Study On The Key Enzyme Of C₃and C₄Pathway In Ulva Prolifera

During the bloom of the massive green algae in June2008, huge amounts offloating Ulva prolifera accumulated along the coast of Qingdao, Whichcaused tremendous damage to the travel and aquaculture. The occurrence of greentide was just at the stage of preparation for the2008Olympic Sailing Game inQingdao, which caused wide attention from domestic and abroad. However, there wasno answer about the reason of the bloom of U. prolifera. U. prolifera couldaccumulate a large biomass in a relatively short time period, suggesting thatphotosynthesis in this organism, particularly its carbon fixation pathway, must be veryefficient.Photosynthesis, which can fix solar energy and transform inorganic into organic,is the planet’s most important chemical reaction. The discovery of C₄pathway in C₃plants and the existence of C₃photosynthesis in specific developmentalstages of some C₄plants, imply that photosynthetic carbon fixationpathway has great plasticity. Recent genome sequencing data suggest relevant genesof C₄pathway have been identified in the green alga Ostreococcus tauri and thediatoms Thalassiosira weissflogii and Phaeodactylum tricornutum. PEPC activity in T.weissfogii increases in cells acclimatized to low CO₂, indicating a probable linkbetween inorganic carbon uptake and PEPC.The maximal photochemical efficiency of PS II （Fv/Fm） and the effective PS IIquantum yield （Y II） of U. prolifera subjected to the different treatments wasmeasured using Dual-PAM-100. Fv/Fm and Y （II） reduced under desiccation, highsalinity （60‰）, low salinity （0‰）, high temperature （35℃） and high light intensity （2000μmol m-2s-1）, which indicated that U. linza was stressed. Fv/Fm and Y （II）decreased quickly under desiccation and high light intensity, which demonstrated theycould caused intense stress for U. prolifera.The transcriptome of U. prolifera was sequenced by454sequencing. Aftertrimming the adapter sequences and removing the short sequences with lowcomplexity and low quality scores,445,787high quality reads were obtained,corresponding to88.5%of the original raw sequences. Reads were assembled into13,426contigs with an average length of1,000bases. Contigs were further assembledinto10,784isotigs with an average length of1,515bases. KEGG analysis showed that9356reads were assigned to the carbon fixation pathway, which encode almost all ofthe enzymes of C₃and C₄pathway.According to partial sequence of PPDK gene from transcriptome sequencing, wedesigned primers and acquired partial sequence of PPDK gene by PCR. Based on thepartial sequences of PPDK, the5’-and3’-ends of the cDNA were obtained by theRACE approach. The full-length of PPDK was3114bp. The ORF of PPDK was2700bp encoding889amino acid residues. The theoretical Mw and pI of the PPDK proteinwere96.43KDa and5.60, respectively. The sequences of PPDK was submitted toGenBank and assigned accession number JN936854. The phylogenetic tree of PPDKshowed the species of C₃, C₃-C₄and C₄existed in both higher plant and algae, whichindicated C₄pathway was multiple independent origin.Relative quantitative PCR were carried out to determine the differences inexpression levels of rbcL and PPDK genes under the different stress treatments. Theexpression levels of both rbcL and PPDK increased under some stress conditions, butPPDK was higher. The activity of Rubisco and PPDK were measured byspectrophotometry in U. prolifera. Rubisco and PPDK have activity under anyconditions and the activity of Rubisco was higher than PPDK. The activity ofRubisco and PPDK increased under certain stress, but decreased beyond acertain range. Studies have shown that U. prolifera have both C₃and C₄pathway andmainly in C₄pathway.