Comparative Study of Starch Phosphorylase Genes and Encoded Proteins in Various Monocots and Dicots with Emphasis on Maize

Abstract

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. "True" orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues.

Keywords: comparative study; cytosolic starch phosphorylase; plastidial starch phosphorylase; starch phosphorylase.

Figure 1

Gene structure of PHO1 and PHO2 from the translation start to stop sites in various monocots and dicots. Symbols include: Sb, Sorghum bicolor; Zm, Zea mays; Si, Setaria italica; Ph, Panicum hallii; Bd, Brachypodium distachyon; Ta, Triticum aestivum; Hv, Hordeum vulgare; Os, Oryza sativa; At, Arabidopsis thaliana; Sl, Solanum lycopersicum; St, Solanum tuberosum; and Ca, Capsicum annuum. Solid boxes indicate the exons, and lines indicate the introns. Colors represent the similarities among respective exons as a reference to O. sativa. The values of zero, one, and two marked above each intron are intron phases.

Figure 2

Percent identity of exons and introns in PHO1 and PHO2 of monocots and dicots with respect to exons and introns of O. sativa.

Figure 3

Representative figure showing regulatory elements identified in the 1 kb upstream region of PHO1 and PHO2. Different color bars represent the major regulatory elements. Symbols include: Sb, Sorghum bicolor; Zm, Zea mays; Si, Setaria italica; Ph, Panicum hallii; Bd, Brachypodium distachyon; Ta, Triticum aestivum; Hv, Hordeum vulgare; Os, Oryza sativa; At, Arabidopsis thaliana; Sl, Solanum lycopersicum; St, Solanum tuberosum; and Ca, Capsicum annuum.

Figure 4

Amino acids sequence similarity of PHO1 and PHO2 among various monocots and dicots with respect to the consensus sequence. A value position of zero indicates the consensus sequence. Similar amino acids are plotted on scales 1–5 in monocots and −1 to −5 in dicots.

Figure 5

The 3D structures of maize PHO1 and PHO2: (A) Amino acid residues and their location involved in the binding of a ligand (PLP); (B) Homo-dimeric 3D structure of the PHO1 of Z. mays. The L80 domain (orange) arranged outside the main protein core; (C) Homo-dimeric 3D structure of the PHO2 of Z. mays; (D) Superimposed structures of the predicted PHO1 of Z. mays (yellow-colored) over the PHO1 of H. vulgare (blue colored); (E) Superimposed structures of the predicted PHO2 of Z. mays (yellow-colored) over the PHO2 of A. thaliana (blue colored).

Figure 6

A phylogenetic tree constructed by the neighbor-joining method using the amino acid sequences of PHO1 and PHO2 to depict the relationship among monocots and dicots. The bootstrap value is calculated based on 1000 replications and displayed on each node. Symbols include: Sb, Sorghum bicolor; Zm, Zea mays; Si, Setaria italica; Ph, Panicum hallii; Bd, Brachypodium distachyon; Ta, Triticum aestivum; Hv, Hordeum vulgare; Os, Oryza sativa; At, Arabidopsis thaliana; Sl, Solanum lycopersicum; St, Solanum tuberosum; and Ca, Capsicum annuum. The branch length indicates the magnitude of genetic changes.

Figure 7

Distribution and expression pattern of the PHO1 and PHO2 transcripts of Z. mays: (A) Tissue-specific expression levels of the PHO1 and PHO2 of Z. mays. The relative expression pattern is shown and the transcript level in the embryo is used as control. The leaf1 is the first leaf harvested from the mature plant when it is flowering. The 15 DAP seeds and dissected endosperm were used to analyze the transcript level; (B) Relative expression levels of the PHO1 and PHO2 of Z. mays in different developmental stages of the endosperm. The transcript level in 10 DAP endosperm was used as a control.

Figure 8

Expression level of the PHO1 and PHO2 of maize under ABA treatment. The 15 DAP middle seed of corn cob was used to treat with ABA in MS medium. Furthermore, 0 h indicates no ABA treatment and is used as a control. (A) Effect on PHO1 transcripts expression. (B) Effect on PHO2 transcripts expression.