Transcript profiling of the anoxic rice coleoptile

Abstract

Rice (Oryza sativa) seeds can germinate in the complete absence of oxygen. Under anoxia, the rice coleoptile elongates, reaching a length greater than that of the aerobic one. In this article, we compared and investigated the transcriptome of rice coleoptiles grown under aerobic and anaerobic conditions. The results allow drawing a detailed picture of the modulation of the transcripts involved in anaerobic carbohydrate metabolism, suggesting up-regulation of the steps required to produce and metabolize pyruvate and its derivatives. Sugars appear to play a signaling role under anoxia, with several genes indirectly up-regulated by anoxia-driven sugar starvation. Analysis of the effects of anoxia on the expansin gene families revealed that EXPA7 and EXPB12 are likely to be involved in rice coleoptile elongation under anoxia. Genes coding for ethylene response factors and heat shock proteins are among the genes modulated by anoxia in both rice and Arabidopsis (Arabidopsis thaliana). Identification of anoxia-induced ethylene response factors is suggestive because genes belonging to this gene family play a crucial role in rice tolerance to submergence, a process closely related to, but independent from, the ability to germinate under anoxia. Genes coding for some enzymes requiring oxygen for their activity are dramatically down-regulated under anoxia, suggesting the existence of an energy-saving strategy in the regulation of gene expression.

Figure 1.

Effects of anoxia on rice coleoptile growth and ADH gene expression. A, Growth pattern of rice coleoptiles in air and anoxia; rice seeds were germinated in air and anoxia and the coleoptile length recorded daily. Data are means of 20 replicates ±sd. B, Pattern of expression of ADH1 (Os11g10480) and ADH2 (Os11g10510) genes in coleoptiles from rice seeds germinated in air and anoxia. Relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at day 2). Data are means of three replicates ±sd. C, Growth pattern of rice coleoptiles and roots from seedlings germinated under aerobic conditions for 4 d and then transferred to anoxia for an additional 48-h period; the coleoptile and root length were recorded during 48-h anoxic treatment as well as in air. Data are means of 20 replicates ±sd. D, Pattern of expression of ADH1 and ADH2 genes in the coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). E, Pattern of expression of ADH1 and ADH2 genes in the leaves, internodes, and roots from rice plants grown in air for 20 d and then transferred to anoxia for an additional 3- to 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values).

Figure 2.

Effects of anoxia on carbohydrate metabolism in rice coleoptiles. Data mining of the transcriptome of aerobic and anoxic coleoptiles allowed the identification of the genes involved in starch and Suc metabolism, sugar transport, and glycolysis. Genes showing a statistically significant change in expression when the aerobic dataset was compared with the anoxic dataset (see “Materials and Methods”) are reported on the metabolic pathway shown in this figure. Dotted lines summarize metabolic steps catalyzed by several enzymes. Red arrows highlight the metabolic steps that, based on transcripts level changes, are strongly up-regulated under anoxia, whereas blue arrows indicate down-regulation. It should be remarked that increased mRNA level relative to an enzymatic step does not necessarily imply increased activity of the enzyme in vivo. The fold change (nX, air versus anoxia) is shown together with the gene abbreviation (see text). Fold changes are means of two biological replicates.

Figure 3.

Effects of anoxia on α-amylase gene expression. A, Pattern of expression of the RAMY3D (Os08g36910) gene in coleoptiles from rice seeds germinated in air and anoxia; relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown (REL, 1 = expression data from the aerobic coleoptile at day 2); data are means of three replicates ±sd. B, Pattern of expression of the RAMY3D gene in the coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). C, Starch, Suc, Fru, and Glc content in the coleoptiles from rice seeds germinated in air and anoxia. Data are mean ± sd of three replicates. D, Effects of Glc (100 mm) and mannitol (100 mm, used as an osmotic control) on the expression of the RAMY3D gene in the coleoptiles dissected from 4-d-old aerobically germinated seedlings; REL, measured by real-time RT-PCR, is shown in the graph (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates ±sd. E, Pattern of expression of the RAMY3D gene in the leaves, internodes, and roots from rice plants grown in air for 20 d and then transferred to anoxia for an additional 3- to 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). F, Starch, Suc, Fru, and Glc content in leaves, internodes, and roots from 20-d-old rice plants treated under anoxia for 3 to 6 h. Data are means of three replicates (±sd did not exceed 20% of the reported data).

