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Soluble solids content (SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC has been shown to be transcriptionally regulated via sugar metabolism. Previous studies have been predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development. However, the specific regulatory mechanism of C2H2 in the accumulation of soluble solids in tomato fruits are yet to be fully understood. This study involved the selection of eight tomato accessions with varying levels of SSC to study the expression of SlC2H2family genes in red ripe fruits. The study found that the levels of SlC2H2-71 expressions were significantly reduced in high-SSC accessions compared to low-SSC accessions. The Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, leading to elevated levels of soluble solids, fructose, glucose, malic, and citric acids in mature red ripe fruits. However, sucrose content in the edited Slc2h2-71 mutant lines generally decreased. RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to sugar and acid metabolic pathways, which was further confirmed by quantitative real time PCR. Specifically, there was an observed increase in the expression of SlLIN5encoding the cell wall invertase (CWIN). The yeast one-hybrid (Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay (EMSA) demonstrated that SlC2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of SlLIN5, culminating in the repression of its transcriptional activity. The activity of acid invertase in the SlC2H2-71knock-out lines exhibited a significantly higher level compared to that observed in the control lines. In summary, the regulation of tomato fruit SSC by SlC2H2-71 involves the inhibition of SlLIN5expression.

Domestication and improvement are important processes that generate the variation in genome and phonotypes underlying crop improvement. Unfortunately, during selection for certain attributes, other valuable traits may be inadvertently discarded. One example is the decline in fruit soluble solids content (SSC) during tomato breeding. Several genetic loci for SSC have been identified, but few reports on the underlying mechanisms are available. In this study we performed a genome-wide association study (GWAS) for SSC of the red-ripe fruits in a population consisting of 481 tomato accessions with large natural variations and found a new quantitative trait locus, STP1, encoding a sugar transporter protein. The causal variation of STP1, a 21-bp InDel located in the promoter region 1124 bp upstream of the start codon, alters its expression. STP1Insertionaccessions with an 21-bp insertion have higher SSC than STP1Deletionaccessions with the 21-bp deletion. Knockout of STP1in TS-23 with high SSC using CRISPR/Cas9 greatly decreased SSC in fruits. In vivoand in vitroassays demonstrated that ZAT10-LIKE, a zinc finger protein transcription factor (ZFP TF), can specifically bind to the promoter of STP1Insertionto enhance STP1expression, but not to the promoter of STP1Deletion, leading to lower fruit SSC in modern tomatoes. Diversity analysis revealed that STP1was selected during tomato improvement. Taking these results together, we identified a naturally occurring causal variation underlying SSC in tomato, and a new role for ZFP TFs in regulating sugar transporters. The findings enrich our understanding of tomato evolution and domestication, and provide a genetic basis for genome design for improving fruit taste.