| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 281, Issue 36, 26260-26267, September 8, 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Biochemistry and Molecular Biology Section, Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
Received for publication, April 13, 2006 , and in revised form, June 26, 2006.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
and 
subunits of cytochrome b559. Insertional inactivation or deletion of the genes for these components results in the complete loss of oxygen evolution activity. Additionally, a number of low molecular mass, intrinsic membrane protein components are associated with PS II (4-6); however, the functions of many of these proteins remain obscure. Although PS II complexes containing only these intrinsic components can evolve oxygen, they do so at low rates (
25-40% of control), are extremely susceptible to photoinactivation, and require high, non-physiological levels of calcium and chloride for maximal activity (1, 3).
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 (for review, see Ref. 7). The 33-kDa component, PsbO protein, is required for stabilization of the manganese cluster during exposure to low chloride concentrations or to exogenous reductants. 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, however, whether the PsbP and PsbQ proteins act in concert in modulating the cofactor requirement or whether each has individual, discrete functions within the photosystem. Miyao and Murata (8) demonstrate that the PsbQ protein enhances oxygen evolution at chloride concentrations of <3 mM. Additionally, they show that the PsbQ protein, in concert with the PsbP component, slow the inactivation of oxygen evolution during chloride depletion and the activation of oxygen evolution during chloride reconstitution. As these authors point out, however, these experiments were performed under non-physiological conditions. It had been demonstrated earlier that the thylakoid membrane is highly permeable to chloride (9) and that the stromal chloride concentration is 30-60 mM (10). Consequently, it is unclear what role the PsbQ component plays under physiological conditions. It should be noted that homologues of the PsbP and PsbQ proteins also exist in cyanobacteria. Deletion of these components in the cyanobacterial system leads to significant alterations in PS II activity (11). Arabidopsis contains two expressed PsbQ proteins (12, 13). These proteins are encoded by two genes, psbQ-1 (At4g21280) and psbQ-2 (At4g05180). The mature proteins are highly conserved among all higher plant species (7).
RNA interference (RNAi) is a post-transcriptional gene-silencing process in which double-stranded RNA induces the degradation of homologous mRNA sequences (14). 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 also become a popular research methodology for investigating the physiological functions of target genes in plants (15). With respect to PS II proteins, RNAi has been used to investigate the in vivo function of the PsbO proteins in Arabidopsis (16) and the PsbP and PsbQ proteins in tobacco (17). The studies performed in tobacco indicate that RNAi suppression of the PsbQ protein leads to no observable phenotype (17).
In this study, we have reported the use of RNAi technology to simultaneously suppress the expression of both psbQ genes in Arabidopsis. 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.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
PCR was performed on a Rapidcycler (Idaho Technology, Inc.) in thin-walled microcentrifuge tubes in 50-µl reactions containing 5 µl of 10x PCR reaction buffer, 1.5 µl of 50 mM MgCl2, 1.5 µl of 2.5 mM dNTP mixture, 3 µl of 10 pM/µl primer mixture, 0.25 µl of 5 units/µl Taq polymerase (Invitrogen), and 25 ng of Arabidopsis genomic DNA in purified water. Cycling parameters were a predenaturation step at 96 °C for 2 min followed by 30 amplification cycles (denatured at 95 °C for 20 s, annealed at 53 °C for 20 s, and extended at 72 °C for 40 s) and a final extension at 72 °C for 7 min. The amplified sequence was cloned into both forward and reverse orientations flanking the Pdk intron of the pHANNIBAL vector (18). After construction and verification by sequencing, the expression region was excised from pHANNIBAL with NotI and then subcloned into pART27 for transformation of the Agrobacterium strain GV3101 by the freeze-thaw method (19). Four-week-old Arabidopsis plants (Col-0) were transformed by the floral dip method as described previously (20). Harvested seeds were surface-sterilized with 50% ethanol and 0.5% Tween 20 for 3 min, washed briefly with 95% ethanol, and then soaked in 70% ethanol for 3 min followed by washing three times with sterile water. The seeds were spread on solid Murashige and Skoog medium containing 0.7% agar, 2% sucrose, 50 mg/liter kanamycin, and 400 mg/liter carbenicillin and then incubated for 2 days at 4 °C in the dark. Germination and the first 10 days of growth occurred under lighted conditions at 28 °C in Petri dishes, and then the plants were transplanted to culture boxes containing solid Murashige and Skoog medium with 2% sucrose, 50 mg/liter kanamycin, and 400 mg/liter carbenicillin. To test for photoautotrophic growth, the plants were transplanted onto medium from which the sucrose was omitted and then transplanted onto soil to obtain seeds. The plants were grown under continuous light at an intensity of 25-40-µmol photons/m2/s.
