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. 2020 Jun;183(2):517-529.
doi: 10.1104/pp.19.01564. Epub 2020 Apr 3.

Dual-Localized Enzymatic Components Constitute the Fatty Acid Synthase Systems in Mitochondria and Plastids

Xin Guan  1   2 , Yozo Okazaki  3 , Rwisdom Zhang  2   4 , Kazuki Saito  3   5 , Basil J Nikolau  6   2   7
Affiliations

Dual-Localized Enzymatic Components Constitute the Fatty Acid Synthase Systems in Mitochondria and Plastids

Xin Guan et al. Plant Physiol. 2020 Jun.

Abstract

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report the identification and characterization of Arabidopsis (Arabidopsis thaliana) genes encoding three enzymes shared between the mitochondria- and plastid-localized type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β-ketoacyl-acyl carrier protein (ACP) reductase and enoyl-ACP reductase, catalyze two of the reactions that constitute the core four-reaction cycle of the FAS system, which iteratively elongates the acyl chain by two carbon atoms per cycle. The third enzyme, malonyl-coenzyme A:ACP transacylase, catalyzes the reaction that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl cofactor of ACP. GFP fusion experiments revealed that the these enzymes localize to both chloroplasts and mitochondria. This localization was validated by characterization of mutant alleles, which were rescued by transgenes expressing enzyme variants that were retargeted only to plastids or only to mitochondria. The singular retargeting of these proteins to plastids rescued the embryo lethality associated with disruption of the essential ptFAS system, but these rescued plants displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, and reduced growth. However, these latter traits were reversible in an elevated-CO2 atmosphere, which suppresses mtFAS-associated photorespiration-dependent chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these nonredundant fatty acid biosynthetic machineries.

