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. 2007 Aug;144(4):1763-76.
doi: 10.1104/pp.107.103762. Epub 2007 Jun 28.

Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance

Frédéric Gévaudant  1 , Geoffrey Duby, Erik von Stedingk, Rongmin Zhao, Pierre Morsomme, Marc Boutry
Affiliations

Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance

Frédéric Gévaudant et al. Plant Physiol. 2007 Aug.

Abstract

The plasma membrane proton pump ATPase (H(+)-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(+)-ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (DeltaPMA4), 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(+)-ATPase activity was markedly increased. This indicates that, in vivo, H(+)-ATPase overexpression is compensated by down-regulation of H(+)-ATPase activity. In contrast, plants that expressed DeltaPMA4 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(+)-ATPase in plant development. The DeltaPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H(+)-ATPase is involved in salt tolerance.

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Figures

Figure 1.
Figure 1.
Growth and ATPase activity of yeast cells expressing wild-type or truncated N. plumbaginifolia H+-ATPase PMA4. A, Growth of yeast strain YAK2 transformed with the wild-type PMA4 gene (wtPMA4) or the truncated PMA4 gene (ΔPMA4). Serial 2-fold dilutions were spotted on a plate at pH 4.0. B, Specific activity of plasma membrane H+-ATPase from YAK2 transformed with the complete PMA4 gene (wtPMA4) or truncated PMA4 gene (ΔPMA4).
Figure 2.
Figure 2.
PMA4 protein levels in transgenic and untransformed tobacco plants. Microsomal proteins (15 μg) from the leaves (A and C) and roots (B and D) of wtPMA4 (lines 28 and 29), ΔPMA4 (lines 41 and 72), or untransformed (wild type) plants were separated on 10% SDS-PAGE, blotted, and subjected to immunodetection with anti-PMA4 or anti-PMA2 antibodies and I125 conjugated protein A for quantification. A and B, Phosphorimage of a representative immunoblot performed as described above. C and D, Graphical representation of the phosphorimager counts corresponding, respectively, to A and B, expressed as a percentage of that for the untransformed plant. The white and black bars for ΔPMA4 indicate, respectively, ΔPMA4 and endogenous PMA4. Error bars indicate the sem.
Figure 3.
Figure 3.
RT-PCR analysis of PMA4 mRNA from transgenic and untransformed plants. A, Typical ethidium bromide-stained gel of RT-PCR assays, performed with single-strand cDNA synthesized from RNA extracted from mature leaves of wtPMA4 (line 28), ΔPMA4 (line 41), and untransformed (wild type) plants. Two pairs of PCR primers were used, one for the PMA4 gene and the other for the internal control gene ATP2.1. PCR products are expected at 1,185 (PMA4) and 652 bp (ATP2.1). The PCR products were electrophoresed on an agarose gel after 24 (lanes 1), 26 (lane 2), 28 (lane 3), or 30 (lane 4) reaction cycles. M indicates the lane containing a SmartLadder marker (Eurogentec). B, Graphical representation of RT-PCR analysis showing PMA4 mRNA levels compared to those in control plants. Bands were quantified with a PhosphorImager. The PMA4/ATP2.1 (internal control) ratio is expressed relative to that for the untransformed plant (wild type). Data represent the mean ± sem for three independent experiments.
Figure 4.
Figure 4.
Time course of pH variation in the bathing medium of leaf discs. A, The lower epidermis of leaf discs of wtPMA4 (line 28), ΔPMA4 (line 41), and untransformed (wild type) plants was stripped off and the discs floated on medium containing 250 mm mannitol, 0.5 mm CaCl2, and 0.25 mm MgCl2, and the pH was recorded over time. B, Rate of pH change. Values represent the means ± sem for two independent experiments each performed as in A with lines 28 and 29 (wtPMA4) and lines 41 and 72 (ΔPMA4).
Figure 5.
Figure 5.
