Domestication of crop plants had effects on human lifestyle and agriculture. However, little is known about the underlying molecular mechanisms accompanying the changes in fruit appearance as a consequence of selection by early farmers. We report the fine mapping and cloning of a tomato (Solanum lycopersicum) fruit mass gene encoding the ortholog of KLUH, SlKLUH, a P450 enzyme of the CYP78A subfamily. The increase in fruit mass is predominantly the result of enlarged pericarp and septum tissues caused by increased cell number in the large fruited lines. SlKLUH also modulates plant architecture by regulating number and length of the side shoots, and ripening time, and these effects are particularly strong in plants that transgenically down-regulate SlKLUH expression carrying fruits of a dramatically reduced mass. Association mapping followed by segregation analyses revealed that a single nucleotide polymorphism in the promoter of the gene is highly associated with fruit mass. This single polymorphism may potentially underlie a regulatory mutation resulting in increased SlKLUH expression concomitant with increased fruit mass. Our findings suggest that the allele giving rise to large fruit arose in the early domesticates of tomato and becoming progressively more abundant upon further selections. We also detected association of fruit weight with CaKLUH in chile pepper (Capsicum annuum) suggesting that selection of the orthologous gene may have occurred independently in a separate domestication event. Altogether, our findings shed light on the molecular basis of fruit mass, a key domestication trait in tomato and other fruit and vegetable crops.

The sizes of plant organs such as fruit and seed are crucial yield components. Tomato KLUH underlies the locus fw3.2, an important regulator of fruit and seed weight. However, the mechanism by which the expression levels of KLUH affect organ size is poorly understood. We found that higher expression of SlKLUH increased cell proliferation in the pericarp within 5 d post-anthesis in tomato near-isogenic lines. Differential gene expression analyses showed that lower expression of SlKLUH was associated with increased expression of genes involved in lipid metabolism. Lipidomic analysis revealed that repression of SlKLUH mainly increased the contents of certain non-phosphorus glycerolipids and phospholipids and decreased the contents of four unknown lipids. Co-expression network analyses revealed that lipid metabolism was possibly associated with but not directly controlled by SlKLUH, and that this gene instead controls photosynthesis-related processes. In addition, many transcription factors putatively involved in the KLUH pathway were identified. Collectively, we show that SlKLUH regulates fruit and seed weight which is associated with altered lipid metabolism. The results expand our understanding of fruit and seed weight regulation and offer a valuable resource for functional studies of candidate genes putatively involved in regulation of organ size in tomato and other crops.

CRISPR/Cas-mediated genome editing is a powerful approach to accelerate yield enhancement to feed growing populations. Most applications focus on "negative regulators" by targeting coding regions and promoters to create nulls or weak loss-of-function alleles. However, many agriculturally important traits are conferred by gain-of-function alleles. Therefore, creating gain-of-function alleles for "positive regulators" by CRISPR will be of great value for crop improvement. CYP78A family members are the positive regulators of organ weight and size in crops. In this study, we engineered allelic variation by editing tomato KLUH promoter around a single-nucleotide polymorphism (SNP) that is highly associated with fruit weight. The SNP was located in a conserved putative cis-regulatory element (CRE) as detected by the homology-based prediction and the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Twenty-one mutant alleles with various insertion and deletion sizes were generated in the LA1589 background. Five mutant alleles (m2+4bp , m3+1bp , m5-1bp , m13-8bp , and m14-9bp ) showed a consistent increase in fruit weight and a significant decrease in the proportion of small fruits in all experimental evaluations. Notably, m2+4bp and m3+1bp homozygote significantly increase fruit weight by 10.7-15.7 and 8.7-16.3%, respectively. Further analysis of fruit weight based on fruit position on the inflorescence indicated that the five beneficial alleles increase the weight of all fruits along inflorescence. We also found that allele types and transcriptional changes of SlKLUH were poor predictors of the changes in fruit weight. This study not only provides a way of identifying conserved CRE but also highlights enormous potential for CRISPR/Cas-mediated cis-engineering of CYP78A members in yield improvement.