Genome-wide identification, characterization and expression pattern analysis of APYRASE family members in response to abiotic and biotic stresses in wheat

Abstract

APYRASEs, which directly regulate intra- and extra-cellular ATP homeostasis, play a pivotal role in the regulation of various stress adaptations in mammals, bacteria and plants. In the present study, we identified and characterized wheat APYRASE family members at the genomic level in wheat. The results identified a total of nine APY homologs with conserved ACR domains. The sequence alignments, phylogenetic relations and conserved motifs of wheat APYs were bioinformatically analyzed. Although they share highly conserved secondary and tertiary structures, the wheat APYs could be mainly categorized into three groups, according to phylogenetic and structural analysis. Additionally, these APYs exhibited similar expression patterns in the root and shoot, among which TaAPY3-1, TaAPY3-3 and TaAPY3-4 had the highest expression levels. The time-course expression patterns of the eight APYs in response to biotic and abiotic stress in the wheat seedlings were also investigated. TaAPY3-2, TaAPY3-3, TaAPY3-4 and TaAPY6 exhibited strong sensitivity to all kinds of stresses in the leaves. Some APYs showed specific expression responses, such as TaAPY6 to heavy metal stress, and TaAPY7 to heat and salt stress. These results suggest that the stress-inducible APYs could have potential roles in the regulation of environmental stress adaptations. Moreover, the catalytic activity of TaAPY3-1 was further analyzed in the in vitro system. The results showed that TaAPY3-1 protein exhibited high catalytic activity in the degradation of ATP and ADP, but with low activity in degradation of TTP and GTP. It also has an extensive range of temperature adaptability, but preferred relatively acidic pH conditions. In this study, the genome-wide identification and characterization of APYs in wheat were suggested to be useful for further genetic modifications in the generation of high-stress-tolerant wheat cultivars.

Keywords: APYRASE; Abiotic and biotic stress; Enzymatic activity; Expression pattern; Wheat.

Figure 1. Phylogenetic analysis of the putative APYs in wheat and other plant species.

The phylogenetic tree was created using the MEGA5 software with maximum Likelihood method. Bootstrap values for 1,000 replicates were indicated. Genes were separated into three groups and marked with different colors (Group I–III), according to the categorization in the phylogenetic tree.

Figure 2. Conserved motif analysis of the wheat APYs.

(A) The motif analysis of the eight APYs was carried out by using the online software MEME suite 5.0.2. (B) and (C) The details of the conserved motifs (1 and 8) of the nine APYs were represented. Different motifs were represented in different colors in the protein.

Figure 3. Secondary structure analysis of the nine wheat APYs (A–I).

Alpha helix was colored in red, Extend strand in blue and random coli in purple. The cross membrane domain was predicted and marked with a red box.

Figure 4. 3D structure analysis of the nine wheat APYs.

The structure models of the nine wheat APYs (A–I) were constructed using the Swiss-model website (https://www.swissmodel.expasy.org/).

Figure 5. Expression pattern of the nine TaAPYs in the root and leaf of the 10-d-old wheat seedlings.

TaACT was used as internal control. Data are presented as means ± SD of three biological replicates.

Figure 6. Expression pattern of the wheat APYs in response to the abiotic stresses.

(A) Heavy metal (200 mM CdCl₂). (B) Drought (300 mM mannitol). (C) Heat (42 °C). (D) Salt (300 mM NaCl). 1, leaf. 2, root. TaACT was used as internal control. Different colors represented decreased or decreased expression level.

Figure 7. Expression pattern of the APYs in response to the Bgt infection.

The expression of the APYs was analyzed separately at 24, 48, 72 and 96 h post Bgt infection. TaACT was used as the internal control. Green and red colors represented decreased or decreased expression level.

Figure 8. Enzymatic activity analysis of recombinant TaAPY3-1.

(A) Scheme of the purified TaAPY3-1 without the membrane spanning domain. (B) SDS-PAGE analysis of the protein purification. (C) Enzymatic activity of TaAPY3-1 in degradation of ATP under different temperature. (D) Activity of TaAPY3-1 under different pH. (E) Enzymatic activity of TaAPY3-1 in degradation of ATP, ADP, TTP, CTP and GTP. (F) Effects of different ions (Ca^{2 +}, Mg^{2 +} and Zn^{2 +}) on the enzymatic activity of TaAPY3-1 in degradation of ATP. (G) Enzymatic activity analysis of TaAPY3-1 with different concentrations of ATP. Data are presented as means ± SD of three biological replicates.