Knock-out of the genes coding for the Rieske protein and the ATP-synthase delta-subunit of Arabidopsis. Effects on photosynthesis, thylakoid protein composition, and nuclear chloroplast gene expression

Abstract

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b(6)/f (cyt b(6)/f) complex and the delta-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b(6)/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor Q(A) in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-delta destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b(6)/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.

Figure 1.

Mutations in the PetC and AtpD loci. a, In petc-1, the PetC gene (At4g03280) is disrupted by an insertion of the autonomous En transposon. The sequences of the empty donor sites of DNA from three independent somatic revertant leaves (REV1-3) were obtained by PCR; uppercase letters indicate plant DNA sequences flanking the En; insertion footprints left at each locus after En transposition are indicated by bold lowercase letters; and bold uppercase letters indicate the target site in the WT. In the other two mutant alleles, a copy of the nonautonomous Ds transposon (petc-2) and the ROK2 T-DNA (petc-3) are inserted 5′ of the start codon of the PetC gene. b, In atpd-1, a copy of the 5.2-kb SKI015 T-DNA is inserted in the promoter region of AtpD (At4g09650). PCR analysis indicated a small deletion of 7 bp adjacent to the right border of the SKI015 insertion. The En, Ds, and T-DNA insertions are not drawn to scale.

Figure 2.

Phenotypes of WT, petc, and atpd plants. Plants grown on Murashige and Skoog medium supplemented with Suc were illuminated with white light or UV light (B-100AP/R, UVP Inc., Upland, CA). a, WT and petc-1, -2, and -3 plants illuminated with white light; b, WT and petc-2 plants under UV light; and c, WT and atpd-1 plants under white light (top) or UV light (bottom).

Figure 3.

AtpD and PetC mRNA and protein levels in WT and mutant plants. a, For northern analysis of the PetC transcript in petc-2 and WT plants, 30-μg samples of total RNA were analyzed using a fragment of the PetC transcript as a probe. A full-length AtpD cDNA was used as a probe for northern analysis of the AtpD transcript in atpd-1 and WT plants. As a loading control, the blots were reprobed with a cDNA fragment derived from the ACTIN1 gene. b, Samples of thylakoid membranes equivalent to 5 μg of Chl from WT and petc-2 or atpd-1 plants were fractionated by denaturing PAGE. Decreasing amounts of WT thylakoid membranes (3.75, 2.5, and 1.25 μg of Chl) were loaded in the lanes marked 0.75×, 0.5×, and 0.25× WT. Filters were probed with antibodies specific for the Rieske protein or the δ-subunit of the cpATPase.

Figure 4.

Protein composition of thylakoid membranes. a, Thylakoid membranes corresponding to 30 μg of Chl from WT, petc-2, and atpd-1 mutant plants were fractionated first by electrophoresis on a non-denaturing lithium dodecyl sulfate-polyacrylamide (PA) gel and then on a denaturing SDS-PA gel. Positions of WT thylakoid proteins previously identified by western analyses with appropriate antibodies are indicated by numbers to the right of the corresponding spots: 1, α- and β-subunits of the ATPase complex; 2, D1-D2 dimers; 3, CP47; 4, CP43; 5, oxygen-evolving complex (OEC); 6, LHCII monomer; 7, LHCII trimer; 8, PSI-D; 9, PSI-F; 10, PSI-C; and 11, PSI-H. The alterations observed in the mutants are quantified in Table I. Samples of thylakoid membranes equivalent to 5 μg of Chl from WT, and petc-2 (b) and atpd-1 (c) plants were fractionated by denaturing PAGE. Decreasing amounts of WT thylakoid membranes (3.75, 2.5, and 1.25 μg of Chl) were loaded in the lanes marked 0.75×, 0.5×, and 0.25× WT. Replicate filters were probed with antibodies raised against the D1 protein of the PSII reaction center, the F subunit of PSI, the Lhca4 protein, the cyt b₆/f subunits cyt b₆, cyt f and suIV, and the α-, β-, a-, and b-subunits of the cpATPase.

Figure 5.

Spectroscopic analyses of WT, petc-2, and atpd-1 plants. a, Effective quantum yield of PSII (ϕ_II) in leaves of atpd-1, petc-2, and WT plants determined at different photon flux densities. Leaves were dark-adapted for 30 min before the measurements were made. Mean values of five independent experiments are shown. Bars indicate sds. b, Thylakoid membrane potential decay of WT and atpd-1 leaves. Maximum transthylakoid proton gradient was reached by exposing 4-week-old leaves of WT and atpd-1 plants to a saturating light pulse for 15 and 25 ms, respectively. The dark-induced thylakoid membrane potential decay was then monitored by recording the absorption at 520 nm. For the absorption measurements, short light pulses at 520 nm were used to prevent actinic effects. The results of one out of five measurements are shown. c, Steady-state levels of oxidized reaction center Chl in PSI (P700⁺) in WT and mutant plants (n = 3 each). The steady-state level of P700⁺ was measured from the change in P700 absorption at 820 nm (ΔA/ΔA_MAX) after 5 min of illumination with actinic light. Mean values are shown, and bars indicate sds.

Figure 6.

Effects of petc-2 and atpd-1 mutations on the accumulation of nuclear transcripts encoding chloroplast proteins. a, Hierarchical clustering of the expression profiles of 344 genes that show significant differential expression in at least five of the six mutants petc-2, atpd-1, psae1-1, psad1-1, psan-1, and psao-1. The cladogram at the top summarizes the degree of relatedness between transcriptome responses. Colors indicate up-regulation (red) or down-regulation (green) of gene expression relative to WT. Gray lines indicate non-significant differential expression. For a more detailed image, including gene accession numbers and annotations, see supplementary material 1. b, Fraction of up- and down-regulated genes in seven major functional categories. A complete list of significantly differentially expressed genes is available as supplementary material 3.