ORANGE interplays with TCP7 to regulate endoreduplication and leaf size

Leaf size is a crucial agronomic trait directly affecting crop yield, which is mainly determined by coordinated cell proliferation, growth, and differentiation. Although endoreduplication is known to be correlated with the onset of cell differentiation and leaf size, the underlying molecular mechanisms are largely unclear. The DnaJ-like zinc finger domain-containing protein ORANGE (OR) was initially demonstrated to confer the massive accumulation of carotenoids in cauliflower curds. However, the cauliflower or mutant also possesses other phenotypes such as smaller curds, smaller leaves with elongated petioles, and delayed flowering. Here, we demonstrated that OR physically interacts with the transcription factor TCP7, which promotes endoreduplication by inducing the expression of the cell cycle gene CYCLIN D 1;1 (CYCD1;1). Overexpression of OR resulted in smaller rosette leaves, whereas the OR-silencing plants had larger rosette leaves than wild-type plants. Our microscopic observations and flow cytometry analysis revealed that the variation in leaf size was a result of different endoreduplication levels. Genetic analyses showed that OR functions antagonistically with TCP7 in regulating the endoreduplication levels in leaf cells. While the expression of OR is induced by TCP7, OR represses the transactivation activity of TCP7 by affecting its binding capability to the TCP-binding motif in the promoter region of CYCD1;1. Through this interaction, OR negatively regulates the expression of CYCD1;1 and reduces the nuclear ploidy level in rosette leaf cells. Our findings provide new insights into the regulatory network of leaf size and also reveal a regulatory circuit controlling endoreduplication in leaf cells.