Screening—The presence of the RNAi construct in the kanamycin-resistant plant lines was confirmed by PCR with primers designed to amplify the cauliflower mosaic virus 35S promoter and target gene region of the introduced DNA. All of the plants that exhibited the kanamycin-resistant phenotype also exhibited the presence of the 1-kbp cauliflower mosaic virus 35S promoter and its conjugated gene region, which was absent in the wild type plants (data not shown). Individual kanamycin-resistant plants were screened for the presence of the PsbQ protein by Western blotting. One leaf was placed in a 1.5-ml microcentrifuge tube and ground to a powder in the presence of liquid nitrogen. After evaporation of the liquid nitrogen, a protein isolation buffer (20 mM Tricine-NaOH, pH 8.4, 10 mM EDTA, 450 mM sorbitol, and 0.1% bovine serum albumin) was added followed by the addition of lithium dodecyl sulfate-PAGE solubilization buffer, and the samples were incubated on ice for at least 15 min. The samples were then centrifuged at 16,000 x g for 5 min before running on a 15% polyacrylamide gel. Western blotting and antibody probing were performed as previously described (16) followed by visualization with a chemiluminescent peroxidase substrate (Super-Signal® West Pico chemiluminescent substrate, Pierce). X-ray film was exposed by the blot and developed photographically. The monoclonal antibody FCC4 directed against the mature spinach PsbQ protein (21) was found to cross-react with both the PsbQ-1 and the PsbQ-2 proteins from Arabidopsis and was used in these studies.
To characterize the RNAi-Q plants in which expression of the PsbQ protein was much lower than in the non-transgenic plants, second generation plants were grown under continuous illumination at a light intensity of 25-40-µmol photons/m2/sec, and a subset of these plants was transferred to low light growth conditions (4-5-µmol photons/m2/s) for varying lengths of time.
Immunological Characterization of Thylakoid Proteins—For a more in-depth analysis of the protein complement of the thylakoid membranes, chloroplasts were isolated from wild type and two mutant plant lines that expressed low levels of the PsbQ protein. These lines were designated RNAi-Q7 and RNAi-Q30, and these plant lines are collectively referred to as PsbQ-deficient plants. Leaves were ground in a glass homogenizer with a chloroplast isolation buffer (300 mM sorbitol, 5 mM MgCl2,5mM EGTA, 5 mM EDTA, 20 mM HEPES/KOH, pH 8.0, and 10 mM NaHCO3), the homogenate was then passed through two layers of Miracloth (Calbiochemcal Co.), and the chloroplasts pelleted by centrifugation at 6,000 x g for 5 min. The chloroplasts were then resuspended in a small amount of isolation buffer, and the chlorophyll concentration was determined by the method of Arnon (22). Lithium dodecyl sulfate-PAGE was performed on a 12.5-20% gradient gel with 3 µg of chlorophyll loaded/lane. Western blotting, blocking, probing with antibodies, and x-ray film exposure were as described above. After development, the x-ray films were scanned with a UMax PowerLook III scanner at 300 dots/inch resolution and an 8-bit color depth.
|
Steady State and Flash Oxygen Yield Analysis—Oxygen evolution studies were performed on thylakoid membrane fragments isolated from wild type and PsbQ-deficient plants. Thylakoid fragments were isolated by grinding 2-5 leaves in a glass homogenizer with 1 ml of a buffer containing 0.45 M sorbitol, 10 mM EDTA, 0.1% bovine serum albumin, 1% polyvinylpyrrolidone, and 20 mM tricine-NaOH, pH 8.4. The homogenate was filtered through 1 layer of Miracloth, and the thylakoid fragments were pelletted by centrifugation at 4 °C at 16,000 x g in a microcentrifuge. Steady state measurements were performed in a Hansatech oxygen polarograph. The chlorophyll concentration for these experiments was between 4 and 13 µg of chlorophyll/ml assayed in 0.3 M sorbitol, 5 mM MgCl2, and 20 mM Tricine-NaOH, pH 7.6, with 200 µM DCBQ added as an electron acceptor. The light intensity for these experiments was 2000-µmol photons/m2/s.
Flash oxygen yield measurements were performed on a bare platinum electrode (Artesian Scientific Co., Urbana IL). Flashes were supplied by an integrated, computer-controlled Xenon flash lamp (20 µs at one-half height). For the measurements of S-state distributions and S-state parameters, thylakoid fragments were pelleted and applied to the electrode as a thin paste. The thylakoids were then incubated for 5 min in the dark, the electrode was polarized at 0.73 V for 10 s, and a series of saturating flashes was applied. Data points were collected at 500-µs intervals during the duration of the flash train. The data were analyzed using a four-step, homogeneous model (23). Five- and six-state models that incorporated either an S-1 state or S-1 and S-2 states, respectively, uniformly failed to fit the data acquired either from the wild type or mutant thylakoids. For experiments that examined the stability of the oxygen-evolving complex, the thylakoid fragments were applied to the electrode as described above and incubated on the electrode (in the absence of polarizing voltage) at room temperature (24 °C) for various lengths of time prior to initiation of the flash yield experiment, either in the dark or under low light conditions (5-µmol photons/m2/sec).
