Table 1.
Physicochemical properties of the members of the apyrase gene family in rice (O. sativa).
Fig 1.
Schematic depiction of phylogenetic relationship, gene structure, and conserved motifs of APY genes.
a- Phylogenetic relationship among OsAPYs generated via maximum likelihood approach. The green, pink and blue boxes represent groups I, II, and III, respectively. b- OsAPY conserved motif distribution. Ten varying-colored boxes represent the motifs. The legend below shows the corresponding motif’s protein sequence. c- Gene structure of APYs. Yellow color boxes specify exons, and introns are depicted by lines. The exon-intron lengths can be calculated following the scale shown below.
Fig 2.
Phylogenetic analysis of the APYs in rice, Arabidopsis, peanut and wheat.
The MEGA X software was employed to generate a phylogenetic tree by utilizing the maximum likelihood approach along with 1000 bootstrap replications. Genes were divided into three different groups, each marked with a different color.
Fig 3.
Duplication events of OsAPYs within rice genome.
Duplication of the OsAPYs was specified by the red lines, and the one among the whole rice genome was denoted by the gray lines. TBTools was used to create the circle plot.
Table 2.
Duplication event between rice APY genes and other species.
Fig 4.
Schematic diagram of predicted cis-elements in OsAPY promoter region.
Upstream nucleotides of the translation initiation point are shown by the scale at the end. Different colored boxes depict different cis-regulatory elements.
Table 3.
List of cis-regulatory elements (CREs) found in the 5′ UTR region of the OsAPYs.
Fig 5.
Distribution of OsAPYs throughout the rice genome.
The figure was generated using MapChart. Chromosome number is presented on top. C depicts the centromere position. The position of every gene can be easily compared following the reference chromosome at the left.
Fig 6.
Identification of potential miRNAs targeting OsAPY genes.
Visual depiction of miRNA-OsAPY interaction is generated via Cytoscape. Arrows depict the regulatory relationship, elliptical shapes of different colors represent the OsAPYs, and the miRNAs are shown in ash-colored boxes.
Fig 7.
Analysis of APY-targeting miRNA expression in different abiotic stresses and tissues.
The expression profile of each miRNA can be analyzed following the scale at the right. A heat map was generated using GraphPad Prism 9.0.0.
Table 4.
Distribution of single amino acid polymorphisms (SAPs) throughout the APYs of chosen rice varieties.
Fig 8.
Secondary structure analysis of the nine rice APYs.
Secondary structures were generated using the STRIDE program, and the corresponding positions of membrane-spanning motifs were identified via the TMHMM server. The cross-membrane domains were marked with a black colored box. Legend at the bottom-right side shows the icon for each secondary structure.
Fig 9.
Tertiary structure analysis of the nine rice APYs.
The online tool Robetta was used to construct the tertiary structure, and these structures were visualized via Biovia Discovery Studio Visualizer.
Fig 10.
Protein-ligand residual interaction analysis of OsAPYs with ATP depicted by aqua and red color, respectively.
Illustration of bonds: carbon-hydrogen bonds- snowy mint lines, conventional hydrogen bonds- green lines, pi-cation bonds- black lines, unfavorable bumps- red lines, unfavorable negative-negative interactions- yellow lines, attractive charge interactions- orange lines, pi-donor hydrogen bonds- deep brown lines, pi-sigma bonds- light purple lines, unfavorable acceptor- acceptor interactions- navy blue lines, pi-alkyl bonds- magenta lines, unfavorable donor-donor interactions- pink lines, pi-anion bonds- deep-sea blue lines, pi-lone pair interactions- lemon green lines, salt bridge- purple lines.
Fig 11.
Heatmap of the expression pattern of the OsAPY genes according to tissue type.
The RNA-seq data collected from the Rice Genome Annotation Project was used to conduct expression analysis. The heat map was generated using GraphPad Prism 9.0.0. Expression profile can be analyzed following the bottom located scale. The red boxes represent high expression rates, greens depict low expression rates, and black boxes signify moderate expression levels.
Fig 12.
Heatmap of OsAPY expression profile in response to various biotic stress conditions.
Expression analysis was done utilizing the RNA-seq values of relative expression from GENEVESTIGATOR and using GraphPad Prism 9.0.0, the heat map was generated. The expression profile could be analyzed according to the scale just at the base. The red boxes represent upregulated expression; greens depict downregulated expression, and black boxes signify no change in expression levels.
Fig 13.
Heatmap of OsAPY expression profile in response to various abiotic stress conditions.
Expression analysis was done using the RNA-seq values of relative expression from GENEVESTIGATOR. The heat map was generated using GraphPad Prism 9.0.0. The expression profile could be analyzed according to the scale just at the base. The red boxes represent upregulated expression; greens depict downregulated expression, and black boxes signify no change in expression levels.
Fig 14.
Analysis of relative expression of OsAPYs under various abiotic stresses.
The Y-axis depicts the relative expression of each gene analyzed via RT-qPCR in the rice plant leaves. The X-axis indicates the various abiotic stress conditions under which the relative expression analysis was done. Technical replication was used to find the mean value of expression at different treatments. MS Office 365 and GraphPad Prism 9.0.0 were used to analyze the data. A one-way ANOVA, followed by a Bonferroni post hoc test, was used to assess the significant differences (P ≤ 0.05). To represent the significant differences, different means were labeled with the different number of asterisks (*). *, **, and *** represent the various level of significance (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 respectively).