The Arabidopsis Abiotic Stress-Induced TSPO-Related Protein Reduces Cell-Surface Expression of the Aquaporin PIP2;7 through Protein-Protein Interactions and Autophagic Degradation

Abstract

The Arabidopsis thaliana multi-stress regulator TSPO is transiently induced by abiotic stresses. The final destination of this polytopic membrane protein is the Golgi apparatus, where its accumulation is strictly regulated, and TSPO is downregulated through a selective autophagic pathway. TSPO-related proteins regulate the physiology of the cell by generating functional protein complexes. A split-ubiquitin screen for potential TSPO interacting partners uncovered a plasma membrane aquaporin, PIP2;7. Pull-down assays and fluorescence imaging approaches revealed that TSPO physically interacts with PIP2;7 at the endoplasmic reticulum and Golgi membranes in planta. Intriguingly, constitutive expression of fluorescently tagged PIP2;7 in TSPO-overexpressing transgenic lines resulted in patchy distribution of the fluorescence, reminiscent of the pattern of constitutively expressed yellow fluorescent protein-TSPO in Arabidopsis. Mutational stabilization of TSPO or pharmacological inhibition of the autophagic pathway affected concomitantly the detected levels of PIP2;7, suggesting that the complex containing both proteins is degraded through the autophagic pathway. Coexpression of TSPO and PIP2;7 resulted in decreased levels of PIP2;7 in the plasma membrane and abolished the membrane water permeability mediated by transgenic PIP2;7. Taken together, these data support a physiological role for TSPO in regulating the cell-surface expression of PIP2;7 during abiotic stress conditions through protein-protein interaction and demonstrate an aquaporin regulatory mechanism involving TSPO. INTRODUCTIONEnvironmental stresses such as drought, salinity, or cold are common limiting factors for plant growth and development. These stresses impose osmotic and oxidative stresses at the cellular level, and a critical function of the phytohormone abscisic acid (ABA) is to mediate the plant response to these insults during vegetative growth (Finkelstein et al., 2002;Nambara and Marion-Poll, 2005;Yamaguchi-Shinozaki and Shinozaki, 2006). The increase in active ABA levels in plant cells during water-related stress regulates the expression of ABA-responsive genes by interacting with cytosolic and/or organelle-bound receptors and downstream effectors modulating the activity of defined transcriptional regulators (Fujii and Zhu, 2009;Ma et al., 2009;Park et al., 2009;Wu et al., 2009;Shang et al., 2010). It is thought that up to 10% of the Arabidopsis thaliana transcriptome is responsive to ABA signaling . Extensive studies of stress and ABA-induced gene expression during vegetative growth revealed two waves of response: an early transient response peaking at ;3 h and a late sustained response from 10 h onward (reviewed in Finkelstein, 2013). Characteristically, the so-called "early" genes encode regulatory proteins, such as transcription factors, protein kinases, and phosphatases, and a set of proteins of unknown function Fujita et al., 2006). The "late" genes are presumed to contribute to plant adaptation to the stress and encode proteins such as ...

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