Interaction between Rice MYBGA and the Gibberellin Response Element Controls Tissue-Specific Sugar Sensitivity of -Amylase Genes

Supplemental Data

Files in this Data Supplement:

• Supplemental Table 1 - Condition and efficiency of RTQ-PCR.
• Supplemental Table 2 - Determination of αAmy3,ƒtalphaƒ«Amy8 and MYBGA mRNA levels in rice embryos and endosperms during germination and seedling development by real-time quantitative RT-PCR analysis.
• Supplemental Text
• Supplemental Figure 1 (Revised) - Expression of αAmy3 and αAmy8 correlates with glucose and GA levels in germinating seeds and developing seedlings. (A) A schematic diagram showing the morphological progression of seedling development, time course of seed imbibition (2 h after hydration), germination (between 2 h and day 1 after imbibition) and seedling elongation (days 2 to 7 after imbibition). The inset in the upper panel shows the enlarged photograph of germinating seeds 0 to 2 days after imbibition. (B) Total soluble sugar concentrations in embryos and endosperms during the time course of germination and seedling development. Embryos and endosperms were separately collected from intact seeds after germination. Fifteen seeds were used for each time point and the experiment was done in triplicate. Sugar concentration was determined as described (Yu et al., 1996). (C) Accumulation of mRNA in embryos and endosperms. Rice seeds were germinated at 30°C in the dark for various lengths of time. Young shoots and roots were removed. Total RNA was purified from embryos and endosperms separately and subjected to real-time quantitative RT-PCR analysis. RNA levels of αAmy3 (panel 1), αAmy8 (panel 2) and MYBGA (panel 3) were quantified and normalized to the level of 18S rRNA for each time point. Error bars indicate the standard error of two replicate experiments.
• Supplemental Figure 1-Revised - Expression of αAmy3 and αAmy8 correlates with glucose and GA levels in germinating seeds and developing seedlings. (A) A schematic diagram showing the morphological progression of seedling development, time course of seed imbibition (2 h after hydration), germination (between 2 h and day 1 after imbibition) and seedling elongation (days 2 to 7 after imbibition). The inset in the upper panel shows the enlarged photograph of germinating seeds 0 to 2 days after imbibition. (B) Total soluble sugar concentrations in embryos and endosperms during the time course of germination and seedling development. Embryos and endosperms were separately collected from intact seeds after germination. Fifteen seeds were used for each time point and the experiment was done in triplicate. Sugar concentration was determined as described (Yu et al., 1996). (C) Accumulation of mRNA in embryos and endosperms. Rice seeds were germinated at 30°C in the dark for various lengths of time. Young shoots and roots were removed. Total RNA was purified from embryos and endosperms separately and subjected to real-time quantitative RT-PCR analysis. RNA levels of αAmy3 (panel 1), αAmy8 (panel 2) and MYBGA (panel 3) were quantified and normalized to the level of 18S rRNA for each time point. Error bars indicate the standard error of two replicate experiments.
• Supplemental Figure 2 - Transient expression assays demonstrated that GA non-responsive αAmy3 SRC is glucose sensitive in both embryos and endosperms while GA responsive αAmy8 SRC/GARC is glucose sensitive only in embryos. (A) Rice embryos and endosperms were co-transfected with plasmids containing αAmy3 SRC-Luc or αAmy8 SRC/GARC-Luc construct. Transfected embryos were incubated for 24 h in a medium containing 10 μM GA (+GA), lacking GA (-GA) or containing 20 μM ABA (+ABA). Transfected endosperms were incubated for 3 days in a buffer containing 10 μM GA (+GA) or lacking GA (-GA). (B) Rice embryos and endosperms were co-transfected with plasmids containing αAmy3 SRC-Lucor αAmy8 SRC/GARCLuc construct, incubated for 24 h in a medium or buffer containing glucose (+Glc) or mannitol (-Glc), and luciferase activity determined. X indicates fold repression or induction of luciferase activity by glucose, GA or ABA. Error bars indicate the standard error of three replicate experiments for each construct.
• Supplemental Figure 3 - In transgenic rice seeds, GA non-responsive αAmy3 is glucose sensitive in both embryos and endosperms while GA responsive αAmy8 SRC/GARC is glucose sensitive only in embryos. Embryos and endosperms were separately collected from T2 seeds of transgenic lines carrying the αAmy3 (-990 to +181)-Luc or αAmy8 (-318 to -89)-35Smp-SRC/GARC-Luc construct. Ten isolated embryos or endosperms from one transgenic line were incubated (A) in a buffer containing 10 μM GA (+GA), lacking GA (-GA) or containing 20 μM ABA or (B) in a medium or buffer containing 100 mM mannitol (-Glc) or 100 mM glucose (+Glc) at 30°C for 24 h, and luciferase activity determined. X indicates fold repression or induction of luciferase activity by glucose, GA or ABA. This experiment was repeated with 5 independent transgenic lines with similar results.
• Supplemental Figure 4 - Glucose repression overrides GA activation of αAmy8 SRC/GARC in embryos while GA activation overrides glucose repression of the same promoter in endosperms. Rice embryos or endosperms were co-transfected with plasmids containing αAmy8 SRC/GARC-Luc construct, divided into four groups, each group was incubated in the presence (+) or absence (-) of glucose with or without GA for 24 h, and luciferase activity determined. The value of luciferase activity in embryos or endosperms in the presence of glucose but absence of GA was assigned as 1X, and other values relative to this value were then calculated. Error bars indicate the standard error of three replicates for each construct.
• Supplemental Figure 5 - TA box enhances promoter activity and glucose sensitivity of αAmy8 SRC/GARC in embryos only. (A) Comparison of nucleotide sequences among the wild type TA boxes of αAmy3 and αAmy8 and modified αAmy8 SRC/GARC containing duplicated TA box (2TA) or mutated TA box (mTA). Nucleotides for the wild type TA box are underlined and for substituted sequences are in lowercase letters. (B) Rice embryos and (C) endosperms were co-transfected with plasmids containingƒnαAmy8 SRC/GARC-Luc, αAmy8 SRC/GARC(2TA)-Luc or αAmy8 SRC/GARC(mTA)-Luc construct, incubated for 24 h in a medium or buffer containing glucose (+Glc) or mannitol (-Glc), and luciferase activity determined. X indicates fold repression of luciferase activity by glucose. Error bars indicate the standard error of three replicate experiments for each construct.
• Supplemental Figure 6 - In transgenic endosperms, G box does not enhance glucose sensitivity of αAmy8 SRC/GARC. T2 seeds of transgenic lines carrying the αAmy8 SRC/GARC-Luc or αAmy8 SRC/GARC(+G)-Luc construct were obtained. (A) embryos and (B) endosperms from ten seeds of one transgenic line were separately collected, incubated at 30°C for 24 h in a medium or buffer containing 100 mM glucose (+Glc) or mannitol(-Glc), and luciferase activity determined. X indicates fold repression of luciferase activity by glucose. Error bars indicate the standard error of three replicate experiments for each transgenic line. This experiment was repeated with 5 independent transgenic lines with similar results.