# Gene transcriptions/GAREs

"In the germinated cereal aleurone layer, gibberellic acids (GA) induce expression of a number of genes encoding hydrolytic enzymes that participate in the mobilization of stored molecules."

## Gibberellic acids

"Gibberellins (GAs) are diterpenoid hormones that play crucial roles in plant growth and development, including seed germination, leaf expansion, stem elongation, and flower and fruit development (Hooley, 1994)."
In "the aleurone layer [...] GAs trigger the expression of hydrolytic enzymes through transcription activation of corresponding genes (Jacobsen et al., 1985)."
An "R2R3-type Myb transcription factor GAMYB (Gubler et al., 1995) [...] induces the expression of genes such as high- and low-pI α-amylases, proteinase, and β-glucanase, through direct binding to [the] GA-responsive element (GARE)".
"Raventós et al. (1998) identified a barley (Hordeum vulgare) transcription repressor (HRT) [a barley zinc-finger protein] capable of binding the GARE region of an α-amylase gene and repressing its activity".
"Functional analyses of a number of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, have identified a GA-responsive complex as a tripartite element containing a pyrimidine box motif 5′-CCTTTT-3′."

## GA responsive complexes

"Although this GARC [GA responsive complex] may not always be tripartite, most often it includes three sequence motifs, the TAACAAA box or GA responsive element (GARE), the pyrimidine box CCTTTT, and the TATCCAC box (Skriver et al., 1991;Gubler and Jacobsen, 1992; Rogers et al., 1994)."
In the diagram on the right are transient "expression analysis of the relative abilities of the RAmy1A promoters containing mutations to direct expression of the luciferase reporter gene in the rice aleurone."
Each line in the diagram refers to the "5'-upstream regulatory sequence (407 bp long; -380 to +27) from the RAmy1A gene [...] fused to a reporter gene cassette containing the firefly luciferase coding region (luc+) and the 3' terminator from the nopaline synthase gene (Nos-t). The positions of predicted promoter motifs are indicated by open ellipses. Their sequences and positions in the RAmy1A promoter (referred to the transcription initiation site) are indicated. Nucleotide substitutions in the mutated versions are indicated by shaded eclipses."
"The rice WRKY transcription factor OsWRKY71 may be a transcriptional repressor in rice aleurone cells. Transient expression of OsWRKY71 specifically represses GA or OsGAMYB-induced Amy32b α-amylase promoter activity in rice aleurone cells (Washio 2003). Consistent with its role as a transcriptional repressor, OsWRKY71 binds specifically to functionally defined TGAC-containing W boxes of the Amy32b promoter in electrophoretic mobility shift assays (Zhang et al. 2004). Mutations of the two W boxes cause no binding of OsWRKY71 to the promoter, releasing the suppression of OsGAMYB-activated Amy32b expression by OsWRKY71, indicating that OsWRKY71 blocks GA signaling by functionally compromising OsGAMYB (Zhang et al. 2004). Another rice WRKY, namely OsWRKY51, interacts with OsWRKY71, as revealed by bimolecular fluorescence complementation (BiFC) assays, and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter (Xie et al. 2006)."
Various "GA-responsive complexes (GARCs) [may] mediate the regulation of GA-regulated gene expression by GAMYB. Several GA-responsive cis-acting elements (GARE) and GARE-like elements (TAACAA/GA, or TAACGTA) have been identified in the promoters of hydrolase genes expressed in the aleurone (Ueguchi-Tanaka et al. 2000; Sutoh and Yamauchi 2003; Washio 2003), expansin genes expressed in internodes (Lee et al. 2001), and many GAMYB-regulated genes expressed in anthers (Tsuji et al. 2006)."
"Although miR159 [a microRNA] has been demonstrated to regulate OsGAMYB expression in anthers, the expression of miR159 is not regulated by GA treatment and the expression is not altered in transgenic lines overexpressing the SLR1 form lacking the DELLA motif (Itoh et al. 2002). In Osgamyb-1 flowers, miR159 expression is not disturbed, suggesting that OsGAMYB is not involved in the regulation of miR159, although a GARE sequence targeted by GAMYB (CAACAAC) is present upstream of the putative cap site of the miR159a transcriptional unit (Tsuji et al. 2006). Thus, miR159 seems not to be regulated by GA signaling."

