Integrated transcriptome and metabolome analysis revealed that flavonoids enhanced the resistance of Oryza sativa against Meloidogyne graminicola

Abstract

Rice is a crucial food crop worldwide, but its yield and quality are significantly affected by Meloidogyne graminicola is a root knot nematode. No rice variety is entirely immune to this nematode disease in agricultural production. Thus, the fundamental strategy to combat this disease is to utilize rice resistance genes. In this study, we conducted transcriptome and metabolome analyses on two rice varieties, ZH11 and IR64. The results indicated that ZH11 showed stronger resistance than IR64. Transcriptome analysis revealed that the change in gene expression in ZH11 was more substantial than that in IR64 after M. graminicola infection. Moreover, GO and KEGG enrichment analysis of the upregulated genes in ZH11 showed that they were primarily associated with rice cell wall construction, carbohydrate metabolism, and secondary metabolism relating to disease resistance, which effectively enhanced the resistance of ZH11. However, in rice IR64, the number of genes enriched in disease resistance pathways was significantly lower than that in ZH11, which further explained susceptibility to IR64. Metabolome analysis revealed that the metabolites detected in ZH11 were enriched in flavonoid metabolism and the pentose phosphate pathway, compared to IR64, after M. graminicola infection. The comprehensive analysis of transcriptome and metabolome data indicated that flavonoid metabolism plays a crucial role in rice resistance to M. graminicola infection. The content of kaempferin, apigenin, and quercetin in ZH11 significantly increased after M. graminicola infection, and the expression of genes involved in the synthetic pathway of flavonoids also significantly increased in ZH11. Our study provides theoretical guidance for the precise analysis of rice resistance and disease resistance breeding in further research.

Keywords: Meloidogyne graminicola; flavonoids; metabolome; resistance of Oryza sativa; transcriptome.

Figure 1

The relative amount of M. graminicola in infected roots of ZH11 and IR64. (A) shows the RT-qPCR results of the amount of M. graminicola in the infected roots of ZH11 and IR64. (B, C) show M. graminicola infection after 7 days; the roots of ZH11 had no root knots, but the roots of IR64 produced root knots, which were enlarged for the clarity of the display. (D, E) show that the roots of ZH11 and IR64 are infected by M. graminicola.

Figure 2

Analysis of transcriptome data from the rice roots for four groups: IR64-ck, IR64-7d, ZH11-ck, and ZH11-7d. (A) MA plots for pairwise expression analysis of transcriptome data of rice roots between IR64-ck and IR64-7d; (B) MA plots for pairwise expression analysis of transcriptome data of rice roots between ZH11-ck and ZH11-7d; (C) Venn diagram showing the upregulated DEGs and downregulated DEGs in comparison groups ZH11-7d vs ZH11-ck and IR64-7d vs IR64-ck.

Figure 3

GO and KEGG analyses of DEGs in comparison groups ZH11-7d vs ZH11-ck. (A) indicates the GO enrichment of DEGs in comparison group ZH11-7d vs ZH11-ck. (B) indicates the KEGG enrichment of DEGs in carbohydrate metabolism in comparison group ZH11-7d vs ZH11-ck. (C) indicates the KEGG enrichment of DEGs in protein metabolism in comparison group ZH11-7d vs ZH11-ck. (D) indicates the KEGG enrichment of DEGs in plant resistance metabolism in comparison group ZH11-7d vs ZH11-ck.

Figure 4

Overview of the widely targeted metabolome analysis of rice roots from ZH11 and IR64 in response to M. graminicola infection. (A) Heatmap visualization of metabolites. The content of each metabolite was normalized to complete linkage hierarchical clustering. Each example was visualized in a single column, and each metabolite is represented by a single row. Red indicated high abundance metabolites, and green indicated low abundance metabolites. (B) PCA analysis of metabolites showed that the difference in metabolites was mainly due to the difference in rice varieties. (C) The volcano plot indicates the differentially accumulated metabolites (DAMs) between ZH11 and IR64 roots infected by M. graminicola. (D) KEGG enrichment pathways of DAMs. The marked red KEGG terms highlight the significant enrichment metabolism pathways.

Figure 5

DEG and DAM enrichment in KEGG pathways. A P-value was calculated by a hypergeometric test, which indicates the degree of enrichment of DEGs or DAMs. The red arrow indicates metabolites enriched in flavone and flavonol biosynthesis (ko00944) and flavonoid biosynthesis (ko00941), and the green arrow indicates genes enriched in flavone and flavonol biosynthesis (ko00944) and flavonoid biosynthesis (ko00941). The dotted line indicates the size or column height when the p-value is 0.05.

Figure 6

The heatmap shows the content of compounds involved in flavonoids metabolism in healthy roots (ZH11-ck) and infected roots (ZH11-7d) of rice ZH11. Metabolites with increased content during nematode infection were M273T294 (Apiforol), M299T712 (Kaempferide), M269T700 (Apigenin), M283T107 (Quercetin), M255T102_1 ((S)-Pinocembrin), M463T183 (Isoquercitrin), M301T531 (Hesperetin), M610T369 (Rutin), M432T456 (Apigenin 7-O-beta-D-glucoside), M286T419 (Kaempferol). In different samples, the metabolic compounds showed similar states for Pearson clustering.

Figure 7

The metabolic pathway of flavonoids and the expression heatmap of the genes involved in this pathway from O. sativa infected by M. graminicola. PAL: phenylalanine ammonia lyase (Os05g0427400, Os04g0518100, Os02g0627100 and Os04g0518400); C4H: cinnamate 4 hydroxylase (Os02g0467600 and Os05g0320700); TAL: tyrosine ammonia lyase; 4CL: coumaric acid COA ligase (Os08g0245200, Os01g0901500 and Os03g0132000); CHS: chalcone synthetase (Os07g0214900, Os03g0245700 and Os07g0526400); CHI: chalcone isomerase (Os11g0116300); FNSII: flavone synthase II (Os04g0101400); F3’H: flavonoid3’-hydroxylasc (Os10g0320100, Os10g0317900 and Os03g0650200); F3H: flavanone-3-hydroxylase (Os04g0581000); FLS: flavonol synthase (Os02g0767300).

Figure 8

The relative expression of genes involved in the flavonoid biosynthesis in ZH11 roots infected by M. graminicola. Expression level was measured by RT-qPCR and the means ± SE from three independent tests were calculated. Asterisks represent significant differences for the defense-related genes using Duncan’s test. (*p <0.05, **p <0.01).