The CsTCP13-CsKNAT1-CsKNAT6 module regulates leaf width through influencing gibberellin biosynthesis in citrus

Abstract

Leaf morphology is an important agronomic trait that affects photosynthetic efficiency and plant architecture. Some KNOXI (Class I KNOTTED1-LIKE HOMEOBOX) or CIN-TCP (CINCINNATA-LIKE TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL FACTORS) transcription factors control leaf development by influencing GA (gibberellin) content. However, the knowledge about their interaction in regulating leaf morphogenesis is still scarce, especially in woody fruit plants such as citrus. In this study, we found that the expression of CsTCP13 (a citrus CIN-TCP gene) dramatically decreased with a decreasing leaf length-width ratio in citrus. Transiently silencing CsTCP13 in citrus produced wider leaves and significantly decreased CsGA20ox1 (a key citrus GA20-oxidase gene for GA biosynthesis) expression and GAs (GA3, GA4, and GA7) contents. Conversely, stable overexpression of CsTCP13 in tobacco generated narrower leaves and significantly increased GA20ox1 expression and GA1 content; it also significantly shortened the distance between veins and reduced the number of epidermal cells per unit area of leaf. Moreover, transactivation assays showed that CsTCP13 had transcriptional activation, and yeast two-hybrid, split-luciferase complementation, and bimolecular fluorescence complementation assays confirmed that CsTCP13 interacted with CsKNAT1 (a citrus KNOXI protein). Furthermore, yeast one-hybrid and dual-luciferase assays validated that CsTCP13 promoted while CsKNAT1 and its interacting protein CsKNAT6 inhibited CsGA20ox1 expression by binding to its promoter. Interestingly, CsKNAT1 or the CsKNAT1-CsKNAT6 complex interacted with CsTCP13 to attenuate its promotion effect on CsGA20ox1 expression. Taken together, our findings revealed a novel regulatory mechanism that CsTCP13 regulates citrus leaf width through directly influencing CsGA20ox1 expression and then GA content, which can be negatively affected by the interaction with CsKNAT1 or the CsKNAT1-CsKNAT6 complex.