An Arabidopsis Mutant Over-Expressing Subtilase SBT4.13 Uncovers the Role of Oxidative Stress in the Inhibition of Growth by Intracellular Acidification.

Gaetano Bissoli,Edgardo A Vilcara,Ramón Serrano,Lourdes Rubio,Enric Sayas,Amelia Felipo,José A Fernández,Jesús Muñoz-Bertomeu,Regina Niñoles,Eduardo Bueso

doi:10.3390/ijms21031173

Abstract

Intracellular acid stress inhibits plant growth by unknown mechanisms and it occurs in acidic soils and as consequence of other stresses. In order to identify mechanisms of acid toxicity, we screened activation-tagging lines of Arabidopsis thaliana for tolerance to intracellular acidification induced by organic acids. A dominant mutant, sbt4.13-1D, was isolated twice and shown to over-express subtilase SBT4.13, a protease secreted into endoplasmic reticulum. Activity measurements and immuno-detection indicate that the mutant contains less plasma membrane H+-ATPase (PMA) than wild type, explaining the small size, electrical depolarization and decreased cytosolic pH of the mutant but not organic acid tolerance. Addition of acetic acid to wild-type plantlets induces production of ROS (Reactive Oxygen Species) measured by dichlorodihydrofluorescein diacetate. Acid-induced ROS production is greatly decreased in sbt4.13-1D and atrboh-D,F mutants. The latter is deficient in two major NADPH oxidases (NOXs) and is tolerant to organic acids. These results suggest that intracellular acidification activates NOXs and the resulting oxidative stress is important for inhibition of growth. The inhibition of acid-activated NOXs in the sbt4.13-1D mutant compensates inhibition of PMA to increase acid tolerance.

Highlights

Alkaline cytosolic pH is a universal feature of actively growing eukaryotic cells [1]
In the present work we describe a novel mutant tolerant to organic acids that, surprisingly, does not reduce intracellular acidification induced by these acids and under normal conditions has a lower pHc than control
We have observed that a decrease of pHc by organic acids induces production of ROS (Reactive Oxygen Species) dependent on two major NADPH oxidases (NOXs and RBOHs, or Respiratory Burst Oxidase Homologues) of Arabidopsis (AtRBOH-D and AtRBOH-F). This activation is greatly reduced in the mutant over-expressing the protease. These results suggest that one mediator of growth inhibition by intracellular acidification is the production of ROS by activation of NOXs and that over-expression of protease SBT4.13 inhibits plasma membrane H+-ATPase (PMA) and acid activation of NOX

Summary

Introduction

Alkaline cytosolic pH (pHc) is a universal feature of actively growing eukaryotic cells [1]. Fermentations under hypoxic conditions produce protons in the oxidation of sugars to organic acids [9,10]. In this respect, carboxylation/decarboxylation reactions have been proposed to constitute a biochemical pH-stat stabilizing pHc [11]. The plasma membrane H+-ATPase (PMA) and the vacuolar H+-ATPase and H+-PPase are responsible for the extrusion of protons from the cytosol and constitute a biophysical pH-stat. The activities of these proton pumps are inhibited under energy deprivation, a condition prevailing during many stresses, and pHc decreases [12]. During water stress abscisic acid increases and inhibits PMA to produce intracellular acidification and growth inhibition [13]

Methods

Results

Conclusion