the G-box are complexes made up of at least two different proteins [119]. A complex array of events is
required for ABA-regulated expression to occur in response to stress and/or developmental cues.
In transgenic tobacco plants it has been shown that expression of drought-regulated genes is prop-
erly regulated in seeds, but not in response to stress [120,121]. When 482 to 184 of rab16Bfrom rice
was translationally fused to GUS, expression was limited to developing seeds and was not induced by
ABA or water stress in vegetative tissues. Expression of a related gene from rice, rab16A, was not de-
tected in seeds or vegetative tissues [120]. These results may indicate that elements recognized in rice for
ABA-regulated expression cannot be used in tobacco. However, another family member derived from
maize,rab17, is correctly expressed in transgenic tobacco plants when 1330 to 29 is included in the
reporter gene fusion constructs [122]. This gene also contains an ABRE, and only when that sequence is
present in deletion constructs is the reporter gene responsive to ABA [122].
Promoter deletion analyses for several additional ABA-regulated genes are in progress [121]. In the
resurrection plant, Craterostigma plantagineum, DNA elements required for regulation of the gene
CDeT27-45, which is similar in amino acid sequence to the cotton gene leal4, were characterized using
promoter deletion analyses. Using a transient expression system and C. plantagineumprotoplasts, a re-
gion between 282 and 197 of the promoter was demonstrated to be required for ABA-regulated ex-
pression. Similar studies were completed on transgenic tobacco plants carrying this gene and it was found
that there was not ABA-induced expression in tobacco leaves, although the genes were expressed during
development in seeds and anthers. In the region required for ABA induction of CDet27-45 expression,
there are no ABRE-like elements. Therefore, although the ABRE is found in many genes that are ex-
pressed during seed development and in response to ABA application, it is not found in all genes that are
in the ABA-requiring category. As another example, le16, a gene expressed in wild-type tomato but not
in the ABA-deficient mutant, does not contain a consensus ABRE [64]. Therefore, it is expected that there
are multiple DNA elements involved in ABA-regulated expression. In addition, genes that are expressed
during drought are also expressed during specific developmental stages and in specific cell types. There-
fore, additional elements are required to control tissue- and organ-specific expression and other specific
aspects of the expression pattern during water deficit.
D. Recognition of ABA at the Cellular Level
Although the responsiveness of the gene is controlled by the DNA elements within the gene, the pathways
that lead to transcription factor binding are also important to understand. For the ABA-requiring and
ABA-responsive genes, there must be an ABA recognition event followed by the activation of a pathway
that leads to gene induction. Cellular conditions that are required for any of these events to occur are not
understood. The response of the cell to ABA may be altered by the physiological state of the cell. For ex-
ample, sensitivity to ABA is altered by the osmotic potential of the cells; there is increased sensitivity to
ABA with increased osmotic stress. In some cases, osmoticum can completely replace exogenous ABA.
ForEmmRNA accumulation in rice cell cultures, increasing concentrations of NaCl increased the accu-
mulation of EmmRNA in response to suboptimal concentrations of ABA [123]. Therefore, the cells’ re-
sponse to ABA may be altered by the water potential or water content of the cell. However, another pos-
sibility should be considered. Because the cells are induced to accumulate ABA in response to osmotic
stress, it becomes difficult to determine if the newly synthesized ABA is contributing to the induction of
genes. The ABA that is synthesized in the cell may not be located in the same compartment within the
cell as ABA that is applied to the cell [124]. Therefore, the plant may be more sensitive to endogenous
ABA than to applied ABA. It is known that high levels of ABA must be applied to elicit a response sim-
ilar to that stimulated by endogenous ABA concentrations.
In addition to understanding the mechanism of gene induction at the gene level, it must also be un-
derstood how the cell recognizes ABA and what signal transduction pathway is taken to gene induction.
It is important to understand the aspects of the ABA molecule that are required for gene induction (Fig-
ure 3). The strategy has been taken to use ABA analogues to identify parts of the ABA molecule that are
required for gene induction. Walker-Simmons et al. [125] compared optically pure ABA analogues in the
induction of rab,Em, and leagroup 3. The induction of rabandleagroup 3 was similar with similar ana-
logues; however, Eminduction differed. These results support the conclusion that there is more than one
mechanism for ABA regulation of gene expression. In the induction of rab16andbasiin barley aleurone
protoplasts, methylation of the carboxyl group had the least effect on the level of gene expression [126].
ABIOTIC STRESSES AND ABSCISIC ACID 747