Interaction between rice MYBGA and the gibberellin response element controls tissue-specific sugar sensitivity of alpha-amylase genes

Abstract

Expression of alpha-amylase genes during cereal grain germination and seedling growth is regulated negatively by sugar in embryos and positively by gibberellin (GA) in endosperm through the sugar response complex (SRC) and the GA response complex (GARC), respectively. We analyzed two alpha-amylase promoters, alphaAmy3 containing only SRC and alphaAmy8 containing overlapped SRC and GARC. alphaAmy3 was sugar-sensitive but GA-nonresponsive in both rice (Oryza sativa) embryos and endosperms, whereas alphaAmy8 was sugar-sensitive in embryos and GA-responsive in endosperms. Mutation of the GA response element (GARE) in the alphaAmy8 promoter impaired its GA response but enhanced sugar sensitivity, and insertion of GARE in the alphaAmy3 promoter rendered it GA-responsive but sugar-insensitive in endosperms. Expression of the GARE-interacting transcription factor MYBGA was induced by GA in endosperms, correlating with the endosperm-specific alphaAmy8 GA response. alphaAmy8 became sugar-sensitive in MYBGA knockout mutant endosperms, suggesting that the MYBGA-GARE interaction overrides the sugar sensitivity of alphaAmy8. In embryos overexpressing MYBGA, alphaAmy8 became sugar-insensitive, indicating that MYBGA affects sugar repression. alpha-Amylase promoters active in endosperms contain GARE, whereas those active in embryos may or may not contain GARE, confirming that the GARE and GA-induced MYBGA interaction prevents sugar feedback repression of endosperm alpha-amylase genes. We demonstrate that the MYBGA-GARE interaction affects sugar feedback control in balanced energy production during seedling growth and provide insight into the control mechanisms of tissue-specific regulation of alpha-amylase expression by sugar and GA signaling interference.

Figure 1.

Schemes of Constructs for αAmy3 and αAmy8 Promoter Analysis. The αAmy3 SRC (–186 to –82 relative to the transcription start site) and αAmy8 SRC/GARC (–318 to –89) were fused upstream of a cauliflower mosaic virus (CaMV) 35S minimal promoter (35Smp)–alcohol dehydrogenase1 (Adh1) intron (In)–Luc–Nos 3′ chimeric gene. Relative positions of cis-acting elements, including GC, G, the GARE, and TA boxes, in promoters are indicated.

Figure 2.

Gain-of-Function Analysis Demonstrates That GARE Confers GA Responsiveness and Glucose Insensitivity to the αAmy8 Promoter in Endosperms. (A) Comparison of nucleotide sequences between the wild-type αAmy8 GARE and the mutated GARE (mGARE). Nucleotides for the GARE are underlined, and substituted nucleotides are shown in lowercase letters. (B) and (C) Rice endosperms were cotransfected with plasmids containing the αAmy8 SRC/GARC-Luc or αAmy8 SRC/GARC(mGARE)-Luc construct, incubated for 3 d in a buffer containing GA (+GA) or lacking GA (−GA) (B) or for 24 h in a buffer containing glucose (+Glc) or mannitol (−Glc) (C), and luciferase activity was determined. X indicates fold induction or repression of luciferase activity by GA or glucose. Error bars indicate se of three replicate experiments for each construct.

Figure 3.

Loss-of-Function Analysis Demonstrates That the GARE Confers GA Responsiveness and Glucose Insensitivity to the αAmy3 Promoter in Endosperms. (A) Comparison of nucleotide sequences among the wild-type αAmy3 SRC (region between the G box and the duplicated TA box) and αAmy3 SRC containing the αAmy8 GARE (+αAmy8 GARE). Nucleotides for the GARE are underlined, substituted sequences are shown in lowercase letters, and flanking sequences 5′ of αAmy8 GARE are indicated by dots. (B) and (C) Rice endosperms were cotransfected with plasmids containing the αAmy3 SRC-Luc or αAmy3 SRC(+αAmy8 GARE)-Luc construct, incubated for 3 d in a buffer containing GA (+GA) or lacking GA (−GA) (B) or for 24 h in a buffer containing glucose (+Glc) or mannitol (−Glc) (C), and luciferase activity was determined. X indicates fold repression or induction of luciferase activity by glucose or GA. Error bars indicate se of three replicate experiments for each construct.

Figure 4.

Glucose Repression Overrides GA Activation of αAmy8 in Embryos, Whereas GA Activation Overrides Glucose Repression of αAmy8 Expression in Endosperms. Rice embryos and endosperms were prepared as for use in the transient expression assays and divided into four groups. Each group was incubated in the presence (+) or absence (−) of glucose with or without GA for 24 h for embryos and for 2 d for endosperms. Total RNA was purified from embryos and endosperms and subjected to gel blot analysis using αAmy3 and αAmy8 gene-specific DNAs (3′ untranslated regions [UTRs]) as probes. Ethidium bromide staining of rRNA was used as the RNA loading control.

Figure 5.

