Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide

Abstract

CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, a plastid-localized putative diiron protein, is required for the synthesis of protochlorophyllide and therefore is a candidate subunit of the aerobic cyclase in chlorophyll biosynthesis. delta-Aminolevulinic acid-fed antisense Arabidopsis plants with reduced amounts of Crd1/CHL27 accumulate Mg-protoporphyrin IX monomethyl ester, the substrate of the cyclase reaction. Mutant plants have chlorotic leaves with reduced abundance of all chlorophyll proteins. Fractionation of Arabidopsis chloroplast membranes shows that Crd1/CHL27 is equally distributed on a membrane-weight basis in the thylakoid and inner-envelope membranes.

Fig. 1.

The oxidative cyclase reaction for the formation of the fifth ring of the Chl molecule. The conversion of MgPMME to divinyl Pchlide proceeds through three sequential two-electron oxidations. The first step requires molecular oxygen as a substrate for hydroxylation, analogous to the methane monooxygenase reaction. There is also an oxygen requirement for the third reaction.

Fig. 2.

Phenotypes of chl27-as Arabidopsis plants. (Ai) A plant line (chl27-as1.7.5) that shows a severe phenotype early after transfer from sucrose-containing plates to soil. (Aii) A line (chl27-as2.38.10) that shows a severe chlorotic phenotype at a later stage of development. (Aiii) A plant line (chl27-as2.38.4) that displays a variegated phenotype. (Aiv) A WT plant transformed with pPZP111 (vector control). (B) Immunoblot detection of CHL27. Plant material was collected from chlorotic lines (1, chl27-as1.7.5; 2, chl27-as2.22.7), variegated lines (3, chl27-as2.22.10; 4, chl27-as2.22.12), green lines (5, chl27-as2.22.1; 6, chl27-as2.22.6), and WT lines (7 and 8). Proteins were resolved by SDS/PAGE, and the abundance of CHL27 was revealed by immunoblotting. Each lane contains 30 μg of total protein.

Fig. 3.

Analysis of porphyrin pigments accumulating in chl27-as lines after feeding with ALA in the dark. (A) Room-temperature fluorescence emission spectra of acetone extracts of chlorotic (yellow) leaves from an antisense line (chl27-as2.38.10) and green leaves from WT plants after overnight incubation with ALA compared to purified standards. (B) HPLC traces of the extracted pigment mixtures relative to known standards. The eluate was monitored at 420 nm for optimal detection of MgPMME and MgP (i, iii, v, and vii) or 440 nm for detection of Pchlide (ii, iv, and vi). (Inset) Traces show spectral absorbance of the major eluate peak, measured from 350 to 595 nm, with the wavelength of maximum absorbance indicated.

Fig. 4.

Suborganellar localization of CHL27. (A) Analysis of Arabidopsis chloroplast fractions. E, envelope; T, thylakoid; S, stroma. (Left) Fractions analyzed after SDS/PAGE by Coomassie blue staining. (Right) Immunoblot detection of CHL27 and OEP21. (B) Distribution of CHL27 between envelope fractions from spinach chloroplasts. Envelope membrane fractions were enriched in inner-envelope membrane (IM) or outer-envelope membrane (OM). T₀,T₁₀₀, and T₆₀₀, envelope fractions were obtained from chloroplasts treated without thermolysin or with 100 or 600 μg/ml thermolysin, respectively. (Left) Coomassie blue staining. (Right) Immunodetection of CHL27, E37, an inner-envelope membrane protein, and E24, an outer-envelope membrane protein. Each lane was loaded with 20 μg of protein. The membrane was incubated with anti-CHL27 (1:1,000), anti-E37 (1:10,000), and anti-E24 (1:5,000).

Fig. 5.

Reduced Chl protein abundance in chl27-as plants. (A) Fluorescence emissions (77 K) measured from 650 to 800 nm from WT (black), antisense lines showing no visible phenotype [green (chl27-as2.22.1) and light green (chl27-as2.22.6)], variegated lines [blue (chl27-as2.22.10) and light blue (chl27-as2.22.12)], and chlorotic [orange (chl27-as1.7.5)] plants. (B) Immunoblot analysis of CHL27-deficient and WT plants. Thylakoid membranes isolated from lines 2–14 (mild variegation) and 1–7 (more severe variegation) were analyzed for the abundance of the indicated thylakoid membrane protein. Each lane was loaded with 3 μg of protein.