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
To screen individual transgenic plants for the presence of the PsbQ proteins, Western blot analysis with a monoclonal antibody that recognizes both the PsbQ-1 and PsbQ-2 proteins was performed. The results from a typical screening experiment are shown in Fig. 1. In the wild type plants, two major protein bands reacted strongly with the monoclonal reagent and represent the putative PsbQ-1 and PsbQ-2 proteins. These two bands migrated with apparent molecular masses of 15.4 and 14.2 kDa, which are comparable to the masses previously observed for these proteins (13). Additionally, plants transformed with RNAi constructs designed to suppress the psbQ-1 gene lost the upper band, and plants transformed with constructs designed to suppress the psbQ-2 gene lost the lower band (data not shown). In the current study, individual transgenic plants exhibited different degrees of suppression of the expression of the two PsbQ proteins. In total, 56 plants were screened for the presence of the PsbQ proteins. The results showed that 42% of the plants had expression levels similar to wild type for the PsbQ-1 and PsbQ-2 proteins, 39% exhibited an intermediate level of expression, and 19% of the transgenic plants exhibited almost complete loss of the PsbQ proteins. These results are consistent with the results obtained in other RNAi studies targeting other proteins. In almost all instances, different RNAi-containing plant lines exhibit different degrees of suppression of the protein targets (16, 24, 25).
Photosynthetic Competence of Plants Grown under Normal and Low Light Conditions—When grown under normal light intensities, the PsbQ-deficient plants exhibited growth rates, leaf color, and overall morphology visually indistinguishable from that of wild type. To assess the effect of the loss of the expression of PsbQ proteins on the functional competence of PS II, chlorophyll fluorescence characteristics were examined in wild type and transgenic plant leaves. Under normal light growth conditions, fluorescence analysis indicated that the PsbQ-deficient plants were very similar to wild type. Few differences were observed during the initial fluorescence rise following a single saturating flash (Fig. 2A), during a fluorescence induction experiment (Fig. 2C and Table 1), or in the fluorescence decay following a single saturating flash either in the absence (Fig. 3A) or presence (Fig. 3C) of 40 µM DCMU. The latter two experiments probed the efficiency of the electron transfer from QA- to QB or the charge recombination between QA- and the S2 state of the oxygen-evolving complex, respectively. The results indicated that, with respect to a number of fluorescence parameters, wild type and the PsbQ-deficient plants were very similar. Additionally, steady state oxygen evolution measurements (Table 1) indicated that the wild type and PsbQ-deficient plants exhibited similar oxygen evolution capabilities. These results verify and extend the observations by Ifuku et al. (17) on transgenic RNAi-containing tobacco plants that were deficient in PsbQ. These authors concluded that, under normal conditions, the absence of the PsbQ protein yielded no observable phenotype.
|
|
|
In Fig. 3D, the decay of fluorescence was monitored after a single saturating flash in the presence of 40 µM DCMU. Because under these conditions electron transfer cannot occur beyond QA, fluorescence decay principally monitors charge recombination between QA- and the S2 state of the oxygen-evolving complex. Our results indicate that this charge recombination in the PsbQ-deficient plants is strongly retarded. The results from these fluorescence decay experiments (minus and plus DCMU) demonstrate that both the reducing and oxidizing sides of the photosystem are compromised in the PsbQ-deficient plants that have been grown at low light intensities.
Our observations on the growth of the PsbQ-deficient plants, their fluorescence characteristics, and their steady state oxygen evolution capability indicate that, in the absence of the PsbQ protein, the plants cannot maintain PS II in a functional state under low light conditions. Ifuku et al. (17) characterize their transgenic PsbQ-deficient tobacco plants under normal and high light conditions. The conditions under which Ifuku et al. conducted their examination would not have allowed them to note the phenotype that we observed.
Alterations of the Thylakoid Protein Complement in PsbQ-deficient Plants—Fig. 4 presents an analysis of the thylakoid proteins isolated from wild type and the two PsbQ-deficient plant lines RNAi-Q7 and RNAi-Q30. In Fig. 4A, the Coomassie Blue protein-staining profile is shown for plants that had been grown under normal light conditions. Essentially no visible differences were evident. In Fig. 4B, immunostaining was used to examine a number of PS II components and control proteins. Under normal illumination conditions, the PsbQ protein was absent from the RNAi-Q7 and RNAi-Q30 plants. None of the other PS II components examined (CP47, CP43, D1, D2, the PsbOs, and PsbP) or the control proteins (cytochrome f, the large subunit of ribulose-1,5-bis-phosphate carboxylase and PsaB) was affected. Upon low light incubation, however, a markedly different protein pattern was observed. Although the thylakoids isolated from wild type plants exhibited few changes when compared with the plants grown under normal light, the two PsbQ-deficient plant lines exhibited profound alterations in their protein complement. The CP47 and D2 proteins were substantially decreased while the CP43, D1, the PsbOs, and PsbP proteins were reduced by varying degrees. Although cytochrome f and the PsaB protein were unaffected, the large subunit of ribulose-1,5-bis-phosphate carboxylase was significantly reduced in one of the PsbQ-deficient plant lines (RNAi-Q30). The significance of this latter observation is unclear at this time.
