The TOR pathway modulates the structure of cell walls in Arabidopsis

Abstract

Plant cell growth is limited by the extension of cell walls, which requires both the synthesis and rearrangement of cell wall components in a controlled fashion. The target of rapamycin (TOR) pathway is a major regulator of cell growth in eukaryotes, and inhibition of this pathway by rapamycin reduces cell growth. Here, we show that in plants, the TOR pathway affects cell wall structures. LRR-extensin1 (LRX1) of Arabidopsis thaliana is an extracellular protein involved in cell wall formation in root hairs, and lrx1 mutants develop aberrant root hairs. rol5 (for repressor of lrx1) was identified as a suppressor of lrx1. The functionally similar ROL5 homolog in yeast, Ncs6p (needs Cla4 to survive 6), was previously found to affect TOR signaling. Inhibition of TOR signaling by rapamycin led to suppression of the lrx1 mutant phenotype and caused specific changes to galactan/rhamnogalacturonan-I and arabinogalactan protein components of cell walls that were similar to those observed in the rol5 mutant. The ROL5 protein accumulates in mitochondria, a target of the TOR pathway and major source of reactive oxygen species (ROS), and rol5 mutants show an altered response to ROS. This suggests that ROL5 might function as a mitochondrial component of the TOR pathway that influences the plant's response to ROS.

Figure 1.

Suppression of the lrx1 Root Hair Phenotype by Mutations in rol5. Seedlings were grown for 5 d ([A] to [E]) and 10 d (F) in a vertical orientation. The wild type (A) developed regular root hairs, whereas root hairs of the lrx1 mutant (B) were severely deformed. The EMS missense allele rol5-1 (C) was complemented with a ROL5 genomic clone, inducing an lrx1-like phenotype (D). The rol5-2 T-DNA knockout mutant (E) also suppressed the lrx1 mutant phenotype. The rol5 mutation (F) leads to shorter roots as shown for rol5-1. Bars = 0.5 mm in (A) to (E) and 10 mm in (F).

Figure 2.

Identification of the ROL5 Locus. (A) The rol5 locus was identified by map-based cloning on the long arm of chromosome 2, south of Athbio2. BAC clones in the region of ROL5 are indicated. For mapping, cleaved-amplified polymorphic sequence and simple sequence length polymorphism markers were established, of which F4I1-Sph and F4I1-Cla were the closest flanking markers identified. (B) The ROL5 gene consists of 10 exons encoding a protein of 355 amino acids. The G-to-A mutation in rol5-1 is located in the second exon and changes Gly-65 to Asp. rol5-2 represents a T-DNA insertion line that interrupts the reading frame at the amino acid codon Glu-170. Gray boxes, exons. (C) RT-PCR experiments on RNA isolated from shoots (S) and roots (R) of 1-week-old seedlings demonstrated that the ROL5 gene is expressed in the wild type and the rol5-1 mutant but not to detectable levels in rol5-2. RT-PCR on the ACTIN2 gene was performed to confirm the use of similar amounts of RNA in the different samples. One of two biological replicates is shown. (D) In roots, ROL5 is predominantly expressed in the elongation zone (ez) and in a striped pattern in the differentiation zone (dz) (top panel). A close-up of the root (GFP fluorescence in the middle panel; bright field in the bottom panel) revealed overlapping GFP fluorescence and root hair formation. Red dots, root hair–forming trichoblasts; arrow, root hair structure. Bar = 0.3 mm. (E) When transiently expressed in Arabidopsis epidermal cells, ROL5-GFP (left panel) and a mitochondrial marker protein (for details, see Methods) fused to red fluorescent protein (middle panel) display overlapping fluorescence patterns (right panel). Bar = 50 μm.

Figure 3.

ROL5 Is Homologous to Ncs6p of Yeast. The alignment of ROL5 with Ncs6p of S. cerevisiae reveals 54% identity and 70% similarity between the two proteins. The Ncs6p-like proteins of different organisms share common motifs that are indicated below the sequences [(CxxC)₂ – SGGxDS – CxxC – GH – PL – C – (CxxC)₂], all of which are conserved between the two proteins. The motif PL is not fully conserved in Ncs6p and ROL5. Sequences important for protein activity (Björk et al., 2007) are boxed. The Gly-65 to Asp mutation in rol5-1 (star) is adjacent to the PP-loop motif SGGxDS, which is important for ATP binding.

Figure 4.

Ncs6p and ROL5 Have Similar Functions. (A) tRNA was extracted from 7-d-old wild-type and rol5-1 seedlings and separated on an acrylamide gel with (left panel) or without (right panel) APM, a compound that interacts with the 2-thiouridine and retards migration in the gel. (B) Wild-type (WT) and Δncs6 mutant yeast was grown in the absence or presence of rapamycin. The Δncs6 mutant grew normally on control medium but was hypersensitive to rapamycin compared with the wild type. Expression of ROL5 but not rol5-1 under the control of the yeast PHOSPHOGLYCERATE KINASE promoter in the Δncs6 mutant suppressed this rapamycin hypersensitivity phenotype. Spots on a line represent serial dilutions (10-fold).

Figure 5.

Rapamycin Treatment Suppresses the lrx1 Root Hair Phenotype. (A) RNA gel blot of wild-type (WT) Arabidopsis transformed with a 35S promoter:FKBP12 construct. (B) lrx1 mutants expressing FKBP12 were sensitive to rapamycin (rapam.) and showed a suppressed lrx1 root hair phenotype (right). In nontransgenic lrx1 mutants, the root hair phenotype was not affected by rapamycin (left). Bar = 0.5 mm.

Figure 6.

Immunolabeling of Cell Walls of rol5-1 and Rapamycin-Treated Wild-Type Seedlings. Immunolabeling of 4-d-old roots with monoclonal antibodies (1→4)-β-d-galactan side chains of RG I (LM5) and glucuronic acid–containing side chains of arabinogalactan proteins (LM2). (A) Compared with the wild type (WT), the rol5-1 mutant root surface revealed reduced detection of the LM5 epitope and stronger detection of the LM2 epitope. (B) Roots of FKBP12-expressing wild-type seedlings grown in the absence (left) and presence (right) of rapamycin. The presence of rapamycin led to a reduced labeling with LM5 and a stronger labeling in proximal parts with LM2. Arrowheads, root apex. Bars = 0.3 mm.

Figure 7.

Altered Response of the rol5-1 Mutant to ROS and ROS Scavenger. Seedlings were grown in liquid culture for 10 d. Under control conditions (A), growth of the wild type and rol5-1 is comparable. rol5-1 seedlings are hypersensitive to H₂O₂ (8 mM), revealed by the reduced growth (B), and hyposensitive to the ROS scavenger CuCl₂ (100 μm), indicated by better growth and the development of green cotyledons (C). col, wild-type Columbia.