A sweeter tomato: cracking the Cis-regulatory code of gene regulation

Throughout the world, the tomato is a valuable and loved vegetable. Its appeal to consumers is important to breeders and growers as well as for promoting consumer health. Tomato sweetness is a trait that consumers generally like. The sugar content, often measured as soluble solids content (SSC or Brix), is an essential contributor to the experience of sweetness. The fruit’s soluble solids are mainly imported as sucrose, which is converted to equimolar amounts of glucose and fructose in the fruit. Sucrose is transported from the sources, e.g. leaves, to fruits (the sink), in a tightly coordinated fashion. Final sugar content in ripe fruit is determined by the production and export of photosynthates from the source and the ability of the fruit to import and accumulate sugars (such as in starch) during growth and finally, release them during ripening. The aim of this thesis was to explore new ways of increasing sugar content using targeted mutagenesis and editing with CRISPR/Cas9. Expression modulation by promoter mutation, the manipulation of post-translational regulation and, gene targeting were explored.

Chapter 2 reviewed and explored the regulation of sugar metabolism in tomato, focussing on genes that would be amenable for editing using CRISPR/Cas to increase sugar content in the fruit. Identifying and understanding the role of the genes underlying the production and distribution of sugars is crucial for crop breeding. From this review, it became clear that both ADP-Glucose Pyrophosphorylase Large subunit 1 (AGPL1) and Cell Wall INvertase 5 (LIN5) are essential genes for sugar accumulation. AGPL1 encodes the large subunit of ADP-glucose pyrophosphorylase (AGPase). This tetrameric AGPase protein complex is involved in starch synthesis in plastids during fruit development. LIN5 hydrolyses sucrose into glucose and fructose. The gradient created is a major driving force for importing sugars into the fruit (sink strength). Both genes underlie a QTL for Brix, with both having an allele from a wild tomato relative conferring the positive effect on sugar content. One of this thesis’ aims was to study and modify the transcriptional regulation of expression of AGPL1 and LIN5. I studied the Cis-regulatory Elements (CREs) involved in the regulation of these two genes. One approach was to identify CREs and their interacting Transcription Factors (TFs) using various in silico and experimental tools. Simultaneously, I applied CRISPR/Cas9 multiplexed mutagenesis to create variation in both promoters. By studying mutants for their effect on the target gene expression, I intended to discover new types of CRE functionalities in an in vivo system. I hypothesized that when a mutation occurred in a CRE it would disrupt a Transcription Factor’s binding. If the disrupted site is the binding site for a repressor, the target gene expression would be expected to increase. By increasing the expression of either LIN5 or AGPL1, we concomitantly tried improving the tomato flavour by increasing sugar content.

An in silico analysis of the AGPL1 promoter in Chapter 3 resulted in identifying conserved and accessible promoter regions, and Yeast-one-hybrid (Y1H) assays identified interacting TFs. I studied the TFs effect on AGPL1 transcription with promoter-reporter assays. Two potential repressors (FRUITFULL 2 and the CCCH-type Zinc Finger C3H13) and several activators (Abscisic Acid Responsive Elements-Binding Factor 1 (AREB1), the homolog of the Arabidopsis B-box zinc-finger TF BBX19, the homolog of the Arabidopsis GATA-motif containing TF GATA9, Jasmonic Acid 1 (JA1), Nuclear Factor-YA10 (NF-YA10), the homolog of the Arabidopsis Telomerase Repeat Binding Factor-Like 3 (TRFL), and the WRKY motif-containing WRKY24, WRKY41 and WRKY81) were found. I used multiplexed mutagenesis with CRISPR/Cas9 on the AGPL1 promoter in cv. Moneyberg to modulate expression in situ and create plants with higher sugar content. Brix was increased in several of the obtained mutant lines, however, always occurring with a decreased fruit weight. For two out of three of the lines with the highest Brix, an increase of AGPL1 expression was demonstrated. 

Chapter 4 focussed on LIN5. Y1H and promoter reporter assays revealed several TFs interacting with the LIN5 promoter. GATA9, the homolog of pepper MYB48, ‘MYB48’, R2R3MYB58, MYB76, and a NAC TF behaved as repressors, while C3H13, NF-YA10 and a homolog of pepper NPY1, ‘NPY1’, increased expression in the promoter reporter assay.  I made promoter mutants and found increased Brix in several of the generated mutant lines. Several of these were characterized in the T­2 generation for LIN5 expression, where I could confirm altered expression in the mutants compared to the wild type. In a promoter reporter assay comparing the mutant promoters with a wild-type promoter, HAT1, MYB HYH and WRKY24 were identified as weak activators of the mutant’s promoters.

The work on the promoters of LIN5 and AGPL1 provided leads for fruit quality improvement and gained us some insight into the potential of genome editing for altering gene expression (up or down-regulation) in crops by targeting candidate gene CREs. My conclusion is that this approach is feasible, although growing and phenotyping mutants is space- and labour-intensive when targeting the promoter in a non-biased manner. Reducing the target area by predicting promising promoter regions would reduce the number of mutants required.

In Chapter 5, I explored the role of Cell-wall Inhibitor of β-fructosidase (CIF1 or INVINH1), a post-translational inhibitor of LIN5, in regulating LIN5 activity. I generated knockout-mutants of CIF1 and studied the effects on final soluble solids content (Brix˚) and invertase activity, as a previous report showed a positive effect of down-regulation by RNAi on sugar content. CRISPR/Cas-generated knockout mutants did have an increased Brix, but their fruit size decreased considerably.

Chapter 6 focused on improving the CRISPR/Cas9-toolbox by applying multiplexed Gene Targeting on LIN5. Three amino acid substitutions in an S. pennellii allele are thought to be responsible for a more active LIN5 protein. My goal was to create these three amino acid changes in our cultivar, cv. Moneyberg via homologous recombination, with a geminiviral-based replicon donor delivery system to simultaneously target all three amino acids. I found that the approach results in Gene Targeting in experiments in protoplasts and in stable transformation, albeit at a very low efficiency (1-3%).