Our goal is to create maize varieties that have enhanced tolerance to a number of abiotic stresses, in order to reduce crop losses and, therefore, the costs of production. Our strategy to increase stress tolerance is based upon a) the well-documented phenomenon that organisms can be "pre-adapted" to withstand higher stress levels by the presence of low-to-moderate levels of protective proteins; and b) the more recent observations that mitochondria integrate stress signals and regulate cell death. Thus, mitochondria must be protected if plants are to survive and recover from severe stress. We hypothesize that a central stress defense in plant cells relies on the concerted action of two stress-induced mitochondrial proteins, HSP22 and AOX. The introduction of constitutively expressed genes for both proteins into maize lines is expected to protect plants by "pre-adapting" them to multiple environmental stresses. Because our method relies on altering the expression patterns of native maize genes, it represents an approach that should mitigate one of the current concerns of anti-GMO activists; i.e., the introduction of genes from other organisms.

Our first year objectives were two-fold: 1) The development of constructs to introduce the hsp22 gene at different levels of constitutive expression (relatively low to relatively high) into maize embryos, and to subsequently generate transgenic plants. 2) Identify a candidate aox gene for manipulation and introduction into plants. Because the alternative oxidase protein is encoded by a small multigene family, we first needed to determine which aox gene is the best candidate for our study. Our first-year goal was to complete sequencing and analysis of this gene family and to generate the constructs for transformation. In subsequent years, we will introduce constitutively expressed genes for both types of protein into maize and test for enhanced resistance to diverse stresses (e.g. heat, chilling and oxidative).

The sequence and analysis of the alternative oxidase genes and their induction in plants carrying mitochondrial mutations is now in press at The Plant Cell. (Karpova, O.V., E.V. Kuzmin, T.E. Elthon and K.J. Newton 2002. Differential expression of alternative oxidase genes in maize mitochondrial mutants. Plant Cell, December issue.) By analyzing the expression of the aox genes in mutants and under stress conditions, we have identified aox2 as the best candidate to encode a stress-protective protein, as it is induced most readily and rapidly in response to heat and oxidative stress. However, both the aox2 and aox3 genes are in the process of being introduced into maize cells.

We have made considerable progress on generating stable transgenic plants expressing HSP22 constitutively. These plants are normal in appearance; thus the constitutive expression of this stress protein does not appear to be harmful to the plant. Seeds from the current plants will be able to be tested for responses to stress and will be ready to cross with the transgenic plants constitutively expressing AOX, when they are ready.

Publications and Presentations

Work sponsored by this project was included in an invited talk presented at the 6th International Congress on Plant Mitochondria in Perth, Australia (July 10-14, 2002) entitled: Mitochondrial mutations affecting function in maize. The analysis of the maize alternative oxidase genes was also included in a seminar given at Kyoto University in Japan (February 15, 2002).

Abstract of Plant Cell paper

Olga V. Karpova, Evgeny V. Kuzmin, Thomas E. Elthon, and Kathleen J. Newton. Differential Expression of Alternative Oxidase Genes in Maize Mitochondrial Mutants. Plant Cell, in press (December 2002 issue).

We have examined the expression of three alternative oxidase genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mtDNA deletions affecting subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mtDNA rearrangements, but are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all non-photosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH-dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be differentially induced in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 was detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.

Details on the generation of constitutively expressing Hsp-22 plants

Using particle-gun bombardment, 165 plants were derived from seven transformation events. Two different constructs were used, one using the 35S promoter for which 92 plants were obtained, and the other using the ubiquitin 1 promoter for which 73 plants were obtained. Of these initial T0 plants, 98 were positive for presence of neomycin phosphotransferase indicating that vector had become integrated into the genome and that the marker protein was being expressed. An attempt was made to self all positive plants and then to outcross them to B73 or other lines when possible. In all, 269 plants were pollinated. The 98 T0 nptII positive plants were all screened with Southerns using a hsp22 probe, and additionally by westerns with a monoclonal antibody against HSP22. From these results, seed from 16 plants with strong levels of HSP22 expression on westerns were planted for the T1 generation. Fifteen seed from each of these 16 lines were planted in the greenhouse and selfed when possible, and also outcrossed to B73 or other lines when feasible. Of these T1 plants, 35 were nptII positive. From these 35 nptII positive plants, 81 pollinations were performed. Seeds from four plants that were selfed were chosen to carry into the T2 generation based upon uniformity of growth, timing of pollen shed and silk production, and seed production and quality.

The T2 lines were all derived from T0 plants that were selfed. In the T1 generation, two sibs were carried forward that were also selfed. The resultant 4 T2 lines are thus of two different genotypes and have two different promoters.

Forty seeds from each of the 4 T2 lines have been planted in the greenhouse and are currently being used for heat stress experiments and for generation of additional seed. All 160 plants have been evaluated for nptII expression and the results indicated the following:

T2-HSP22 ubi 1 parent was heterozygous

T2-HSP22 ubi 2 parent was heterozygous

T2-HSP22 35S 1 parent was heterozygous

T2-HSP22 35S2 parent was homozygous