The plasma membrane H + -ATPase of maize embryos localizes in regions that are critical during the onset of germination Consuelo Enrı ´quez-Arredondo a , Sobeida Sa ´nchez-Nieto a , Erika Rendo ´n-Huerta b , Diego Gonza ´lez-Halphen c , Marina Gavilanes-Ruı ´z a, * , David Dı ´az-Pontones d a Departamento de Bioquı ´mica, Facultad de Quı ´mica, Conj. E. UNAM. Cd. Universitaria, Coyoaca ´n, 04510 Me ´xico, D.F., Me ´xico b Departamento de Bioquı ´mica, Facultad de Medicina, UNAM. Cd. Universitaria, Coyoaca ´n, 04520 Me ´xico, D.F., Me ´xico c Departamento de Gene ´tica Molecular, Instituto de Fisiologı ´a Celular, UNAM. Cd. Universitaria, Coyoaca ´n, 04520 Me ´xico, D.F., Me ´xico d Departamento de Ciencias de la Salud, Divisio ´n de Ciencias Biolo ´gicas y de la Salud, Universidad Auto ´noma Metropolitana Iztapalapa, Apartado Postal 55535, Me ´xico 09340, D.F., Me ´xico Received 26 January 2005; accepted 28 January 2005 Available online 22 March 2005 Abstract The H + -ATPase is a plasma membrane enzyme that hydrolyzes ATP and translocates protons from the cytosol to the apoplast space. The resulting acidification is used to promote stomata opening, pH homeostasis, secondary transport of nutrients, and cell elongation. The latter two processes are critical in germination, since radicle protrusion, requires the growth of embryo cells. Earlier studies have shown that the plasma membrane H + -ATPase is already active during the first hours of imbibition. Here, the localization of this enzyme was studied in maize embryos by histochemical and immunochemical techniques at 2 h of imbibition. The enzyme is mainly located in regions of the embryo that require high transport activity or that participate in cell elongation: the scutellum, the root and the plumule. The cells that were more immunolabeled were those located in the epidermis, in the parenchyma and in the elements of the vascular bundles. These results suggest that the plasma membrane H + -ATPase levels present in the first hours of imbibition are preserved and/or are newly synthesized to fulfill the demands of transport and elongation of the embryo on the onset of seed germination. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: H + -ATPase; Histochemistry; Immunostaining; Maize; Plasma membrane; Zea mays 1. Introduction The plasma membrane (PM) H + -ATPase is a protein with a molecular mass of 100 kDa, whose amino acid sequence has been deduced from the corresponding gene [1,2]. Secondary structure analyses predict that the polypeptide contains 10–12 transmembrane stretches that are thought to be involved in H + transport [3]. A large hydrophilic loop, exposed to the cytoplasm, contains the catalytic site where ATP is hydrolyzed. The enzyme pumps H + out from the cell, generating a transmembrane proton electrochemical gra- dient that is used to drive solute translocation, therefore regulating pH, stomata opening, and cell nutrition [4,5]. Essential nutrients, such as nitrate and sulfate, are taken into the cell against their concentration and electrical gradients by H + -coupled anion symporters [6]. Also, the translocation of organic compounds from source-tissues to sink-tissues involves energy-dependent steps, carried out by various H + - coupled symporters for amino acids and sucrose [7,8]. It has also been proposed that the apoplast acidification con- tributes to pH regulation and cell wall loosening, facilitating cell elongation [9,10]. The PM H + -ATPase participates as a primary pump in multiple functions, and is therefore a key element in plant cell physiology [5,11,12]. Tissue localization studies have shown that the expres- sion of PM H + -ATPase isoforms is differentially regulated depending on the tissue and the stage of plant development www.elsevier.com/locate/plantsci Plant Science 169 (2005) 11–19 Abbreviations: MBS; n-maleimidebenzoyl-N-hydroxysuccinimide ester; PBS; phosphate buffered saline; PFA; paraformaldehyde; PM; plasma membrane * Corresponding author. Tel.: +52 5 622 5275; fax: +52 5 622 5329. E-mail address: gavilan@servidor.unam.mx (M. Gavilanes-Ruı ´z). 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.01.021