Comparative transcriptomic and physiological analyses of weedy rice and cultivated rice to identify vital differentially expressed genes and pathways regulating the ABA response

Abstract

Weedy rice is a valuable germplasm resource characterized by its high tolerance to both abiotic and biotic stresses. Abscisic acid (ABA) serves as a regulatory signal in plant cells as part of their adaptive response to stress. However, a global understanding of the response of weedy rice to ABA remains to be elucidated. In the present study, the sensitivity to ABA of weedy rice (WR04-6) was compared with that of temperate japonica Shennong9816 (SN9816) in terms of seed germination and post-germination growth via the application of exogenous ABA and diniconazole, an inhibitor of ABA catabolism. Physiological analysis and a transcriptomic comparison allowed elucidation of the molecular and physiological mechanisms associated with continuous ABA and diniconazole treatment. WR04-6 was found to display higher ABA sensitivity than SN9816, resulting in the rapid promotion of antioxidant enzyme activity. Comparative transcriptomic analyses indicated that the number of differentially expressed genes (DEGs) in WR04-6 seedlings treated with 2 μM ABA or 10 μM diniconazole was greater than that in SN9816 seedlings. Genes involved in stress defense, hormone signal transduction, and glycolytic and citrate cycle pathways were highly expressed in WR04-6 in response to ABA and diniconazole. These findings provide new insight into key processes mediating the ABA response between weedy and cultivated rice.

Figure 1

Germination rate (%) of WR04-6 and SN9816 seeds for different concentrations of ABA or diniconazole. (a) Germination rate (%) of WR04-6 seeds after ABA treatment. (b) Germination rate (%) of SN9816 seeds after ABA treatment. (c) Germination rate (%) of WR04-6 seeds after diniconazole treatment. (d) Germination rate (%) of SN9816 seeds after diniconazole treatment. Different letters above the bars indicate significant differences in the germination rate for different concentrations of ABA or diniconazole (one-way ANOVA. p < 0.05). Error bars represent SEs (n = 3).

Figure 2

The growth and development of WR04-6 and SN9816 seedlings were retarded when they were continuously treated with ABA or diniconazole. (a) Growth of WR04-6 and SN9816 seedlings after 7 d and 15 d of continuous treatment with ABA. Bar = 2 cm. (b) Comparison of plant height (mm) of WR04-6 and SN9816 seedlings after treatment with different concentrations of ABA for 7 days and 15 days. (c) Growth of WR04-6 and SN9816 seedlings with continuous diniconazole treatment for 7 d and 15 d. Bar = 2 cm. (d) Comparison of plant height (mm) of WR04-6 and SN9816 seedlings after treatment with different concentrations of diniconazole for 7 and 15 days. Error bars represent SEs (n = 3). Different letters over the bars indicate treatments that are significantly different (p < 0.05).

Figure 3

Evaluation of MDA concentration and antioxidant enzyme activity in WR04-6 and SN9816 seedlings after treatment with 2 μM ABA or 10 μM diniconazole. (a) MDA content. (b) SOD activity. (c) POD activity. (d) CAT activity. Error bars represent SEs (n = 3). Different letters indicate significant differences between treatments. ns indicates no difference (p < 0.05).

Figure 4

Plot of differentially expressed genes (DEGs) from different comparisons of samples from WR04-6 and SN9816 plants after ABA and diniconazole treatments. (a) Numbers of DEGs resulting from pairwise comparison of samples between WR04-6 and SN9816 after ABA and diniconazole treatments. A in WR_A or SN_A indicates ABA treatment, D in WR_D or SN_D indicates diniconazole treatment, and CK in WR_CK or SN_CK indicates the control group without treatment. (b) Congruence of the up-regulation of DEGs from pairwise comparisons of samples after treatment with ABA or diniconazole in WR04-6 and SN9816 plants. Red represents DEGs uniquely expressed in each treatment for WR04-6 and SN9816 plants; green represents DEGs uniquely expressed in WR_A and WR_D; yellow represents DEGs uniquely expressed in SN_A and SN_D; purple represents DEGs uniquely expressed in WR_A and SN_A; Blue-grey represents DEGs uniquely expressed in WR_D and SN_D; and pink represents DEGs uniquely expressed after both ABA and diniconazole treatments in the two varieties. (c) GO enrichment analysis of the differentially expressed genes presented as a heat map. (d) KEGG enrichment analysis of differentially expressed genes presented as a bubble chart.

Figure 5

DEGs relevant to peroxisome, glutathione metabolism, and hormone signaling after treatment with ABA or diniconazole. (a) Change in transcript expression levels associated with peroxisomes in WR04-6 and SN9816 plants after ABA and diniconazole treatment. (b) Expression patterns of genes involved in the glutathione metabolism pathway in the four pairwise transcriptome comparisons. (c) DEGs involved in hormone signaling in response to ABA and diniconazole treatment in WR04-6 and SN9816 plants. Gene expression levels were transformed by log₂(FPKM) values. Red and blue represent up-regulation and down-regulation, respectively. A in WR_A or SN_A indicates ABA treatment, D in WR_D or SN_D indicates diniconazole treatment, and CK in WR_CK or SN_CK indicates the control group without treatment.

Figure 6

Expression profiles of defense-related genes in WR04-6 and SN9816 plants after exogenous treatments. Comparative analysis of pathogen interactions (a), MAPK signaling pathway transduction (b), and the expression of transcription factor related genes (c) in WR04-6 and SN9816 plants after exogenous treatment. Gene expression levels were transformed by log₂(FPKM) values. Red and blue represent up-regulation and down-regulation, respectively. A in WR_A or SN_A indicates ABA treatment, D in WR_D or SN_D indicates diniconazole treatment, and CK in WR_CK or SN_CK indicates the control group without treatment.

Figure 7

DEGs relevant to glycolytic metabolism and the citrate cycle after treatment with ABA or diniconazole. (a) Change in transcript expression levels associated with glycolysis and citrate cycle pathways in WR04-6 and SN9816 plants after ABA and diniconazole treatment. (b) Expression patterns of genes involved in glycolysis and citrate cycle pathways in the four pairwise transcriptome comparisons.

Figure 8

Comparison of relative expression level of twelve genes by RNA-Seq and RT-qPCR. (a) up-regulated genes; (b) down-regulated genes. Black and red bas represent relative expression level from FPKM and RT-qPCR, respectively. Error bars represent SEs (n = 3).