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Under nitrogen (N)-limited conditions, the non-N2-fixing cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) actively grows during early stages of starvation by performing photosynthesis but gradually stops the growth and eventually enters dormancy to withstand long-term N limitation. During N limitation, Synechocystis 6803 cells degrade the large light-harvesting antenna complex phycobilisomes (PBSs) presumably to avoid excess light absorption and to reallocate available N to essential functions for growth and survival. These two requirements may be driving forces for PBS degradation during N limitation, but how photosynthesis and cell growth affect PBS degradation remains unclear. To address this question, we examined involvements of photosynthesis and cell growth in PBS degradation during N limitation. Treatment of photosynthesis inhibitors and shading suppressed PBS degradation and caused non-bleaching of cells during N limitation. Limitations of photosynthesis after initial gene responses to N limitation suppressed PBS degradation, implying that photosynthesis affects PBS degradation in a post-translational manner. In addition, limitations of cell growth by inhibition of peptidoglycan and fatty acid biosynthesis, low growth temperature and phosphorous starvation suppressed PBS degradation during N limitation. Because decreased photosynthetic activity led to decreased cell growth, and vice versa, photosynthesis and cell growth would inseparably intertwine each other and affect PBS degradation during N limitation in a complex manner. Our data shed light on the coordination mechanisms among photosynthesis, cell growth and PBS degradation during N limitation.

The lipid bilayer matrix of the thylakoid membrane of cyanobacteria and chloroplasts of plants and algae is mainly composed of uncharged galactolipids, but also contains anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) as major constituents. The necessity of PG for photosynthesis is evident in all photosynthetic organisms examined to date, whereas the requirement of SQDG varies with species. In plants, although PG and SQDG are also found in non-photosynthetic plastids, their importance for the growth and functions of non-photosynthetic organs remains unclear. In addition, plants synthesize another anionic lipid glucuronosyldiacylglycerol (GlcADG) during phosphorus starvation, but its role in plant cells is not elucidated yet. To understand the functional relationships among PG, SQDG, and GlcADG, we characterized several Arabidopsis thaliana mutants defective in biosynthesis of these lipids. The mutants completely lacking both PG and SQDG biosynthesis in plastids showed developmental defects of roots, hypocotyls, and embryos in addition to leaves, which suggests that these lipids are pleiotropically required for the development of both photosynthetic and non-photosynthetic organs. Furthermore, our analysis revealed that SQDG, but not GlcADG, is essential for complementing the role of PG, particularly in photosynthesis under PG-deficient conditions such as phosphorus starvation.

Thylakoid membranes, the site of photochemical and electron transport reactions of oxygenic photosynthesis, are composed of a myriad of proteins, cofactors including pigments, and glycerolipids. In the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803, the size and function of thylakoid membranes are reduced under nitrogen (N) starvation but are quickly recovered after N addition to the starved cells. To understand how the functionality of thylakoid membranes is adjusted in response to N status in Synechocystis sp. PCC 6803, we examined changes in thylakoid components and the photosynthetic activity during the N starvation and recovery processes. In N-starved cells, phycobilisome content, photosystem II protein levels and the photosynthetic activity substantially decreased as compared with those in N-sufficient cells. Although the content of chlorophyll (Chl) a, total protein and total glycerolipid also decreased under the N-starved condition based on OD730 reflecting cell density, when based on culture volume, the Chl a and total protein content remained almost constant and total glycerolipid content even increased during N starvation, suggesting that cellular levels of these components decrease under the N-starved condition mainly through dilution due to cell growth. With N addition, the photosynthetic activity quickly recovered, followed by full restoration of photosynthetic pigment and protein levels. The content of phosphatidylglycerol (PG), an essential lipid constituent of both photosystems, increased faster than that of Chl a, whereas the content of glycolipids, the main constituents of the thylakoid lipid bilayer, gradually recovered after N addition. The data indicate differential regulation of PG and glycolipids during the construction of the photosynthetic machinery and regeneration of thylakoid membranes. Of note, addition of PG to the growth medium slightly accelerated the Chl a accumulation in wild-type cells during the recovery process. Because PG is required for the biosynthesis of Chl a and the formation of functional photosystem complexes, rapid PG biosynthesis in response to N acquisition may be required for the rapid formation of the photosynthetic machinery during thylakoid regeneration.

Access to glycopeptides with C-terminal thioester functionality is essential for the synthesis of large glycopeptides and glycoproteins through the use of native chemical ligation. Toward that end, we have developed a concise method for the synthesis of a glycopeptide thioester having an intact complex-type dibranched disialyl-oligosaccharide. The synthesis employed a coupling reaction between benzylthiol and a free carboxylic acid at the C-terminus of a glycopeptide in which the peptide side chains are protected. After construction of glycopeptide on the HMPB-PEGA resin through the Fmoc-strategy, the protected glycopeptide was released upon treatment with acetic acid/trifluoroethanol and then the C-terminal carboxylic acid was coupled with benzylthiol at -20 degrees C in DMF. For this coupling, PyBOP/DIPEA was found to be the best for the formation of the thioester, while avoiding racemization. Finally, the protecting groups were removed in good yield with 95% TFA, thus affording a glycopeptide-thioester having an intact and homogeneous complex-type disialyl-oligosaccharide.

Summary A new bioactive peptide was isolated from frog brain using a bioassay for chick rectum relaxant activity. Amino acid sequence of this peptide was determined to be Gly-Tyr-Ser-Arg-Gly-Cys-Phe-Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ala- Phe-Ser-Gly-Leu-Gly-Cys, in which two cysteines were linked by a disulfide bond. The peptide was found to belong structurally to the natriuretic peptide family and to exert diuretic-natriuretic activity as well as hypotensive activity when injected into rats. The peptide showed a high homology to recently identified porcine C-type natriuretic peptide (CNP) and a pharmacological spectrum highly similar to porcine CNP. Thus, the peptide was designated frog C-type natriuretic peptide (frog CNP). Frog CNP may participate in the central control of body fluid homeostasis, since its tissue concentration is high in brain.