Ton Winkelmolen

Plant architecture is a highly dynamic trait that can be influenced by many factors. Because of this, evolutionary adaptation created an immense variation in plant architecture across the plant kingdom. Also during domestication, several plant architectural traits were targeted to improve human usability of crops. Several important traits are tangled on a molecular level, meaning that changing one trait, often changes other traits as well. This is also the case in barley, where the transcription factor VRS5, regulates both tiller development and lateral spikelet development. Both of these processes are important yield related traits in barley, and their genetic connection makes it difficult to manipulate these traits independently. The main objective in this thesis was to provide molecular insights that can help untangle these traits, with the focus on VRS5.

As a transcription factor, VRS5 can directly bind to DNA and regulate the expression of downstream targets. Understanding what these targets are, can help to better understand how VRS5 exactly functions and regulates these different processes. In Chapter 2, we identified these direct targets in barley by combining two techniques. Using DNA-affinity purification followed by sequencing (DAP-seq), we identified the genome-wide binding sites of VRS5. Next to that, we compared gene expression in vrs5 mutants to wild-type at four developmental stages using RNA sequencing. By combining the genes that are differentially expressed in vrs5 and the loci where VRS5 binds, we obtained a set of direct VRS5 targets that are thus bound by and regulated by VRS5. We investigated four different developmental stages and therefore we could identify which of these targets mostly play a role early in development, and presumably in tillering, while others mostly play a role later in development, when the inflorescence is developing.

Next, we looked in more detail at VRS5 as a protein in Chapter 3. We found that in barley two different protein isoforms of VRS5 are formed; VRS5α and VRS5β. We wanted to know if these isoforms function differently, and by CRISPR mutagenesis we made mutations causing only one of the protein isoforms to be formed. After investigating these lines, we found that it is possible to uncouple the tillering from the row-type function. While complete knock-out lines of vrs5 have both an increased number of tillers and six-rowed to intermediate spike phenotype, the vrs5α mutants only show increased tillering, but a two-rowed spike. We showed that these phenotypical differences are likely caused by differences in DNA binding of the isoforms in complex with another transcription factor, TB2.

One of the direct targets of VRS5 identified in Chapter 2, VRS1 was already known as thus far the most downstream regulator of lateral spikelet development. Despite this role in such an agronomically important trait, little is known of the molecular function of VRS1. In Chapter 4, we identified the consensus DNA binding motif of VRS1, using DAP-seq. In combination with an already available RNA-seq dataset of a VRS1 allele, we also identified some potential direct downstream targets of VRS1. One of these targets was SOC1-like, of which we made CRISPR mutants. These mutants showed significantly smaller lateral spikelets compared to WT. Analysis of natural variation in SOC1-like revealed that there has been a strong selection for a frameshift mutation of SOC1-like in six-rowed barleys.

Many of the experiments performed in this thesis would be impossible without having an efficient CRISPR based mutagenesis protocol. In Chapter 5, we showed how we greatly improved the editing efficiency of Cas9 in barley by using several methods shown to improve editing efficiency. With this improved system we were able to generate multiple knock-out lines and even double knock-outs in primary transformants already.

Finally, in Chapter 6 I combined the obtained results and place them in a broader scientific perspective. I highlight the conserved function of VRS5 in regulating tillering, while some interesting VRS5 targets that might regulate lateral spikelet development are discussed. In conclusion, with the molecular insights provided in this thesis, I showed several potential ways to help untangle yield-related traits in barley, specifically via VRS5.