Monogalactosyldiacylglycerol Facilitates Synthesis of Photoactive Protochlorophyllide in Etioplasts

Abstract

Cotyledon cells of dark-germinated angiosperms develop etioplasts that are plastids containing unique internal membranes called prolamellar bodies (PLBs). Protochlorophyllide (Pchlide), a precursor of chlorophyll, accumulates in PLBs and forms a ternary complex with NADPH and light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR), which allows for the rapid formation of chlorophyll after illumination while avoiding photodamage. PLBs are 3D lattice structures formed by the lipid bilayer rich in monogalactosyldiacylglycerol (MGDG). Although MGDG was found to be required for the formation and function of the thylakoid membrane in chloroplasts in various plants, the roles of MGDG in PLB formation and etioplast development are largely unknown. To analyze the roles of MGDG in etioplast development, we suppressed MGD1 encoding the major isoform of MGDG synthase by using a dexamethasone-inducible artificial microRNA in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. Strong MGD1 suppression caused a 36% loss of MGDG in etiolated seedlings, together with a 41% decrease in total Pchlide content. The loss of MGDG perturbed etioplast membrane structures and impaired the formation of the photoactive Pchlide-LPOR-NADPH complex and its oligomerization, without affecting LPOR accumulation. The MGD1 suppression also impaired the formation of Pchlide from protoporphyrin IX via multiple enzymatic reactions in etioplast membranes, which suggests that MGDG is required for the membrane-associated processes in the Pchlide biosynthesis pathway. Suppressing MGD1 at several germination stages revealed that MGDG biosynthesis at an early germination stage is particularly important for Pchlide accumulation. MGDG biosynthesis may provide a lipid matrix for Pchlide biosynthesis and the formation of Pchlide-LPOR complexes as an initial step of etioplast development.

Figure 1.

Effect of MGD1 suppression on galactolipid biosynthesis in etiolated seedlings. A, Quantitative reverse transcription-PCR analysis of MGD1 mRNA levels in 4-d-old etiolated seedlings of amiR-MGD1 under +DEX and −DEX conditions. Data are presented as fold difference from the −DEX control after normalizing to the control gene ACTIN8. Data are means ± se from 13 (L4w) or three (L4g) independent experiments. B, Accumulation of MGDG and DGDG in 4-d-old etiolated seedlings of amiR-MGD1 L4w. C and D, Fatty acid composition of MGDG (C) and DGDG (D) in 4-d-old etiolated seedlings of amiR-MGD1 L4w. In B to D, data are means ± se from three independent experiments. Asterisks indicate significant differences from the −DEX control (*, P < 0.05; **, P < 0.01; and ***, P < 0.001, Student’s t test).

Figure 2.

Contribution of MGDG to the accumulation of Pchlide and carotenoids in etiolated seedlings. A, Pchlide content in 4-d-old etiolated seedlings of amiR-MGD1 grown under −DEX and +DEX conditions. Total and nonphotoactive Pchlide were extracted before and after flash treatment, respectively. Data are means ± se from 12 (L4w) or 15 (L4g) independent experiments. The amount of photoactive Pchlide was estimated by subtracting the amount of nonphotoactive Pchlide from total Pchlide. B, Pchlide content in 4-d-old etiolated L4w seedlings treated with DEX at different times after seeding. L4w plants were treated with DEX from the beginning of seeding (+DEX), 1 d after seeding (1-d DEX), or 2 d after seeding (2-d DEX). Seedlings grown in the absence of DEX were analyzed as the untreated control (−DEX). Data are means ± se from seven to 12 independent experiments. Different letters indicate significant differences (P < 0.05, Tukey-Kramer multiple comparison test). C, Pchlide accumulation in etiolated amiR-MGD1 L4w seedlings grown for 2 to 5 d under −DEX and +DEX conditions. Data are means ± se from six to 12 independent experiments. D, Immunoblot analysis of total LPOR proteins (∼37 kD) in 4-d-old etiolated seedlings of amiR-MGD1 L4w. As a loading control, Ponceau-stained proteins between ∼25 and ∼50 kD blotted onto a membrane are shown. Representative data from three biologically independent experiments are shown. E, Total carotenoid content in 4-d-old etiolated seedlings of amiR-MGD1 L4w. Data are means ± se from eight independent experiments. In A, C, and E, asterisks indicate significant differences from the −DEX control (**, P < 0.01; and ***, P < 0.001, Student’s t test).

Figure 3.

