Auxin treatments reduce citrus fruit splitting by increasing cell wall loosening and expansion in the peel

Fruit splitting in citrus fruits is a physiological disorder caused by an imbalance in the growth of the peel and pulp, which typically occurs at the end of the cell expansion stage. This disorder can be mitigated by applying growth regulators. The most effective treatment is to apply 2,4-dichlorophenoxyacetic acid (2,4-D) at the beginning of the cell expansion stage — i.e. two months before splitting occurs. However, the reason why the treatment is effective two months after application remains unclear. The main hypothesis proposes anatomical changes in the peel that can induce long-term increased resistance to internal pulp pressure. These changes could include an increase in cell number, an increase in cell size, or both. In this study, we examine the mechanism by which 2,4-D reduces fruit splitting by up to 45 % in citrus through the analysis of ultra-histological (TEM) and molecular changes in the exocarp. The results demonstrate that 2,4-D exerts a direct localised effect on the outer exocarp, significantly increasing cell size and cell wall thickness by 38.7 % and 41.7 % respectively, rather than affecting cell number. This is associated with upregulation of cell wall loosening-related enzymes, including endoglucanases, expansins, polygalacturonases, and pectin methylesterases. Our results indicate that the long-term efficacy of 2,4-D in preventing citrus splitting is due to its localised impact on the morphology of the outer exocarp cell walls. This has practical implications for the timing and method of treatment. • auxin (2,4-D) treatments induce physiological and anatomical changes to increase peel resistance two months ahead of citrus fruit splitting • the anatomical effect is localized in the cell wall of the 10 outermost cell layers of the peel • auxin increases cell size and cell wall width, rather than cell number, through upregulation of endoglucanases, expansins, polygalacturonases, and pectinesterases