Comparative Analysis of AGPase Genes and Encoded Proteins in Eight Monocots and Three Dicots with Emphasis on Wheat

Abstract

ADP-glucose pyrophosphorylase (AGPase) is a heterotetrameric enzyme with two large subunits (LS) and two small subunits (SS). It plays a critical role in starch biosynthesis. We are reporting here detailed structure, function and evolution of the genes encoding the LS and the SS among monocots and dicots. "True" orthologs of maize Sh2 (AGPase LS) and Bt2 (AGPase SS) were identified in seven other monocots and three dicots; structure of the enzyme at protein level was also studied. Novel findings of the current study include the following: (i) at the DNA level, the genes controlling the SS are more conserved than those controlling the LS; the variation in both is mainly due to intron number, intron length and intron phase distribution; (ii) at protein level, the SS genes are more conserved relative to those for LS; (iii) "QTCL" motif present in SS showed evolutionary differences in AGPase belonging to wheat 7BS, T. urartu, rice and sorghum, while "LGGG" motif in LS was present in all species except T. urartu and chickpea; SS provides thermostability to AGPase, while LS is involved in regulation of AGPase activity; (iv) heterotetrameric structure of AGPase was predicted and analyzed in real time environment through molecular dynamics simulation for all the species; (v) several cis-acting regulatory elements were identified in the AGPase promoters with their possible role in regulating spatial and temporal expression (endosperm and leaf tissue) and also the expression, in response to abiotic stresses; and (vi) expression analysis revealed downregulation of both subunits under conditions of heat and drought stress. The results of the present study have allowed better understanding of structure and evolution of the genes and the encoded proteins and provided clues for exploitation of variability in these genes for engineering thermostable AGPase.

Keywords: ADP_Glucose_PP domain; AGPase; expression analysis; genes structure; ligand binding; molecular dynamics simulation; promoter analysis.

Figure 1

Gene structure for AGPase LS from translation start to stop sites in eight monocots (including all the wheat homoeologues) and three dicots. Solid boxes indicate exons and lines indicate introns. Exons are color coded based on the sequence similarity with the respective exons in the AGPase LS gene of maize (used as reference). Intron phases 0, 1, and 2 are marked above each intron.

Figure 2

Gene structure for AGPase SS from translation start to stop sites in eight monocots (including all the wheat homoeologues) and three dicots. Solid boxes indicate exons and lines indicate introns. Exons are color coded based on the sequence similarity with the respective exons in the AGPase SS gene of maize (used as reference). Intron phases 0, 1, and 2 are marked above each intron.

Figure 3

Percent similarity of exons and introns in LS and SS of monocots and dicots with respect to exons and introns of maize LS and SS AGPase genes.

Figure 4

Representative figure showing regulatory elements identified in 1 kb upstream region of AGPase LS. Different symbols indicate major regulatory elements identified. TATA box (), CAAT box (), light responsive response elements (), abiotic stresses responsive elements (), endosperm expression responsive elements ().

Figure 5

Amino acid sequence similarity of AGPase LS among 8 monocots (including wheat homoeologues on group 1 chromosomes) and 3 dicots with respect to consensus sequence. Position 0 (on y-axis) indicates amino acid consensus sequence. Presence of similar amino acids against consensus is plotted on a scale of 1–10 in monocots (blue) and 1–3 in dicots (red).

Figure 6

Amino acid sequence similarity of AGPase SS among 8 monocots (including wheat homoeologues on group 7 chromosomes) and 3 dicots with respect to consensus sequence. Position 0 (on y-axis) indicates amino acid consensus sequence. Presence of similar amino acids against consensus is plotted on a scale of 1–10 in monocots (blue) and 1–3 in dicots (red).

Figure 7

Representative figure showing superimposed structure of the predicted wheat AGPase heterotetramer (orange colored) over potato AGPase homotetramer (cyan colored).

Figure 8

3D structure of wheat AGPase LS protein. The amino acids and their positions in the protein involved in ligand binding are shown in blue. Green sphere represent metallic heterogen (Mg⁺⁺) involved in ligand binding.

Figure 9

Phylogenetic tree obtained by neighbor-joining method using amino acid sequences of proteins encoded by genes for AGPase LS+SS to depict the relationship among monocots and dicots. The branch length represents magnitude of genetic change.

Figure 10

Representative figure showing in silico expression of wheat AGPase LS and SS where = transcript (probset id: Ta.2797.2.S1_x_at) encoding AGPase LS and = transcript (probset id: Ta.242.1.S1_at) encoding AGPase SS. (A) Expression during major abiotic stresses (drought and heat) where fold change is significant at p ≥ 0.05; Numbers on Y-axis indicate 11 different microarray experiments; (B) Expression in different tissues and (C) Expression at different plant developmental stages.

Figure 11

Expression analysis based on wheat transcriptome data in different tissues (grain, leaf, root, spike and stem) and their development stages. (A) Expression analysis of homoeologous genes for AGPase LS (Transcript id's: Traes_1AL_A1B2A8EB0.1, Traes_1BL_190920E1E.1 and Traes_1DL_844FE40E6.1) on group 1 chromosomes of wheat, and (B) Expression analysis of homoeologous genes for AGPase SS (Transcript id's: Traes_7AS_1B2A8C929.2, Traes_7BS_4FBE4B00A.2, and Traes_7DS_02539EB3B.1) on group 7 chromosomes of wheat.

Figure 12

Expression analysis based on wheat transcriptome data under heat and drought stress conditions. (A) Expression analysis of homoeologous genes for AGPase LS (Transcript id's: Traes_1AL_A1B2A8EB0.1, Traes_1BL_190920E1E.1 and Traes_1DL_844FE40E6.1) on group 1 chromosomes of wheat (B) Expression analysis of homoeologous genes for AGPase SS (Transcript id's: Traes_7AS_1B2A8C929.2, Traes_7BS_4FBE4B00A.2, and Traes_7DS_02539EB3B.1) on group 7 chromosomes of wheat.