A Novel Maize Dwarf Resulting from a Gain-of-Function Mutation in a Glutamate Receptor Gene

Plant height is an important agronomic trait and a major target for crop improvement. Owing to the ease of detection and measurement of plant stature, as well as its high heritability, several height-related mutants have been reported in maize. The genes underlying a few of those mutants have also been identified, with a majority of them related to the biosynthesis or signaling of two key phytohormones - gibberellins (GAs) and brassinosteroids (BRs). However, most other maize dwarfing mutants, and especially those that result from gain-of-function mutations, remain uncharacterized. The present study was undertaken to characterize a novel dominant dwarfing mutant, named D13. This mutant appeared in the M1 population of the inbred B73 that was generated by mutagenesis with ethyl methanesulfonate (EMS). Like most other maize dwarfing mutants, the reduction in D13 height was largely due to the compression of the internodes. However, unlike the GA or BR mutants, D13 had no defects in the female or male inflorescences. Further, in contrast to the GA and BR mutants, the mesocotyl elongation during etiolation was not impacted in D13. D13 seedlings developed red coloration in two to three lowermost leaves. In addition, D13 also showed enhanced tillering when the phenotype was very severe. The size of the shoot apical meristem of D13 was reduced slightly, and significant aberrations in the structure of vascular bundles in the mutant were observed. All anatomical and phenotypic features of D13 were highly exaggerated in homozygous state, indicating the partially dominant nature of the D13 mutation. Interestingly, the heterozygous mutants showed remarkable variation in their phenotype, which was maintained across generations. Moreover, the D13 phenotype was found to be sensitive to the genetic background, being completely suppressed in Mo17, Oh7B, enhanced in CML322, P39 and changed to different degrees in others. To identify the genetic defect responsible for the D13 mutant phenotype, a map-based cloning approach was used, which identified a single basepair change from G to A (G2976A) in the coding region of a glutamate receptor gene (Zm00001d015007). The G2976A missense mutation resulted in the replacement of alanine with threonine at the location 670. The replaced alanine is highly conserved in glutamate receptors across all domains of life from cyanobacteria to plants to mammals, suggesting a causal relationship between the G2976A substitution and the D13 phenotype. To validate this relationship, a targeted EMS-based mutagenesis approach was used to knock-out (inactivate) the D13 mutant allele. A suppressor mutant was found in which the D13 mutant phenotype reverted to the normal tall phenotype. The sequence of the revertant allele, designated D13*, revealed that the original D13 mutant allele underwent a second G to A mutation (G1520A) to change glycine into aspartic acid at position 473. This intragenic second-site mutation in the D13 allele suppressed the function of the D13 allele, thereby preventing it from interfering with the function of the wild type allele. To further unveil the genes and underlying mechanisms that enable the D13 mutant to confer a dwarf phenotype, transcriptomic and metabolomic analyses of D13mutants were conducted and compared to the wild type sibs