LCAA, a novel factor required for magnesium protoporphyrin monomethylester cyclase accumulation and feedback control of aminolevulinic acid biosynthesis in tobacco

Abstract

Low Chlorophyll Accumulation A (LCAA) antisense plants were obtained from a screen for genes whose partial down-regulation results in a strong chlorophyll deficiency in tobacco (Nicotiana tabacum). The LCAA mutants are affected in a plastid-localized protein of unknown function, which is conserved in cyanobacteria and all photosynthetic eukaryotes. They suffer from drastically reduced light-harvesting complex (LHC) contents, while the accumulation of all other photosynthetic complexes per leaf area is less affected. As the disturbed accumulation of LHC proteins could be either attributable to a defect in LHC biogenesis itself or to a bottleneck in chlorophyll biosynthesis, chlorophyll synthesis rates and chlorophyll synthesis intermediates were measured. LCAA antisense plants accumulate magnesium (Mg) protoporphyrin monomethylester and contain reduced protochlorophyllide levels and a reduced content of CHL27, a subunit of the Mg protoporphyrin monomethylester cyclase. Bimolecular fluorescence complementation assays confirm a direct interaction between LCAA and CHL27. 5-Aminolevulinic acid synthesis rates are increased and correlate with an increased content of glutamyl-transfer RNA reductase. We suggest that LCAA encodes an additional subunit of the Mg protoporphyrin monomethylester cyclase, is required for the stability of CHL27, and contributes to feedback-control of 5-aminolevulinic acid biosynthesis, the rate-limiting step of chlorophyll biosynthesis.

Figure 1.

A, Growth phenotypes of tobacco wild type (WT) and the four LCAA antisense lines in the order of increasing strength of the phenotype. Plants were grown in the greenhouse at 250 µE m⁻² s⁻¹ actinic light intensity. B, Northern blot showing the correlation between the visible phenotype and LCAA mRNA accumulation.

Figure 2.

Sequence alignment showing the evolutionary conservation of LCAA in higher plants, mosses, eukaryotic algae, and cyanobacteria. The homologs from tobacco, Arabidopsis, and C. reinhardtii are nucleus encoded and therefore possess a putative chloroplast transit peptide at their N terminus. The LCAA homologs of the moss P. patens, the red alga P. purpurea, the brown alga F. vesiculosus, and P. chromatophora are all plastid encoded and therefore lack an N-terminal transit peptide. Additionally, sequences from two cyanobacteria (Synechococcus sp. WH8102 and N. punctiforme PCC 73102) were included. Identical amino acids are marked by stars, conserved substitutions by colons, and semiconserved substitutions by dots. The seed sequence of the Ycf54 domain (DUF2488) is underlined.

Figure 3.

Targeting of LCAA to chloroplasts. Fusions of GFP with the Arabidopsis and the tobacco LCAA genes were transiently transformed into tobacco protoplasts and expressed under the control of the strong constitutive cauliflower mosaic virus 35S promoter. Confocal laser scanning microscopy and overlays of GFP and chlorophyll a fluorescence revealed that both the tobacco and Arabidopsis proteins are targeted to the chloroplast, while the unfused GFP control is clearly retained in the cytosol.

Figure 4.

Accumulation of photosynthetic complexes and antenna proteins, as quantified by immunoblot analyses using antibodies against essential subunits of the different photosynthetic complexes (A) and representative antenna proteins of PSII and PSI (B). A, Samples were loaded on an equal chlorophyll basis, and for semiquantitative analysis, 200%, 150%, and 100% of the wild-type sample (WT) were loaded in lanes 1 to 3 for comparison. For PSII, the essential PSII reaction center subunits PsbD (D2 protein) and PsbE, the extrinsic PsbO subunit of the oxygen-evolving complex, and the nonessential PsbS protein, which is required for photoprotective nonphotochemical quenching of excess excitation energy, were analyzed. Cytochrome b₆f complex accumulation was probed with antibodies against cytochrome f (PetA) and the Rieske iron-sulfur protein (PetC). PSI accumulation was probed with antibodies against the essential PsaA and PsaB reaction center subunits. ATP synthase abundance was probed with an antibody against the essential AtpB subunit. B, To determine LHC protein accumulation, a dilution series of 25%, 50%, and 100% of the wild-type sample was loaded in lanes 1 to 3 for semiquantitative analysis. For the PSII antenna, accumulation of the Lhcb1 and Lhcb2 subunits was tested. For the PSI antenna, accumulation of Lhca1, Lhca2, and Lhca4 was determined.

Figure 5.

Chlorophyll a fluorescence data to analyze antenna function. A, 77K chlorophyll a fluorescence emission spectra. The emission maximum of PSII-LHCII at 686 nm was normalized to 1. B, Light-response curve of qL. The fully oxidized state of the PSII acceptor side was normalized to 1, and the fully reduced state was normalized to 0. C, Light-response curve of qN. WT, Wild type.

Figure 6.

ALA synthesis rate and accumulation of key enzymes of tetrapyrrole biosynthesis. A, ALA synthesis rates as determined in tobacco leaf discs after incubation in 40 mm levulinic acid under continuous white light (120 µE m⁻² s⁻¹) at 22°C for 4 h. LCAA mutants possess a higher ALA synthesis rate than control tobacco plants. B, Immunoblots against representative enzymes of the tetrapyrrole biosynthesis pathway. Total protein was extracted from leaves, and equal protein amounts were loaded. The measured enzymes are GluTR, GSAT, the Mg chelatase subunit CHLD, the CHLH subunit of the Mg chelatase, the CHLM subunit of the Mg protoporphyrin methyltransferase, the CHL27 subunit of the MgProtoMME cyclase, and POR. Additionally, LIL3 and NTRC were included. LIL3 stabilizes the geranylgeranyl reductase involved in the late steps of chlorophyll biosynthesis, and NTRC protects the MgProtoMME cyclase against oxidative stress. LCAA antisense lines contain elevated LIL3 and NTRC contents, which parallel the increased contents of GluTR, CHLH, CHLM, and POR. WT, Wild type.

Figure 7.

qPCR analyses to quantify transcript accumulation levels of genes involved in tetrapyrrole biosynthesis, photosynthetic light reactions, and the antioxidative system of chloroplasts. Detailed information on primer design and on the closest Arabidopsis homologs of the analyzed genes is provided in Supplemental Table S1. The top panel shows genes involved in tetrapyrrole biosynthesis displayed in the sequence of the chlorophyll biosynthesis pathway. The bottom panel shows genes for light-harvesting complex proteins, photosynthetic complex subunits, and antioxidative enzymes in the chloroplast. WT, Wild type.

Figure 8.

Bimolecular fluorescence complementation was applied to analyze the protein-protein interaction of LCAA and CHL27 in chloroplasts. The left column shows restored Venus fluorescence resulting from the interaction of candidate proteins. The middle column shows chlorophyll fluorescence representing chloroplasts. The right column shows merged images demonstrating the colocalization of restored Venus fluorescence and chlorophyll fluorescence. These results show the interaction of LCAA with CHL27 and the homodimerization of LCAA. However, no homodimerization of CHL27 was detected.