Role of MGDG in the formation of the Pchlide-LPOR-NADPH complex and processes after illumination. A, C, D, and E, In situ 77K fluorescence spectra under 440-nm excitation in etiolated cotyledons of amiR-MGD1 L4w grown for 4 d under +DEX and −DEX conditions. Samples were frozen in liquid nitrogen without flash treatment (A), immediately after a 0.7-ms flash (C), and after additional dark incubation for 20 min (D) or 2 h (E) following flash treatment. Representative data from three or more biologically independent experiments are shown. Vertical dotted lines in A and E represent the peak wavelength of fluorescence from photoactive Pchlide (P₆₅₃) in −DEX seedlings. B, +DEX minus −DEX difference spectra in continuous dark (Dark) or 2 h of dark after flash irradiation (Flash + 2 h dark). Data are means of eight (Dark) or three (Flash + 2 h dark) independent experiments. The arrow indicates the fluorescence peak from the dimeric Pchlide-LPOR-NADPH complex at ∼645 nm. F, Pchlide content in 4-d-old etiolated amiR-MGD1 L4w seedlings dark incubated for 20 min after flash treatment. Data are means ± se from five to seven independent experiments. The amount of photoactive Pchlide was estimated by subtracting the amount of nonphotoactive Pchlide from total Pchlide. Asterisks indicate significant differences from each form of Pchlide of the −DEX control (*, P < 0.05 and ***, P < 0.001, Student’s t test).

Figure 4.

Ultrastructure of etioplasts in cotyledon cells of 4-d-old etiolated amiR-MGD1 L4w seedlings. A and C, Images of whole etioplasts in cotyledons grown under −DEX (A) and +DEX (C). Bars = 1 μm. B and D, Magnified images of PLB lattices in A (B) and C (D). Bars = 200 nm. For more images, see Supplemental Figure S6. E to K, Quantitative data of circularity index (E) and area (F) of PLBs, area of a single PLB unit (G), relative sd value (sd/average) of the unit area in a PLB (H), length of PTs (I), and circularity index (J) and area (K) of etioplasts. The horizontal line in each box represents the median value of the distribution. The top and bottom of each box represent the upper and lower quartiles, respectively. The whiskers represent the range. Data were obtained from 46 different etioplasts. In H, the relative sd value was calculated from 20 units of the PLB in each etioplast. The distribution of the PLB unit area in each etioplast is shown in Supplemental Figure S7. Asterisks indicate significant differences from the −DEX control (*, P < 0.05; **, P < 0.01; and ***, P < 0.001, Welch’s t test).

Figure 5.

Effect of MGD1 suppression on Pchlide biosynthesis in the dark. A to C, Accumulation of porphyrin pigments in etiolated amiR-MGD1 L4w seedlings fed ALA for 1.5 h (A) and 24 h (B) and L4g seedlings fed ALA for 24 h (C). Seedlings were grown in the dark under +DEX or −DEX conditions for 4 d before ALA feeding. D and E, Accumulation of porphyrin pigments in 4-d-old etiolated wild-type (WT), chlm, and chl27 seedlings fed ALA for 1.5 h (D) and 24 h (E). In A to E, data are means ± se from three to six independent experiments. ND, Not detected; Trace, trace amount. F, Quantitative reverse transcription-PCR analysis of the mRNA expression of genes involved in Pchlide biosynthesis in L4w and L4g seedlings grown in the dark for 4 d. mRNA levels in +DEX seedlings are presented as fold differences from the −DEX controls (broken line) after normalizing to the control gene ACTIN8. Data are means ± se from 10 (L4w) or three (L4g) independent experiments. In A, B, C, and F, asterisks indicate significant differences from the −DEX control (*, P < 0.05 and ***, P < 0.001, Student’s t test). In D and E, different letters indicate significant differences (P < 0.05, Tukey-Kramer multiple comparison test).

Figure 6.

Quantitative reverse transcription-PCR analysis of mRNA levels of photosynthesis-associated and reactive oxygen species-responsive genes in amiR-MGD1 L4w etiolated seedlings grown for 4 d under −DEX and +DEX conditions. A, Genes encoded in the plastid genome. B, Photosynthesis-associated and reactive oxygen species-responsive genes encoded in the nucleus. In A and B, mRNA levels are presented as fold difference from the −DEX control after normalizing to the control gene ACTIN8. Data are means ± se from 10 independent experiments. None of the genes showed significant differences between +DEX and −DEX seedlings (P > 0.05, Student’s t test).

Figure 7.

Roles of MGDG in Pchlide biosynthesis and the formation of photoactive Pchlide-LPOR-NADPH complexes during etioplast development. Arrowheads indicate enzymatic steps in the Pchlide biosynthesis pathway from ALA. Most of the Pchlide synthesized in etioplasts forms the photoactive ternary complex with LPOR and NADPH, and the photoactive complex exists as the dimer or further aggregates into oligomeric complexes. MGDG is required for the Pchlide biosynthesis pathway from Proto IX to Pchlide (arrow 1), the formation of the photoactive Pchlide-LPOR-NADPH ternary complex (arrow 2), and the oligomerization of the ternary complex (arrow 3).