URBANA, ILLINOIS

University of Illinois

GAINESVILLE, FLORIDA

University of Florida

 Transposon tagging of nuclear genes that control mitochondrial gene expression --Gabay-Laughnan, S, Chase, CD The S system of cytoplasmic male sterility (CMS-S) in maize presents an unprecedented opportunity for the identification, cloning and functional characterization of nuclear genes regulating mitochondrial gene expression in a higher eukaryote. In this system, expression of a novel chimeric gene in the mitochondria results in the collapse of starch-filling pollen and, consequently, a male-sterile phenotype. Loss-of-function mutations in nuclear genes required for mitochondrial gene expression behave as restorer-of-fertility (Rf) alleles, blocking expression of the chimeric mitochondrial gene and the male sterility trait. Such mutations are visible in pollen because it is haploid. Rf alleles also block expression of essential mitochondrial genes These mutations are tolerated in pollen because late-stage pollen development and pollen germination do not require high levels of mitochondrial gene expression or function.

Based upon these observations, we propose that Rf alleles for CMS-S maize can be generated by transposon mutagenesis and cloned via transposon tagging. These mutations should be recovered at high frequency due to the large number of nuclear genes involved in mitochondrial gene expression. The recovery of new Rf alleles by transposon mutagenesis is being approached by screening populations of male-sterile, S-cytoplasm plants carrying active Ac-Ds or I-En (Spm) transposons. Pollen from plants with male-fertile tassels or tassel sectors will be used to fertilize the ears of CMS-S tester plants for recovery of the new Rf alleles.

To demonstrate the feasibility of this transposon mutagenesis, we conducted a preliminary screen of 1,241 CMS-S plants carrying an active Ac element in chromosome 9 and a Ds reporter at the a1 locus (a1-m4). This screen was carried out by Chase and Gabay-Laughnan in Gabay-Laughnan�s 1999 summer nursery at the University of Illinois, Champaign-Urbana. The screening population was developed in the Mo17 inbred background, and the plants were derived from spotted kernels indicative of Ac activity. Twenty-four plants had visible sectors of male fertility on otherwise male-sterile tassels. Pollen samples from each sector were examined through a field microscope and observed to consist of starch-filled and collapsed grains in roughly equal proportions. This demonstrated that each sector resulted from a nuclear mutation. (Sectors resulting from cytoplasmic mutants consist entirely of starch-filled pollen). Of the 24 sectors, 12 were large enough to be used for testcrosses. Testcrosses from 10 of the 12 plants resulted in seed set. In CMS-S maize, only Rf pollen is functional. All of the testcross progeny are therefore expected to carry an Rf allele. This will be confirmed in our 2000 summer nursery.

Our preliminary observations indicate that we have recovered 10 independent Rf alleles from 1,241 plants. This high rate of mutation is consistent with the large number of nuclear genes known to regulate mitochondrial gene expression in yeast. Most independent Rf alleles are expected to result from mutations at different nuclear loci. This will be confirmed through allelism tests. The high rate of mutation in the Mo17-S a1-m4 plants contrasts with the mutation rate in Mo17-S plants lacking an active Ac. Over the past five years, we have examined over 2,000 Mo17-S plants and recovered only two sectors of male fertility. The Ac-Ds system is almost certainly responsible for the Rf alleles recovered in the Mo17-S a1-m4 materials. This will enable us to recover molecular clones of these alleles.

In summary, observations made in Gabay-Laughnan�s summer nursery provide a strong indication that we will efficiently recover new Rf alleles from CMS-S plants carrying active transposable element systems. If, as expected, the majority of mutations are transmitted to the next generation and result from mutations at different loci, the recovery of mutants will not be a limiting factor in our study. We are hoping to clone on the order of 10 different rf loci. Given the high-throughput methods that are now being applied to clone transposon-tagged loci, our target is very reasonable. Indeed it may be possible to clone many more loci.

The value of the molecular clones we will recover is significantly enhanced by our ability to examine the effects of mutations at rf loci on the expression of mitochondrial genes in developing maize pollen. The mutants and gene sequences derived from this project will provide an invaluable resource for the future genetic and molecular dissection of mitochondrial function in higher organisms.
 
 

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