Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1140–1145, February 1999 Plant Biology ARG1 (Altered Response to Gravity) encodes a DnaJ-like protein that potentially interacts with the cytoskeleton JOHN C. SEDBROOK,RUJIN CHEN, AND PATRICK H. MASSON* Laboratory of Genetics, University of Wisconsin-Madison, 445 Henry Mall, Madison, WI 53706 Edited by Maarten Koornneef, Wageningen Agricultural University, Wageningen, The Netherlands and approved December 4, 1998 (received for review September 1, 1998) ABSTRACT Gravitropism allows plant organs to direct itropism and lack the hormonal-response phenotypes found in their growth at a specific angle from the gravity vector, mutants affected in polar auxin transport and organ bending. promoting upward growth for shoots and downward growth We also demonstrate that ARG1 encodes a DnaJ-like protein for roots. Little is known about the mechanisms underlying whose structure suggests an interaction with the cytoskeleton. gravitropic signal transduction. We found that mutations in the ARG1 locus of Arabidopsis thaliana alter root and hypocotyl MATERIALS AND METHODS gravitropism without affecting phototropism, root growth Plant Stocks and Manipulation. Wild-type A. thaliana seeds responses to phytohormones or inhibitors of auxin transport, of the ecotype Wassilewskija (WS) were provided by Timothy or starch accumulation. The positional cloning of ARG1 Caspar (DuPont). Wild-type Landsberg erecta (Ler), Columbia revealed a DnaJ-like protein containing a coiled-coil region (Col), and No-O seeds, as well as seeds from the Feldmann homologous to coiled coils found in cytoskeleton-interacting collection of T-DNA insertional mutants (16), were provided proteins. These data suggest that ARG1 participates in a by the Arabidopsis Biological Resource Center (ABRC; Ohio gravity-signaling process involving the cytoskeleton. A com- State University, Columbus, Ohio). arg1–1 and arg1–2 were bination of Northern blot studies and analysis of ARG1-GUS isolated from the DuPont and Feldmann collections of T-DNA fusion-reporter expression in transgenic plants demonstrated insertional mutants, respectively, by using the reorientation that ARG1 is expressed in all organs. Ubiquitous ARG1 and root-waving assays described in refs. 17 and 18. expression in Arabidopsis and the identification of an ortholog All techniques and incubation conditions aimed at growing in Caenorhabditis elegans suggest that ARG1 is involved in and manipulating A. thaliana seeds, seedlings, and plants were other essential processes. described in ref. 19. Quantification of Root and Hypocotyl Gravitropism. Seed- Plants use the gravity vector as a directional guide to growth, lings were grown embedded in vertically oriented 0.8% agar- thereby positioning roots and shoots below and above ground. containing germination media [GM, half-strength Murashige– The complexity of this process is evident when one recognizes Skoog (MS) salts and 1.5% sucrose (20)] in square Petri dishes that primary and lateral organs must have distinct responses to wrapped in aluminum foil (to confer darkness) and incubated gravity to become spatially separated. Furthermore, roots and within a Conviron (Asheville, NC) TC16 growth chamber shoots grow in opposite directions within the gravity field in an (22°C, 75% relative humidity). Plates were unwrapped and environmentally regulated manner (1). pictures were taken and digitized. Digitized images were used Gravitropism was first identified nearly 200 years ago by to determine the angle from vertical of a surface tangential to Knight (2) and was later characterized by Darwin (3). To date, the hypocotyl tip below the apical hook, as described (19). few details of the underlying mechanisms have been resolved. Hypocotyl response to gravistimulation was analyzed essen- Higher plants have been shown to perceive gravity primarily by tially as described above, except that plates were rotated 90° the sedimentation of starch-containing amyloplasts, which are after 4 days of growth and incubated for 3 more days before located in the columella cells of root tips as well as in the starch being analyzed. Root gravitropism was analyzed as described sheath cells of shoots (4–6). An unknown process converts the (19). physical movement of amyloplasts into a physiological signal. Analysis of Hypocotyl Phototropism. The kinetics of hypo- It has been hypothesized that the second messenger Ca21 as cotyl phototropism were determined by growing wild-type and well as the cytoskeleton are involved in gravitropic signal mutant seedlings on vertically oriented 0.8% agar-containing transduction (7–9). GM plates wrapped in black paper and aluminium foil. After After stimulation, a concentration gradient of auxin and 