Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a

Abstract

Chlorophyll b is an ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated six chlorophyll b-less mutants by insertional mutagenesis by using the nitrate reductase or argininosuccinate lyase genes as tags and examined the rearrangement of mutant genomes. We found that an overlapping region of a nuclear genome was deleted in all mutants and that this encodes a protein whose sequence is similar to those of methyl monooxygenases. This coding sequence also contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. The results demonstrate that a chlorophyll a oxygenase is involved in chlorophyll b formation. The reaction mechanism of chlorophyll b formation is discussed.

Figure 1

HPLC analysis of chlorophyll from the transformant mutants. Chlorophylls of transformants colonies were extracted with 80% acetone and were determined by HPLC. Four chlorophyll b-less mutants (cbs1–cbs4) and two mutants (cbs5 and cbs6) were isolated by using pMN24 and p389, respectively.

Figure 2

Southern blot analysis of genomic DNA from the chlorophyll b-less mutants. DNA from the mutants were digested by SacI and hybridized with radioactive probe for DNA fragment b as indicated in Fig. 3.

Figure 3

Restriction map of wild-type genomic DNA and deletion maps for mutants. Different genomic DNA clones were isolated that covered the deleted region of cbs3. Deletions in each of the mutants, indicated by heavy lines, were determined with probes consisting of 13 different genomic DNA fragments clones from wild type. In some cases, the ends of the deletions were not determined (shown by dotted lines). Open and closed boxes represent the cDNA sequence and exons in the gene, respectively. The location of a TATA box (TATAAAT), an initiation codon (ATG), a termination codon (TAG), and a polyadenylation signal sequence (TGTAA) are marked by the vertical arrows. E, EcoRI; H, HindIII; N, NotI; S, SacI; A(n), a poly(A) tail; a, DNA fragment for the isolation of cDNA; b, DNA fragment for the Southern blot analysis in this figure; c, DNA fragment for the complementation of Fig. 4.

Figure 4

Rescue of the chlorophyll b-less phenotype by transformation of wild-type CAO gene. An 8-kb genomic DNA fragment was subcloned into plasmid pSP109 and the resultant plasmid (pSP109-E8) was linearized with ScaI. Strain cbs3 was transformed with the 2 μg of linearized plasmids and selected on plates containing 2 μg/ml phleomycin. Pigments were extracted from the transformant (b3-8E), cbs3, and 5D with 80% acetone and determined by HPLC.

Figure 5

(A) Predicted amino acid sequence of the chlorophyll a oxygenase protein with specific binding sites underlined and expanded in B. (B) Amino acid sequence alignments of the Rieske [2Fe-2S] center and mononuclear iron-binding regions of CAO, toluenesulfonate methyl monooxygenase (TsaM), and vanillate demethylase (VanA). Residues for the binding domain are shaded.

Figure 6

Northern blot analysis of CAO transcripts. Poly(A)⁺ RNA isolated from wild-type and mutant cells were examined by RNA gel blot analysis with radioactive CAO probe.

Figure 7

Proposed chlorophyll cycle, whereby interconversion of chlorophyll a and chlorophyll b levels could be regulated via 7-hydroxymethyl chlorophyll.