Our data shows that in the RY/G cluster, two RY-repeats interact with a perfect G-box to provide ABA-independent transactivation by ABI3. In addition, the tetrameric RY/G construct displayed a low (twofold) increase in activity upon treatment with ABA ( Figure 6), suggesting that the RY/G complex is moderately responsive to ABA. However, the relative weakness of the response indicates that, in the napA promoter, the main ABRE is within the B-box.
We have earlier shown that the B-box tetramer is a seed-specific ABRE ( Ezcurra et al., 1999 ). Our present analysis provides a molecular basis to this function by demonstrating that ABI3 mediates the ABA responsiveness of the B-box. Moreover, ABI3 activates the B-box even in the absence of ABA. In the composite B-box, the distB element mediates seed specificity by functioning as an ABI3-dependent ABRE, and by providing ABI3 regulation even in the absence of added ABA. In addition, the adjacent proxB element is also involved in napA regulation, because mutation in the native context severely impairs the activation by ABI3 in the presence and absence of ABA treatment ( Figure 2).
Our results showing that the distB tetramer does not respond to ABA in the absence of ABI3 are in contrast with results by others ( Ono et al., 1996 ; Skriver et al., 1991 ; Vasil et al., 1995 ), showing that tetramers of Em1a-type ABREs are responsive to ABA independently of ABI3. However, the type of cell lines used in those studies express Vp1 ( Hollung et al., 1997 ; Nakagawa et al., 1996 ), and thus it is probable that the reported ABI3-independent ABA responses actually reflect endogenous VP1 activity. Another Em1a-type ABRE displaying ABI3-independent response to ABA is Hex3 ( Table 1). Hex3 was reported to mediate seed specificity and ABA response in vegetative tissues of transgenic tobacco ( Lam and Chua, 1991). However, the response was measured in only one transgenic plant, and it was observed when the Hex3 tetramer was fused to the −46 35S minimal promoter, but not when it was fused to the −90 35S truncated promoter. Moreover, the response appeared weak as judged by an S1 protection assay, and it markedly declined after 2 h of ABA treatment, as opposed to Rab 21, which still increased after 12 h treatment ( Mundy and Chua, 1988). In contrast, it was recently reported that a tetramer of an Em1a-type G-box, Box1 ( Table 1), mediates strict seed-specific expression, but is not responsive to ABA in seedlings of transgenic tobacco ( Ishige et al., 1999 ). This suggests that Box1 functions as yet another ABI3-dependent ABRE. Thus, because all results showing VP1-independent response to ABA of Em1a-type tetramers are, in one way or another, questionable, we conclude that tetramers of Em1a-type ABREs are ABI3/VP1-dependent, and, in consequence, seed-specific. It has been established that the promoter unit necessary and sufficient to mediate the ABA response consists of an ABRE and a closely linked sequence, called a coupling element ( Shen et al., 1996 ). Further, a coupling element may be replaced by another ABRE ( Hobo et al., 1999a ; Ono et al., 1996 ), and it was suggested that coupling elements might also mediate tissue specificity to the hormone response ( Rogers and Rogers, 1992). Then, in the tetrameric construct, the additional ABREs might mediate ABI3-dependence to the ABA response. Further, in the context of their corresponding promoters, the ABREs interact with nearby coupling elements and mediate, accordingly, ABI3-dependent (as in napA) or ABI3-independent (as in rab genes) response to ABA (reviewed by Singh, 1998). Interestingly, in the context of napA, the distB ABRE interacts with proxB and the RY/G complex, and mediates the ABI3-dependent ABA response. However, when fused to the 35S enhancer the B-box tetramer mediates ABA response in vegetative tissues ( Ezcurra et al., 1999 ).