RRC ID 31370
Author Segami S, Makino S, Miyake A, Asaoka M, Maeshima M.
Title Dynamics of vacuoles and H+-pyrophosphatase visualized by monomeric green fluorescent protein in Arabidopsis: artifactual bulbs and native intravacuolar spherical structures.
Journal Plant Cell
Abstract We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H(+)-pyrophosphatase (H(+)-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H(+)-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H(+)-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H(+)-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H(+)-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.
Volume 26(8)
Pages 3416-34
Published 2014-8-1
DOI 10.1105/tpc.114.127571
PII tpc.114.127571
PMID 25118245
PMC PMC4371836
MeSH Arabidopsis / metabolism* Arabidopsis / ultrastructure Arabidopsis Proteins / analysis Arabidopsis Proteins / metabolism* Cell Membrane / metabolism Dimerization Green Fluorescent Proteins / analysis Inorganic Pyrophosphatase / analysis Inorganic Pyrophosphatase / metabolism* Models, Biological Recombinant Fusion Proteins / analysis Vacuoles / metabolism* Vacuoles / ultrastructure
IF 9.618
Times Cited 54
Arabidopsis / Cultured plant cells, genes sja05800