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. 1995 Dec;7(12):2053–2067. doi: 10.1105/tpc.7.12.2053

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

N D DeWitt 1, M R Sussman 1
PMCID: PMC161061  PMID: 8718619

Abstract

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.

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Selected References

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  1. Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 1984 Nov 26;12(22):8711–8721. doi: 10.1093/nar/12.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bouche-Pillon S., Fleurat-Lessard P., Fromont J. C., Serrano R., Bonnemain J. L. Immunolocalization of the Plasma Membrane H+ -ATPase in Minor Veins of Vicia faba in Relation to Phloem Loading. Plant Physiol. 1994 Jun;105(2):691–697. doi: 10.1104/pp.105.2.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boutry M., Michelet B., Goffeau A. Molecular cloning of a family of plant genes encoding a protein homologous to plasma membrane H+-translocating ATPases. Biochem Biophys Res Commun. 1989 Jul 31;162(2):567–574. doi: 10.1016/0006-291x(89)92348-6. [DOI] [PubMed] [Google Scholar]
  4. DeWitt N. D., Harper J. F., Sussman M. R. Evidence for a plasma membrane proton pump in phloem cells of higher plants. Plant J. 1991 Jul;1(1):121–128. doi: 10.1111/j.1365-313x.1991.00121.x. [DOI] [PubMed] [Google Scholar]
  5. Ewing N. N., Bennett A. B. Assessment of the number and expression of P-type H(+)-ATPase genes in tomato. Plant Physiol. 1994 Oct;106(2):547–557. doi: 10.1104/pp.106.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ewing N. N., Wimmers L. E., Meyer D. J., Chetelat R. T., Bennett A. B. Molecular Cloning of Tomato Plasma Membrane H-ATPase. Plant Physiol. 1990 Dec;94(4):1874–1881. doi: 10.1104/pp.94.4.1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harper J. F., Manney L., DeWitt N. D., Yoo M. H., Sussman M. R. The Arabidopsis thaliana plasma membrane H(+)-ATPase multigene family. Genomic sequence and expression of a third isoform. J Biol Chem. 1990 Aug 15;265(23):13601–13608. [PubMed] [Google Scholar]
  8. Harper J. F., Manney L., Sussman M. R. The plasma membrane H(+)-ATPase gene family in Arabidopsis: genomic sequence of AHA10 which is expressed primarily in developing seeds. Mol Gen Genet. 1994 Sep 28;244(6):572–587. doi: 10.1007/BF00282747. [DOI] [PubMed] [Google Scholar]
  9. Harper J. F., Surowy T. K., Sussman M. R. Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1234–1238. doi: 10.1073/pnas.86.4.1234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Houlné G., Boutry M. Identification of an Arabidopsis thaliana gene encoding a plasma membrane H(+)-ATPase whose expression is restricted to anther tissue. Plant J. 1994 Mar;5(3):311–317. doi: 10.1111/j.1365-313x.1994.00311.x. [DOI] [PubMed] [Google Scholar]
  11. Katz D. B., Sussman M. R., Mierzwa R. J., Evert R. F. Cytochemical localization of ATPase activity in oat roots localizes a plasma membrane-associated soluble phosphatase, not the proton pump. Plant Physiol. 1988 Mar;86(3):841–847. doi: 10.1104/pp.86.3.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  13. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Lůcas W. J., Wolf S. Plasmodesmata: the intercellular organelles of green plants. Trends Cell Biol. 1993 Sep;3(9):308–315. doi: 10.1016/0962-8924(93)90013-q. [DOI] [PubMed] [Google Scholar]
  16. Palmgren M. G., Christensen G. Complementation in situ of the yeast plasma membrane H(+)-ATPase gene pma1 by an H(+)-ATPase gene from a heterologous species. FEBS Lett. 1993 Feb 15;317(3):216–222. doi: 10.1016/0014-5793(93)81279-9. [DOI] [PubMed] [Google Scholar]
  17. Palmgren M. G., Christensen G. Functional comparisons between plant plasma membrane H(+)-ATPase isoforms expressed in yeast. J Biol Chem. 1994 Jan 28;269(4):3027–3033. [PubMed] [Google Scholar]
  18. Parets-Soler A., Pardo J. M., Serrano R. Immunocytolocalization of Plasma Membrane H-ATPase. Plant Physiol. 1990 Aug;93(4):1654–1658. doi: 10.1104/pp.93.4.1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Samuels A. L., Fernando M., Glass A. D. Immunofluorescent Localization of Plasma Membrane H-ATPase in Barley Roots and Effects of K Nutrition. Plant Physiol. 1992 Aug;99(4):1509–1514. doi: 10.1104/pp.99.4.1509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Serrano R., Monk B. C., Villalba J. M., Montesinos C., Weiler E. W. Epitope mapping and accessibility of immunodominant regions of yeast plasma membrane H(+)-ATPase. Eur J Biochem. 1993 Mar 15;212(3):737–744. doi: 10.1111/j.1432-1033.1993.tb17712.x. [DOI] [PubMed] [Google Scholar]
  21. Stadler R., Brandner J., Schulz A., Gahrtz M., Sauer N. Phloem Loading by the PmSUC2 Sucrose Carrier from Plantago major Occurs into Companion Cells. Plant Cell. 1995 Oct;7(10):1545–1554. doi: 10.1105/tpc.7.10.1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Uknes S., Dincher S., Friedrich L., Negrotto D., Williams S., Thompson-Taylor H., Potter S., Ward E., Ryals J. Regulation of pathogenesis-related protein-1a gene expression in tobacco. Plant Cell. 1993 Feb;5(2):159–169. doi: 10.1105/tpc.5.2.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang Q., Monroe J., Sjölund R. D. Identification and characterization of a phloem-specific beta-amylase. Plant Physiol. 1995 Nov;109(3):743–750. doi: 10.1104/pp.109.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]

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