Dissection of Genetic Variation in Maize Underlying Photosynthetic Traits
Forward genetics has been vital in revealing the identity and function of most plant genes. However, detecting phenotypically relevant variation outside the coding region of genes and distinguishing it from neutral changes is not trivial. One way to facilitate this is by employing genetic sensors that reveal even minute variation in the trait. Our lab has designed a novel approach in which the phenotype of a mutant is used as a reporter to identify modifier loci present naturally in the germplasm. It has been dubbed MAGIC, for Mutant-Assisted Gene Identification and Characterization. I used a MAGIC screen in maize to explore natural variation underlying an important component of photosynthesis. I hoped that it might lead to the revelation and genetic understanding of new traits or ideas capable of enhancing the photosynthetic output of this crop. For this MAGIC screen, I used a semi-dominant mutant allele of the gene oil yellow1 (oy1) that encodes one of the three subunits (subunit I) of magnesium chelatase (MgChl). MgChl is a multimeric enzyme complex that catalyzes the first committed step to chlorophyll biosynthesis. The semi-dominant Oy1-N1989 allele used in this work encodes a mutant protein that acts as a competitive inhibitor of MgChl. This mutant permitted rapid phenotyping of leaf color as a gauge or reporter for variation in chlorophyll accumulation. In the first set of experiments, an Oy1-N1989-mediated MAGIC screen was conducted on two bi-parental mapping populations derived from a cross of B73 with Mo17, two inbred lines that significantly suppress and enhance the mutant phenotype of Oy1-N1989, respectively. Both populations led to the identification of a single, large-size QTL that mapped in the vicinity of the reporter locus itself. I named this QTL very oil yellow1 (vey1). In the second set of experiments, I crossed Oy1-N1989 to an association mapping panel of more than 300 inbred lines and evaluated the severity of the mutant phenotype in the resulting testcross populations. The subsequent genome-wide association (GWAS) of the phenotypic and genotypic datasets also identified vey1 as the key factor responsible for variation in the Oy1-N1989 phenotype. In addition, examination of various expression datasets derived from a RIL population and diverse maize lines predicted vey1 to be a cis-acting regulatory sequence polymorphism present in specific wild-type alleles of oy1. Molecular analysis confirmed this notion and showed that vey1 skews the accumulation of OY1 transcripts, thereby explaining how vey1 modifies the phenotype of Oy1-N1989 mutants. The genetic background also impacted the flowering time of Oy1-N1989 mutants. A positive relationship was observed between the severity of the mutant phenotype and the time it took to flower. Mutant plants with low chlorophyll levels were delayed in flowering by up to two weeks. Interestingly, vey1 again came through as the single modifier for flowering time variation in both the QTL and association mapping. Thus, vey1 not only impacted the photosynthetic output of the mutant plant but also modified its transition to flowering, thereby putting on a firm foundation of the hypothesis that photosynthates mediate flowering time regulation in maize. These results were confirmed using an alternate approach that manipulated photosynthesis. This suggests that a threshold level of a photosynthate(s) may be needed as a signal for maize to switch to flowering. The identity of the signal(s) that regulates flowering time in the material described in this study remains unknown, but I believe my findings will provide an impetus to explore the nature of this signal(s) and the mechanism by which it may regulate flowering. The intracellular signaling between different organelles in a cell is essential for cellular homeostasis. Nuclear genes regulate the chloroplast development. Various abiotic stresses are perceived by the chloroplast, which leads to the production of a plastid signal(s) that controls the expression of different stress-responsive genes in the nucleus. The signaling pathway initiated by the chloroplast to regulate the expression of nuclear genes is called retrograde signaling (RS). The biochemical pathway leading to RS between the plastid and the nucleus is not characterized in maize. Understanding of any biochemical pathway requires either isolation of mutants using a mutagenized population or functional characterization of mutant alleles in known genes of the pathway from other plant species. In Arabidopsis, one of the subunits of MgChl - GENOMES UNCOUPLED5 (GUN5) - has been shown to be perturbed in RS. Besides, the co-factor GUN4 of MgChl has also been identified as a component of RS. I used Mutator (Mu) transposon insertional alleles of gun4 and gun5 to look into the role of these genes in RS. (Abstract shortened by ProQuest.)
Johal, Purdue University.
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