ZF protein C2H2-71 regulates soluble solids content by inhibiting LIN5 in tomato

Abstract

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 SlC2H2 family 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 SlLIN5 encoding 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 SlC2H2-71 knock-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 SlLIN5 expression.