Novel Regulation of Aquaporins during Osmotic Stress<sup><a href="#fn1" rid="fn1" class=" fn">1</a></sup>
Abstract
Aquaporin protein regulation and redistribution in response to osmotic stress was investigated. Ice plant (Mesembryanthemum crystallinum) McTIP1;2 (McMIPF) mediated water flux when expressed in Xenopus leavis oocytes. Mannitol-induced water imbalance resulted in increased protein amounts in tonoplast fractions and a shift in protein distribution to other membrane fractions, suggesting aquaporin relocalization. Indirect immunofluorescence labeling also supports a change in membrane distribution for McTIP1;2 and the appearance of a unique compartment where McTIP1;2 is expressed. Mannitol-induced redistribution of McTIP1;2 was arrested by pretreatment with brefeldin A, wortmannin, and cytochalasin D, inhibitors of vesicle trafficking-related processes. Evidence suggests a role for glycosylation and involvement of a cAMP-dependent signaling pathway in McTIP1;2 redistribution. McTIP1;2 redistribution to endosomal compartments may be part of a homeostatic process to restore and maintain cellular osmolarity under osmotic-stress conditions.
It seems intuitively obvious that plant water channels, aquaporins (AQP), ought to play a dynamic role in maintaining cellular water homeostasis under conditions that necessitate modifications in water flux. Changes in water uptake and allocation would be required to balance alterations in the cellular osmotic potential and, therefore, AQP activity and/or expression should be tightly regulated.
Environmental stimuli, including drought, dehydration, desiccation, and salinity, as well as a rise in abscisic acid (ABA), which accompanies the perception of osmotic stress, have been shown to regulate the expression of both tonoplast (vacuolar membrane; TP) and plasma membrane (PM) AQP at the transcript level (for review, see Maurel et al., 2002).
Recent evidence supports a direct role for aquaporins in plant water relations and provides information on their involvement in drought stress tolerance. Manipulation of PM intrinsic protein (PIP) transcript levels through overexpression (Aharon et al., 2003), or through gene silencing by antisense suppression (Kaldenhoff et al., 1998; Martre et al., 2002; Siefritz et al., 2002) or T-DNA insertion (Javot et al., 2003), resulted in changes in root hydraulic conductivity, transpiration rates, and cellular osmotic water potential and, in some cases, directly affected the plant's ability to recover from water deficit conditions (Martre et al., 2002; Siefritz et al., 2002; Aharon et al., 2003).
Few studies have addressed the direct regulation of AQP expression by osmotic stress at the protein level, and fewer still have focused on AQP dynamic behavior. However, when factors such as mRNA stability and, conceivably, altered turnover under stress conditions are considered, evidence for transcriptional regulation may be less compelling than observations indicating that protein amounts are directly affected.
In the ice plant (Mesembryanthemum crystallinum), regulation of AQP protein amount by salinity stress was observed using peptide-specific antibodies. McTIP1;2 amounts in leaf TP decreased during salt stress and levels of McPIP2;1 (McMIPC) in root PM fractions were induced, while McPIP1;4 (McMIPA) showed no change (Kirch et al., 2000). For McTIP1;2, the decrease in protein amount in the presence of NaCl was contrasted by an increase in the presence of mannitol (Vera-Estrella et al., 2000), suggesting precise deciphering and discrimination of osmotic and ionic signaling pathways.
Little is known about the synthesis of AQP and the pathway delivering them to their destination membranes. Endosomal trafficking of plant AQP may play a role in the regulation and turnover of these proteins at their target membrane under conditions of osmotic stress. In animals, regulatory cycling between membranes has been demonstrated for several AQP. Vasopressin-induced increases in water permeability of kidney-collecting duct cells have been shown to occur via shuttling of AQP2 from intracellular vesicles to the apical PM through exocytosis (Nielsen et al., 1995; for review, see Knepper and Inoue, 1997). Also, acetylcholine-induced increases in cytoplasmic Ca appeared to trigger the translocation of AQP5 from intracellular membranes to the apical PM of rat parotid tissue (Ishikawa et al., 1999). In the ice plant, changes in membrane distribution of the TP AQP McTIP1;2, correlated with changes in osmotic potential, have been suggested (Barkla et al., 1999) and observed (Vera-Estrella et al., 2000). Here, we further characterize regulation of the ice plant AQP McTIP1;2 under osmotic stress, and begin to dissect the mechanisms responsible for the stress-induced changes in McTIP1;2 membrane distribution.
Acknowledgments
We thank Drs. Ramon Serrano (Valencia, Spain), Phil Rea (Philadelphia), Karl-Josef Dietz (Bielefeld, Germany), and John C. Rogers (Pullman, WA) for antibodies against PMA1 (PM H-ATPase), PAB-HK (vacuolar H-pyrophosphatase), VMA-E (vacuolar H-ATPase), and BP80, respectively, and Christophe Maurel for the AtTIP1;1 construct. We also thank Enrique Balderas for help with the oocytes, Chris Michalowski for help with the cDNA constructs, and Xochitl Alvarado for help with the confocal microscope.
Notes
This work was supported by the Consejo Nacional de Ciencia y Tecnología (grant nos. 31794N to R.V.-E. and 33054N to O.P.) and by the National Science Foundation International Program (USA-Mexico; to O.P. and H.J.B.).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.044891.