Figure 4.

Effects of anoxia on expansin gene expression. A, Pattern of expression of the expansin genes differentially regulated by anoxia; data are expressed as a heat map showing the signal intensity from the microarray experiment (data are means of two GeneChip experiments) performed on coleoptiles from rice seeds germinated in air and anoxia for 4 d. Fold change is shown on the right side of the heat map (FDR P value < 0.01). TIGR gene codes are as follows: EXPA2 = Os01g60770; EXPA7 = Os03g60720; EXPB2 = Os10g40710; EXPB6 = Os10g40700; EXPB11 = Os02g44108; and EXPB12 = Os03g44290. B, Pattern of expression of expansin genes EXPA2, EXPA4 (Os05g39990), EXPA7, and EXPB12 in the coleoptiles from rice seeds germinated in air and anoxia; relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown (REL, 1 = expression data from the aerobic coleoptile at day 2); data are means of three replicates ±sd. C, Pattern of expression of the expansin genes EXPA2, EXPA4, EXPA7, and EXPB12 in the coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). D, Pattern of expression of the expansin genes EXPA2, EXPA4, EXPA7, and EXPB12 in the leaves, internodes, and roots from rice plants grown in air for 20 d and then transferred to anoxia for an additional 3- to 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values).

Figure 5.

Effects of anoxia on HSP gene expression. A, Pattern of expression of HSP genes differentially regulated by anoxia; data are expressed as a heat map showing the signal intensity from the microarray experiment (data are means of two GeneChip experiments) performed on coleoptiles from rice seeds germinated in air and anoxia for 4 d. HSP categories (left side of the heat map) are as described by Wang et al. (2004). Fold change is shown on the right side of the heat map (FDR P value < 0.01). B, Pattern of expression of Os02g52150 in the coleoptiles from rice seeds germinated in air and anoxia; relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown (REL, 1 = expression data from the aerobic coleoptile at day 2); data are means of three replicates ±sd. C, Pattern of expression of At5g59720 in Arabidopsis shoots and roots (10-d-old seedlings germinated in air and then transferred to anoxia for up to 6 h). REL, measured by real-time RT-PCR, is shown in the graph (REL, 1 = expression data from the Arabidopsis aerobic leaves at t = 0); data are means of three replicates ±sd. D, Pattern of expression of Os02g52150 in the coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown in the heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). E, Effects of Glc (100 mm) and mannitol (100 mm, used as an osmotic control) on the expression of the HSP gene Os02g52150 in the coleoptiles dissected from 4-d-old aerobically germinated seedlings; REL, measured by real-time RT-PCR, is shown in the graph (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates ±sd. F, Pattern of expression of the HSP gene Os02g52150 in the leaves, internodes, and roots from rice plants grown in air for 20 d and then transferred to anoxia for an additional 3- to 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values).

Figure 6.

