Global Transcriptome Analysis of Rice Seedlings in Response to Extracellular ATP

doi:10.1016/j.rsci.2025.03.002

Abstract

Abstract:

Herbivorous insects and pathogens cause severe damage to rice tissues, affecting yield and grain quality. Damaged cells trigger downstream defense responses through various signals. Extracellular ATP (eATP), a signaling molecule released during mechanical cell damage, is considered a constitutive damage-associated molecular pattern (DAMP), which is crucial for initiating plant defense responses. Thus, understanding how rice plants cope with DAMPs such as eATP is essential. Here, we found that exogenous ATP affected rice growth and development, cell wall composition, chloroplast development, and cell death. Subsequent global transcriptome analysis revealed that several pathways were involved in the eATP response, including genes related to cell surface receptors, cell wall organization, chlorophyll biosynthesis, heat and temperature stimulation, epigenetic regulation, and reactive oxygen species metabolism. Cell surface receptors, including members of the lectin receptor-like kinases (LecRKs), were found to participate in the eATP response. We further investigated ATP-induced genes in T-DNA activation mutants of OsLecRKs, demonstrating their involvement in eATP signaling in rice. This study confirms a DAMP-mediated transcriptional response in plants and provides novel candidates for advancing resistant rice breeding against insect herbivores and pathogens.

Key words: extracellular ATP, damage-associated molecular pattern, RNA-sequencing, transcriptome analysis, rice

Chaemyeong Lim, Sae Hyun Lee, Haeun Lee, So-Yon Park, Kiyoon Kang, Hyeryung Yoon, Tae-Jin Yang, Gary Stacey, Nam-Chon Paek, Sung-Hwan Cho. Global Transcriptome Analysis of Rice Seedlings in Response to Extracellular ATP[J]. Rice Science, 2025, 32(3): 380-399.

Figures/Tables 8

Fig. 1. Phenotypes of rice seedlings under different extracellular ATP (eATP) concentration treatments. A‒D, Whole-plant phenotype (A), leaf and root morphology (B), total chlorophyll content (C), and shoot and root lengths (D) under different ATP concentrations (0, 1, and 2 mmol/L). Data represent Mean ± SD (n = 8 in C and 10 in D). Different lowercase letters above bars indicate significant differences according to one-way analysis of variance followed by Duncan’s test (P < 0.05). Asterisks denote significant differences compared with mock control (Student’s t-test: **, P < 0.01).E, Cellulose (calcofluor white) and lignin (basic fuchsin) staining in rice leaves treated with 2 mmol/L ATP at 12 d after treatment (DT). Scale bars, 100 µm. BF, Bulliform cell; CC, Collenchyma; PAR, Parenchyma; VB, Vascular bundle.F, Time-course analysis of MPK3/6 phosphorylation in rice leaves treated with 500 µmol/L ATP. Leaves from 5-day-old wild type (WT) seedlings were sprayed with ATP, and phosphorylation was detected using anti-phospho-p44/p42 MAPK antibody. Coomassie brilliant blue (CBB) staining (bottom panel) confirmed equal protein loading. Data represent Mean ± SD from six biological replicates. WT plants (japonica cv. Dongjin) were hydroponically grown in full-strength Yoshida solution for 5 d under long-day (LD) conditions (14 h light at 30 ºC /10 h dark at 25 ºC). Seedlings were then transferred to Yoshida solution containing 0, 1, or 2 mmol/L ATP and maintained under LD conditions for 12 d. Phenotypic analyses were performed at 12 DT. All experiments were repeated three times with consistent results.

Fig. 2. Experimental designs and differentially expressed genes (DEGs) identified in rice plants exposed to eATP treatment. A and B, Venn diagrams of DEGs in leaf (A) and root (B) in different comparisons (treated vs control). Numbers of up- and downregulated genes are indicated numerically and by heatmap in each Venn diagram section. Sampling was conducted at 5 d after germination using leaves or roots of wild type seedlings grown in Yoshida solution in a growth chamber under long-day conditions (14 h of light at 30 ºC/10 h of dark at 25 ºC). Plants were treated 1 mmol/L ATP solutions at 0, 0.5, and 24 h for RNA-seq analysis.

Fig. 3. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in rice leaves and roots. A and B, 0.5 h vs 0 h (A) and 24 h vs 0 h (B) of leaf comparison.C and D, 0.5 h vs 0 h (C) and 24 h vs 0 h (D) of root comparison. padj, Adjusted P-value. The gene number and the adjusted P-value for the top 20 most enriched GO in the differentially up- and downregulated genes are represented by bar size and color, respectively.

