Ethylene Mediates Alkaline-Induced Rice Growth Inhibition by Negatively Regulating Plasma Membrane H+-ATPase Activity in Roots

Abstract

pH is an important factor regulating plant growth. Here, we found that rice was better adapted to low pH than alkaline conditions, as its growth was severely inhibited at high pH, with shorter root length and an extreme biomass reduction. Under alkaline stress, the expression of genes for ethylene biosynthesis enzymes in rice roots was strongly induced by high pH and exogenous ethylene precursor ACC and ethylene overproduction in etol1-1 mutant aggravated the alkaline stress-mediated inhibition of rice growth, especially for the root elongation with decreased cell length in root apical regions. Conversely, the ethylene perception antagonist silver (Ag⁺) and ein2-1 mutants could partly alleviate the alkaline-induced root elongation inhibition. The H⁺-ATPase activity was extremely inhibited by alkaline stress and exogenous ACC. However, the H⁺-ATPase-mediated rhizosphere acidification was enhanced by exogenous Ag⁺, while H⁺ efflux on the root surface was extremely inhibited by exogenous ACC, suggesting that ethylene negatively regulated H⁺-ATPase activity under high-pH stress. Our results demonstrate that H⁺-ATPase is involved in ethylene-mediated inhibition of rice growth under alkaline stress.

Keywords: ACC; H+-ATPase; Oryza sativa; ethylene; growth inhibition; high-pH stress.

FIGURE 1

Phenotypic characterization of rice grown under alkaline conditions. Plants (cv. Nipponbare) were grown hydroponically for 7 days with full-strength nutrition at pH 5.5. The plants were transplanted into the full-strength nutrition at pH 6, 7, or 8. After 30 days, the plants were evaluated to investigate the phenotypic response to various pH. (A) Whole plants, (B) root morphology, (C) microscopy of newly grown roots after 7-day treatment, (D) dry weight per plant, (E) newly grown root length and (F) length of the apical unbranched zone. Values are the means (n = 6) and error bars denote the SE. Different letters represent statistical significance among treatments (P < 0.05).

FIGURE 2

Expression of the genes encoding ethylene biosynthesis enzymes in adventitious roots. Plants (cv. Nipponbare) were grown hydroponically for 7 days in full-strength nutrition at pH 5.5. The plants were transplanted into full-strength nutrition at pH 6 or 8. After 3 days, the roots were evaluated to explore expression of the genes encoding ethylene biosynthesis enzymes. RT-qPCR analyses of the ACS genes (A) and the ACO genes (B). Values are the means (n = 3), and error bars denote the SE. Asterisk indicates significance at the 10% level.

FIGURE 3

Effects of an ethylene precursor, ACC, on rice growth and root elongation under different pH conditions. After 5 days of germination, plants (cv. ZH11) were grown hydroponically under different pH conditions with 0, 1, and 10 μM ACC treatments. After 10 days, the plants phenotypes were investigated. (A) Whole plants grown under different treatments, (B) dry weight per plants (n = 6), (C) newly grown adventitious roots, (D) the length of the apical unbranched zone (n = 12), (E) paraffin section of the cell size in the root mature zone (10 mm from apex), and (F) average length of cortical cells (n = 50) in the root mature zone (10 mm from apex). Error bars denote the SE. Different letters represent statistical significance among treatments (P < 0.05).

FIGURE 4

Ethylene is involved in alkaline stress-induced inhibition of rice growth. (A) Rice growth and (B) plant height of WT (cv. ZH11) and mutant etol1-1 plants under different pH conditions in a hydroponic system. The ZH11 and etol1-1 plants were grown hydroponically for 7 days under different pH conditions. To study the effect of alkaline stress on root elongation, the seeds were surface-sterilized for 15 min with 0.5% NaClO (w/v), rinsed completely with ultrapure water, incubated at 30°C for 3 days, and planted on solid agarose solid medium containing one-half-strength MS at pH 6 or 8. (C) The root elongation and (D) primary root length of WT plants (cv. Nipponbare and ZH11) and mutants (ein2-1 and eto1-1) under alkaline stress. Values are the means (n = 6) and error bars denote the SE. Asterisk indicates significance at the 10% level.

FIGURE 5

Positive effect of H⁺-ATPase on rice growth. After 7 days of germination, plants of Nipponbare (WT) and OX-A8 plants were grown hydroponically for 7 days under different pH conditions. (A) Root growth of WT and OX-A8 plants at pH 6. (B,C) Net H⁺ fluxes rate of WT and OX-A8 plants on the root surface 10 mm from the root tips at pH 6. (D) The growth of WT and OX-A8 plants at pH 6 or 8. (E) Newly grown root length and (F) dry weight per WT and OX-A8 plant under different pH conditions. Values are the means and error bars denote the SE. Asterisk indicates significance at the 10% level.

FIGURE 6

Net H⁺ flux rate of roots responding to different pH conditions. After 7 days of germination, Nipponbare (WT) plants were grown hydroponically for 3 days under different pH conditions. (A) The non-invasive micro-test technology (NMT) was used to measure the root H⁺ fluxes 10 mm from the root apex. (B,C) Net H⁺ flux rate of roots (WT) responding to different pH conditions (n = 8). (D) H⁺-ATPase activity in roots under different pH conditions. (E) Relative expression of the genes encoding H⁺-ATPase in roots. Values are the means and error bars denote the SE.

FIGURE 7

Rice plants under alkaline stress with auxin (IAA) and fusicoccin (FC) treatments. Plants (cv. Nipponbare) were subjected to alkaline stress (pH 8) with FC (a PM ATPase stimulator; 10 μM) or exogenous auxin (IAA; 10 μM) for 5 days in hydroponic culture. (A) The plant growth under alkaline stress (pH 8) with exogenous FC and IAA. (B) Plant height and (C) newly grown root length were investigated after 5 days of treatment. (D) Responses of the root net IAA fluxes rate to different external pH conditions. (E) The correlation between H⁺ flux rate and IAA flux rate. Values are the means (n = 6) and error bars denote the SE. Different letters represent statistical significance among treatments (P < 0.05).

FIGURE 8

The involvement of ethylene in alkaline stress-induced inhibition of root H⁺-ATPase. (A) The initial pH of the medium was adjusted to 8 and the medium acidification was visualized using the pH indicator bromocresol purple (0.04 g L^-1) for treatments at pH 8 with ACC (1 and 10 μM) and AgNO₃ (10 μM). (B) WT (cv. Nipponbare) and mutant ein2-1 plant growth under an alkaline condition (pH 8) with AgNO₃ (10 μM) and NaVO₃ (100 μM). (C) Root morphology and (D,E) Net H⁺ fluxes rate under a normal condition (pH 6) with ACC (10 and 50 μM). Values are the means (n = 8), and error bars denote the SE. Different letters represent statistical significance among treatments (P < 0.05).