Gene expression profiles deciphering rice phenotypic variation between Nipponbare (Japonica) and 93-11 (Indica) during oxidative stress

Abstract

Rice is a very important food staple that feeds more than half the world's population. Two major Asian cultivated rice (Oryza sativa L.) subspecies, japonica and indica, show significant phenotypic variation in their stress responses. However, the molecular mechanisms underlying this phenotypic variation are still largely unknown. A common link among different stresses is that they produce an oxidative burst and result in an increase of reactive oxygen species (ROS). In this study, methyl viologen (MV) as a ROS agent was applied to investigate the rice oxidative stress response. We observed that 93-11 (indica) seedlings exhibited leaf senescence with severe lesions under MV treatment compared to Nipponbare (japonica). Whole-genome microarray experiments were conducted, and 1,062 probe sets were identified with gene expression level polymorphisms between the two rice cultivars in addition to differential expression under MV treatment, which were assigned as Core Intersectional Probesets (CIPs). These CIPs were analyzed by gene ontology (GO) and highlighted with enrichment GO terms related to toxin and oxidative stress responses as well as other responses. These GO term-enriched genes of the CIPs include glutathine S-transferases (GSTs), P450, plant defense genes, and secondary metabolism related genes such as chalcone synthase (CHS). Further insertion/deletion (InDel) and regulatory element analyses for these identified CIPs suggested that there may be some eQTL hotspots related to oxidative stress in the rice genome, such as GST genes encoded on chromosome 10. In addition, we identified a group of marker genes individuating the japonica and indica subspecies. In summary, we developed a new strategy combining biological experiments and data mining to study the possible molecular mechanism of phenotypic variation during oxidative stress between Nipponbare and 93-11. This study will aid in the analysis of the molecular basis of quantitative traits.

Figure 1. Differential responsiveness of rice japonica variety (Nipponbare) and indica variety (93-11) to MV treatment.

A. 93-11 (up) and Nipponbare (down) sprouted seeds were mock-treated (water) or treated with 10 µM MV. B. The chlorophyll content of seedling plants after treatment.

Figure 2. Summary of the transcription profiles of different rice cultivars (Nipponbare vs 93-11) under 10 µM MV treatment.

A. The Venn diagram illustrates probe level expression affected by two factors -cultivar (Nipponbare vs. 93-11) and treatment (10 µM MV vs. mock). The number of probes showing a significant change at P< = 0.01 is shown. B. The cultivar and treatment effects of Core Intersectional Probesets (CIPs). The blue circles at top-right represent the CIPs preferentially expressed in Nipponbare and up-regulated by MV treatment. The brown triangles at bottom-left represent the CIPs preferentially expressed in 93-11 and down-regulated by MV treatment. The yellow squares at upper-left represent the CIPs preferentially expressed in Nipponbare and down-regulated by MV treatment. The green diamonds at bottom-right represents the CIPs preferentially expressed in 93-11 and up-regulated by MV treatment.

Figure 3. The expression pattern of the Core Intersectional Probesets (CIPs) in highlighted gene superfamilies.

A. The probe sets were derived from peroxidases, GSTs, UGTs, P450s, UDP-glucoronosyl transferase, and NB-ARC domain-containing proteins. B. The probe sets were from transporters, kinases, and TFs. The color scale (representing expression level) is shown at bottom-right. Red indicates a higher expression level, whereas blue indicates a lower expression level. White indicates the average expression level.

Figure 4. InDel analysis of selected genes between indica and japonica varieties.

Lanes 1–5: PCR products obtained using genomic DNA of indica varieties: 93-11, 37760, 03A-11, TP34, and ShuiYuan349, respectively. Lanes 6–10: PCR products obtained using genomic DNA of japonica varieties: Nipponbare, IR66746-76-3-2, REIMEL, ShangZhou10, and YunFeng7, respectively. LOC_Os11g12340–disease resistance protein RPM1; LOC_Os02g56700–Cinnamoyl-CoA reductase (OsCCR10); LOC_Os11g10550–NBS-LRR disease resistance protein; LOC_Os07g33690–NBS-LRR type disease resistance protein Hom-F; LOC_Os10g38360–glutathione S-transferase.

Figure 5. The sketch map for GSTs in the 20.15 to 20.17 Mb region of chromosome 10.

This map highlights three glutathione S-transferase genes within the CIP list, LOC_Os10g38340, LOC_Os10g38350, and LOC_Os10g38360, including their locations, transcript direction, the positions of indel regions different between japonica and indica, motifs in promoter regions, and expression patterns.

Figure 6. The expression pattern of selected phenylpropanoid pathway related genes in 10 µM MV treated seedling plants from rice japonica variety (Nipponbare) and indica variety (93-11).

The black bars indicate relative expression (fold-change by real-time PCR) of the gene in Nipponbare samples. The grey bars indicate relative expression (fold-change by real-time PCR) of the gene in 93-11 samples. Error bars represent the standard error of three replicates. LOC_Os05g12240–Chalcone synthase 8 (CHS8); LOC_Os10g12080–Cytochrome P450 98A15p (C3H); LOC_Os02g56460–Cinnamoyl-CoA reductase (OsCCR1); LOC_Os02g56700–Cinnamoyl-CoA reductase (OsCCR10).