Abscisic acid (ABA), like other plant growth substances, has multiple functions in plant growth and development. It is involved in such diverse physiological processes as closure of stomata, and seed maturation, dormancy, and germination. ABA also functions in response to environmental stresses, such as drought, cold, heat, and salinity through its ability to induce expression of specific genes whose products ameliorate the effects of such stresses. Biosynthesis of ABA is stimulated during water stress, so there is a large increase in ABA content during drought. Following rehydration, ABA is rapidly degraded to phaseic acid (PA). To understand the many roles of ABA in plants, it is essential to know how its concentration is regulated at the tissue and cellular levels. ABA is synthesized from epoxy-carotenoids (C40) by oxidative cleavage to form xanthoxin (C15) and C25-apo-aldehydes. Xanthoxin is further converted via ABA-aldehyde to ABA. In previous work, a gene encoding the cleavage enzyme, 9-cis-epoxy-carotenoid dioxygenase (NCED), was cloned from bean and avocado. Analysis of expression of this gene at the mRNA and protein levels showed that the cleavage reaction is the limiting step in ABA biosynthesis, being strongly induced in dehydrated bean leaves and ripening avocado fruit. At present, the endogenous carotenoid substrate of the cleavage reaction is not known. Carotenoids are part of the pigment-protein complexes in the thylakoid membranes of the chloroplast. In future work, thylakoid membranes will be fractionated and analyzed to determine which component provides the substrate for the cleavage reaction, and to isolate native cleavage enzyme from dehydrated leaves. The promoter of the NCED gene from bean will be fused to the reporter gene encoding green-fluorescent protein and introduced into Arabidopsis. In these transgenic plants, expression of the cleavage enzyme will be monitored at the organ and cellular levels to determine when and where the cleavage enzyme is expressed. In further work, the gene encoding the cleavage enzyme will be expressed in transgenic plants under the control of a constitutive or inducible promoter to determine whether the ABA level can be increased permanently with a constitutive promoter, or transiently with an inducible promoter. Manipulation of ABA metabolism may produce plants with raised ABA levels that are better adapted than normal plants to grow under stressful conditions. However, ABA causes its own deactivation by inducing ABA 8'-hydroxylase activity, which converts ABA to PA. It is conceivable, therefore, that in plants overexpressing NCED, ABA homeostasis is maintained due to ABA deactivation.
