Chlororespiratory reduction 6 is a novel factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

Abstract

The chloroplast NAD(P)H dehydrogenase (NDH) complex is involved in photosystem I cyclic electron transport and chlororespiration in higher plants. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction 6 (crr6) mutant lacking NDH activity was identified by means of chlorophyll fluorescence imaging. Accumulation of the NDH complex was impaired in crr6. Physiological characterization of photosynthetic electron transport indicated the specific defect of the NDH complex in crr6. In contrast to the CRR7 protein that was recently identified as a potential novel subunit of the NDH complex by means of the same screening, the CRR6 protein was stable under the crr2 mutant background in which the NDH complex does not accumulate. The CRR6 gene (At2g47910) encodes a novel protein without any known motif. Although CRR6 does not have any transmembrane domains, it is localized in the thylakoid membrane fraction of the chloroplast. CRR6 is conserved in phototrophs, including cyanobacteria, from which the chloroplast NDH complex has evolutionally originated, but not in Chlamydomonas reinhardtii, in which the NDH complex is absent. We believe that CRR6 is a novel specific factor for the assembly or stabilization of the NDH complex.

Figure 1.

Monitoring of NDH activity using chlorophyll fluorescence analysis. A, Schematic model of NDH function. The NDH complex functions in electron transport from an unidentified electron donor, possibly NAD(P)H or ferredoxin (Fd) to PQ. PQ reduction was monitored by chlorophyll fluorescence emitted from PSII. PQ reduction in the dark depends on NDH activity and can be monitored as a transient increase in chlorophyll fluorescence after AL illumination. PC, Plastocyanin; FNR, ferredoxin-NADP⁺ oxidoreductase. B, Analysis of the transient increase in chlorophyll fluorescence after turning off AL. The bottom curve indicates a typical trace of chlorophyll fluorescence in the wild type (WT). Leaves were exposed to AL (50 μmol photons m⁻² s⁻¹) for 5 min. AL was turned off and the subsequent change in chlorophyll fluorescence level was monitored. Insets are magnified traces from the boxed area. The fluorescence levels were normalized by F_m levels. ML, Measuring light; SP, saturating pulse of white light; crr6 + CRR6, crr6 complemented by introduction of the wild-type genomic CRR6.

Figure 2.

In vivo analysis of electron transport activity. A, Light-intensity dependence of ETR. ETR was depicted relative to Φ_PSII × light intensity (μmol photons m⁻² s⁻¹). sds are <10% of values (n = 5). B, Light-intensity dependence of NPQ of chlorophyll fluorescence. sds are <20% of values (n = 5). crr6 + CRR6, crr6 transformed with the genomic wild-type CRR6.

Figure 3.

Protein-blot analysis of the NDH complex. Immunodetection of an NDH subunit, NdhH, and a subunit of the Cyt b₆f complex, Cyt f. Proteins were extracted from the thylakoid membrane fraction of the chloroplasts. Lanes were loaded with the protein samples corresponding to 0.2 μg chlorophyll for Cyt f and 5 μg chlorophyll for NdhH (100%) and the series of dilutions indicated. crr6 + CRR6, crr6 transformed by wild-type genomic CRR6.

Figure 4.

Positional cloning of crr6. A, Structure of CRR6. Exons (boxes) and an intron (horizontal thin line) were determined by direct sequencing of the reverse transcription-PCR products. The position of the crr6 mutation is indicated. B, Alignment of CRR6 homolog sequences. The predicted cleavage site of the target signal (TargetP) is indicated by a vertical arrow. AtCRR6, Arabidopsis; OsCRR6, rice; Slr1097, Synechocystis sp. PCC 6803; All5169, Anabaena sp. PCC 7120; Tll1292, Thermocynechococcus elongatus BP-1. The position of the crr6 mutation is indicated by an asterisk.

Figure 5.

Protein-blot analysis of HA-tagged CRR6. A, Immunodetection of CRR6 protein using a monoclonal antibody against the HA tag. Chloroplast preparations were further fractionated to obtain a thylakoid membrane fraction and a stromal fraction. Large subunit of Rubisco (RbcL) and Cyt f were detected as the control for the fractionation. B, CRR6 protein was absent in the mitochondrial fraction. The lanes were loaded with protein samples corresponding to 5 μg (CRR6-HA), 5 μg (NdhH), 0.2 μg (Cyt f), and 0.01 μg (RbcL) chlorophyll. The crude mitochondrial protein was loaded so that it corresponded to 0.2 μg chlorophyll of the thylakoid fraction. Alternative oxidase was detected as a marker of the mitochondrial fraction. crr6 + CRR6-HA, crr6 transformed by genomic CRR6 fused to the HA epitope tag. Three independent lines (nos. 1–3) were analyzed.

Figure 6.

Immunodetection of CRR6-HA protein under the crr2-2 mutant background. The lanes were loaded with the thylakoid membrane protein samples corresponding to 0.2 μg chlorophyll for Cyt f and 5 μg chlorophyll for CRR6-HA. crr2-2 crr6 + CRR6-HA, crr2-2 crr6 transformed by the chimeric gene encoding CRR6-HA. Three independent lines (nos. 1–3) were analyzed and then used for crosses with crr6 to generate crr2-2/+ crr6 + CRR6-HA (heterozygous for crr2-2, homozygous for crr6, and containing the transgene).