Oryza sativa H+-ATPase (OSA) is Involved in the Regulation of Dumbbell-Shaped Guard Cells of Rice

Abstract

The stomatal apparatus consists of a pair of guard cells and regulates gas exchange between the leaf and atmosphere. In guard cells, blue light (BL) activates H(+)-ATPase in the plasma membrane through the phosphorylation of its penultimate threonine, mediating stomatal opening. Although this regulation is thought to be widely adopted among kidney-shaped guard cells in dicots, the molecular basis underlying that of dumbbell-shaped guard cells in monocots remains unclear. Here, we show that H(+)-ATPases are involved in the regulation of dumbbell-shaped guard cells. Stomatal opening of rice was promoted by the H(+)-ATPase activator fusicoccin and by BL, and the latter was suppressed by the H(+)-ATPase inhibitor vanadate. Using H(+)-ATPase antibodies, we showed the presence of phosphoregulation of the penultimate threonine in Oryza sativa H(+)-ATPases (OSAs) and localization of OSAs in the plasma membrane of guard cells. Interestingly, we identified one H(+)-ATPase isoform, OSA7, that is preferentially expressed among the OSA genes in guard cells, and found that loss of function of OSA7 resulted in partial insensitivity to BL. We conclude that H(+)-ATPase is involved in BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species.

Keywords: Dumbbell-type stomata; H+-ATPase; Light-induced stomatal opening; Rice.

Fig. 1

H⁺-ATPase is involved in the regulation of rice dumbbell-type stomata. (A) Representative images of open and closed stomata of 5-day-old rice seedlings. Scale bars = 5 μm. (B) The percentage of opened stomata observed under various conditions. Mean ± SD (n = 3; at least 50 stomata were observed for each replicate). FC, 10 μM fusicoccin for 3 h; RL+BL, 150 μmol m⁻² s⁻¹ red light and 50 μmol m⁻² s⁻¹ blue light for 4 h; RL+BL+VD, RL+BL treatment with 1 mM vanadate; RL+BL+ABA, RL+BL treatment with 20 μM ABA. Asterisks indicate statistical differences (P < 0.05) based on the Student’s t-test.

Fig. 2

Sequential and phylogenetic analysis of H⁺-ATPases in rice. (A) Sequence alignment of the C-terminal inhibitory domain of Oryza sativa H⁺-ATPases (OSAs) and Arabidopsis AHA2. The 10th transmembrane domain and the inhibitory motif (Region-I and Region-II) within the C-terminal inhibitory domain are shown. Blue arrowheads below the sequence indicate amino acids that are critical for the function of the inhibitory domain of AHA2 (Axelsen et al. 1999). Red arrowheads over the sequence indicate phosphorylation target sites in AHA2 (Fugisang et al. 2007, Niittylä et al. 2007, Haruta et al. 2014). (B) Phylogenetic tree of H⁺-ATPases of rice, maize (ZmHAs), Arabidopsis (AHAs), tobacco (PMAs), M. polymorpha (MpHAs), P. patens (PpHA) and C. reinhardtii (CrHA). The phylogenetic tree was constructed using the full-length amino acid sequences of H⁺-ATPases. The scale bar indicates 0.04 amino acid substitutions per site. Blue, red and green nodes represent H⁺-ATPases of dicots, monocots and others, respectively. CrHA was used as an outgroup. Daggers indicate non-pT-type H⁺-ATPases. Roman numerals indicate subfamilies defined by Arango et al. (2003). PMA5, PMA7, MpHA1 and MpHA5 were not incorporated into this analysis because full-length sequences were not available.

Fig. 3

Presence of phosphoregulation of the penultimate threonine in pT-type OSAs. (A) Western blot showing the specificity of antibodies (anti-CAT and anti-pThr) against protein extracts of rice leaf blades harvested under light. Molecular weights are shown on the left. The positions of OSAs are indicated by arrows. (B) Penultimate threonine phosphorylation in H⁺-ATPase of rice. Rice seedlings at 5 d after germination were adapted to dark for >12 h prior to 10 μM FC (+) or mock (–) treatment. Top column, immunoblot using anti-CAT and anti-pTh. Bottom column, graphs quantifying the relative phosphorylation level (anti-pThr detection level normalized by the anti-CAT detection level). Mean ± SD (n = 3).

Fig. 4

Expression pattern of OSAs in rice seedling. Tissue-specific expression pattern of OSAs in tissues obtained from 5-day-old seedlings were examined using RT–PCR analysis. Expression levels of 25SrRNA were used as a control. GCEF, guard cell-enriched fraction; MCP, mesophyll cell protoplasts.

Fig. 5

Detection of pT-type OSAs in rice guard cells. Subcellular localization of pT-type OSAs in guard cells was confirmed by immunostaining using (A, C) pre-immune serum or (B, D) anti-pThr against longitudinal sections of rice leaf blades. (A, B) The bulbous end and (C, D) central part of guard cells, respectively, were observed. Pre-immune serum was used as a negative control. Red and blue signals indicate phosphorylated penultimate threonine in OSAs and cell wall autofluorescence, respectively. A set of guard cells is represented by arrowheads in each panel. Scale bars = 5 μm.

Fig. 6

osa7 displays defects in light-induced stomatal opening. (A) Schematic genome structure of OSA7. Lines indicate untranslated regions and introns. Exons are drawn as white boxes. Positions of the TOS17 insertion are shown. (B) Expression of OSA7 in leaf blades of the wild type and osa7 observed by Northern blot analysis. The arrow and arrowheads indicate the position of the native form and truncated form of OSA7 mRNA, respectively. rRNA confirms the equal loading of the total RNA. Data represent the results of two biological replicates. (C) Detection of OSA7 protein in leaf blades of the wild type and osa7. The arrow indicates the position of OSA7. Antibody recognizing actin protein (Anti-Actin) was used to confirm equal loading of total protein. Data represent the results of two biological replicates. (D) Transpiration rate of wild-type and osa7 leaf blades evaluated under daylight. Mean ± SD (n ≥ 7). The P-value indicates the Student’s t-test. (E) Light-induced increases in stomatal conductance in the wild type(left) and osa7 (right). Leaf blades of dark-adapted rice seedlings were subjected to measurement of stomatal conductance (top panels) and photosynthetic rate (bottom panels). Points of red light (RL) and blue light (BL) irradiation are indicated by arrows. Data represent the results of three biological replicates, each measured in the same day. RL, 700 μmol m⁻² s⁻¹ red light; BL, 3 μmol m⁻² s⁻¹ blue light. (F) Graphs quantifying the increase in the stomatal conductance by RL (left) and BL superimposed on RL (right), respectively. Stomatal conductance increase by RL was calculated by subtracting the stomatal conductance in dark conditions from stable stomatal conductance by RL. The increase in stomatal conductance induced by BL was calculated by subtracting the stable stomatal conductance by RL from the maximum stomatal conductance by BL in the background of RL. Mean ± SD (n = 3). P-values indicate the Student’s t-test.