The function of starch phosphorylase has long been debated on the regulation of starch metabolism during the growth and development of plants. In this study, we isolated starch phosphorylase genes (Pho1 and Pho2) from barley, characterized their gene and protein structures, predicated their promoter's cis-elements and analyzed expression patterns. Multiple alignments of these genes showed that (1) both Pho1 and Pho2 genes possess 15 exons and 14 introns in all but three of the species analyzed, Aegilops tauschii (for Pho1 which contains 16 exons and 15 introns), potato (for Pho1b which contains 14 exons and 13 introns), and Triticum uraru (for Pho2 which contains 15 exons and 14 introns); (2) the exon-intron junctions of Pho1 and Pho2 flanking the ligand-binding sites are more conservative than the other regions. Analysis of protein sequences revealed that Pho1 and Pho2 were highly homologous except for two regions, the N terminal domain and the L78 insertion region. The results of real-time quantitative PCR (RT-qPCR) indicated that Pho2 is mainly expressed in germinating seeds, and the expression of Pho1 is similar to that of starch synthesis genes during seed development in barley. Microarray-based analysis indicated that the accumulation of Pho1 or Pho2 transcripts exhibited uniform pattern both in various tissues and various stages of seed development among species of barley, rice, and Arabidopsis. Pho1 of barley was significantly down-regulated under cold and drought treatments, and up-regulated under stem rust infection. Pho2 exhibited similar expression to Pho1 in barley. However, significant difference in expression was not detected for either Pho1 or Pho2 under any of the investigated abiotic stresses. In Arabidopsis, significant down-regulation was detected for Pho1 (PHS1) under abscisic acid (ABA) and for Pho2 (PHS2) under cold, salt, and ABA. Our results provide valuable information to genetically manipulate phosphorylase genes and to further elucidate their regulatory mechanism in the starch biosynthetic pathway.
Keywords: Microarray analysis; Promoter prediction; Real-time quantitative PCR; Starch phosphorylase.