Identification of AGPase proteins
Forty-five AGPase domain-containing proteins from I. batatas (26 accessions), I. trifida (10 accessions), and I. triloba (9 accessions) were identified and used for various analyses (Table 1). The sizes of these proteins were distinctly different; the amino acids ranged from 165 to 525 and the molecular weights (MW) ranged from 18.35 to 58.19 kDa.
The isoelectric point (pI), which represents the average pH of the molecule without a net electrical charge or electrically neutrality, was 4.71–9.53 in all categories. The average pI of I. batatas, I. trifida, and I. triloba AGPase were 6.83, 7.11, and 6.47, respectively. The instability index (II), which represents the stability and instability of a polypeptide at ≤ 40 and > 40, respectively, indicated 40 or less in AGPase of I. batatas. In contrast, some AGPases of the I. trifida and I. triloba were 40 or more. The aliphatic index (AI), which represents the relative volume of the aliphatic side chains of a polypeptide, was similar in the three species, but there were differences between subunits of I. batatas AGPase. Higher AI values were observed for the small subunits than the large subunits of I. batatas AGPase. The grand average of hydropathy (GRAVY), which was analyzed to determine the hydropathy of AGPase, showed that I. batatas had different characteristics from the other two species. All I. batatas AGPases showed negative values, whereas some of the I. trifida and I. triloba AGPases had positive values.
Conserved domain analysis
Six types of conserved domains that showed different distributions were included in the AGPase proteins of these three species (Fig 1b, Supplementary Table 1). Most of the I. trifida and I. triloba AGPase had only the NTP_transferase domain and some had two conserved domains: NTP_transferase at the N-terminal and Hexapep or Cpn60_TCP1 at the C-terminal. On the other hand, the I. batatas AGPase proteins had four types of conserved domains (NTP_transferase, LbH_G1P_AT_C, ADP_Glucose_PP, and Glyco_tranf_GTA_type); each of them had two conserved domains. All of the I. batatas AGPase proteins had the LbH_G1P_AT_C domain at the C-terminals, but the N-terminals differed according to the subunit. The N terminal of all large subunits of I. batatas AGPase proteins has the NTP_transferase domain only except for CAB51610.1, whereas all small subunits have ADP_Glucose_PP domain except for CAB55496.1, AAA19648.1, and CAA86726.1. The proteins with this exception all had partial sequences and had the Glyco_tranf_GTA_type domain at the C-terminals instead.
The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei 1987). Fig 1a presents the optimal tree with the sum of the branch length = 29.09. This analysis involved 45 amino acid sequences and 512 positions. The conserved domains were labeled on the amino acid sequences (Fig 1a). The length and type of the domain were different for each species. Based on the phylogenetic tree, AGPase proteins from these species were classified into two large subunit groups and two small subunit groups.