Thesis presented March 28, 2019

Abstract:
The Evening Complex (EC), a three protein complex comprising EARLY FLOWERING 3 (ELF3), ELF4 and LUX ARRYTHMO (LUX), is a key component of the plant circadian clock and an important regulator of genes important for thermosensitive growth, including PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Studies have shown that EC activity is temperature dependent, with increased repressive activity at lower temperatures. However, the molecular mechanisms for EC complex formation, DNA-binding and thermosensitive activity were not known. In order to address this, a series of in vitro, structural and in planta experiments were performed.

All three proteins of the EC were recombinantly produced and purified to homogeneity. These proteins were used to reconstitute the EC in vitro and to study its DNA-binding activity. The role of all three proteins in complex formation and activity were determined. LUX acts as the driver of DNA-binding via its MYB DNA-binding domain and targets the EC to its cognate sites. ELF3 acts as a scaffold for EC formation by binding both LUX and ELF3. However, the LUX-ELF3 complex poorly binds to DNA. ELF4 is required to restore DNA-binding of the complex. To further explore the DNA-binding specificity determinants, the DNA-binding domain (DBD) of LUX was expressed, purified and crystallized. The crystal structure of the LUX DBD in complex with two different DNA oligonucleotides reveals the residues critical for base read-out. The majority of these residues contact the major groove and are part of a plant-specific signature motif SH(A/L)QK(F/Y). In addition, an arginine residue (Arg 146) in the flexible N-terminal region of the protein acts as a clamp with contacts in the minor groove. Based on these structural studies, an arginine to alanine mutation (R146A) was made which had decreased DNA-binding affinity but retained specificity as determined in vitro via band shift assays as compared to the wild type protein. Transgenic experiments were used to determine the effect of the R146A mutation in planta. As predicted, this mutation resulted in a phenotype between wild type and an EC knock out mutant. This suggests that by altering the DNA binding affinity of LUX, the activity of the entire EC can be tuned in the plant. PIF4 expression levels were measured in the mutant and were shown to be elevated with respect to wild type but less affected than in a lux mutant, further supporting the decreased repressive activity of the EC due to the R146A mutation in LUX.

To further explore PIF4 regulation, CRISPR-Cas9 was used to target different cis-elements in the PIF4 promoter including the LUX Binding Site (LBS) and a G-box element. These mutations had opposite effects on plant growth and thermoresponse with the LBS mutant exhibiting elongated hypocotyls and an early flowering phenotype at 22°C as compared to wt. The G-box mutant on the contrary exhibited shortened hypocotyls and a late flowering phenotype at 27°C as compared to wt. This suggests that altering cis-elements in the PIF4 promoter may be a way to reprogram plant growth and thermoresponse at different temperatures.

Taken together, these results provide different strategies to affect plant growth under different non-stress ambient temperature regimes through either structure-based protein engineering as shown for LUX R146A mutation or via genome editing of specific regulatory elements known to affect growth and thermoresponse such as the LBS and G-box in the PIF4 promoter. With the increase in global temperatures due to climate change and the deleterious effects this has on crop productivity, the ability to predictably alter plant growth and thermoresponse is an attractive way to address this global challenge. These results provide a potential foundation for future applications in bioengineering of important crop species.

On-line thesis.