toc120-3 (SALK_017374) and ppi3-1 were provided by Paul Jarvis (Constan et al., 2004; Kubis et al., 2004), and toc120-3 was backcrossed to WS wild type before use. mar2-1 was previously described as a mutation in the TOC132 gene (AT2G16640) (Stanga et al., 2009). To comply with the Arabidopsis nomenclature, we have renamed this allele toc132-4^mar2−1 in this manuscript. The arg1-2 and arg1-2 toc132-4^mar2−1 mutants have also been described previously (Sedbrook et al., 1999; Stanga et al., 2009).

The seeds were sterilized by washing with 95% ethanol. They were plated on half-strength buffered Linsmaier and Skoog medium containing macro- and micro-nutrients, vitamins, and 1.5% sucrose (Caisson Laboratories, North Logan, UT) supplemented with 1.5% agar type E (Sigma-Aldrich, St. Louis, MO) unless otherwise indicated. The seedlings were grown in a Conviron (Asheville, NC) TC16 growth chamber set at 22°C and a 16 h light/8 h dark cycle. The light intensity was 50–70 μmol m⁻²s⁻¹ and was provided by cool white fluorescent bulbs (Grainger, Lake Forest, IL).

The bases encoding the Toc132 GTP-binding and membrane domains (bases 1365–3618 from the start codon) were amplified from Col-0 DNA with the addition of a start codon. This region was cloned in between the AttL1 and AttL2 sites in the Gateway entry vector pENTR/D-TOPO (Life Technologies, Carlsbad, CA). An LR reaction was then performed to transfer this region into the binary vector pMDC32, which placed Toc132GM under the control of the CaMV 35S promoter and the NOS terminator (Curtis and Grossniklaus, 2003; Xu and Li, 2008).

This construct was sequenced and introduced into arg1-2 toc132-4^mar2−1 plants using the Agrobacterium-mediated floral dip method (Clough and Bent, 1998). T1 transformants were selected and self-pollinated (Harrison et al., 2006), and T3 seeds likely to carry two copies of the transgene as determined by antibiotic resistance were used.

The seeds were embedded within the medium described above supplemented with 0.7% agar. After at least 2 days of stratification, the seedlings were grown vertically as described above for 8 days. The plates were then turned horizontally, and photographs were taken at select time points. The root tip angle of each seedling at each time point was measured using Adobe Photoshop.

WS and toc132-4^mar2−1 seedlings were grown vertically for 2 weeks on medium supplemented with 0.6% agarose with one genotype grown on medium containing natural abundance ammonium nitrate and potassium nitrate (Sigma-Aldrich) and the other grown on ¹⁵N-enriched ammonium nitrate and potassium nitrate (Cambridge Isotope Laboratories, Tewksbury, MA) as previously described (Kline et al., 2010; Minkoff et al., 2012). The roots from approximately 300 seedlings per sample were dissected, combined, frozen in liquid nitrogen, and ground using a Mixer Mill 200 (Retsch, Haan, Germany). This process was repeated at non-overlapping times for a total of three trials. For each trial, each genotype was grown on both nitrogen sources for a total of six samples, each containing WS wild-type and toc132-4^mar2−1 tissue. Therefore, for each trial, one sample contained WS seedlings grown on natural abundance nitrogen media and toc132-4^mar2−1 seedlings grown on ¹⁵N-enriched media, while the second sample contained toc132-4^mar2−1 seedlings grown on natural abundance nitrogen media and WS seedlings grown on ¹⁵N-enriched media. For each sample and in each trial, the proteins were extracted, trypsin digested, subjected to a solid-phase extraction, and analyzed on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher, Waltham, MA) as previously described (Minkoff et al., 2012). Mascot software v2.2.2 (Perkins et al., 1999) was used to compare the mass spectrometra to sequences present in The Arabidopsis Information Resource protein database (Lamesch et al., 2012). The settings for Mascot searches included permitting up to two missed cleavages. Deamidation of asparagine and glutamine residues and oxidation of methionine residues were set as variable modifications. Cysteines were searched in carbamidomethylated form as a fixed modification. The precursor mass tolerance was set to 20 ppm (allowing for the selection of precursors from the monoisotopic or first or second ¹³C isotopes), and fragment tolerance was set to 0.5 Da. The Mascot output was filtered to a 1.0% false discovery rate using a concatenated decoy database strategy with an in-house written script. Census software (Park et al., 2008) and additional in-house scripts (http://www.biotech.wisc.edu/sussmanlab/research/supporting_Minkoff_2013) were used to make quantitative ratio comparisons between the ¹⁴N- and ¹⁵N-labeled signals, and the ratios were normalized to 1 based on the median of each trial. ¹⁴N/¹⁵N (light/heavy) ratios were determined for each peptide and were then averaged for all the peptides in each protein. The ¹⁴N/¹⁵N ratios of the samples in which WS wild type was labeled with ¹⁴N were compared to the ¹⁴N/¹⁵N ratios of the samples in which WS wild type was labeled with ¹⁵N using a Student's T-Test and a significance level of p < 0.1. For the proteins in which one of these ratios was greater than 1.15 and the other was less than 0.85, the ratio between the genotypes was calculated by taking the inverse of the ¹⁴N/¹⁵N ratio for the samples in which toc132-4^mar2−1 was labeled with ¹⁵N and averaging the six toc132-4^mar2−1/WS values. The data are available in Data Sheet 1.

