Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit

Abstract

Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and <i>β</i>-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of <i>SlARF4</i>. <i>SlARF4</i> was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (<i>arf4</i>) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The <i>arf4</i> mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in <i>arf4</i> mutants than in wildtype plants under water stress; furthermore, <i>arf4</i> mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in <i>arf4</i> mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in <i>arf4</i> mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and <i>arf4</i> mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes' promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of <i>SlABI5/ABF</i> and <i>SCL3</i>, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.