Involvement of a Putative Bipartite Transit Peptide in Targeting Rice Pheophorbide a Oxygenase into Chloroplasts for Chlorophyll Degradation during Leaf Senescence

Leaf senescence is one of the major factors contributing to the productivity and the grain quality in crops. The regulatory mechanism of leaf senescence remains largely unknown. Here, we report the identification and characterization of a rice e_aarly senescence 1 （easl） mutant, which displayed an early leaf senescence phenotype, accompanying by dwarfism and reduced tiller number, eventually leading to the reduction of grain yield. Map-based cloning revealed that the nuclear gene EAS1 encodes a pheophorbide a oxygenase （PaO）, a key enzyme for chlorophyll breakdown. A highly conserved Thr residue of PaO was mutated into Ile in the easl mutant. Phylogenetic analysis indicates that PaO is an evolutionarily conserved protein, and EAS1 is 68% identical to the Arabidopsis ACCERLERATED CELL DEATH （ACD1） protein. Unlike ACD1 that contains a single transit peptide, EAS1 contains two putative transit peptides at its N-ter- minus, which are essential for its functionality, suggesting that targeting of EAS1 to the chloroplast is likely mediated by a putative bipartite transit peptide. Consistently, only a short version of EAS1 lacking the first putative transit peptide, but not the full-length EAS1, was capable of rescuing the Arabidopsis acdl mutant phenotype. These results suggest that rice EASI represents a functional PaO, which is involved in chlorophyl/degradation and may utilize a unique mechanism for its import into the chloroplast.

THE OPTICAL PROPERTIES OF PHEOPHORBIDE A

Photochemical therapy has become a new area in treating malignant tumour with photosensitive medicines. In recent years, a new photosensitive medicine phcophorbide A (PhA) is developed, with curative effect ten times as high as that of Hematoporphyrin. In this note, the method of preparing PhA from pteridophyte is reported, the electronic spectra of PhA is qualitatively analyzed by HMO figure theory and the mechanism of photochemically treating cancer is discussed.

Characteristics of photosensitization of Pheophorbide a in liposomal media

Pheophorbide a (PPa), a decomposition product of chlorophyll a, is a photosensitizer. The photosensiti-zation mechanisms (Type Ⅰ and Type Ⅱ) of PPa in simple buffer solutions and in buffer solutions containing double-layered DPPC liposomes have been studied using techniques of ESR, spin-trapping, spin-counteraction and laser flash photolysis . The results showed that adding DPPC liposomes to the buffer solution caused an increase of efficiency of generating 1O2 and PPa - by photoactivating PPa. The increase could be ascribed to the disaggregation of hydrophobic PPa caused by the addition of liposomes and the protective effect of liposomal media on the triplet state of PPa. It is concluded that the photosensitization of PPa in liposomal systems is different from that in simple aqueous solutions, and shows higher efficacy. The results will be useful to elucidating the mechanisms of photodynamic therapy of cancer.

Characterization of Amphidinium(Amphidiniales,Dinophyceae)species from the China Sea based on morphological,molecular,and pigment data

Amphidinium species are amongst the most abundant benthic dinoflagellates in marine intertidal sandy ecosystems.Some of them are able to produce a variety of bioactive compounds that can have both harmful effects and pharmaceutical potentials.The diversity of Amphidinium in shallow waters along the Chinese coast was investigated by isolating single cells from sand,coral,and macroalgal samples collected from 2012 to 2020.Their morphologies were subjected to examination using light microscopy(LM)and scanning electron microscopy(SEM).A total of 74 Amphidinium strains were morphologically identified,belonging to 11 species:A.carterae,A.gibbosum,A.operculatum,A.massartii,A.cf.massartii,A.fijiensis,A.pseudomassartii,A.steinii,A.thermaeum,A.theodori,A.tomasii,as well as an undefined species.The last seven species have not been previously reported in Chinese waters.Amphidinium carterae subcladesⅠ,Ⅱ,andⅣwere found in the South China Sea,while subcladeⅢwas only found in the Yellow Sea.Threadlike body scales were observed on the surface of subcladesⅢ and Ⅳ,supporting the idea that A.carterae might contain several different species.Large subunit ribosomal RNA(LSU rRNA)sequencesbased phylogeny revealed two groups(GroupsⅠandⅡ)within Amphidinium,which is consistent with the relative position of sulcus(in touch with cingulum or not).In addition,large differences in morphology and molecular phylogeny between A.operculatum(the type species of Amphidinium)and other species,suggest that a subdivision of Amphidinium might be needed.The pigment profiles of all available strains were analyzed by high performance liquid chromatography(HPLC).Eleven pigments,including peridinin,diadinoxanthin,diatoxanthin,pheophorbide(and pheophorbide a),antheraxanthin,β-carotene,and four different chlorophylls were detected.The high pheophorbide/pheophorbide a ratio in Amphidinium implies that it may be a good candidate as a natural source of photosensitizers,a well-known anticancer drug.