How Many and Which Genes for Stress Tolerance

Experimentation must continue that elucidates mechanisms by which plants achieve stress tolerance. Especially instructive is the comparison of mechanisms gleaned from model systems, yeast and the ice plant, for example, with stress responses present in stress sensitive plants. Additionally, information from various genome analysis projects, genomic sequences and sequences of expressed sequence tags (ESTs) for stress responsive genes will have to be collected and integrated. The cell-, tissue-, and developmental stage-specific expression of stress relevant transcripts and proteins needs to be analyzed in more detail. Finally, technological breakthroughs are imperative regarding targeted gene elimination and gene replacement as well as techniques for the insertion of multiple genes into chromosomes, either through further development of the established vector and gene delivery systems, or by novel techniques such as the construction of artificial, additional chromosomes.

Metabolic engineering projects with a focus on stress tolerance have resulted in partially stress protected transgenic models, mainly tobacco and Arabidopsis (Table 19.2, summarized in Nelson et al12 and Bohnert and Sheveleva68). Some dicot crop species have been transformed with the objective of learning about stress tolerance, but very few transgenic analyses have targeted monot crops such as rice or corn. For example, rice has been transformed to achieve the overexpression of an LEA (late embryogenesis abundant) protein.89 The transgenic plants indeed showed higher osmotic stress tolerance. One must view all generated lines as tester lines; to our knowledge none of these lines has been sufficiently tested under field conditions. In fact, we think field testing would be premature for two reasons. The single gene transfers carried out in the past are unlikely to be sufficient for effective stress protection, and the transgenes are constitutively expressed. Salt tolerant yeast, the ice plant and celery (which

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Fig. 19.1. Activity of the ice plant MipB promoter in transgenic Arabidopsis. A promoter-luciferase fusion gene was introduced into Arabidopsis thaliana (Columbia) and homozygous lines were selected. Individual seedlings were germinated on agar, sprayed with luciferase substrate and photographed. The activity of the promoter under optimal growth conditions (panel A) is compared to the activity after seedlings experienced 20 minutes of drought (panel B), i.e., after the seedlings had been placed on the laboratory bench for 20 minutes. Quantitation of the signal indicated that luciferease activity increased by 10 to 12-fold under drought conditions. A less pronounced increase, by 3-fold, was obtained when seedlings were incubated with 10 mM calcium chloride. Sodium chloride (150 mM), ABA (100 ^M) and mannitol (300 mM) had no effect.

naturally accumulates mannitol), for example, induce responses following stress. Stress defense mechanisms are not constitutively present. Also, it seems unlikely that single gene transfers, which have been carried out in the past, will be sufficient. The brief discussion provided earlier seems to indicate that a large number of genes might have to be transferred to achieve osmotic stress tolerance for the protection of crop plants.

Our discussion has concentrated on the biochemical and physiological mechanisms that constitute tolerance to stress. Alternatively, it is possible to focus on stress perception and signal transduction pathways, leading to the induction or enhancement of stress tolerance proteins or pathways that are present in all plants.16,90-93 Mutants of Arabidopsis which reveal stress related signal transduction chains have recently become available,91,94 and their biochemical dissection is under way.95-97 It is therefore conceivable that within a very short time we will have genes available that are high in the hierarchy of regulators. Enhancement of a few genes might then be sufficient for the induction of resident stress tolerance mechanisms.

The best strategy may be a combination of genes to strengthen both stress signal transduction and to add new downstream biochemical protective pathways. One argument favoring proceeding in this way is that crop plants do not possess a complete repertoire of biochemical mechanisms; it seems that some pathways and their genes are missing. Also, we argue that there may have been inadvertent, breeding-associated selection against the induction of stress response mecha-nisms—or example, by selection for ABA insensitivity which would tend to lead to sto-matal opening even under low water conditions. Signal transduction chains could have become inoperative and would not induce stress responses that might have been present before domestication. The combination of genes would first contain genes that enhance the plant equivalent genes of the yeast HOG and calcineurin-related signal transduction pathways (see Nelson et al12). Genes for pathways that enhance the scavenging of radical oxygen species, osmotic adjustment, and controlled uptake of water and ions should be included in any transgenic stress engineering strategy. It is difficult to gauge the usefulness of the LEA genes, which certainly have resulted in increased tolerance in a few model experiments, because there is no mechanistic understanding of their action. Thus, the next generation of transgenic tester lines might include on the order of 20 genes, the transfer of which can be accommodated even by present technologies.

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