Diurnal oscillations in steady‐state mRNA levels and transcription rates were measured for seven transcripts (five of which encode chloroplast‐localized proteins) in tomato seedlings: photosystem I and photosystem II chlorophyll a/b binding proteins (CAB/I and CAB/II), small subunit of RuBisCO (RBCS), actin, subunit II of the photosystem I reaction center (PSAD), subunit I of the photosystem II oxygen‐evolving enzyme (OEE1), and a biotin‐binding protein of unknown function. CAB/II mRNA levels were found to oscillate greater than 20‐fold, showing a peak at noon, while only marginal diurnal oscillations are seen in RBCS transcripts. The oscillations are at least partially controlled at the transcriptional level. Transcription rates of both CAB/II and RBCS, measured by nuclear run‐on experiments, were found to oscillate, with a peak around 8 a.m. Transcription rates of the ‘biotin’ clone also oscillated, with a peak around noon. Transfer of plants to constant darkness or constant light conditions alters the amplitude of the transcriptional oscillation, but does not abolish it, suggesting that it is at least partially controlled by a circadian clock. The oscillations are still visible after three days in complete darkness, and have a period very close to 24 h. The oscillator phase can be reset by out‐of‐phase light treatment.

We report here the isolation and nucleotide sequence of a complete cDNA clone encoding a photosystem I (PS I) polypeptide that is recognized by a monoclonal antibody made against photosystem II (PS II) chlorophyll a/b-binding (CAB) proteins. The deduced sequence of this PS I protein shows 30% overall identity to PS II CAB sequences, and two long segments within this protein show 50% and 65% identity to the corresponding segments in the PS I CAB polypeptides. Even though the sequence of this PS I CAB protein is substantially divergent from PS II CAB sequences, their hydropathy plots are very similar and suggest they all traverse the thylakoid membrane three times. A segment of the PS I CAB polypeptide shows similarity to the functionally analogous (3 subunits of the antenna proteins of purple bacteria. In contrast, no homology was observed between these bacterial proteins and PS II CAB polypeptides. MATERIALS AND METHODSFractionation of PS I and PS 11. Tomato (Lycopersicon esculentum) PS I and PS II were fractionated according to Mullet et al. (14) with the exception that the Triton X-100 concentration was changed to 0,5% (wt/vol) for the solubilization of the thylakoid membrane. Under these conditions, the chlorophyll concentration was 0.8 mg/ml.Polyacrylamide Gel Electrophoresis. Protein samples were subjected to NaDodSO4/polyacrylamide gel electrophoresis in 13% gels under reducing conditions (13,15 Immunoblots. Protein was transferred to nitrocellulose paper at pH 8.3 by applying a voltage gradient of 30 V/cm for 1 hr (17). Transfer was-followed by a 30-min incubation in Tris-buffered saline (TBS; 10 mM Tris HCI, pH 7.5/150 mM NaCl) containing 2% (wt/vol) nonfat dry milk powder. Blots were incubated with antibodies for 16 hr at 40C and washed successively with TBS, TBS containing 0.1% Nonidet P-40, and TBS. Blots incubated with monoclonal antibodies were further incubated with goat anti-mouse immunoglobulin conjugated to alkaline phosphatase as described by Darr et al. (13)

Chronic treatment of cultured cells with very low levels of azide (I 50 <10 M) leads to slow (t1 ⁄2 ‫؍‬ 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azidemediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl 2 , CuHis) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either Cu A or Cu B . In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa 3 content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties.

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Various chimeric precursors and deletions of the 33 kd oxygen‐evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose‐1,5‐bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N‐terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C‐terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.