Expression of a Constitutively Activated Plasma Membrane H⁺-ATPase Alters Plant Development and Increases Salt Tolerance

Abstract

The plasma membrane proton pump ATPase (H 1 -ATPase) plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. Its activity is regulated by a C-terminal autoinhibitory domain that can be displaced by phosphorylation and the binding of regulatory 14-3-3 proteins, resulting in an activated enzyme. To better understand the physiological consequence of this activation, we have analyzed transgenic tobacco (Nicotiana tabacum) plants expressing either wild-type plasma membrane H 1 -ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (DPMA4), generating a constitutively activated enzyme. Plants showing 4-fold higher expression of wtPMA4 than untransformed plants did not display any unusual phenotype and their leaf and root external acidification rates were not modified, while their in vitro H 1 -ATPase activity was markedly increased. This indicates that, in vivo, H 1 -ATPase overexpression is compensated by down-regulation of H 1 -ATPase activity. In contrast, plants that expressed DPMA4 were characterized by a lower apoplastic and external root pH, abnormal leaf inclination, and twisted stems, suggesting alterations in cell expansion. This was confirmed by in vitro leaf extension and curling assays. These data therefore strongly support a direct role of H 1 -ATPase in plant development. The DPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H 1 -ATPase is involved in salt tolerance.The H 1 -ATPase transports protons out of the cell across the plasma membrane, thus establishing the proton electrochemical gradient that contributes to the maintenance of the intracellular and extracellular pHs and drives secondary transport of ions and metabolites. As solute transport is directly related to osmotic water movement, the H 1

The Effect of a Genetically Reduced Plasma Membrane Protonmotive Force on Vegetative Growth of Arabidopsis

“…The proton pump is the only means to recharge the system by converting the chemical energy of ATP to the PMF; thus, it is thought to play a fundamental role in diverse processes, including pathogen perception, hormone-mediated cell elongation, nutrient acquisition, root gravitropism, stomatal regulation, and salinity stress resistance (Michelet and Boutry, 1995;Schaller and Oecking, 1999;Palmgren, 2001;Hager, 2003;Fuglsang et al, 2007;Liu et al, 2009). Consistent with these predicted roles, mutant tobacco (Nicotiana tabacum) or Arabidopsis plants with altered H + -ATPase expression exhibit several phenotypes related to transport processes (Zhao et al, 2000;Vitart et al, 2001;Gévaudant et al, 2007).…”

Exclusion of a Proton ATPase from the Apical Membrane Is Associated with Cell Polarity and Tip Growth inNicotiana tabacumPollen Tubes

“…Whereas wild-type pollen germination rate is enhanced by ;25% in the presence of fusicoccin, the germination rate of LAT52:NtAHA-GFP seems unaffected by the toxin ( Figure 6F). This result suggests that either the chimeric protein is already in an always-active state due to a structural modification of the autoinhibitory C terminus by the GFP fusion, such as what happens when the C terminus of these proteins is truncated (Gevaudant et al, 2007), or the GFP fusion is preventing pump activation via C-terminal phosphorylation and/or binding of 14-3-3 proteins (Palmgren, 2001), which would result in a chimeric pump with reduced or absent activity. It was shown that a similar H þ -ATPase-GFP fusion protein from N. plumbaginifolia (PMA4-GFP) was unable to complement a yeast mutant strain where the main plasma membrane H þ -ATPase was deleted (Lefebvre et al, 2004).…”

The Arabidopsis Chaperone J3 Regulates the Plasma Membrane H+-ATPase through Interaction with the PKS5 Kinase

“…These H + -ATPases play regulatory roles in signal transduction, during cell expansion, turgor regulation, in the regulation of intracellular pH, and in the response of the plant to increases in soil salinity (Palmgren, 2001;Rober-Kleber et al, 2003;Fuglsang et al, 2007;Gevaudant et al, 2007;Merlot et al, 2007). A number of factors, including hormones (auxin and abscisic acid [ABA]), calcium, blue light, and fungal elicitors, have been shown to elicit changes in cellular pH through regulation of the PM H + -ATPase (Kinoshita et al, 1995;Xing et al, 1997;Kim et al, 2001;Brault et al, 2004;Zhang et al, 2004).…”