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Figures

Figure 1.
Figure 1.
Subcellular localization of potential mtFAS proteins. Shown are confocal fluorescence micrographs of roots and leaf mesophyll cells, imaging the emission of GFP, MitoTracker Orange, chlorophyll autofluorescence, and the merged images. Fluorescence micrographs are from nontransgenic wild-type control plants (WT) and transgenic plants carrying the p35S::GFP control, p35S::mtPPT1-240-GFP, or p35S::UGP31-600-GFP transgenes (A); p35S::mtER1-1122-GFP, p35S::mtER1-300-GFP, or p35S::mtER301-1122-GFP transgenes (B); p35S::pt/mtMCAT1-1179-GFP, p35S::pt/mtMCAT1-216-GFP, or p35S::pt/mtMCAT205-1179-GFP transgenes (C); p35S::pt/mtKR1-957-GFP, p35S::pt/mtKR1-234-GFP, or p35S::pt/mtKR214-957-GFP transgenes (D); and p35S::pt/mtER1-1167-GFP, p35S::pt/mtER1-261-GFP, or p35S::pt/mtER262-1167-GFP transgenes (E).
Figure 1.
Figure 1.
Subcellular localization of potential mtFAS proteins. Shown are confocal fluorescence micrographs of roots and leaf mesophyll cells, imaging the emission of GFP, MitoTracker Orange, chlorophyll autofluorescence, and the merged images. Fluorescence micrographs are from nontransgenic wild-type control plants (WT) and transgenic plants carrying the p35S::GFP control, p35S::mtPPT1-240-GFP, or p35S::UGP31-600-GFP transgenes (A); p35S::mtER1-1122-GFP, p35S::mtER1-300-GFP, or p35S::mtER301-1122-GFP transgenes (B); p35S::pt/mtMCAT1-1179-GFP, p35S::pt/mtMCAT1-216-GFP, or p35S::pt/mtMCAT205-1179-GFP transgenes (C); p35S::pt/mtKR1-957-GFP, p35S::pt/mtKR1-234-GFP, or p35S::pt/mtKR214-957-GFP transgenes (D); and p35S::pt/mtER1-1167-GFP, p35S::pt/mtER1-261-GFP, or p35S::pt/mtER262-1167-GFP transgenes (E).
Figure 1.
Figure 1.
Subcellular localization of potential mtFAS proteins. Shown are confocal fluorescence micrographs of roots and leaf mesophyll cells, imaging the emission of GFP, MitoTracker Orange, chlorophyll autofluorescence, and the merged images. Fluorescence micrographs are from nontransgenic wild-type control plants (WT) and transgenic plants carrying the p35S::GFP control, p35S::mtPPT1-240-GFP, or p35S::UGP31-600-GFP transgenes (A); p35S::mtER1-1122-GFP, p35S::mtER1-300-GFP, or p35S::mtER301-1122-GFP transgenes (B); p35S::pt/mtMCAT1-1179-GFP, p35S::pt/mtMCAT1-216-GFP, or p35S::pt/mtMCAT205-1179-GFP transgenes (C); p35S::pt/mtKR1-957-GFP, p35S::pt/mtKR1-234-GFP, or p35S::pt/mtKR214-957-GFP transgenes (D); and p35S::pt/mtER1-1167-GFP, p35S::pt/mtER1-261-GFP, or p35S::pt/mtER262-1167-GFP transgenes (E).
Figure 1.
Figure 1.
Subcellular localization of potential mtFAS proteins. Shown are confocal fluorescence micrographs of roots and leaf mesophyll cells, imaging the emission of GFP, MitoTracker Orange, chlorophyll autofluorescence, and the merged images. Fluorescence micrographs are from nontransgenic wild-type control plants (WT) and transgenic plants carrying the p35S::GFP control, p35S::mtPPT1-240-GFP, or p35S::UGP31-600-GFP transgenes (A); p35S::mtER1-1122-GFP, p35S::mtER1-300-GFP, or p35S::mtER301-1122-GFP transgenes (B); p35S::pt/mtMCAT1-1179-GFP, p35S::pt/mtMCAT1-216-GFP, or p35S::pt/mtMCAT205-1179-GFP transgenes (C); p35S::pt/mtKR1-957-GFP, p35S::pt/mtKR1-234-GFP, or p35S::pt/mtKR214-957-GFP transgenes (D); and p35S::pt/mtER1-1167-GFP, p35S::pt/mtER1-261-GFP, or p35S::pt/mtER262-1167-GFP transgenes (E).
Figure 1.
Figure 1.
Subcellular localization of potential mtFAS proteins. Shown are confocal fluorescence micrographs of roots and leaf mesophyll cells, imaging the emission of GFP, MitoTracker Orange, chlorophyll autofluorescence, and the merged images. Fluorescence micrographs are from nontransgenic wild-type control plants (WT) and transgenic plants carrying the p35S::GFP control, p35S::mtPPT1-240-GFP, or p35S::UGP31-600-GFP transgenes (A); p35S::mtER1-1122-GFP, p35S::mtER1-300-GFP, or p35S::mtER301-1122-GFP transgenes (B); p35S::pt/mtMCAT1-1179-GFP, p35S::pt/mtMCAT1-216-GFP, or p35S::pt/mtMCAT205-1179-GFP transgenes (C); p35S::pt/mtKR1-957-GFP, p35S::pt/mtKR1-234-GFP, or p35S::pt/mtKR214-957-GFP transgenes (D); and p35S::pt/mtER1-1167-GFP, p35S::pt/mtER1-261-GFP, or p35S::pt/mtER262-1167-GFP transgenes (E).
Figure 2.
Figure 2.
Genetic and biochemical characterization of mtFAS gene candidates. A, Genetic complementation of yeast mtFAS mutants (mct1, oar1, and etr1) by expression of Arabidopsis mtFAS candidate genes (AT2G30200, AT1G24360, AT3G45770, and AT2G05990); expression of the wild-type yeast homologs (MCT1, OAR1, and ETR1) served as a positive control. Gene expression was transcriptionally controlled by the PGK promoter (pPGK) and terminator (tPGK). The mitochondrial presequence of yeast COQ3 protein was fused to the N terminus of each protein to ensure mitochondrial localization. All yeast strains, carrying the indicated expression cassettes, were grown on medium containing either glycerol or Glc as the sole carbon source, and a dilution series served as the inoculum for each strain. B, In vitro characterization of the catalytic capability of purified recombinant Arabidopsis mtER and pt/mtER enzymes. Substrate concentration dependence of the enoyl reductase activity was assayed with increasing concentrations of either trans-Δ2-10:1-CoA or trans-Δ2-16:1-CoA as substrate. The tabulated Michaelis-Menten kinetic parameters were calculated from three to six replicates for each substrate concentration.
Figure 2.
Figure 2.
Genetic and biochemical characterization of mtFAS gene candidates. A, Genetic complementation of yeast mtFAS mutants (mct1, oar1, and etr1) by expression of Arabidopsis mtFAS candidate genes (AT2G30200, AT1G24360, AT3G45770, and AT2G05990); expression of the wild-type yeast homologs (MCT1, OAR1, and ETR1) served as a positive control. Gene expression was transcriptionally controlled by the PGK promoter (pPGK) and terminator (tPGK). The mitochondrial presequence of yeast COQ3 protein was fused to the N terminus of each protein to ensure mitochondrial localization. All yeast strains, carrying the indicated expression cassettes, were grown on medium containing either glycerol or Glc as the sole carbon source, and a dilution series served as the inoculum for each strain. B, In vitro characterization of the catalytic capability of purified recombinant Arabidopsis mtER and pt/mtER enzymes. Substrate concentration dependence of the enoyl reductase activity was assayed with increasing concentrations of either trans-Δ2-10:1-CoA or trans-Δ2-16:1-CoA as substrate. The tabulated Michaelis-Menten kinetic parameters were calculated from three to six replicates for each substrate concentration.
Figure 3.
Figure 3.
In vivo physiological characterization of the pt/mtMCAT and pt/mtKR genes. A, Morphological phenotypes of the pt/mtmcat and pt/mtkr mutants complemented by full-length or truncated transgenes. Plants were grown in either ambient air or in a 1% (v/v) CO2 atmosphere. B, Western-blot analysis of the H subunit of GDC detected with either anti-H-protein antibodies or anti-lipoic acid antibodies, detecting the lipoylation status of the H-protein. C, Gly accumulation. Plants of the indicated genotypes were grown in either ambient air or in a 1% CO2 atmosphere.
Figure 4.
Figure 4.
Physiological characterization of the pt/mtER and mtER genes. A, Morphological phenotypes of single or double mutants of the pt/mtER and mtER genes. Plants were grown in either ambient air or in a 1% (v/v) CO2 atmosphere. B, Western-blot analysis of the H subunit of GDC detected with anti-H-protein antibodies, and the lipoylation status of the H-protein detected with anti-lipoic acid antibodies. Plants were grown in a 1% (v/v) CO2 atmosphere. C, Gly accumulation. Plants of the indicated genotypes were grown in either ambient air or in a 1% (v/v) CO2 atmosphere. WT, Wild type.
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