Alternative Oxidase Inhibition Impairs Tobacco Root Development and Root Hair Formation

Abstract

Alternative oxidase (AOX) is the terminal oxidase of the mitochondrial respiratory electron transport chain in plant cells and is critical for the balance of mitochondrial hemostasis. In this study, the effect of inhibition of AOX with different concentrations of salicylhydroxamic acid (SHAM) on the tobacco root development was investigated. We show here that AOX inhibition significantly impaired the development of the main root and root hair formation of tobacco. The length of the main root of SHAM-treated tobacco was significantly shorter than that of the control, and no root hairs were formed after treatment with a concentration of 1 mM SHAM or more. The transcriptome analysis showed that AOX inhibition by 1 mM SHAM involved in the regulation of gene expression related to root architecture. A total of 5,855 differentially expressed genes (DEGs) were obtained by comparing SHAM-treated roots with control. Of these, the gene expression related to auxin biosynthesis and perception were significantly downregulated by 1 mM SHAM. Similarly, genes related to cell wall loosening, cell cycle, and root meristem growth factor 1 (RGF1) also showed downregulation on SHAM treatment. Moreover, combined with the results of physiological measurements, the transcriptome analysis demonstrated that AOX inhibition resulted in excessive accumulation of reactive oxygen species in roots, which further induced oxidative damage and cell apoptosis. It is worth noting that when indoleacetic acid (20 nM) and dimethylthiourea (10 mM) were added to the medium containing SHAM, the defects of tobacco root development were alleviated, but to a limited extent. Together, these findings indicated that AOX-mediated respiratory pathway plays a crucial role in the tobacco root development, including root hair formation.

Keywords: alternative oxidase; auxin; respiration; root architecture; tobacco.

Figure 1

Effects of salicylhydroxamic acid (SHAM) treatment on root formation of tobacco. (A) Comparison of tobacco root development under normal conditions (CK) and different concentrations of SHAM. (B) Comparison of root length in the absence (CK) and presence of SHAM treatments. The data are the means ± SD of three independent experiments from at least 30 seedlings. The significant differences between the SHAM-treated samples and the control samples are denoted by asterisks, ^∗p < 0.05. (C) Microscopic comparison of root phenotypes in the absence (CK) and presence of SHAM treatments. Seedlings on 8th day of growth were used for observation and comparison.

Figure 2

Comparison of oxidative damage and respiration rate between the SHAM-treated seedlings and the control (CK). (A,B) Changes of total respiration (V_t) and alternative pathway respiration (V_alt) in the absence (CK) or presence of SHAM treatments. (C) Determination of superoxide ion with 0.5 mM nitrotetrazolium blue chloride (NBT) for 2 h. (D) Quantification of NBT staining intensity in the main root zone using ImageJ software; AU, arbitrary units. (E) Comparison of H₂O₂ content between SHAM-treated seedlings and the control (CK). Seedlings on 8th day of growth were used for determination. The data are the means ± SD of three independent experiments from at least 30 seedlings. The significant differences are denoted by different letters. n.a., not available.

Figure 3

Comparison of cell death between the SHAM-treated seedlings and the control (CK). (A) Cell death was determined with trypan blue, and the representative roots are shown. (B) The microscopic images of roots after propidium iodide (PI) staining and the representative roots are shown. Seedlings on 8th day of growth were used for trypan blue and PI staining. Scale bar, 200 μm.

Figure 4

Overview of changes in gene expression profiles of tobacco on SHAM treatment. (A) Heat map diagrams showed the relative expression levels of total differentially expressed genes (DEGs). (B) Number of DEGs includes up- and downregulated genes on SHAM treatment, p_adj < 0.05. (C) Number of DEGs (fold change ≥2) in SAHM vs. CK. log₂FC, log₂FoldChange. (D) Gene Ontology (GO) terms enriched in SHAM vs. CK. (E) KEGG pathway enriched in SHAM vs. CK.

Figure 5

Heat map diagrams of DEGs related to carbon metabolism (A), biosynthesis of amino acids (B), glyoxylate and dicarboxylate metabolism (C), and cysteine and methionine metabolism (D) in response to SHAM treatment. DEGs of |log₂FC ≥ 1| are shown in the image. log₂FC, log₂FoldChange. The red-, blue-, and green-colored triangles or rectangles represent DEGs that appear in different KEGG pathways.

Figure 6

Heat map diagrams of relative expression levels of reactive oxygen species (ROS) scavenging-related genes in response to SHAM treatment. (A) SOD, superoxide dismutase; (B) POD, peroxidase; (C) APX, ascorbate peroxidase; (D) GPX, glutathione peroxidase; (E) CAT, catalase; (F) GST, glutathione S-transferase; (G) PAO, polyamine oxidase; and (H) PPO, polyphenol oxidase. log₂FC, log₂FoldChange.

Figure 7

Effects of SHAM treatment on the gene expression of auxin biosynthesis and signaling transduction pathway. DEGs related to auxin biosynthesis (A), auxin receptor (B), auxin inducible (C), auxin transport (D), and auxin response (E) are shown. TAA1, tryptophan aminotransferase; TIR1, transport inhibitor response 1; ABP, auxin binding protein; SAUR, small auxin-up RNA; ABCB, ABC transporter B family member; MFS, multidrug resistance protein; GH3, indole-3-acetic acid-amido synthetase GH3; and ARF, auxin response factor. log₂FC, log₂FoldChange.

Figure 8

Comparison of DEGs related to ethylene biosynthesis and signaling transduction pathway on SHAM treatment. ACO, 1-aminocyclopropane-1-carboxylate oxidase; ETR, ethylene receptor; EIN3, ethylene insensitive 3; and ERF, ethylene response factor. log₂FC, log₂FoldChange.

Figure 9

Relative expression levels of apoptosis-related genes in response to SHAM treatment. DEGs related to apoptosis (A) and their quantitative analysis by qRT-PCR. (B). log₂FC, log₂FoldChange.

Figure 10

Effects of adding methyl jasmonate (MeJA) on the root development of SHAM treatment. The root development on the 8th day of samples treated with 1 mM SHAM (CK) and treated with SHAM plus different concentrations of MeJA are shown.

Figure 11

Effects of adding indoleacetic acid (IAA) and dimethylthiourea (DMTU) on the root development of SHAM treatment. The root development on the 8th day of samples treated with 1 mM SHAM and treated with SHAM plus different concentrations of IAA (A) and DMTU (B) are shown. In addition, the representative root hairs were observed with a microscope. Scale bar, 500 μm.