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. 2018 Dec;178(4):1568-1583.
doi: 10.1104/pp.18.01042. Epub 2018 Oct 11.

ARSENATE INDUCED CHLOROSIS 1/ TRANSLOCON AT THE OUTER ENVOLOPE MEMBRANE OF CHLOROPLASTS 132 Protects Chloroplasts from Arsenic Toxicity

Peitong Wang  1 , Xi Chen  2 , Xuan Xu  1 , Chenni Lu  1 , Wei Zhang  2 , Fang-Jie Zhao  3
Affiliations

ARSENATE INDUCED CHLOROSIS 1/ TRANSLOCON AT THE OUTER ENVOLOPE MEMBRANE OF CHLOROPLASTS 132 Protects Chloroplasts from Arsenic Toxicity

Peitong Wang et al. Plant Physiol. 2018 Dec.

Abstract

Arsenic (As) is highly toxic to plants and detoxified primarily through complexation with phytochelatins (PCs) and other thiol compounds. To understand the mechanisms of As toxicity and detoxification beyond PCs, we isolated an arsenate-sensitive mutant of Arabidopsis (Arabidopsis thaliana), arsenate induced chlorosis1 (aic1), in the background of the PC synthase-defective mutant cadmium-sensitive1-3 (cad1-3). Under arsenate stress, aic1 cad1-3 showed larger decreases in chlorophyll content and the number and size of chloroplasts than cad1-3 and a severely distorted chloroplast structure. The aic1 single mutant also was more sensitive to arsenate than the wild type (Columbia-0). As concentrations in the roots, shoots, and chloroplasts were similar between aic1 cad1-3 and cad1-3 Using genome resequencing and complementation, TRANSLOCON AT THE OUTER ENVOLOPE MEMBRANE OF CHLOROPLAST132 (TOC132) was identified as the mutant gene, which encodes a translocon protein involved in the import of preproteins from the cytoplasm into the chloroplasts. Proteomic analysis showed that the proteome of aic1 cad1-3 chloroplasts was more affected by arsenate stress than that of cad1-3 A number of proteins related to chloroplast ribosomes, photosynthesis, compound synthesis, and thioredoxin systems were less abundant in aic1 cad1-3 than in cad1-3 under arsenate stress. Our results indicate that chloroplasts are a sensitive target of As toxicity and that AIC1/Toc132 plays an important role in protecting chloroplasts from As toxicity.

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Figures

Figure 1.
Figure 1.
Phenotype of aic1 cad1-3. A, Phenotypic comparison of Columbia-0 (Col-0), cad1-3, and aic1 cad1-3 grown on one-half-strength Murashige and Skoog (1/2 MS) medium containing 0 or 2 μm As(V) for 10 d. Bars (red) = 1 cm. B to D, Fresh weight (B), primary root length (C), and chlorophyll content (D) of seedlings germinated and grown on 1/2 MS medium containing 0, 2, 4, 6, or 8 μm As(V) for 10 d. Data are means ± se (n = 3) for fresh weight and chlorophyll content analysis and (n = 8) for primary root length. Different letters indicate statistically significant differences within treatments (P < 0.05). FW, Fresh weight.
Figure 2.
Figure 2.
As uptake, elemental concentrations in chloroplasts, and number and structure of chloroplasts in aic1 cad1-3 and cad1-3. A, As concentrations in the roots and shoots of plants grown in one-fifth-strength Hoagland hydroponic medium containing 5 μm As(V) for 3 d. DW, Dry weight. B, Chloroplast elemental concentrations in cad1-3 and aic1 cad1-3. Intact chloroplasts were isolated from plants grown on 1/2 MS medium with 2 μm As(V) for 15 d. C, Transmission electron micrographs of chloroplasts from cad1-3 and aic1 cad1-3. Plants were grown on 1/2 MS medium containing 0 or 8 μm As(V) for 10 d. Bars = 1 μm. D, Number of chloroplasts. Plants were grown on 1/2 MS medium containing 0 or 3 μm As(V) for 15 d. Data are means ± se (n = 3). Different letters indicate statistically significant differences within treatments (P < 0.05). FW, Fresh weight.
Figure 3.
Figure 3.
Cloning and complementation analysis of the mutant gene. A, Genomic structure of TOC132 and the mutation site. B, DNA sequence of the point mutation in toc132. The red box indicates the mutation position. C and D, Photographs (C) and chlorophyll content (D) of cad1-3, aic1 cad1-3, and two independent complementation lines. Seedlings were grown on 1/2 MS medium containing 4 μm As(V) for 10 d. Bar (red) = 1 cm. Data are means ± se (n = 3). Different letters indicate statistically significant differences between genotypes (P < 0.05). FW, Fresh weight. E, Genotype analysis to confirm the complementation lines. dCAPS primer was used to amplify the PCR products of TOC132 from cad1-3, aic1 cad1-3, and the complementation lines. The PCR products were digested with HindIII.
Figure 4.
Figure 4.
Characterization of toc132 single mutants. A, Diagram of the T-DNA insertion mutant of the TOC132 gene. UTR, Untranslated Region. B, Semiquantitative reverse transcription (RT)-PCR analysis of TOC132 expression in the T-DNA insertion mutant with ACTIN2 as a reference gene. C, Phenotypic comparison of Col-0, toc132, and aic1 grown on 1/2 MS medium containing 0 or 200 μm As(V) for 10 d. Bars (red) = 1 cm. D, Fresh weight of plants shown in C. Data are means ± se (n = 3). Different letters indicate statistically significant differences within treatments (P < 0.05).
Figure 5.
Figure 5.
Phenotypes and profiling of chloroplast protein expression of cad1-3 and aic1 cad1-3. A, Phenotypes of plants grown on 1/2 MS medium containing 0 or 3 μm As(V) for 15 d. B, Numbers of differentially expressed proteins within cad1-3 (WT) or aic1 cad1-3 (Mu) under 3 μm As(V) (+As) compared with the normal condition (−As). C, Numbers of differentially expressed proteins between cad1-3 (WT) and aic1 cad1-3 (Mu) under 0 (−As) or 3 μm As(V) (+As) condition.
Figure 6.
Figure 6.
FBPase activity in cad1-3 and aic1 cad1-3. Plants were grown on 1/2 MS medium containing 0 or 4 μm As(V) for 10 d. Data are means ± se (n = 3 biological replicates). Different letters indicate statistically significant differences within treatments (P < 0.05). FW, Fresh weight.
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