The Coordinated Upregulated Expression of Genes Involved in MEP, Chlorophyll, Carotenoid and Tocopherol Pathways, Mirrored the Corresponding Metabolite Contents in Rice Leaves during De-Etiolation

Abstract

Light is an essential regulator of many developmental processes in higher plants. We investigated the effect of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1/2 genes (OsHDR1/2) and isopentenyl diphosphate isomerase 1/2 genes (OsIPPI1/2) on the biosynthesis of chlorophylls, carotenoids, and phytosterols in 14-day-old etiolated rice (Oyza sativa L.) leaves during de-etiolation. However, little is known about the effect of isoprenoid biosynthesis genes on the corresponding metabolites during the de-etiolation of etiolated rice leaves. The results showed that the levels of α-tocopherol were significantly increased in de-etiolated rice leaves. Similar to 1-deoxy-D-xylulose-5-phosphate synthase 3 gene (OsDXS3), both OsDXS1 and OsDXS2 genes encode functional 1-deoxy-D-xylulose-5-phosphate synthase (DXS) activities. Their expression patterns and the synthesis of chlorophyll, carotenoid, and tocopherol metabolites suggested that OsDXS1 is responsible for the biosynthesis of plastidial isoprenoids in de-etiolated rice leaves. The expression analysis of isoprenoid biosynthesis genes revealed that the coordinated expression of the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, chlorophyll, carotenoid, and tocopherol pathway genes mirrored the changes in the levels of the corresponding metabolites during de-etiolation. The underpinning mechanistic basis of coordinated light-upregulated gene expression was elucidated during the de-etiolation process, specifically the role of light-responsive cis-regulatory motifs in the promoter region of these genes. In silico promoter analysis showed that the light-responsive cis-regulatory elements presented in all the promoter regions of each light-upregulated gene, providing an important link between observed phenotype during de-etiolation and the molecular machinery controlling expression of these genes.

Keywords: carotenoids; coordinated gene expression; isoprenoids; light-upregulated genes; rice (Oryza sativa L.); secondary metabolites.

Figure 1

Tocopherol composition and content in the etiolated leaves of 14-day-old rice plants during de-etiolation. Values represent the mean of five biological replicates and bars represent standard deviations. Asterisks indicate statistically significant differences compared with the control (0 h illumination) according to the Student’s t-test: * p < 0.05.

Figure 2

Functional analysis of the OsDXS gene family. (A) Color complementation and enhanced β-carotene accumulation due to the expression of OsDXS1, OsDXS2, and OsDXS3, respectively, in E. coli engineered for β-carotene biosynthesis. The plate was divided into four sections, which were inoculated separately with bacteria carrying pACCAR16∆crtX and pUC8-OsDXS1 (16∆crtX-OsDXS1), pACCAR16∆crtX and pUC8-OsDXS2 (16∆crtX-OsDXS2), pACCAR16∆crtX and pUC8-OsDXS3 (16∆crtX-OsDXS3), or pACCAR16∆crtX and pUC8 (empty vector as a control) (16∆crtX-pUC8). (B) Production of β-carotene production by E. coli strains expressing OsDXS1/2/3. Lane 16∆crtX-pUC8, E. coli strain expressing pACCAR16ΔcrtX and pUC8; Lane 16∆crtX-OsDXS1, E. coli strain expressing pACCAR16ΔcrtX and pUC8-OsDXS1; Lane 16∆crtX-OsDXS2, E. coli strain expressing pACCAR16ΔcrtX and pUC8-OsDXS2; Lane 16∆crtX-OsDXS3, E. coli strain expressing pACCAR16ΔcrtX and pUC8-OsDXS3. Values represent the mean of five biological replicates and error bars represent standard deviations. Double asterisks indicate highly significant differences compared with the control (p < 0.01, Student’s t-test).

Figure 3

Relative expression levels of the MEP pathway and the MVA pathway genes in etiolated rice leaves during de-etiolation at various times after the onset of irradiation with white light.

Figure 4

Relative expression levels of carotenoid pathway genes in etiolated rice leaves during de-etiolation at different times after the onset of irradiation with white light.

Figure 5

Relative expression levels of tocopherol pathway genes in etiolated leaves during de-etiolation at different times after the onset of irradiation with white light. Abbreviations: DMGGBQ, 2,3-dimethyl-6-geranylgeranylbenzoquinol; DMPBQ, 2,3-dimethyl-6-phytylbenzoquinol; MGGBQ, 2-methyl-6-geranylgeranylbenzoquinol; MPBQ, 2-methyl-6-phytylbenzoquinol; OsGGR, geranylgeranyl reductase gene; OsHGGT, rice homogentisate geranylgeranyltransferase gene; OsVTE1, tocopherol cyclase gene; OsVTE2, homogentisate phytyltransferase gene; OsVTE4, γ-tocopherol methyltransferase gene; OsVTE5, phytol kinase gene; PPP, phytyl pyrophosphate; α-Toc, α-Tocopherol; β-Toc, β-Tocopherol; γ-Toc, γ-Tocopherol; δ-Toc, δ-Tocopherol; α-T3, α-Tocotrienol; β-T3, β-Tocotrienol; γ-T3, γ-Tocotrienol; δ-T3, δ-Tocotrienol.

Figure 6

Relative expression levels of chlorophyll biosynthesis pathway genes in etiolated rice leaves during de-etiolation at different times after the onset of irradiation with white light.