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More than one hundred mutants of Synechocystis sp. PCC 6803 impaired in photoautotrophic growth were generated by in vitro random PCR mutagenesis targeted to a region of the psbAII gene corresponding to a 210 amino acid (Ser148–Ala357) segment of the D1 protein. The 90 random mutants that could translate the full-length D1 protein carried 1–9 (on average 3.0) amino acid substitutions in the targeted region. Mutations were often found in the obligate photoheterotrophic strains at specific residues that have been reported or speculated to be important in the function of PSII, such as Y161, H198, H272, E333 and H337. This verifies the usefulness of the present method to identify functionally important residues in PSII. Other residues that were often mutated in the strains with impaired photoautotrophy included non-charged residues around the lumenal edges of transmembrane helices C, D and E, such as I192 and N296. Eleven mutants carried a single-point mutation in residues, such as Q165, Q187, W278, A294 and N298, and these identified the functional importance of these residues, most of which were on the donor side of PSII. A preliminary characterization of some of the mutants obtained in this study is provided.

A cDNA encoding a putative RNA and/or DNA helicase has been isolated from Arabidopsis thaliana cDNA libraries. The cloned cDNA is 5166 bases long, and its largest open reading frame encodes 1538 amino acids. The central region of the predicted protein is homologous to a group of nucleic acid helicases from the DEAD/H family. However, the N- and C-terminal regions of the Arabidopsis cDNA product are distinct from these animal DEIH proteins. We have found that the C-terminal region contains three characteristic sequences: (i) two DNA-binding segments that form a probe helix (PH) involved in DNA recognition; (ii) an SV40-type nuclear localization signal; and (iii) 11 novel tandem-repeat sequences each consisting of about 28 amino acids. We have designated this cDNA as NIH (nuclear DEIH-boxhelicase). Functional character-ization of a recombinant fusion product containing the repeated region indicates that NIH may form homodimers, and that this is the active form in solution. Based on this information and the observation that the sequence homology is limited to the DEAH regions, we conclude that the biological roles of the plant helicase NIH differ from those of the animal DEIH family.

Random mutations were introduced into the DNA fragment of the psbA2 gene of Synechocystis sp. PCC 6803, which encodes the carboxyl-terminal 178 amino acid region of the D1 protein of the PSII reaction center, by in vitro random mutagenesis to obtain D1 species resistant to herbicides and to understand the protein-herbicide interactions. The mutants were screened on the criterion of resistance to either 1 microM DCMU or 10 microM atrazine. In these mutants, amino acid substitutions were distributed throughout the entire area of the targeted region in the D1 protein. However, in every mutant, except for one case, the substitution was present in the region described as the "herbicide-binding niche", i.e., between Phe211 and Leu275, although some amino acid substitutions which were not previously described were found at residues known to be involved with herbicide affinity. Thus, the result of random mutagenesis basically supports the validity of the proposed structural model for the D1 protein, as well as of the herbicide-binding niche. Preliminary characterization of the herbicide-resistant mutants obtained in this study has also been conducted.

Mechanisms underlying suppressed levels of transcripts for plastid photosynthesis genes in nongreen tissues such as roots and calli were analyzed in Arabidopsis thaliana, a plant suitable for further genetic dissection. A region encoding promoters of rbcL, the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, and the atpB/E operon for the [beta] and [epsilon] subunits of coupling factor one were cloned and sequenced. Transcripts for rbcL, atpB/E, and psbA, the gene for the D1 protein in the photosystem II reaction center, were barely detectable in roots of A. thaliana, whereas 16S rRNA was detected at a low level. The run-on transcription experiment revealed that expression of rbcL, atpB/E, and psbA was regulated at transcription. The copy number of plastid DNA in roots was one-fifth that in green leaves on the basis of total cellular DNA, suggesting that in the latter the DNA copy-number regulation also exists in plastid gene expression. Digestion of DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and subsequent polymerase chain reaction, as well as in vitro transcription of plastid DNAs with Escherichia coli RNA polymerase, resulted in no evidence of regulation by DNA modification. In spite of predominant suppression of expression of rbcL, atpB/E, and psbA at transcription in roots and calli, 16S rRNA levels were decreased because of low RNA stability.

Transient absorbance changes of the primary electron donor chlorophyll a (P680) and acceptor pheophytin a (H) were measured at 77 K by nanosecond laser spectroscopy in the Dl-D2-cytochrome bSS9 photosystem II reaction center complex containing dibromomethylisopropyl benzoquinone (DBMIB). After the laser excitation of the reaction center in the presence of DBMIB, only the P680+(DBMIB) state was detected. P680+ mainly decayed with a t 1/e of 11 ms. In the absence of DBMIB, the excitation produced the P680+H~ radical pair. The radical pair produced the triplet state (P680T) with a t 1/e of 50 ns, and P680T then decayed with a tVt of 2.1 ms. It was concluded that H~ was oxidized by DBMIB in a time range faster than the detecting time resolution (3.5 ns) even at 77 K. The rapid oxidation of H~ by DBMIB was also confirmed by the suppression of delayed fluorescence with a decay t1/e of 50 ns. The P680+(DBMIB")/P680(DBMIB) difference spectrum exhibited a Q, band with a peak at 682 nm with a shoulder at 673 nm. The spectral shape was almost temperature insensitive between 77 and 265 K. The feature of this spectrum in the wavelength range between 330 and 720 nm was compared with that of P680 T/P680 or H"/H at 77 K.

The D1 and D2 subunits of the reaction center of photosystem II are intrinsic proteins. A procedure was developed for the separation and purification of these two proteins on a large scale from the photosystem II reaction center complex of spinach by high-performance liquid chromatography on a gel-permeation column. The purified D1 and D2 proteins were subjected to determination of their carboxy-terminal sequences by digestion of the proteins with carboxypeptidase Y

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F(I), F(II), and F(III), corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at -196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F(II) (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at -196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F(I), F(II), and F(III) at -196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F(I) showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F(II) and F(III) in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F(I) and F(II) caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at -196 C with an increase in the yield of fluorescence especially in the case of F(I).