The D1 protein of the photosystem II (PSII) complex in the thylakoid membrane ofoxygenic photosynthetic organisms is synthesized as a precursor polypeptide (pDl) with a C-terminal extension. Posttransational processing ofthe pDl protein is essential to estbfsh water oxidation activity of the PSII complex. We have recently identified a gene, cipA, a mutation in which resulted in a loss of PSII activity in the cyanobacterium Synechocystis sp. PCC 6803. To study the function of the CtpA protein, we inactivated the ctpA gene by inserting a kanamycin-resistance gene into its coding sequence. The resultant mutant strain, T564, had no PSII-mediated water oxidation activity, but it had normal cytochrome b6fand photosystem I activities. Measurements ofthermoluminescence profiles and rates of reduction of 2,6-dichilorophenolindophenol indicated that PSI1 complexes in the mutant cells had functional reaction centers that were unable to accept electrons from water. Immunoblot analysis showed that D1, D2, CP47, CP43, and the a subunit of cytochrome bwg, five Integral membrane proteins of PSI1, were present in T564 cells. Interestingly, the D1 protein in the mutant cells was 2 kDa larger than that in wild-type cells, due to the presence of a C-terminal extension. We conclude that the CtpA protein is a processing enzyme that cleaves off the C-terminal extension of the D1 protein. Interestingly, the CtpA protein shows scant sequence similarity to the interphotoreceptor retinoid-binding proteins in the bovine, human, and insect eye systems.
The psbL gene is a member of the psbEFLJ gene cluster in the cyanobacterium Synechocystis sp. PCC 6803 and higher plants. psbL, a 4.5 kDa protein encoded by this gene, is a component of the photosystem II complex. The amino acid sequence of this protein indicates that it has a single membrane-spanning a-helical domain. We have used a targeted mutagenesis technique to delete the coding region of the psbL gene in Synechocystis 6803. The resultant mutant strain T345 did not show any PSII-mediated oxygen evolution activity and, as a result, could not grow under photoautotrophic conditions. However, it had normal PSI activity. The chlorophyll to phycobilin ratio in the T345 cells was significantly lower than that in the wild type cells. Fluorescence emission spectra (77 K) of the mutant cells showed the absence of a 695 nm band that usually originates from the PSII complex. Binding assays with radioactive diuron demonstrated that the mutant cells did not have any herbicide binding activity. However, immunostaining experiments showed that both the D 1 (the herbicide binding protein) and the D 2 proteins of the PSII reaction center were present at > 25 % of their normal levels in the thylakoid membranes of the T345 mutant cells. Our data indicate that the PsbL protein is essential for the normal functioning of PSII.
A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 mM adenine sulfate +2.46 mM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 mM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60±65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.
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