Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen

Abstract

Stomata in the plant epidermis open in response to light and regulate CO₂ uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H⁺-ATPase in guard cells. Overexpression of PM H⁺-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H⁺-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H⁺-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H⁺-ATPase in guard cells promotes the growth of perennial woody plants.

Keywords: PM H+-ATPase; PUMP; guard cell; hybrid aspen; stomatal conductance.

Figure 1

Amino acid sequence similarity and gene expression of Populus PM H⁺-ATPases. (A) Amino acid sequence alignment of P. trichocarpa H⁺-ATPases with the C-terminal inhibition domain of Arabidopsis PM H⁺-ATPase (AHA2). The 10th transmembrane domain and the inhibitory motifs (regions I and II) in the C-terminal inhibitory domain are shown. Identical and similar amino acid residues are highlighted by black and grey backgrounds, respectively. Blue arrowheads below the sequence alignment indicate amino acids important for the function of the inhibitory domain of AHA2 (Axelsen et al., 1999) Red arrowheads above the sequence alignment indicate phosphorylation target sites of AHA2 (Fuglsang et al., 2007; Niittylä et al., 2007; Haruta et al., 2014). (B) Phylogenetic tree of PM H⁺-ATPases in A. thaliana and P. trichocarpa. Phylogenetic trees were reconstructed by the neighbour-joining (NJ) and maximum-likelihood (ML) methods based on the alignment of full-length amino acid sequences. The phylogenetic topology was the same in trees reconstructed by the NJ and ML methods. Bootstrap values were calculated by the NJ method with 1,000 replications (left) and by the ML method with 1,000 replications (right). Roman numerals indicate classes, as defined by Arango et al. (2003). (C) The expression pattern of P. trichocarpa H⁺-ATPases in xylem, phloem, leaf, shoot, and root tissues. The raw count data set obtained by tissue-specific RNA sequencing (GSE81077, Shi et al., 2017) was reanalysed to calculate normalised read counts as gene expression level of each gene. Error bars represent the SD with three sample replicates.

Figure 2

Promoter activity of AtGC1 in hybrid aspen and generation of AtGC1::AHA2 transgenic hybrid aspens. (A) Histochemical GUS analysis of AtGC1::GUS transgenic hybrid aspens. Images are of the abaxial side of the leaf. A high-magnification image is shown in the right panel. Scale bar=100μm (left panel) and 20μm (right panel). (B) Expression level of AHA2 and P. tremula×P. tremuloides (Pt×t) H⁺-ATPase in transgenic hybrid aspens and wild type (WT). The expression of AHA2 and Pt × tHA2 was analysed by reverse transcription PCR. Ubiquitin 11 (UBQ, Takata et al., 2009) was used as the internal control. (C) Immunohistochemical analysis of PM H⁺-ATPase in guard cells of transgenic hybrid aspens and WT. Isolated abaxial leaf epidermis was immunolabeled with antiserum raised against the catalytic domain of AHA2. Fluorescence (upper panel) and bright-field images (lower panel) were captured by a fluorescence microscope. Arrowheads indicate guard cells. Scale bar=50μm. (D) Immunofluorescence intensity in guard cells of transgenic hybrid aspens and WT. Fluorescence intensities in transgenic plants were normalised to those in WT plants. Data are means±SD of three independent measurements. Asterisks denote a mean significantly higher than the WT (set to 1.0; Student’s t test followed by the Benjamini and Hochberg multiple test correction; ^**p<0.01 and ^*p<0.05).

Figure 3

Gas exchange properties of AtGC1::AHA2 transgenic and wild-type (WT) plants. (A) Light responses of stomatal conductance and (B) the photosynthetic rate in transgenic and WT plants. Measurements were conducted under dark conditions followed by 1,000μmol.m⁻².s⁻¹ light. Black arrows indicate the time of light-on. Data were plotted every 30s. Measurements were conducted on three different plants for each transgenic event. Error bars represent SE and are not shown if smaller than the symbols.

Figure 4

Growth and biomass production of AtGC1::AHA2 transgenic and wild-type (WT) plants. (A) Growth phenotypes of 63-day-old transgenic and WT plants. Scale bar=10cm. (B) Height (circles) and diameter (squares) of the transgenic and WT plants against time over 63days of growth. (C) Growth rates and biomass production of transgenic and WT plants. Values are means±SD (n=4). Double asterisk indicates p<0.01 by Student’s t test followed by the Benjamini and Hochberg multiple test correction.