|
In any event, it is clear that the absence of the PsbQ protein leads to a profound alteration in the PS II protein complement when plants are incubated under low light conditions. These results, when coupled with the functional measurements described in the previous section, indicate that the absence of the PsbQ protein leads to the loss of the assembly/stability and function of PS II under low light growth conditions. The low light sensitivity of the PsbQ-deficient mutants is quite unusual. Only plants deficient in the PsbR protein, which appears to be an assembly factor for PS II, exhibit a similar phenotype, although at higher light intensities than reported here (28).
Alterations of the Oxygen-evolving Complex in PsbQ-deficient Plants Grown under Normal Light Intensities—Although PsbQ-deficient plants grown at normal light intensities exhibited normal growth, color, fluorescence, and oxygen evolution characteristics, we hypothesized that loss of the PsbQ protein could result in defects in the oxygen-evolving complex that would not necessarily be evident in fluorescence or steady state oxygen evolution experiments. Consequently, we examined the stability and function of the oxygen-evolving complex in plants grown under normal light conditions. Our initial attempts to determine S2 and S3 state lifetimes in thylakoids isolated from the PsbQ-deficient plants failed. This was puzzling, because these experiments were normally quite easy to perform with thylakoids isolated from wild type plants. These measurements require the oxygen-evolving complex to be stable for 20-40 min at 22 °C. We therefore examined the stability of the oxygen-evolving complexes in thylakoids isolated from wild type and the PsbQ-deficient plants. In these experiments, we monitored the flash oxygen yield arising from the third flash in a series of 16 saturating flashes. Between these flash series, the thylakoids were incubated on the platinum electrode (polarizing current off), either in the dark or at 5-µmol photons/m2/s for varying lengths of time. The results obtained are shown in Fig. 5A. Within the limits of error, the thylakoids isolated from wild type plants contained oxygen-evolving complexes that were quite stable and exhibited little change during either light or dark incubation. The oxygen-evolving complexes contained in the thylakoids isolated from the PsbQ-deficient plants, however, were very unstable. These lost their ability to evolve oxygen, with half-times of 12.5 and 5 min in the dark and light, respectively. These results indicate that, even though the PsbQ-deficient plants appeared to assemble normal amounts of PS II reaction centers (Fig. 4), these centers are quite unstable with respect to their ability to carry out oxygen evolution.
|
|
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
We hypothesize that, under normal growth conditions, PS II repair mechanisms (possibly photoactivation) can compensate for the observed instability of the oxygen-evolving complex. However, under low light conditions, the repair rate is insufficient to compensate for this defect, and PS II complexes are ultimately lost from the thylakoid membrane. We are currently testing this hypothesis in our PsbQ-deficient plant lines.
|
FOOTNOTES |
|---|
1 To whom correspondence should be addressed: Biochemistry and Molecular Biology Section, Dept. of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803. Tel.: 225-578-1555; Fax: 225-578-7258; E-mail: btbric{at}lsu.edu.
2 The abbreviations and trivial names used are: PS II, photosystem II; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; Tricine, N-[2-hydroxy-1,1-bis-(hydroxymethyl)ethyl]glycine; RNAi, RNA interference; DCBQ, 2,6-dichloro p-benzoquinone.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Ishihara, A. Takabayashi, K. Ido, T. Endo, K. Ifuku, and F. Sato Distinct Functions for the Two PsbP-Like Proteins PPL1 and PPL2 in the Chloroplast Thylakoid Lumen of Arabidopsis Plant Physiology, November 1, 2007; 145(3): 668 - 679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Yi, S. R. Hargett, H. Liu, L. K. Frankel, and T. M. Bricker The PsbP Protein Is Required for Photosystem II Complex Assembly/Stability and Photoautotrophy in Arabidopsis thaliana J. Biol. Chem., August 24, 2007; 282(34): 24833 - 24841. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
, wild type; •, PsbQ-deficient (n = 3-5). Error bars are ±1 S.D. In some instances, the error bars are smaller than the symbols.
, wild type; 
and •, PsbQ-deficient (n = 5). Error bars are ±1 S.D. B, typical flash oxygen yield pattern obtained during a train of 16 saturating flashes. Symbols are as described for A.