Seedlings were grown as described for the mass spectrometry experiment using medium supplemented with natural abundance ammonium nitrate and potassium nitrate. The roots were dissected with a scalpel, and RNA was prepared using the QIAGEN RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The RNA samples were treated with RQ1 DNase (Promega, Madison, WI) according to the instructions. This procedure was conducted at three non-overlapping times for a total of three biological replicates. cDNA synthesis and quantitative real-time PCR (qRT-PCR) were conducted simultaneously using a qScript™ One-Step qRT-PCR Kit (Quanta Biosciences, Gaithersburg, MD) as recommended. Four technical replicates per sample were included. The samples were run on a Roche Light Cycler 480 and analyzed using LinRegPCR (Ramakers et al., 2003). Reference genes with similar expression levels to the candidate genes were chosen (Czechowski et al., 2005); At1g58050 was used for AT4G23690 and GDH3, At4g27960 was used for MAJOR LATEX PROTEIN LIKE 1, and At1g13320 was used for all other genes.

If Toc132 specifically acts as a ligand as proposed in the direct interaction model, mutations in other TOC complex receptors are unlikely to enhance the arg1 gravitropic defect. However, if Toc132 mediates the gravitropic response through its role in plastid protein import, then mutations in other TOC complex receptor genes such as TOC120 and TOC34/PPI3 may also enhance the arg1 mutant phenotype. Therefore, we created arg1-2 toc120-3 and arg1-2 ppi3-1 plants and analyzed their gravitropic responses. Although the roots of these double mutants did not grow in completely random directions as did arg1-2 toc132-4^mar2−1 and arg1-2 mar1-1 roots (Stanga et al., 2009), they showed significantly more variable root growth on hard agar surfaces relative to the single mutants (Figure 2). This result suggests that mutations in multiple TOC complex receptors can enhance the arg1 gravitropic defect, and therefore, protein import via the TOC complex is important for gravitropism. These data indicate that the direct interaction model is unlikely.

If the direct interaction model is not supported and Toc132 does not act directly as a ligand, we expect a Toc132 construct that lacks the acidic region but still retains the GTP-binding and membrane domains (Toc132GM) to restore the gravitropic response in an arg1 toc132 mutant background to that of arg1 single mutants. Indeed, such a truncated protein is likely to retain its function in protein import into plastids (Inoue et al., 2010) even though it lacks the acidic domain. This domain is the region most likely to act as a ligand because it protrudes into the cytosol and is the most divergent among the TOC complex receptors. We expressed TOC132GM in arg1-2 toc132-4^mar2−1 mutant plants under the constitutive CaMV 35S promoter and found that the transformed seedlings showed similar root reorientation kinetics to arg1-2 single mutants (Figure 3). This result demonstrates that the acidic domain of Toc132 is not required for a gravitropic response, and it is consistent with both the targeted and the indirect interaction models.

We next sought to distinguish between the targeted and indirect interaction models. In the targeted interaction model, the signal transducer imported by the TOC complex triggers signal transduction upon amyloplast sedimentation, whereas the indirect interaction model postulates a role for this plastid-localized transducer that may not rely on amyloplast sedimentation. Therefore, to determine whether the TOC complex functions in conjunction with amyloplast sedimentation, we generated toc132-4^mar2−1 pgm1-1 double mutants. pgm1-1 single mutants lack starch, and their amyloplasts do not sediment, although they still display some response to gravity (Caspar and Pickard, 1989; Kiss et al., 1989). We found that toc132-4^mar2−1 pgm1-1 double mutants displayed stronger gravitropic defects than pgm1-1 single mutants (Figure 4). This result is not directly compatible with the targeted interaction model, in which we would expect no enhancement of the gravitropic defect, although it does not rule it out either as the double mutant still retains some gravitropic response.

To identify candidate proteins that might not be properly imported into plastids in toc132 mutants, we analyzed the wild-type and toc132-4^mar2−1 proteomes of whole root tissue. A similar approach was recently used to investigate the levels of plastid-associated proteins using toc159 whole leaf tissue (Bischof et al., 2011). We expected proteins that were not properly imported into plastids in toc132 mutants to be degraded or to accumulate and therefore to show differences in expression between the two genotypes. We identified only one protein present at different levels between wild type and toc132-4^mar2−1 mutants that was highly likely to localize to plastids (Baginsky and Gruissem, 2009), NUCLEOSIDE DIPHOSPHATE KINASE 3 (NDPK3) (Table 1). However, we found 25 nucleus-encoded proteins present at different levels between wild type and toc132-4^mar2−1 that localize to other regions of the cell or whose localizations are unknown. Sixteen of these proteins were more abundant in toc132-4^mar2−1, and nine were less abundant (Table 1). Nine of the proteins that were more abundant in toc132-4^mar2−1 and seven of those that were less abundant are annotated as functioning in stress responses (Provart and Zhu, 2003).

We then used qRT-PCR to determine if some of the genes encoding these proteins are differentially expressed in toc132-4^mar2−1 compared to wild type. The primers are shown in Table 2. Of the eight genes selected for analysis, four showed differences at the transcript level in the same directions predicted by the proteomic analysis (Figure 5).