## Cis-acting elements

"Some other cis-acting elements, such as pyrimidine boxes (GGTTTT) and TAT boxes (TATCCAT), are usually present in the vicinity of the GARE sequence of genes regulated by GA in cereal aleurone cells (Gubler and Jacobsen 1992; Cercos et al. 1999; Tsuji et al. 2006). For example, GARE and a novel CARE (CAACTC regulatory elements) elements are present in the promoter of rice RAmy1A (Ueguchi-Tanaka et al. 2000; Sutoh and Yamauchi 2003). Cis-element analyses have shown that the OsGAMYB protein activates RAmy1A expression through interaction with GARE in the promoter (Washio 2003). In addition, GARE and CARE are also present in a cysteine proteinase gene REP-1, which is expressed in rice aleurone and is induced by GAs and repressed by ABA. These two elements have been identified as necessary and sufficient for conferring GA inducibility of the REP-1 promoter. Mutations of CARE in the promoters of RAmy1A and REP-1 result in loss of GA inducibility and GAMYB transactivation, suggesting that CARE is a regulatory element for the GA-inducible expression of hydrolase genes in germinating seeds (Sutoh and Yamauchi 2003)."

## Consensus sequences

"GAMYB is also known to bind the sequence TAACAGAC in vitro, which is the GARE found in barley low-pI α-amylase promoters (Gubler et al., 1995)."
The "TAACAAA box [is the] GA responsive element (GARE)".
"GARE-like elements (TAACAA/GA, or TAACGTA) have been identified in the promoters of hydrolase genes expressed in the aleurone (Ueguchi-Tanaka et al. 2000; Sutoh and Yamauchi 2003; Washio 2003)".

## GARE sampling of A1BG promoters

For the Basic programs (starting with SuccessablesGARE.bas) written to compare nucleotide sequences with the sequences on either the template strand (-), or coding strand (+), of the DNA, in the negative direction (-), or the positive direction (+), including extending the number of nts from 958 to 4445, the programs are, are looking for, and found:
* negative strand in the negative direction (from ZSCAN22 to A1BG) is SuccessablesGARE--.bas, looking for 3'-TAACAAA-5', 0,
* negative strand in the positive direction (from ZNF497 to A1BG) is SuccessablesGARE-+.bas, looking for 3'-TAACAAA-5', 0,
* positive strand in the negative direction is SuccessablesGARE+-.bas, looking for 3'-TAACAAA-5', 0,
* positive strand in the positive direction is SuccessablesGARE++.bas, looking for 3'-TAACAAA-5', 0,
* complement, negative strand, negative direction is SuccessablesGAREc--.bas, looking for 3'-ATTGTTT-5', 0,
* complement, negative strand, positive direction is SuccessablesGAREc-+.bas, looking for 3'-ATTGTTT-5', 0,
* complement, positive strand, negative direction is SuccessablesGAREc+-.bas, looking for 3'-ATTGTTT-5', 0,
* complement, positive strand, positive direction is SuccessablesGAREc++.bas, looking for 3'-ATTGTTT-5', 0,
* inverse complement, negative strand, negative direction is SuccessablesGAREci--.bas, looking for 3'-TTTGTTA-5', 1, 3'-TTTGTTA-5', 230,
* inverse complement, negative strand, positive direction is SuccessablesGAREci-+.bas, looking for 3'-TTTGTTA-5', 0,
* inverse complement, positive strand, negative direction is SuccessablesGAREci+-.bas, looking for 3'-TTTGTTA-5', 0,
* inverse complement, positive strand, positive direction is SuccessablesGAREci++.bas, looking for 3'-TTTGTTA-5', 0,
* inverse, negative strand, negative direction, is SuccessablesGAREi--.bas, looking for 3'-AAACAAT-5', 0,
* inverse, negative strand, positive direction, is SuccessablesGAREi-+.bas, looking for 3'-AAACAAT-5', 0,
* inverse, positive strand, negative direction, is SuccessablesGAREi+-.bas, looking for 3'-AAACAAT-5', 1, 3'-AAACAAT-5', 230,
* inverse, positive strand, positive direction, is SuccessablesGAREi++.bas, looking for 3'-AAACAAT-5', 0.