The AtCAO gene, encoding chlorophyll a oxygenase, is required for chlorophyll b synthesis in Arabidopsis thaliana

Abstract

Chlorophyll b is synthesized from chlorophyll a and is found in the light-harvesting complexes of prochlorophytes, green algae, and both nonvascular and vascular plants. We have used conserved motifs from the chlorophyll a oxygenase (CAO) gene from Chlamydomonas reinhardtii to isolate a homologue from Arabidopsis thaliana. This gene, AtCAO, is mutated in both leaky and null chlorina1 alleles, and DNA sequence changes cosegregate with the mutant phenotype. AtCAO mRNA levels are higher in three different mutants that have reduced levels of chlorophyll b, suggesting that plants that do not have sufficient chlorophyll b up-regulate AtCAO gene expression. Additionally, AtCAO mRNA levels decrease in plants that are grown under dim-light conditions. We have also found that the six major Lhcb proteins do not accumulate in the null ch1-3 allele.

Figure 1

Amino acid alignment of AtCAO and CAO. Identical amino acids are indicated by lines between sequences. The top box is the Rieske binding site, the middle box is the mononuclear nonheme Fe-binding site, and the bottom box is the unique conserved site. The V274 that is converted to E in the ch1-2 allele is underlined, and the region with the double underline is deleted in ch1-3.

Figure 2

Relative AtCAO and Lhcb1 mRNA levels in wt plants and lines with increased a/b ratios and in wt plants grown in dim light. Quantified and normalized mRNA levels are shown relative to wt plants for each experiment. Dark gray boxes represent AtCAO mRNA levels, whereas the light gray boxes represent Lhcb1 mRNA levels. “60” indicates 60 μmol⋅m⁻²⋅sec⁻¹, “5” indicates 5 μmol⋅m⁻²⋅sec⁻¹ and “5–60” indicates samples that had been in 5 μmol⋅m⁻²⋅sec⁻¹ for 3 days and then transferred back to 60 μmol⋅m⁻²⋅sec⁻¹ for the indicated time.

Figure 3

Second-dimension denaturing gel electrophoreses of wt and ch1-3 thylakoid membranes and immunoblot analysis with anti-CP43. The nondenaturing green-gel is shown at Top. The indicated pigmented bands were electrophoresed on denaturing gels, stained with Coomassie, and are shown in the Middle. Bottom is an immunoblot of the denaturing gels reacted with the anti-CP43 antibody.

Figure 4

Immunoblots probed with specific Lhcb antibodies. Thylakoid proteins from wt , ch1-2, and ch1-3 plants were loaded on an equal protein basis and reacted with the Lhcb antibodies described in Materials and Methods. A Coomassie-stained gel is shown below the immunoblots as a control for protein loading.