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.

The Arabidopsis thaliana SICKLE (SIC) protein is proline-rich but largely uncharacterized. sic-1 mutants have a miRNA deficiency, alterations in transcript splicing, and increased intron accumulation. sic-3 mutants have global changes in transcript splicing and circadian clock defects likely caused by altered splicing of circadian clock transcripts. Here we show that 13 of 20 putative SIC-interacting proteins are splicing-associated, and that Arabidopsis SIC and DBR1 interact directly. DBR1 is the only lariat debranching enzyme in Arabidopsis and knockout mutants are embryo lethal. The weak dbr1-2 mutant accumulates intron lariats, which interfere with miRNA processing, and has morphological phenotypes similar to sic-1. Human cells deficient in DBR1 also have lariat accumulation and this causes exon skipping. Here we show that sic-1, sic-3, and a new dbr1 mutant allele, dbr1-3, accumulate lariats like the dbr1-2 mutant, and that sic-3 and dbr1-2 have similar changes in splicing, including increased intron retentions. Therefore, lariat accumulation in the sic-1 allele may be responsible for its miRNA deficiencies and related morphological phenotypes, as well as the alterations in transcript splicing. We present a unified theory for sic mutant phenotypes based on SIC-DBR1 interaction and sic intron lariat accumulation.

The circadian clock is a key mechanism for plants to anticipate and respond to daily and seasonal cycles of light and temperature. Cues of temperature and light cycles set the timing of circadian rhythms through the process of entrainment, yet the period length of rhythms remains constant because of temperature compensation. The molecular pathways that sense and respond to temperature are not well understood in plants. Recent studies in Arabidopsis thaliana suggest that alternative splicing of circadian clock transcripts is a fundamental mechanism that links ambient temperature perception with circadian clock rhythms. Splice variants of circadian clock transcripts are observed in response to temperature changes in the environment, but the source of splice variants and their effect on the circadian clock are not known. In addition, the core circadian clock genes LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) play an important role in modifying the circadian clock’s response to temperature.

Here, we describe characterization of a new mutant allele of the Arabidopsis SICKLE (SIC) gene, sic-3, and the existing sic-1 allele with respect to their effects on the Arabidopsis circadian clock. sic was first identified as, warm acute response of PRR7 (warp2). sic has a unique temperature-specific defect in circadian clock function and in alternative splicing of circadian clock transcripts. sic exhibits low-amplitude or arrhythmic circadian rhythms under cool ambient temperature cycles, but not under light-dark entrainment. sic mutants also lengthen free running period in a manner consistent with impaired temperature compensation. sic mutant alleles accumulate splice variants of core circadian clock gene transcripts, including LHY and CCA1, which is exacerbated by cool temperatures. sic mutant alleles also modify transcription of circadian clock genes, particularly in cool temperatures. The cca1 lhy double mutant is epistatic to sic, indicating that control of LHY and CCA1 splicing by SIC is needed for circadian clock function. Furthermore, the double and triple mutant combinations of sic- 3 with cca1-1 and lhy-20 indicate that CCA1 and LHY function redundantly to modify temperature compensation, and that this regulation is dependent on SIC in cool temperatures. Finally, expression of LHY protein is altered in sic mutants in a temperature-dependent manner.

SIC is a nuclear and cytoplasmic protein of unknown function, previously implicated in the control of alternative splicing, microRNA (miRNA) biogenesis, and stress responses (Zhan et al. 2012). SIC has homologous proline/serine rich proteins present only in angiosperms. The SIC protein interacts with spliceosome-associated proteins, including protein arginine methyltransferases PRMT4a and PRMT4b, as well as members of the NineTeen Complex (NTC), including the lariat debranching enzymeDBR1. The NTC is a multiprotein complex involved in transcription, transcription coupled DNA repair, and alternative splicing. This suggests that SIC maintains spliceosome efficiency at low temperatures through interaction with splicing factors, and this aspect of pre-RNA metabolism is critical for low temperature adaptation and for the circadian clock’s response to temperature cues.

We propose the following model: alternative splicing converts circadian clock pre- mRNA transcripts to mature mRNA in a temperature-dependent manner, and the resulting splice variant pool determines the proper accumulation and timing of circadian clock proteins, especially LHY and CCA1. This form of post-transcriptional circadian clock regulation is crucial for temperature compensation and thermocycle entrainment in Arabidopsis. Where SIC fits into this model is unknown, but our data suggests two possibilities: 1) SIC modifies PRMT4a and PRMT4b activity, which then affects spliceosome activity, and/or 2) SIC modifies the NTC to regulate transcription, transcription coupled DNA repair, and alternative splicing. All of these processes are temperature-dependent, and it is clear that the activity of SIC is most important in cool temperatures. SIC’s affect on LHY and CCA1 alternative splicing appears to play a large role in regulating temperature compensation at cool temperatures. Discovery of the biochemical and molecular activities of SIC is certain to reveal important details about the functional link between alternative splicing and temperature responses within the Arabidopsis circadian clock, and holds the potential to provide new insights into the molecular processes involved in pre-mRNA processing.

Together, RecQ helicase and topoisomerase III (Topo III) of Escherichia coli comprise a potent DNA strand passage activity that can catenate covalently closed DNA (Harmon, F. G., DiGate, R. J., and Kowalczykowski, S. C. (1999) Mol. Cell 3, 611-620). Here we directly assessed the structure of the catenated DNA species formed by RecQ helicase and Topo III using atomic force microscopy. The images show complex catenated DNA species involving crossovers between multiple double-stranded DNA molecules that are consistent with full catenanes. E. coli single-stranded DNA-binding protein significantly stimulated both the topoisomerase activity of Topo III alone and the DNA strand passage activity of RecQ helicase and Topo III. Titration data suggest that an intermediate of the RecQ helicase unwinding process, perhaps a RecQ helicase-DNA fork, is the target for Topo III action. Catenated DNA is the predominant product under conditions of molecular crowding; however, we also discovered that RecQ helicase and single-stranded DNA-binding protein greatly stimulated the intramolecular strand passage ("supercoiling") activity of Topo III, as revealed by changes in the linking number of uncatenated DNA. Together our results demonstrate that RecQ helicase and Topo III function together to comprise a potent and concerted single-strand DNA passage activity that can mediate both catenation-decatenation processes and changes in DNA topology.

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