4 days of growth (time 0), the plates were unwrapped, photo- apoplastic Ca21 is believed to develop across the root tip and graphed, and rewrapped on all sides but one. The unwrapped ' m z 22z 21 to be transmitted to the distal and main elongation zones, side was exposed to a horizontal light source ( 1 E m sec where it would promote differential cellular elongation on from cool-white fluorescent tubes). At the specified times, the opposite flanks, resulting in organ bending (10–12). The AUX1 plates were unwrapped, photographed, and rewrapped to and AGR1 genes appear to encode components of the auxin continue the experiment. The angles between planes tangen- influx and efflux carriers involved in the signal-transmission tial to the hypocotyl tips and vertical planes were measured and phase of gravitropism (13–15). analyzed as described above. To gain insight into the poorly understood transduction phase of gravitropism, we identified and characterized two This paper was submitted directly (Track II) to the Proceedings office. mutant alleles at a new locus in Arabidopsis thaliana named Abbreviations; CAPS, cleaved amplified polymorphic sequence; RFLP, restriction fragment length polymorphism; Col, Columbia; Ler, ARG1 (Altered Response to Gravity). We show that arg1 Landsberg erecta; WS, Wassilewskija, GUS, b-glucuronidase; IAA, mutants are specifically altered in root and hypocotyl grav- indoleacetic acid; cM, centimorgan; 2,4-D, 2,4-dichlorophenoxyacetic acid; YAC, yeast artificial chromosome; BAC, bacterial artificial The publication costs of this article were defrayed in part by page charge chromosome. Data deposition: The sequence reported in this paper has been payment. This article must therefore be hereby marked ‘‘advertisement’’ in deposited in the GenBank database (accession no. AF089810). accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. e-mail: pmasson@ PNAS is available online at www.pnas.org. macc.wisc.edu. 1140 Downloaded by guest on September 28, 2021 Plant Biology: Sedbrook et al. Proc. Natl. Acad. Sci. USA 96 (1999) 1141 Analysis of Starch Accumulation in Wild-Type and Mutant the polymorphism. The ETR1 fragment length polymorphism, Columella and Hypocotyl Endodermis Cells. Wild-type, located 1.4 kb upstream from the ETR1 translational start site, arg1–1, and arg1–2 seedlings were grown for 7 days on the was identified by sequencing the cloned ETR1 upstream surface of a vertical 0.8% agar-containing GM medium in the sequences from the WS and Col ecotypes (31). PCR primers light (19). They were then stained for 15 minutes with the were designed flanking that polymorphism (59-GTA TCT iodine/potassium iodide (IKI) solution described in ref. 21 and GCC CCC ACT CTT-39 and 59-AGC CTA TCT CGA ACT analyzed under a Nikon Optiphot-2 microscope equipped with GAA TC-39). Template DNA for PCR analysis was isolated Nomarski optics (22). pgm mutant seedlings were used as from cotyledon tissue by using the method described in ref. 32. negative-staining controls in these experiments (23). Chromosome Walking. A contig of bacterial artificial chro- Analysis of Root Growth in the Presence or Absence of mosome (BAC) genomic DNA clones derived from the Texas Phytohormones or Auxin-Transport Inhibitors. Wild-type and A&MUniversity BAC library (obtained from the ABRC, mutant seedlings were grown on vertically oriented 0.8% Ohio State University) and overlapping the ARG1 locus was agar-containing GM plates in the Conviron for 4 days before constructed by using standard procedures (33–35). DNA frag- being transferred onto vertically oriented 0.8% agar- ments were subcloned and analyzed as described (25, 26, 28). containing GM plates supplemented with the phytohormones The DNA clones were purified from bacteria by using the or auxin-transport inhibitors cited in the text at the indicated QIAprep spin miniprep kit (Qiagen, Chatsworth, CA). concentrations. Phytohormones and auxin-transport inhibitors Complementation of arg1–2 by a Cloned Wild-Type were purchased from Sigma, prepared as 10 mM stock solu- Genomic DNA Fragment. BAC DNA fragments were sub- tions in diluted NaOHyEtOH or water (15), and added to the cloned into the Agrobacterium pBIN19 binary vector and pH-buffered medium at the concentrations defined in the text. transformed into Agrobacterium strain GV3101. arg1–2 plants Plates were incubated vertically under constant light ('75 were transformed with these clones by using vacuum infiltra- mEzm22zsec21; E, Einstein 5 1 mol of photons) at 22°C for 5 tion, and the primary transformants were identified by kana- days, with pictures taken every 12 hours. Root lengths were mycin selection (36). T2 seeds were harvested from the measured on digitized images and statistically analyzed (19). primary