The Plant Cell This article is published in The Plant Cell Online, The Plant Cell Preview Section, which publishes manuscripts accepted for publication after they have been edited and the authors have corrected proofs, but before the final, complete issue is published online. Early posting of articles reduces normal time to publication by several weeks. The Plant Cell Preview, www.aspb.org © 2003 American Society of Plant Biologists 1 of 14 ARG1 Is a Peripheral Membrane Protein That Modulates Gravity-Induced Cytoplasmic Alkalinization and Lateral Auxin Transport in Plant Statocytes Kanokporn Boonsirichai, a,1 John C. Sedbrook, a,2 Rujin Chen, a,3 Simon Gilroy, b and Patrick H. Masson a,4 a Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706 b Biology Department, Pennsylvania State University, University Park, Pennsylvania 16802-5301 ARG1 (ALTERED RESPONSE TO GRAVITY) is required for normal root and hypocotyl gravitropism. Here, we show that tar- geting ARG1 to the gravity-perceiving cells of roots or hypocotyls is sufficient to rescue the gravitropic defects in the corre- sponding organs of arg1-2 null mutants. The cytosolic alkalinization of root cap columella cells that normally occurs very rapidly upon gravistimulation is lacking in arg1-2 mutants. Additionally, vertically grown arg1-2 roots appear to accumulate a greater amount of auxin in an expanded domain of the root cap compared with the wild type, and no detectable lateral auxin gradient develops across mutant root caps in response to gravistimulation. We also demonstrate that ARG1 is a pe- ripheral membrane protein that may share some subcellular compartments in the vesicular trafficking pathway with PIN auxin efflux carriers. These data support our hypothesis that ARG1 is involved early in gravitropic signal transduction within the gravity-perceiving cells, where it influences pH changes and auxin distribution. We propose that ARG1 affects the local- ization and/or activity of PIN or other proteins involved in lateral auxin transport. INTRODUCTION Higher plant organs sense gravity primarily through the sedi- mentation of starch-filled amyloplasts in specialized cells called statocytes (Caspar and Pickard, 1989; Kiss et al., 1989; Kuznetsov and Hasenstein, 1996; Blancaflor et al., 1998). These cells constitute the columella of the root cap and the endoder- mal layer of the shoot. Amyloplast displacement and sedimen- tation is thought to activate signal transduction pathways that lead to the asymmetric redistribution of the plant hormone auxin across the stimulated organ (reviewed by Masson et al., 2002). A greater amount of auxin accumulates in the bottom flank of the organ, where it inhibits cell elongation in the root and promotes it in the shoot. As a result of this growth differen- tial, the root curves downward and the shoot curves upward (reviewed by Masson et al., 2002). In roots, the amyloplasts appear to sediment within a net- work of fine actin filaments that are tethered through the corti- cal endoplasmic reticulum (ER) and anchored at the plasma membrane (Yoder et al., 2001). Mechanical perturbation of the cytoskeleton and/or the ER was proposed to activate a gravity signal transduction pathway through the regulation of mem- brane channels (Sievers and Busch, 1992). Consistent with this model, transient changes in Ca 2+ fluxes and alkalinization of the columella cytoplasm have been observed in response to gravistimulation (Scott and Allen, 1999; Fasano et al., 2001; Plieth and Trewavas, 2002). Using fluorescent cytosolic pH reporters, Scott and Allen (1999) and Fasano et al. (2001) reported a transient pH increase in the S2 and S3 layers of the columella cells upon gravistimu- lation. Within 30 s, the cytosolic pH increased from 7.2 to 7.5. This phenomenon was accompanied by a decrease in the apo- plastic pH around the columella cells (Fasano et al., 2001). Ap- plication or injection of pH-modifying agents into these cells ei- ther enhanced or reduced the gravitropic curvature (Scott and Allen, 1999; Fasano et al., 2001). Furthermore, the pgm-1 mu- tant of Arabidopsis, which exhibits reduced gravitropism as a result of the absence of starch in its gravity-perceiving amylo- plasts, also showed a reduction and/or a delay in the onset of gravity-induced cytosolic and apoplastic pH changes (Fasano et al., 2001). Together, these observations indicate that changes in the cellular and apoplastic pH of columella cells play a vital role in root gravitropism. Possibly, they condition the root cap for the establishment of lateral polarity, such as the gradient of auxin, necessary for normal gravitropism. Auxin influx and efflux carriers mediate the distribution of auxin in the root and the shoot. Some of these carriers show polar localization at the plasma membrane in certain tissues. The AUX1 gene encodes a transmembrane component of the influx machinery present in cells of the protophloem, the col- umella, the lateral root cap, and the epidermis of the root elon- gation zones (EZs) (Bennett et al., 1996; Marchant et al., 1999; Swarup et al., 2001). Likewise, Arabidopsis PIN genes encode putative transmembrane components of the efflux machinery (reviewed by Friml and Palme, 2002). When mutated, AUX1, 1 Current address: Office of Atoms for Peace, 16 Vibhavadi Rangsit Road, Chatuchak, Bangkok 10900, Thailand. 2 Current address: Department of Biological Sciences, Illinois State Uni- versity, Campus Box 4120, Normal, IL 61790-4120. 3 Current address: Plant Biology Division, Samuel Roberts Noble Foun- dation, 2510 Sam Noble Parkway, Ardmore, OK 73401. 4 To whom correspondence should be addressed. E-mail phmasson@ wisc.edu; fax 608-262-2976. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.015560.