silver-enhanced and viewed by reflection confocal laser scanning microscopy (A and B) and transmission electron microscopy (C and D). WT parenchymal walls are strongly labeled (green in A; black particles in C); whereas, in contrast, the LM6 arabinan epitope abundance in the equivalent T7.2 walls is greatly reduced and localized to the primary wall (arrowheads in D) and absent from the middle lamella (asterisks in C and D). Pd and Ctx indicate periderm and cortical regions, respectively; C and D show perimedullary walls. Scale bars represent 200 μm (in A and B) and 2 μm (in C and D).
Tables
- Table I.
Sugar compositions (mol%) of RGI material obtained from tubers of two pGED/ST∷ARA transformants (T7.2, T5.2) and wild type (WT)
WT T7.2 T5.2 Fuc 0.2 ± 0.2 0.2 ± 0.1 0.1 ± 0.1 Ara 15.8 ± 1.2 4.9 ± 0.3 5.2 ± 0.5 Rha 5.7 ± 2.0 6.1 ± 1.4 6.2 ± 0.3 Gal 64.6 ± 4.1 77.0 ± 3.5 76.3 ± 4.3 Glc 0.3 ± 0.2 0.4 ± 0.2 0.3 ± 0.2 Xyl 0.4 ± 0.3 0.1 ± 0.1 0.1 ± 0.1 GalA 13.0 ± 2.5 11.3 ± 1.5 11.8 ± 1.1 Data (±sd) are the average of three independent experiments.