Fig. 4. Gene expression patterns of cellulose/lignin-biosynthesis, chlorophyll metabolism, reactive oxygen species (ROS) scavenging, heat response, temperature stimulation, epigenetic pathway, and inorganic ion transport and metabolism related pathways in response to eATP treatment. A‒G, Cellulose synthesis-related genes (A), lignin synthesis-related genes (B), ROS scavenging-related genes (C), chlorophyll metabolism-related genes (D), heat response and temperature stimulation related genes (E), epigenetic pathway related genes (F), and inorganic ion transport and metabolism-related genes (G). Expression fold changes (FC) are presented with a heat map. ‘1’ represents control vs 0.5 h; ‘2’ represents 0.5 h vs 24 h, and ‘3’ represents control vs 24 h.

Fig. 5. Phylogenetic tree, gene expression, and domain compositions of rice LecRKs. A, The maximum likelihood phylogenetic tree with the coding sequence of LecRKs. The classified clades are shown as colored circles. Arabidopsis AtP2K1 and AtP2K2 are indicated by green dots. OsLecRK7 and OsSIT2 are indicated by blue and red dots, respectively.B, Detailed information of Clade III. C, TPM (Transcripts per million) values of LecRK genes according to ATP treatment.

Fig. 6. OsLecRK7 is involved in eATP-mediated chlorophyll biosynthesis and cell wall metabolism. A, Relative transcript levels of OsLecRK7 in the leaves of wild type (WT) and OsLecRK7-D (enhancer-trapped T-DNA insertional activation line) plants in response to 1 mmol/L eATP. B, Schematic representation of the OsLecRK7 locus (Os07g0129900) and its T-DNA insertional activation line OsLecRK7-D. The upside-down triangle indicates the position of the T-DNA insertion (OsLecRK7-D, PFG_3A-52532). Black box represents the exon and two white boxes represent 5′-the untranslated region and 3′-untranslated region. C, Expression of OsLecRK7 was measured in the leaves of WT and OsLecRK7-D plants grown in a plant growth chamber for 10 d. Asterisks indicate statistically significant differences between WT and OsLecRK7-D plants, as determined by Student’s t-test (***, P < 0.001). D, Comparison of WT and OsLecRK7-D plants at 0 and 12 d after eATP treatment (DT).E, Leaf greenness phenotypes of 2 mmol/L ATP-treated leaves of WT and OsLecRK7-D at 12 DT. F, Changes in total chlorophyll content of WT and OsLecRK7-D at 12 DT. G, Expression of OsHEMA, OsCHLH, and OsPORA was responsive to 1 mmol/L ATP treatment. The leaves of 5-day-old WT plants were exposed to 1 mmol/L ATP for 0, 0.5, and 24 h. The relative expression levels of OsHEMA, OsCHLH, and OsPORA were normalized to OsUBQ5 (Os01g22490). Mean and standard deviation values were obtained from three biological replicates. Analysis of variance with Tukey’s multiple comparison test was used to determine statistical significance, with different lowercase letters above bars indicating statistically significant differences (P < 0.05).

Fig. 7. OsSIT2 is involved in chlorophyll biosynthesis mediated by eATP. A, Relative transcript levels of OsSIT2 in the leaves of wild type (WT) and OsSIT2-D (enhancer-trapped T-DNA insertional activation line) plants in response to 1 mmol/L eATP.B, Schematic representation of the OsSIT2 locus (Os04g0531400) and its T-DNA insertional activation line OsSIT2-D. The upside-down triangle indicates the position of the T-DNA insertion (OsSIT2-D, PFG_3A-10378). The black box represents the exon and the two white boxes represent the 5′-untranslated region and 3′-untranslated region. C, Expression of OsSIT2 was measured in the leaves of WT and OsSIT2 plants grown in a plant growth chamber for 10 d. Asterisks indicate statistically significant differences between WT and OsSIT2-D plants, as determined by Student’s t-test (**, P < 0.01). D, Comparison of WT and OsSIT2-D plants at 0 and 12 d after eATP treatment (DT). E, Leaf greenness phenotypes of 2 mmol/L ATP-treated leaves of WT and OsSIT2-D at 12 DT. F, Changes in total chlorophyll content of WT and OsSIT2-D at 12 DT. G, Expression of OsHEMA, OsCHLH, and OsPORA was responsive to 2 mmol/L ATP treatment. The leaves of 5-day-old WT plants were exposed to 1 mmol/L ATP for 0, 0.5, and 24 h. The relative expression levels of OsHEMA, OsCHLH, and OsPORA were normalized to the OsUBQ5 (Os01g22490). Mean and standard deviation values were obtained from three biological replicates. Analysis of variance with Tukey’s multiple comparison test was used to determine statistical significance, with different lowercase letters above bars indicating statistically significant differences (P < 0.05).

Fig. 8. A schematic representation of proposed effects of leaf and root on molecular processes in eATP-treated rice plants. Red and blue arrows indicate upregulated and downregulated genetic pathways in response to eATP, respectively.

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