Mitochondrial beta-cyanoalanine synthase is essential for root hair formation in Arabidopsis thaliana

Abstract

Cyanide is stoichiometrically produced as a coproduct of the ethylene biosynthesis pathway and is detoxified by β-cyanoalanine synthase enzymes. The molecular and phenotypical analysis of T-DNA insertion mutants of the mitochondrial β-cyanoalanine synthase CYS-C1 suggests that discrete accumulation of cyanide is not toxic for the plant and does not alter mitochondrial respiration rates but does act as a strong inhibitor of root hair development. The cys-c1 null allele is defective in root hair formation and accumulates cyanide in root tissues. The root hair defect is phenocopied in wild-type plants by the exogenous addition of cyanide to the growth medium and is reversed by the addition of hydroxocobalamin or by genetic complementation with the CYS-C1 gene. Hydroxocobalamin not only recovers the root phenotype of the mutant but also the formation of reactive oxygen species at the initial step of root hair tip growth. Transcriptional profiling of the cys-c1 mutant reveals that cyanide accumulation acts as a repressive signal for several genes encoding enzymes involved in cell wall rebuilding and the formation of the root hair tip as well as genes involved in ethylene signaling and metabolism. Our results demonstrate that mitochondrial β-cyanoalanine synthase activity is essential to maintain a low level of cyanide for proper root hair development.

Figure 1.

Root Phenotype of the cys-c1 Mutant. (A) and (B) Bright-field image of 2-week-old wild-type and cys-c1 mutant seedlings, respectively, growing on MS medium. (C) and (D) Confocal fluorescent and transmitted light images of a wild-type root stained with propidium iodide for cell wall imaging, respectively. (E) and (F) Confocal fluorescent and transmitted light micrographs of a cys-c1 root stained with propidium iodide, respectively. (G) and (H) Bright-field image of 2-week-old wild-type and mutant seedlings, respectively, grown on MS medium containing 50 μM ACC. (I) and (J) Magnified images of (G) and (H).

Figure 2.

Characterization of the Complemented cys-c1Mutant Lines. (A) β-Cyanoalananine synthase activity of the complemented cys-c1:P35S-CYSC1.1 and cys-c1:P35S-CYSC1.24 lines was quantified in root tissue of 4-week-old plants by measuring the formation of sulfide. One unit of activity corresponds to the formation of 1 nmol min⁻¹ of sulfide. Values are means ± sd of three independent experiments. (B) Root phenotype of the cys-c1 and complemented mutant lines. Bright-field image of 2-week-old seedlings growing on MS medium. a, The wild type; b, cys-c1 mutant line; c, complemented cys-c1:P35S-CYSC1.1 mutant line; d, complemented cys-c1:P35S-CYSC1.24 mutant line.

Figure 3.

Cyanide Determination in Root and Leaf Tissues. (A) Two-week-old seedlings grown on vertical MS medium were collected and root and leaf tissues were assayed for cyanide content. (B) Whole 2-week-old seedlings grown on vertical MS medium in the absence or presence of 10, 25, or 50 μM ACC were also collected and their cyanide content was determined. Values are means ± sd of three independent experiments. ***P < 0.001.

Figure 4.

Cyanide Effect on Root Hair Formation. Seedlings from wild-type and β-cyanoalanine mutant lines grown for 5 d on vertical MS medium were transferred to MS medium containing cyanide and grown for two additional days. (A) and (B) Root hair phenotype of wild-type plants grown in the absence or the presence of 50 μM KCN, respectively. (C) and (D) Root hair phenotype of cys-c1 mutant plants grown in the absence or the presence of 50 μM KCN, respectively. (E) and (F) Root hair phenotype of cys-d1 mutant plants grown in the absence or the presence of 50 μM KCN, respectively. (G) and (H) Root hair phenotype of cys-d2 mutant plants grown in the absence or the presence of 50 μM KCN, respectively.

Figure 5.

Hydroxocobalamin Effect on Root Hair Formation and ROS Localization at the Root Hair Tips. (A) and (B) Root hair phenotype of wild-type and cys-c1 mutant plants, respectively, germinated and grown for 5 d in the absence of hydroxocobalamin. (C) and (D) Root hair of wild-type and cys-c1 mutant plants, respectively, germinated and grown for 5 d in the presence of 5 mM hydroxocobalamin. (E) and (F) Five-day-old wild-type and cys-c1 roots stained with NBT for superoxide detection. (G) and (H) Five-day-old wild-type and cys-c1 mutant roots grown on hydroxocobalamin medium stained with NBT.

Figure 6.

Respiration Rates and Mitochondrial Localization in Root Hair. (A) Respiration rate in the wild type and cys-c1 mutant line in 15-d-old root tissues. Cyanide-independent and alternative oxidase respiration was determined in the presence of 0.5 mM KCN or 4 mM SHAM, respectively. The results are expressed as the mean ± sd from at least three replica samples. (B) Transcription level of the alternative oxidase gene AOX1a in 15-d-old wild-type and cys-c1 mutant root tissue was analyzed by quantitative real-time RT-PCR using the primers qAOX1a-F and qAOX1a-R (see Supplemental Table 4 online). The expression level was normalized to that of the constitutive UBQ10 gene by subtracting the CT value of UBQ10 from the CT value of the gene (ΔCT). Values are means ± sd from three replica samples. (C) Visualization of stained mitochondria with MitoTracker Deep Red 633 dye. a and d, Transmitted light imaging of 5-d-old root tissues from wild-type or cys-c1 mutant plants, respectively. b and e, Single optical sections of stained roots from wild-type or cys-c1 mutant plants, respectively. c and d, Maximum projection of 18 optical sections of stained roots from wild-type or cys-c1 mutant plants, respectively.

Figure 7.

Graphic Display of Hierarchical Cluster Analysis of cys-c1 Downregulated Genes in Seedlings Treated with Ethylene Inhibitors or Precursors. Hierarchical clustering analysis was applied to a set of 52 downregulated genes in the cys-c1 mutant, as well as ACC-, AVG-, and AgNO₃-treated seedlings. Each column represents the time course and treatment samples indicated at its top, and each row refers to a gene. A dendrogram representing hierarchical relationships among treatments is shown, and the scale at the top marks the correlation coefficient represented by the length of the branches that connect pairs of nodes. The color scale indicates the log₂ level of expression above (red) or below (blue) the median. The data sets of the ACC time course and of the ethylene inhibitor treatments are from the AtGenExpress Consortium, NASCarray experiment reference numbers 172 and 188, respectively.

Figure 8.

Ethylene Production in the cys-c1 Mutant. Ethylene accumulation was determined in wild-type (continuous line) and cys-c1 mutant (discontinuous line) seedlings grown for 19 d on vertical MS plates by gas chromatography. The accumulation of ethylene was determined at 3, 24, and 48 h after the vials were sealed. Values are means ± sd of at least 20 samples per plant line. *P < 0.05.