ACRE, a class of AP2/ERF transcription factors, activates the expression of sweet potato ß-amylase and sporamin genes through the sugar-responsible element CMSRE-1

Sugars, synthesized by photosynthesis in source organs, are loaded and utilized as an energy source and carbon skeleton in sink organs, and also known to be important signal molecules regulating gene expression in higher plants. The expression of genes coding for sporamin and β-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately induced by sugars. We previously reported on the identification of the carbohydrate metabolic signal-responsible element-1 (CMSRE-1) essential for the sugar-responsible expression of two genes. However, transcription factors that bind to this sequence have not been identified. In this study, we performed yeast one-hybrid screening using the sugar-responsible minimal promoter region of the ß-amylase gene as bait and a library composed only transcription factor cDNAs of Arabidopsis. Two clones, named Activator protein binding to CMSRE-1 (ACRE), encoding AP2/ERF transcription factors were isolated. ACRE showed transactivation activity of the sugar-responsible minimal promoter in a CMSRE-1-dependent manner in Arabidopsis protoplasts. Electric mobility shift assay (EMSA) using recombinant proteins and transient co-expression assay in Arabidopsis protoplasts revealed that ACRE could actually act to the CMSRE-1. Among the DEHYDRATION -RESPONSIVE ELEMENT BINDING FACTOR (DREB) subfamily, almost all homologs including ACRE, could act on the DRE, while only three ACREs could act to the CMSRE-1. Moreover, ACRE-homologs of Japanese morning glory also have the same property of DNA-binding preference and transactivation activity through the CMSRE-1. These findings suggested that ACRE plays an important role in the mechanism regulating the sugar-responsible gene expression through the CMSRE-1 conserved across plant species. Supplementary Information The online version contains supplementary material available at 10.1007/s11103-024-01450-z.

The asterisks represents the 14th valine (Val14) and the 19th glutamic acid (Glu19) , and the 37th alanine in the AP2 domain, shown to be important for the DNA-binding activity of DREB1A and DREB2A, and both DREB and ERF subfamilies (Cao et al. 2001;Sakuma et al. 2002;Liu et al. 2006).

Supplemental Figure S2 Purification of the recombinant MBP-ACRE fusion proteins.
Purification of the recombinant MBP-ACRE fusion protein was performed in accordance with the instruction manual for pMAL protein fusion and purification system (New England BioLabs).Equal amounts corresponding to the crude extract were applied for the resuspended pellets of E. coli cells (P), supernatant after extraction (S), and elute (E), used for EMSA.The red arrowhead indicates the band corresponding to the MBP-fusion protein.Leaf-petioles of 3-week-old seedlings were incubated in water (H) or 6% sucrose (S) for 24 h, then they were sprayed with luciferin solution prior to detection of luminescence with a high resolution photon counting camera.Leaf-petioles of 3-week-old Arabidopsis seedlings incubated in water for 24 h and then in water or 5% sucrose for a further time indicated and then subjected to RT-qPCR analysis of transcripts.The means of the amounts relative to PP2A (At1g13320) in three independent samples from three leaves, are shown with standard deviation.
Expression levels (/PP2A) Expression levels (/PP2A) Expression levels (/PP2A) tree of the DREB subfamily of Arabidopsis thaliana and the ACRE homologs of morning glory.The amino acid sequences of AP2 domain were aligned by ClustalW on the DDBJ website (https://clustalw.ddbj.nig.ac.jp/).(a) The phylogenetic tree was constructed using the NJ method.Classifications of groups by Sakuma et al. (2002) and Nakano et al. (2006) are indicated in parentheses with a dash line.(b) Alignment of amino acid residues corresponding to AP2 domain.Dashes indicate gaps in the amino acid sequences introduced to optimize alignment.The dark gray and light gray background represents fully and highly conserved amino acid residues, respectively.The yellow background represents conserved leucine residues in ACREs and InACREs that can bind to CMSRE-1.
the DRE, but not the CMSRE1-like element of ß-amylase of Arabidopsis.(a) MBP protein alone does not have binding activity.(b) The DNA-binding activity of MBP-fusion proteins with ACRE was analyzed by EMSA using BA (B), SPO (S), DRE (D), and AtBA oligonucleotides (A) as probes.Asterisk and arrowhead indicated by F show the position of the shifted band and free probe, respectively.(c) The sequences of AtBA oligonucleotides used for EMSA.
The effect of amino acid substitution at AP2 domain to transactivation.(a) Amino acid residues of AP2 domain for ACRE2, DREB1A and their amino acid substituted forms.(b) Transactivation of BA-35S46:LUC (top) and DRE-35S46:LUC (bottom) by the co-expression of wild type or amino acid substituted ACRE2 and DREB1A.The LUC activity in each assay was normalized to the hRLUC activity, and normalized activity (LUC/hRLUC) is expressed relative to values obtained with the empty vector.Results represent the means of six independent experiments, with the error bar representing SD.Values that were significantly different from one another according to Tukey's multiple comparison test are indicated by different letters (P < 0.05).Purification of the recombinant MBP-InACRE fusion proteins.Purification of the recombinant MBP-InACRE fusion protein was performed as shown in figure S2.Equal amounts corresponding to the crude extract were applied for resuspended pellets of E. coli cells (P), supernatant after extraction (S), and path-through after binding with amylose resin (PT), elute (E), used for EMSA, and resuspended amylose resin (B).The red arrowhead indicates the band corresponding to the MBP-fusion protein.Effect of ACRE over-expression on the sugar-responsible gene expression (a) Expression of Atß-amylase gene in the ACRE-and ACREL1-overexpression lines.Leaf-petioles of 3-week-old seedlings were isolated and incubated in water (blue bars) or 5% sucrose (orange bars) for 2 days and then subjected to RT-qPCR analysis of Atß-Amy transcripts.Measured values of two to three independent plants in each class are indicated separately as levels relative to PP2A (At1g13320).For each plant, two samples consisting of three leaves were averaged and shown with standard deviation.(b) Transactivation of Atß-Amy(-451):LUC by co-expression of ACRE2 in Arabidopsis protoplasts.The average of relative activity (LUC/hRLUC) in 4 independent experiments is shown with the error bar representing standard deviation.Asterisks indicate significant change at P < 0.05 (Student's T test) against the control.Luminescence image of LUC activity in the ACRE-and ACREL1overexpressing sGsL lines.
of ACRE genes in response to sugar supply.