Effects of anoxia on ERF gene expression. A, Pattern of expression of ERF genes differentially regulated by anoxia; data are expressed as a heat map showing the signal intensity and fold change from the microarray experiment (data are means of two GeneChip experiments) performed on the coleoptiles from rice seeds germinated in air and anoxia for 4 d. ERF categories (left side of the heat map) are as described by Nakano et al. (2006). Fold change is shown on the right side of the heat map (FDR P value < 0.01). See Nakano et al. (2006) or Supplemental Table S2 for corresponding TIGR gene codes. B, Pattern of expression of the ERF genes in coleoptiles from rice seeds germinated in air and anoxia; relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown (REL, 1 = expression data from the aerobic coleoptile at day 2); data are means of three replicates ±sd. C, Pattern of expression of rice ERF genes in the coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). D, Pattern of expression of the ERF genes in Arabidopsis seedlings (4-d-old seedlings germinated in air and then transferred to anoxia for up to 6 h). REL, measured by real-time RT-PCR, is shown in the graphs (REL, 1 = expression data from the Arabidopsis aerobic seedlings at t = 0); data are means of three replicates ±sd. E, Pattern of expression of rice ERF genes in the leaves, internodes, and roots from rice plants grown in air for 20 d and then transferred to anoxia for an additional 3- to 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). F, Effects of Glc (100 mm) and mannitol (100 mm, used as an osmotic control) on the expression of rice ERF genes in coleoptiles dissected from 4-d-old aerobically germinated seedlings; REL, measured by real-time RT-PCR, is shown in the graph (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates ±sd.

Figure 7.

Effects of anoxia on XIP gene expression. A, Pattern of expression of XIP genes differentially regulated by anoxia; data are expressed as a heat map showing the signal intensity from the microarray experiment (data are means of two GeneChip experiments) performed on coleoptiles from rice seeds germinated in air and anoxia for 4 d. Fold change is shown on the right side of the heat map (FDR P value < 0.01). B, Pattern of expression of the Os11g47560 gene in coleoptiles from rice seeds germinated in air and anoxia; relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown in the graph (REL, 1 = expression data from the aerobic coleoptile at day 2); data are means of three replicates ±sd. C, Pattern of expression of the Os11g47560 gene in coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). D, Pattern of expression of the Os11g47560 gene in the leaves, internodes, and roots from rice plants grown in air for 20 d and then transferred to anoxia for an additional 3- to 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). E, Effects of Glc (100 mm) and mannitol (100 mm, used as an osmotic control) on the expression of the Os11g47560 gene in the coleoptiles dissected from 4-d-old aerobically germinated seedlings; REL, measured by real-time RT-PCR, is shown in the graph (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates ±sd.

Figure 8.

Effects of anoxia on P450 gene expression. A, Pattern of expression of P450 genes differentially regulated by anoxia; data are expressed as a heat map showing the signal intensity from the microarray experiment (data are means of two GeneChip experiments) performed on the coleoptiles from rice seeds germinated in air and anoxia for 4 d. Fold change is shown on the right side of the heat map (FDR P value < 0.01). B, Pattern of expression of the Os11g18570 gene in the coleoptiles from rice seeds germinated in air and anoxia; relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown in the graph (REL, 1 = expression data from the aerobic coleoptile at day 2); data are means of three replicates ±sd. C, Pattern of expression of the Os11g18570 gene in coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). D, Pattern of expression of the Os11g18570 gene in the leaves, internodes, and roots from the rice plants grown in air for 20 d and then transferred to anoxia for an additional 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values).

Figure 9.

Effects of anoxia on catalase gene expression. A, Pattern of expression of the Os02g02400 gene in the coleoptiles from rice seeds germinated in air and anoxia; relative expression level (REL), measured by real-time reverse transcription (RT)-PCR, is shown in the graph (REL, 1 = expression data from the aerobic coleoptile at day 2); data are means of three replicates ±sd. B, Pattern of expression of the Os02g02400 gene in the coleoptiles and roots from rice seeds germinated in air for 4 d and then transferred to anoxia for an additional 48-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic coleoptile at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values). C, Pattern of expression of the Os02g02400 gene in the leaves, internodes, and roots from rice plants grown in air for 20 d and then transferred to anoxia for an additional 6-h period; REL, measured by real-time RT-PCR, is shown as a heat map (REL, 1 = expression data from the aerobic leaf at the beginning of the experiment, t = 0); data are means of three replicates (see Supplemental Table S3 for ±sd values).