Uncommon properties of lipid biosynthesis of isolated plastids in the unicellular red alga Cyanidioschyzon merolae

Abstract

Red algae are a large group of photosynthetic eukaryotes that diverged from green algae over one billion years ago, and have various traits distinct from those of both green algae and land plants. Although most red algae are marine species (both unicellular and macrophytic), the Cyanidiales class of red algae includes unicellular species which live in hot springs, such as Cyanidioschyzon merolae, which is a model species for biochemical and molecular biological studies. Lipid metabolism in red algae has previously been studied in intact cells. Here, we present the results of radiolabeling and stable isotope labeling experiments in intact plastids isolated from the unicellular red alga C. merolae. We focused on two uncommon features: First, the galactose moiety of monogalactosyldiacylglycerol was efficiently labeled with bicarbonate, indicating that an unknown pathway for providing UDP-galactose exists within the plastid. Second, saturated fatty acids, namely, palmitic and stearic acids, were the sole products of fatty acid synthesis in the plastid, and they were efficiently exported. This finding suggests that the endoplasmic reticulum is the sole site of desaturation. We present a general principle of red algal lipid biosynthesis, namely, 'indigenous C18 fatty acids are neither desaturated nor directly utilized within the plastid'. We believe that this is valid in both C. merolae lacking polyunsaturated fatty acids and marine red algae with a high content of arachidonic and eicosapentaenoic acids.

Keywords: Cyanidioschyzon merolae; isolated plastids; radiolabeling experiment; red alga; stable isotope; stearoyl‐ACP desaturase.

Figure 1

Purity of isolated intact plastids of Cyanidioschyzon merolae and intact spinach chloroplasts. (A, B) Nomarski differential interference images. (C, D) Fluorescence images stained with 4′,6‐diamidino‐2‐phenylindole. Bars = 5 μm.

Figure 2

Incorporation of [¹⁴C]bicarbonate into polar lipids in isolated plastids. (A) Autoradiogram of polar lipids separated by TLC after labeling with [¹⁴C]bicarbonate. Position of representative lipid classes found in the cell is shown on the right. (B) Time course of incorporation of [¹⁴C]bicarbonate into polar lipids. Labeling experiments were repeated three times, and representative results are shown. We show here ‘PA(+PI)', because PA and phosphatidylinositol (PI) are not separated by the TLC system (the first dimension in the two‐dimensional system in ref. 7) that we used in the present study, but PI is not expected to be a component of isolated plastids.

Figure 3

Incorporation [¹⁴C] and [¹³C] bicarbonate into MGDG in isolated plastids. (A) Flow diagram of analysis of radioactive MGDG. (B) Detection of radioactivity in FAMEs. (C) Detection of radioactivity in galactose and glycerol. (D) Detection of radioactivity in polar groups of DGDG and SQDG. Gal, galactose; Gly, glycerol; SQ, sulfoquinovose. Radiolabeling experiments were repeated three times, and representative results are shown. (E, F) Isotopomer distribution of MGDG synthesized after [¹³C]bicarbonate labeling in intact cells (E) and isolated plastids (F). Unlabeled (or natural abundance) and labeled ¹³C isotopomer populations are shown in light gray and dark gray, respectively. The error bars represented the SD (n = 3).

Figure 4

Subcellular localization of enzymes involved in UDP‐galactose synthesis. (A) Schematic diagram of GFP constructs. A DNA fragment of the N‐terminal peptide of each enzyme was inserted into pCEG1 vector and cloned. GFP‐fusion proteins were transiently expressed under the control of APCC promoter in Cyanidioschyzon merolae cells. EGFP; enhanced green fluorescence protein gene, NOS; nopaline synthase gene terminator, P_APCC; promoter of APCC gene of C. merolae. (B) Immunofluorescence micrographs of C. merolae cells transiently expressing GFP‐fusion protein. Enzyme names are listed in Table 4. Bar = 2 μm.

