A role for the TOC complex in Arabidopsis root gravitropism

Abstract

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes.

Figure 1.

arg1-2 mar1-1 and arg1-2 mar2-1 roots and hypocotyls grow in random directions. A, Light-grown seedlings (n = 31–39) grown on the surface of inclined 1.5% agar GM plates. B and C, Root (B) and hypocotyl (C) angles relative to the gravity vector were measured and placed into one of six bins. The area of each wedge is proportional to the number of plants observed in each 60° bin; the total area for each pie chart is 1 (gray circle). For both roots and hypocotyls, mar1-1, mar2-1, and Ws grow most frequently within 30° of the vertical, while a higher proportion of arg1-2 roots and hypocotyls deviates from this range. Double mutants grow randomly.

Figure 2.

Molecular mapping of mar1-1 and mar2-1. A, mar1-1 is linked to TOPP5. Recombinant breakpoint analysis narrowed the region to between MMM3 and MMM2. The number of recombinants between marker pairs is indicated by gray numbers. B, Sequencing revealed a C-T transition in TOC75-III. The region of TOC75-III that encompasses the mutation (star) is highly conserved throughout plants. It lies in a conserved seven-amino acid (aa) motif (bar) predicted to form a β-barrel. C, mar2-1 maps between nga1145 and nga1126. mar2-1 has a C-T mutation in TOC132 that creates a premature stop codon. D, 35S-driven TOC132 cDNA expression restores the Arg-1 phenotype to arg1-2 mar2-1 plants. Shown are T3 plants homozygous for the transgene compared with arg1-2 and untransformed control plants. The growth conditions were the same as those described in the legend to Figure 1. E, The toc132-3 allele also displays random root growth only when in the arg1-2 background.

Figure 3.

mar2-1 is a protein-null allele of TOC132. Anti-TOC132 antibody recognizes TOC132 in protein extracts from Ws and arg1-2 but not in mar2-1 or mar2-1 arg1-2 extracts. Stars represent nonspecific binding. The anti-actin positive control is shown below.

Figure 4.

mar2-1 resembles Ws, and mar1-1 has pleiotropic defects. A, mar2-1 roots respond like Ws to 90° gravistimulation, whereas mar1-1 roots reorient more slowly toward the gravity vector compared with Ws but not as slowly as arg1-2. B, Dark-grown mar2-1 hypocotyls also respond to gravistimulation like Ws. C, mar2-1 and mar2-1 arg1-2 hypocotyls (n = 38–45) grown on the surface of 0.8% agar GM plates both develop wild-type phototropism. Error bars, which are sometimes masked by the symbols, represent se. Stars represent significant t-test values (P < 0.05) compared with Ws.

Figure 5.

Starch content is similar across genotypes. A, Iodine staining of approximately 20 roots per genotype shows similar starch accumulation in all roots, while arg1-2 and arg1-2 mar2-1 have expanded domains of starch accumulation in the root cap, as expected (Harrison and Masson, 2008). B, Total starch of etiolated seedlings was measured and standardized to Ws = 100%. The t-test values show that Ws and arg1-2 are not statistically different (P > 0.05) from each other, while ACG21 accumulates significantly less starch (star). Error bars represent se. C, Transmission electron microscopy images demonstrate the presence of large starch granules in all amyloplasts. Shown are representatives of 10 images per genotype. No obvious structural defects were detected; the variation in plastid starch content shown between genotypes is similar to that detected between plastids within genotypes. Bar = 1 μm.

Figure 6.

mar2-1 arg1-2 plastid movement following 90° reorientation is like that of the wild type. A, Amyloplasts within central S2 columella cells were analyzed by placing live, growing roots (Ws, n = 7; arg1-2, n = 9; ACG20, n = 5) on a vertically oriented rotatable stage. Images were collected in 10-s intervals following 90° reorientation. B, The distance from the former cell bottom to the leading plastid was measured for each time point on the x axis. C, The distance from the new cell bottom to the top-most plastid was measured for each time point on the y axis. Error bars represent the se, and stars and crosses represent significant t-test values (P < 0.05) compared with Ws at each time point.

Figure 7.

A, mar2-1 also enhances the root defect of arl2-3. B, toc120-3 does not enhance the root defect of arg1-2. Seedlings were grown on the surface of inclined GM growth media plates containing 1.5% agar.

Figure 8.

Possible models of TOC132 action in gravitropism. A, TOC132 may mediate the insertion into the outer membrane of amyloplasts of a protein (Y) that, upon gravity-induced amyloplast sedimentation to the lateral side of the statocytes, may interact with a plasma membrane- or endoplasmic reticulum-associated transducer (X), regulating its signal-transducing activity. B, Alternatively, TOC132 may interact directly with X upon gravity-induced amyloplast sedimentation, thereby triggering signal transduction. In both models, ARG1 and ARL2 (not shown here) would modulate the location and/or activity of transducer X at the sensitive membrane.