Figure 2.

EM Analysis of the Effects of ConcA on MVBs.

(A) ConcA treatment reduces the number of MVBs in the cell. Roots of four independent plants were analyzed either in the absence or presence of ConcA by counting the number of MVBs in 100 sectioned cells per root. Per 100 control cells, 220 ± 30 MVBs were identified, whereas after ConcA treatment, the number of MVBs was fivefold lower (40 ± 15). Error bars indicate the sd.

(B) Quantitative analysis of ConcA effects on the endogenous VSR BP80 and the Rab GTPase ARA7 in an mRFP-ARA7–expressing Arabidopsis line. The Golgi localization of BP80 and mRFP-ARA7 was analyzed in roots of four independent plants either in the absence or presence of ConcA by counting the labeling on 50 randomly chosen Golgi stacks per root. Under standard conditions, BP80 and mRFP-ARA7 do not localize to the Golgi (11% ± 4% and 15% ± 2% of Golgi labeling, respectively), whereas in the presence of ConcA, both proteins also significantly localize to the Golgi stacks (59% ± 5% and 58% ± 4% of Golgi labeling, respectively). Error bars indicate the sd.

(C) IEM localization of the endogenous BP80 in Arabidopsis roots after high-pressure freezing, freeze substitution and Lowicryl HM20 resin embedding. The VSR BP80 localizes to both the TGN (closed arrowheads) and MVBs (open arrowheads).

(D) and (E) Upon ConcA treatment, BP80 is detected at the Golgi stack ([D]; arrows) and at enlarged vesicles in the surrounding area ([E]; closed arrowheads). Although ConcA reduces the number of MVBs, those MVBs that are still present show unaltered BP80 labeling ([E]; open arrowheads).

(F) The Rab GTPase mRFP-ARA7 localizes to the limiting membrane of MVBs (arrowheads).

(G) After ConcA treatment, mRFP-ARA7 localizes to both swollen vesicles (arrowheads) and the Golgi stack (arrows).

(H) In the presence of ConcA, mRFP-ARA7 is detected at the limiting membrane of the remaining MVBs (open arrowheads).

Bars = 200 nm