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J Exp Bot. 2013 Jul;64(10):2817-29. doi: 10.1093/jxb/ert125. Epub 2013 May 16.

ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies.

Author information

1
School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

Abstract

Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1-2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP-ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP-ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP-ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.

KEYWORDS:

ARA7(Q69L); MVB enlargement; homotypic fusion; multivesicular body; pre-vacuolar compartment; transgenic Arabidopsis cells.

PMID:
23682115
PMCID:
PMC3697957
DOI:
10.1093/jxb/ert125
[Indexed for MEDLINE]
Free PMC Article

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