UC Berkeley

This work addresses the function of two circadian clock genes in maize, namely gigantea1 (gi1) and gi2, as well as the larger question of the role that circadian clock genes play in grass species. Previous work on the plant circadian clock has primarily focused on the model genetic system, Arabidopsis thaliana, and the current conception of the clock has been constructed on the basis of this work. Arabidopsis is, however, evolutionary distant from monocot species, such as the grass Zea mays (maize), that are grown as crops. Furthermore, many crop plants have convoluted domestication histories that have resulted in complex genomes containing remnants of entire duplicated genomes. Circadian clock genes are proposed to be preferentially retained after whole genome duplications, and to evolve altered roles when duplicate gene copies persist in genomes.

GIGANTEA (GI) is a plant-specific gene that is conserved across vascular plants and plays a central role within plant circadian and developmental processes. Most plants only have one copy, but maize has two, which are both expressed. One approach taken to investigate the roles of the gi genes was to generate gi mutants in maize and evaluate their impacts on clock gene expression, developmental phenotypes, and disease resistance. This analysis showed that gi1 and gi2 have differential effects on clock gene expression, gi2 may play a role in disease resistance, and that phenotypic effects of either mutant are minor compared to the Arabidopsis gi mutants.

A second approach was to heterologously express maize GI proteins in yeast to identify protein interaction partners. First, a set of predicted protein interaction partners were computationally identified on the basis of known Arabidopsis GI interactors. Both maize GI proteins were found to interact with homologs of known Arabidopsis interactors in maize, and in some cases, GI1 and GI2 had different interaction strengths. Second, GI1 and GI2 were used as baits in yeast two-hybrid screens against a library generated from maize cDNA to identify putative novel interacting partners. The screen identified a number of novel interaction partners, and found that each GI preferentially interacts with a different set of protein types.

Finally, evolutionary trees were elucidated in order to computationally identify orthologs of known circadian clock genes in all three species. In conjunction with this, a comprehensive RNA-Seq timecourse of reference inbred lines for maize, sorghum, and Setaria was performed. This allowed the identification of a number of genes likely involved in the maize circadian clock. Preliminary computational analysis of this extensive dataset indicates that circadian orthologs between the species retain similar phases. For individual genes, altered expression patterns have been found between the species, which could indicate functional innovation.

These lines of evidence suggest that genes within the circadian clock have evolved novel properties across plant species. The work presented here shows that even conserved clock genes have altered functionality in monocots. The catalog of maize circadian genes will provide a starting point for further research of the maize circadian clock. Through evolution and domestication, important monocot crop species contain multiple copies of core circadian genes, meaning that crop clocks have diverged from the model based on the Arabidopsis circadian clock. The circadian clock plays important roles in growth, stress responses, and defense against pathogens, and so continued research directly focused on crop circadian clocks will aid agricultural efforts to breed resilient high-yield crops.