Research On The Key Genes In The Biosynthetic Pathway Of Floridoside In Pyropia Haitanensis

Glycerol-3-phosphate (G3P) is proposed to be a novel regulator in plant defensesignaling. Floridoside (2-O-glycerol-α-D-galactopyranoside), an important downstreamproduct of G3P, is the main photosynthetic product and osmolyte in red algae Pyropiahaitanensis, and plays an important role in the adaptation to external environment. In thisresearch, G3P and floridoside contents of P. haitanensis were quantitatively analyzed. Resultsshowed that G3P levels were significantly increased after induction by flg22as well asincreased in the recovery culture after heat shock. G3P contents were almost6.92timeshigher than those of the control after3hours induction of flg22. Floridoside contents werereduced by nearly24%under2hours induction of flg22, as well as decreased after30minutesunder heat shock, while it became increased and reached1.92-fold higher than the controlafter3hours of recovery culture. These results suggested that G3P and floridoside wereassociated with the defense response and heat stress response in P. haitanensis. The glycerolkinase (also designated as NHO1) and NAD-dependent G3P dehydrogenase (GPDH), two keyenzymes involving in the G3P biosynthesis, were cloned from red algae P. haitanensis(denoted as PhNHO1and PhGPDH). PhNHO1had a complete open reading frame (ORF) of1728bp encoding for a protein of576amino acids and contained a conserved ATP-Bindingmotif. The ORF of PhGPDH is1158bp encoding for a386amino acids protein and carried aconserved NAD-binding domian. Both of them shared high sequence identity and structuralconservation with their orthologs, especially the orthologs from red algae. Phylogeneticanalysis indicated that red algal orthologs were closer to animals than other algae and higherplants. The expression levels of the two genes were up-regulated induced by both flg22andheat shock. Changes of PhGPDH were more rapid and intense, suggesting that it is the majorcontributor of total G3P pool that participates in stress response. In addition, three members oftrehalose-6-phosphate synthase (TPS) gene family were cloned and denoted as PhTPS1,PhTPS2and PhTPS4. The ORF of PhTPS1is3459bp encoding for a protein of1153aminoacids; The ORF of PhTPS2is4041bp encoding for a protein of1347amino acids; The ORF of PhTPS4is3021bp encoding for a protein of1007amino acids. All of them contained aconserved TPS and a TPP domain, among which PhTPS4also had a starch-binding domain(CBM20). They all shared high sequence identity (about60%) with their orthologs from redalgae. Phylogenetic analysis showed that red algal orthologs clustered well into its own groupand had the earliest origin of various groups. The four TPS genes were all up-regulated underflg22, heat shock and desiccation conditions, and dramatic changes were detected underdesiccation conditions. Changes of PhTPS4were the most rapid and intense under the samecondition. To investigate the function of TPS genes, the prokaryotic expression system ofPhTPS1and PhTPS4were built. The purified PhTPS4protein was used to catalyze substratesand the products were analyzed via HPLC-MS. An ion m/z335.09representing the productfloridoside-P was detected and two characteristic fragment ions were also detected viaESI-MS. Thus, PhTPS4may perform the function of floridoside synthesis in P. haitanensis. Insummary, G3P metabolism and floridoside play important roles in stress response in P.haitanensis. Both PhNHO1and PhGPDH contribute to G3P accumulation. Furthermore,PhTPS4may be involved in the synthesis of floridoside in P. haitanensis, and theup-regulated expression of TPS genes may be related to the subsequent floridosideaccumulation.