A cytochrome b5-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii

Abstract

Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn-2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b(5) domain, distinguishing it from other known plastid desaturases. Cytochrome b(5) is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b(5) domain of CrΔ4FAD showed that it is functional in vitro. Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas. Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.

Fig 1

Fatty acyl profiles of MGDG (A) and TAG (B) isolated from Chlamydomonas strains dw15 (filled bars) and UVM4 (shaded bars). Hexadecatetraenoic acid (16:4) is the major fatty acid in the sn-2 position of MGDG but was also recovered in TAG (5% in dw15; 8% in UVM4). Lipids were separated by thin-layer chromatography, converted to their fatty acid methyl esters, and quantified by GC-flame ionization detection. Fatty acids are displayed by the number of carbons, followed by a colon and the number of double bonds. The numbers following Δ indicate the positions of the double bonds in the acyl chain. Means and standard deviations of three biological replicates are shown.

Fig 2

Protein topology prediction for CrΔ4FAD. The hydropathy plot (A) was generated using the TOPPRED program (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::toppred) and is based on the algorithm of von Heijne (46). (B) Schematic overview of CrΔ4FAD topology. cTP, chloroplast transit peptide; Cytb5, cytochrome b₅ domain; TMD, membrane-spanning domains, each composed of two transmembrane helices. The three histidine boxes and the heme binding motif are indicated.

Fig 3

CrΔ4FAD-GFP localizes to the chloroplast in Chlamydomonas. Free GFP is localized in the cytosol of Chlamydomonas strain UVM4 (A), in contrast to CrΔ4FAD-GFP, which is found in the plastid (B). Bars, 5 μm.

Fig 4

Knockdown and overexpression of CrΔ4FAD in Chlamydomonas. (A) Knockdown lines (dw15-Δ4-KD) show lower CrΔ4FAD expression than the empty-vector control (dw15). CrΔ4FAD expression is elevated in strain UVM4-Δ4-GFP, expressing CrΔ4FAD-GFP. UVM4-GFP (UVM4 expressing GFP alone) was used as a control. The expression of the control genes MGD1, DGD1, and RACK1 was not affected in any of the lines. cDNA was prepared from equal amounts of total RNA and was used as the PCR template with differing cycle numbers depending on the gene: 34 for CrΔ4FAD, 30 for MGD1 and DGD1, and 24 for the control gene RACK1. (B) Immunoblot showing the expression of the CrΔ4FAD-GFP fusion construct in three independent lines. Chlamydomonas cells were harvested, mixed with sodium dodecyl sulfate sample buffer, and directly loaded onto a denaturing gel. Proteins were then transferred to a polyvinylidene difluoride membrane and were subjected to immunoblot analysis using an anti-GFP antibody. (C) The fatty acid composition of Chlamydomonas cells overexpressing CrΔ4FAD is changed: the levels of 16:4 and 18:3^Δ9,12,15 are higher in overexpression lines (shaded bars) than in the empty-vector control (filled bars). (D) Total fatty acid composition in CrΔ4FAD knockdown lines. Knockdown lines (shaded bars) show lower levels of 16:4 and 18:3^Δ9,12,15 acyl groups than does the empty-vector control (filled bars). For panels C and D, total fatty acids were converted to their FAMEs and were quantified by GC-flame ionization detection. Significant differences in fatty acid composition between the two lines are marked by asterisks (P, <0.05 by a nonpaired two-sample t test).

Fig 5

CrΔ4FAD expression specifically affects the MGDG content (A), but not the DGDG content (B), of Chlamydomonas. The total fatty acid content remains unchanged (C), but the higher total-galactolipid content in the overexpression lines leads to an increased chlorophyll content (D). Lipids for panels A to C were isolated from a defined amount of cells, separated by TLC, and quantified as FAMEs. Pigments (D) were isolated, and their absorbance spectra were detected in 80% acetone. Asterisks indicate significant differences between knockdown or overexpression lines and their corresponding controls (P, <0.05 by a nonpaired two-sample t test). Data were obtained from at least six independent experiments. Strains are explained in the legend to Fig. 4.

Fig 6

Redox difference spectra of dithionite-reduced (A to C) and dithiothreitol-reduced (D to F) recombinant cytochrome b₅. (A to C) Spectra were calculated from the absorbance of air-oxidized and dithionite-reduced DsRED fusion proteins. DsRED-CrΔ4Cytb5 (A), DsRED-CrFAD13Cytb5 (B), and DsRED-AtCytb5 (C) show difference maxima at 425 (424), 528 (527), and 558 (559) nm, respectively, in line with previous observations (12, 40). (D to F) Spectra were calculated from the absorbance of air-oxidized and DTT-reduced DsRED fusion proteins. The characteristic shape of the cytochrome b₅ difference spectrum is still maintained in DsRED-CrFAD13Cytb5 (E) and DsRED-AtCytb5 (F), although the difference between oxidized and DTT-reduced forms is much less pronounced than that with dithionite. In contrast, DsRED-CrΔ4Cytb5 cannot be reduced with DTT (D), indicating that CrΔ4Cytb5 requires a stronger reductant than DTT [ΔE, −330 mV, comparable with that for NADH; ΔE(dithionite), −660 mV].