A eukaryotic factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

Abstract

The NAD(P)H dehydrogenase (NDH) complex in chloroplasts mediates photosystem I cyclic and chlororespiratory electron transport. Eleven chloroplast genes and three nuclear genes have been identified as encoding Ndh subunits, but the entire subunit composition is still unknown. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction (crr3) mutant was isolated based on its lack of transient increase in chlorophyll fluorescence after actinic light illumination; this was due to a specific defect in accumulation of the NDH complex. The CRR3 gene (At2g01590) encodes a novel protein containing a putative plastid-targeting signal and a transmembrane domain. Consistent with the gene structure, CRR3 localized to the membrane fraction of chloroplasts. In addition to the essential function of CRR3 in stabilizing the NDH complex, the NDH complex is also required for the accumulation of CRR3. These results suggest that CRR3 interacts with the NDH complex in the thylakoid membrane. In contrast to other subunits in the chloroplast NDH complex, CRR3 is not conserved in cyanobacteria from which the chloroplast NDH complex is believed to have originated. We propose that CRR3 is a subunit of the NDH complex, which is specific to the chloroplast.

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, Fd-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; crr3 + CRR3, crr3 complemented by introduction of wild-type genomic CRR3.

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 < 15% of values at the maximal light intensity (n = 5). The curves of wild type and crr3 almost overlap. B, Light-intensity dependence of NPQ of chlorophyll fluorescence. sds < 10% of values at the maximal light intensity (n = 5). crr3 + CRR3, crr3 transformed with genomic wild-type CRR3.

Figure 3.

Protein-blot analysis of the NDH complex. Immunodetection of an NDH subunit, NdhH, and a subunit of the cyt b₆f complex, Cytf. Proteins were extracted from the thylakoid membrane fraction of the chloroplasts. Lanes were loaded with the protein samples corresponding to 0.5 μg chlorophyll for Cytf and 5 μg chlorophyll for NdhH (100%) and the series of dilutions indicated. crr3 + CRR3, crr3 transformed by wild-type genomic CRR3.

Figure 4.

Positional cloning of crr3. A, Structure of CRR3. Exons (boxes) and introns (horizontal lines) were determined by direct sequencing of the reverse transcription-PCR products. Position of the crr3 mutation (asterisks) is indicated. B, Alignment of CRR3 homolog sequences. Predicted cleavage site of the target signal (TargetP) is indicated by a vertical arrow. Arabidopsis CRR6 (At) and its rice homolog (Os) are aligned. The rice sequence was predicted from the genome information. The position of the crr3 mutation is indicated by an asterisk. A horizontal bar indicates the transmembrane domain.

Figure 5.

Protein-blot analysis of CRR3. A, Immunodetection of CRR3 protein using a polyclonal antibody against recombinant CRR3. Chloroplast preparations were further fractionated to obtain a membrane fraction and a stromal fraction. Large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RbcL) and the Cytf were detected as the control for fractionation. Lanes were loaded with protein samples corresponding to 2.5 μg (CRR3), 0.5 μg (Cytf), and 0.01 μg (RbcL) chlorophyll. crr3 + CRR3, crr3 transformed by genomic CRR3.

Figure 6.

Immunodetection of CRR3 against mutant backgrounds lacking the NDH complex. A, Analysis of CRR3 stability in crr and pgr5 mutants. B, Analysis of CRR3 and NdhH levels in crr4-3 and crr4-4. Lanes were loaded with the thylakoid membrane protein samples corresponding to 0.5 μg chlorophyll for Cytf, 2.5 μg chlorophyll for CRR3, and 5 μg chlorophyll for NdhH as 100% and a series of dilutions indicated.