Elimination of Rice MYBGA Expression Permits αAmy8 to Respond to Glucose in Endosperms. (A) Screening of the Tos17-tagged rice mutant gamyb-2 identifies homozygous and heterozygous mutants. PCR with primers Myb12-5′ and Myb11-3′ produced a product of 150 bp from wild-type (wt) rice genomic DNA, and PCR with primers Myb12-5′ and LTR4A produced a product of 250 bp from the rice genomic DNA–Tos17 junction region (mt). M, molecular weight marker; +/+, wild type; +/−, heterozygous for the Tos17 tag; −/−, homozygous for the Tos17 tag. (B) RT-PCR analysis of the expression of αAmy3, αAmy8, MYBGA, and Act1 (as an internal control) in endosperms of wild-type and gamyb-2 mutant rice (cv Nipponbare). Endosperms from the wild type or the gamyb-2 mutant (−/−) were divided into fours groups. Each group containing three endosperms was incubated in the presence (+) or absence (−) of 200 mM glucose with or without 10 μM GA for 2 d. Total RNA was purified from endosperms and subjected to RT-PCR analysis. The number of PCR cycles is indicated at right. ND, not determined. (C) Quantitative (real-time) RT-PCR analysis of αAmy3 and αAmy8 expression using total RNAs prepared as described for (B). RNA levels of αAmy3 and αAmy8 were quantified and normalized to the level of 18S rRNA. The highest αAmy3 mRNA level was assigned as 100, and levels of expression in other samples were calculated relative to this value. The αAmy8 mRNA levels in gamyb-2 endosperms could be 4 orders of magnitude lower than in wild-type endosperms; therefore, relative mRNA levels are shown on log plots. Error bars indicate se for three replicate experiments.

Figure 6.

α-Amylase Gene Promoters Actively Expressed in Endosperms Contain the GARE. Total RNAs were isolated from embryos collected on day 1 (panel 1) and from endosperms collected on day 5 (panel 2) after seed imbibition and used for the synthesis of ³²P-labeled cDNA probes. In the slot-blot analysis, 5 μg of plasmid DNAs containing each α-amylase gene was applied in each slot and hybridized with the cDNA probes. The cis-acting elements in individual α-amylase gene promoters are illustrated in panel 3. These cis-acting elements were numbered relative to the translation start site (ATG) of individual α-amylase genes, and the distances between the GARE and the TA box are indicated (panel 4). G, ACGT core–containing G box; Pyr, pyrimidine box.

Figure 7.

MYBGA Renders αAmy8 SRC/GARC Insensitive to Glucose in Embryos Specifically through the GARE. Rice embryos were cotransfected with effector, reporter, and control plasmids. The effector construct contained the Ubi-MYBGA chimeric gene. The reporter constructs contained αAmy8 SRC/GARC-Luc, αAmy8 SRC/GARC(mTA)-Luc, αAmy8 SRC/GARC(mGARE)-Luc, or αAmy3 SRC-Luc chimeric genes. Transfected embryos were incubated for 24 h in a buffer containing glucose (+Glc) or mannitol (−Glc), and their luciferase activities were determined. X indicates fold repression of luciferase activity by glucose. Error bars indicate se of three replicate experiments for each construct.

Figure 8.

Overexpression of MYBGA Renders αAmy8 Insensitive to Glucose in Embryos. (A) Starch agar plate α-amylase activity assays identified transgenic rice endosperms overexpressing MYBGA. Sixteen isolated endosperms from wild-type or transgenic rice were placed on one starch agar plate. Panel 1, wild-type endosperms expressed α-amylases in starch agar containing 1 μM GA; panel 2, wild-type endosperms did not express α-amylases in starch agar lacking GA; panel 3, endosperms from one transgenic line containing Ubi-MYBGA construct: five endosperms expressed low levels of α-amylases, and one endosperm expressed a high level of α-amylases. (B) RT-PCR analysis of the expression of αAmy3, αAmy8, and endogenous and overexpressed MYBGA. Embryos from the wild type and a transgenic line were incubated at 30°C in the presence (+) or absence (−) of 100 mM glucose for 24 h. Total RNA was purified from embryos and subjected to RT-PCR analyses. (C) Quantitative (real-time) RT-PCR analysis of the expression of αAmy3 and αAmy8 using total RNAs prepared as described for (B). RNA levels of αAmy3 and αAmy8 were quantified and normalized to the level of 18S rRNA. The highest αAmy3 or αAmy8 mRNA level was assigned as 100, and other levels of expression were calculated relative to this value. Error bars indicate se for three replicate experiments.

Figure 9.

Schemes Illustrating the Interactions among Sugars, GAs, MYBGA, and α-Amylases in Rice during Germination and Seedling Development. (A) In the wild-type endosperm, sugar repression of α-amylase expression is inhibited by MYBGA, which interacts with the GARE in α-amylase gene promoters. (B) In the gamyb-2 mutant endosperm, the absence of MYBGA leads to the sugar repression of α-amylase expression.