Abstract
RNA interference was used to simultaneously suppress the expression of the two genes that encode the PsbQ proteins of Photosystem II (PS II) in Arabidopsis thaliana, psbQ-1 (At4g21280) and psbQ-2 (At4g05180). Two independent PsbQ-deficient plant lines were examined. These plant lines produced little detectable PsbQ protein. Under normal growth light conditions, the wild type and mutant plants were visually indistinguishable. Additionally, analysis of steady state oxygen evolution rates and chlorophyll fluorescence characteristics indicated little alteration of photosynthetic capacity in the mutant plants. No loss of other PS II proteins was evident. Interestingly, flash oxygen yield analysis performed on thylakoid membranes isolated from the mutant and wild type plants indicated that the oxygen-evolving complex was quite unstable in the mutants. Furthermore, the lifetime of the S2 state of the oxygen-evolving complex appeared to be increased in these plants. Incubation of the wild type and mutant plants under low light growth conditions led to a significantly stronger observed phenotype in the mutants. The mutant plants progressively yellowed (after 2 weeks) and eventually died (after 3-4 weeks). The wild type plants exhibited only slight yellowing after 4 weeks under low light conditions. The mutant plants exhibited a large loss of a number of PS II components, including CP47 and the D2 protein, under low light conditions. Additionally, significant alterations of their fluorescence characteristics were observed, including an increased FO and decreased FV, yielding a large loss in PS II quantum efficiency (FV/FM). Analysis of QA- decay kinetics in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea indicated a defect in electron transfer from QA- to QB, whereas experiments performed in the presence of this herbicide indicated that the recombination rate between QA- and the S2 state was strongly retarded. These results indicate that the loss of the PsbQ protein induces significant changes in Photosystem II function, particularly in low light-grown plants, and that the PsbQ protein is required for photoautotrophic growth under low light conditions.
Highlights
In higher plants, three extrinsic proteins, with apparent molecular masses of 33, 24, and 17 kDa, are required for high rates of oxygen evolution at physiological inorganic cofactor concentrations
Seeds from wild type and from plants transformed with the RNA interference (RNAi)-Q construct were distributed on agar plates containing Murashige and Skoog medium supplemented with 50 g/ml kanamycin
The results showed that 42% of Tricine-NaOH, pH 7.6, with 200 M DCBQ added as an elec- the plants had expression levels similar to wild type for the tron acceptor
Summary
Three extrinsic proteins, with apparent molecular masses of 33, 24, and 17 kDa, are required for high rates of oxygen evolution at physiological inorganic cofactor concentrations (for review, see Ref. 7). The 24- and the 17-kDa proteins (termed the PsbP and PsbQ proteins, respectively) appear to modulate the calcium and chloride requirements for efficient oxygen evolution These three extrinsic components interact with intrinsic membrane proteins and possibly with each other to yield fully functional oxygen-evolving complexes. It is unclear, whether the PsbP and PsbQ proteins act in concert in modulating the cofactor requirement or whether each has individual, discrete functions within the photosystem. RNAi has been successfully applied as a powerful gene-silencing tool in a variety of organisms, including Caenorhabditis elegans and Drosophila melanogaster, and in mouse oocytes It has become a popular research methodology for investigating the physiological functions of target genes in plants [15]. Our results indicate that the PsbQ protein is required for the stabilization of oxygen-evolving PS II complexes under normal illumination conditions and for PS II function/stability and photoautotrophic growth under low light conditions
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