Spatial Coordination of Chloroplast and Plasma Membrane Activities in Chara Cells and Its Disruption through Inactivation of 14-3-3 Proteins

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A. A. Bulychev^1*, P. W. J. van den Wijngaard², and A. H. de Boer²

¹Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-1115; E-mail: bulychev@biophys.msu.ru

²Department of Developmental Genetics, Vrije Universiteit, Faculty of Earth and Life Sciences, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; E-mail: ahdeboer@bio.vu.nl

^* To whom correspondence should be addressed.

Received March 19, 2004; Revision received June 25, 2004

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In Chara corallina cells exposed to continuous light, external pH (pH_o) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H⁺-pumping and H⁺-conductance are coupled in the different bands. First, PM H⁺-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H⁺ fluxes across the PM. Next, H⁺-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH_o by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H⁺-pump upon the detachment of 14-3-3 proteins from the H⁺-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH_o and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H⁺-ATPase disrupts the balanced operation of PM H⁺-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.

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KEY WORDS: Chara corallina, plasma membrane H⁺-pump, chlorophyll fluorescence, photosynthetic electron transport, 14-3-3 proteins, fusicoccin

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