Visualization of the external pH along the roots of wtPMA4 (line 29), ΔPMA4 (lines 72 and 41), and untransformed (wild type) plants. Plants were grown in hydroponic conditions for 10 d, then incubated for 1 h in an agarose gel film containing bromocresol purple. The yellow color corresponds to zones where the pH has been acidified.
Figure 6.
Figure 6.
ATPase and proton transport activity of plasma membranes from wtPMA4 (lines 28 and 29), ΔPMA4 (lines 41 and 72), and untransformed (wild type) leaves. A, Vanadate-sensitive ATPase activity determined by measuring Pi release colorimetrically. B, Initial rate of H+ pumping of the H+-ATPase determined by the ACMA fluorescence quenching technique.
Figure 7.
Figure 7.
Morphological phenotypes of ΔPMA4 and untransformed (wild type) plants. A, Abnormal leaf bending. An untransformed (wild type) plant and a ΔPMA4 plant (line 41) were transferred to soil. After 3 weeks, the same plants were photographed at the indicated time. The wild-type plant shows normal development, while the ΔPMA4 plant displays intermittently bended leaves. Below is an enlargement of the ΔPMA4 plant showing an upsidedown leaf. Images are representative of several plants of lines 41 and 72. B, Twisted stem and leaves. The untransformed (wild type) plant grows straight and its leaves display normal bending. The two ΔPMA4 plants (lines 41 and 72) have a twisted stem and abnormally bending leaves. C, Twisted stem. The leaves were removed from the stems of mature plants to display their curvature. Stems from untransformed (wild type) plants display a low and regular node bending. Stems from ΔPMA4 plants (lines 41 and 72) display large and irregular bending. Bar = 10 cm. D, The ΔPMA4 flower (line 72) is pale and the ends of the petals show no indentation. The pollen yield is lower than for the wild-type plant. E, Pods are formed on the wild-type flower stem while ΔPMA4 flowers drop off. F, Western-blotting analysis of a microsomal fraction (15 μg proteins) from the upper (colored) part of the petal (P) and from anthers (A) of an untransformed plant (wild type) and a ΔPMA4 plant (line 72). Immunodetection was performed with anti-PMA4 or anti-PMA2 antibodies. Similar data (D–F) were obtained for line 41.
Figure 8.
Figure 8.
In vitro expansion of leaf discs and curvature of leaf strips. A, Area increase of leaf discs. Leaf discs (1 × 1 cm) from mature leaves were incubated for 24 h as indicated in “Materials and Methods” and the leaf disc area expressed as a percentage of the initial leaf disc area was calculated (mean + sem, n = 5, three independent experiments). B, Curvature of leaf strips. Leaf strips were incubated for 24 h as indicated in “Materials and Methods.” Images were taken after 24 h and the strip curvature measured (mean + sem, n = 10, five independent experiments).
Figure 9.
Figure 9.
Phenotype analysis of transgenic and control plants grown on salt medium. A and B, Cumulative germination rates of ΔPMA4 (lines 51 an 72) and untransformed (wild type) seeds in a medium with (B) or without (A) 200 mm NaCl. Means ± sem (n = 50, four independent experiments) and sigmoidal regression curves (R2 > 0,98) are presented. For the heterozygous ΔPMA4 line, kanamycin-sensitive seedlings were not taken into account. C to E, Phenotype of wild type (C) and ΔPMA4 line 51 (D) and line 72 (E) seedlings on 200 mm NaCl. Plants were photographed 25 d after the first plants germinated (bar, 1 cm). ΔPMA4 plants were grown on medium supplemented with kanamycin. Kanamycin-sensitive plants are white. F, Fresh weight of wild-type and ΔPMA4 plantlets grown in the same conditions as in C to E. G and H, Root growth under saline conditions. Ten days after germination on standard Murashige and Skoog medium, the plantlets were transferred for 14 d to Murashige and Skoog medium with or without 200 mm NaCl. G, Representative picture of the phenotype (bar, 1 cm). H, Means ± sem (n = 5, three independent experiments); the black bars are the results in Murashige and Skoog and the white bars those in Murashige and Skoog + 200 mm NaCl. [See online article for color version of this figure.]
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