Figure 5

Working metabolic map of galactolipid synthesis in Cyanidioschyzon merolae. UDP‐galactose is synthesized by two pathways in plastid and cytosol. Dashed lines indicate transport of substrates between plastid and other compartments. UDP‐galactose synthesized by the two pathways is used as a substrate for galactolipid synthesis. G3P is synthesized from triose‐phosphate by plastid‐type glycerol 3‐phosphate dehydrogenase (GPDH; CMR476C, 33). Two cytosolic enzymes, phosphoglycerate mutase and enolase, are required to synthesize acetyl‐CoA from triose‐phosphate (as in land plants). Glc1P, glucose‐1‐phosphate; PEP, phosphoenolpyruvate; Triose‐P, triose‐phosphate; UDP‐Gal, UDP‐galactose; UDP‐Glc, UDP‐glucose. Enzyme names involved in UDP‐galactose synthesis are shown in Table 4.

Figure 6

Incorporation of [2‐¹⁴C] and [2‐¹³C]acetate into fatty acids in isolated Cyanidioschyzon merolae plastids and spinach chloroplasts. (A, B) RP‐TLC separation of FAMEs derived from the total lipids after incorporation of [2‐¹⁴C]acetate in isolated C. merolae plastids (A) and spinach chloroplasts (B). (C, D) Effect of dark (C) and cerulenin addition (D) on the incorporation of [2‐¹⁴C]acetate into fatty acids in isolated plastids of C. merolae. (E) Time course of incorporation of [2‐¹⁴C]acetate into FAMEs in C. merolae plastids. (F) Comparison of fatty acid labeling, after 60‐min incorporation of [2‐¹⁴C]acetate in isolated C. merolae plastids and spinach chloroplasts. Radiolabeling experiments were repeated three times, and representative results are shown. (G) Incorporation of [2‐¹³C]acetate (2 mm) into fatty acids of total lipids in isolated plastids for 1 h. Unlabeled (or natural abundance) and labeled populations of ¹³C isotopomers are shown in light gray and dark gray, respectively. The inset shows an enlargement of ¹³C isotopomer distribution. The error bars represented the SD (n = 3).

Figure 7

Fatty acid export in isolated plastids from Cyanidioschyzon merolae. (A) Two‐step TLC separation of lipid classes in the supernatant and precipitate fractions. First, polar lipids were separated by developing halfway the TLC plate with acetone/toluene/methanol/water (8 : 3 : 2 : 1, by volume). After drying, nonpolar lipids were further developed to the top with n‐hexane/diethyl ether/acetic acid (80 : 30 : 1, by volume). (B) Time course of relative incorporation of [2‐¹⁴C]acetate into polar lipids and FFAs in supernatant and precipitate fractions. The error bars represented the SD (n = 3). (C) Incorporation of [2‐¹⁴C]acetate into FAMEs of plastid membrane lipids. After methanolysis, FAMEs were extracted and fractionated by RP‐TLC. Labeling experiments were repeated three times, and representative results are shown.

Figure 8

Essential similarity of lipid biosynthesis in Cyanidioschyzon merolae (A) and marine red algae (B). In both models, ‘indigenous C18 fatty acids are neither desaturated nor directly utilized within the plastid' is a common principle. Panel A summarizes major findings in the present study. For simplicity, only C18 fatty acids are shown. The 18:0 synthesized by the fatty acid synthase in the plastid is exported to the cytosol and subsequently incorporated into phosphatidylcholine (PC) via CoA thioester in the ER. Then, the acyl groups are desaturated to produce 18:2. Genomic data indicate that desaturation of 18:0‐CoA to 18:1‐CoA is also possible in ER. In this case, 18:1‐PC is formed, and then desaturated. Panel B is a model in marine red algae, such as Pyropia and Porphyridium mainly based on 1, which contain 20:4 or 20:5 as major fatty acids in MGDG and other plastid lipids. For simplicity, only 20:4 and MGDG are shown. The 18:0 synthesized within the plastid is exported and ultimately incorporated into PC. During the process, desaturation and elongation take place in acyl‐CoA, but the exact sequence of process is not clear. 20:1 is further desaturated to 20:4, and possibly 20:5 in either PC or CoA. Finally, 20:4 (or 20:5) is incorporated into plastid lipids. The last step of desaturation is also likely to occur in the plastid. Apparently very different composition of fatty acids in MGDG or other plastid lipids can be understood according to the common principle ‘no desaturation or direct utilization of C18 acids synthesized in the plastid'. UDP‐Gal, UDP‐galactose.