Repression of ARF10 by microRNA160 plays an important role in the mediation of leaf water loss.

Solanum lycopersicum auxin response factor 10 (SlARF10) is post-transcriptionally regulated by Sl-miR160. Overexpression of a Sl-miR160-resistant SlARF10 (mSlARF10) resulted in narrower leaflet blades with larger stomata but lower densities. 35S:mSlARF10-6 plants with narrower excised leaves had greater water loss, which was in contrast to the wild type (WT). Further analysis revealed that the actual water loss was not consistent with the calculated stomatal water loss in 35S:mSlARF10-6 and the WT under the dehydration treatment, indicating that there is a difference in hydraulic conductance. Pretreatment with abscisic acid (ABA) and HgCl2 confirmed higher hydraulic conductance in 35S:mSlARF10, which is related to the larger stomatal size and higher activity of aquaporins (AQPs). Under ABA treatment, 35S:mSlARF10-6 showed greater sensitivity, and the stomata closed rapidly. Screening by RNA sequencing revealed that five AQP-related genes, fourteen ABA biosynthesis/signal genes and three stomatal development genes were significantly altered in 35S:mSlARF10-6 plants, and this result was verified by qRT-PCR. The promoter analysis showed that upregulated AQPs contain AuxRE and ABRE, implying that these elements may be responsible for the high expression levels of AQPs in 35S:mSlARF10-6. The three most upregulated AQPs (SlTIP1-1-like, SlPIP2;4 and SlNIP-type-like) were chosen to confirm AuxRE and ABRE function. Promoters transient expression demonstrated that the SlPIP2;4 and SlNIP-type-like AuxREs and SlPIP2;4 and SlTIP1-1-like ABREs could significantly enhance the expression of the GUS reporter in 35S:mSlARF10-6, confirming that AuxRE and ABRE may be the main factors inducing the expression of AQPs. Additionally, two upregulated transcription factors in 35S:mSlARF10-6, SlARF10 and SlABI5-like were shown to directly bind to those elements in an electromobility shift assay and a yeast one-hybrid assay. Furthermore, transient expression of down-regulated ARF10 or up-regulated ABI5 in tomato leaves demonstrated that ARF10 is the direct factor for inducing the water loss in 35S:mSlARF10-6. Here, we show that although SlARF10 increased the ABA synthesis/signal response by regulating stomatal aperture to mitigate water loss, SlARF10 also influenced stomatal development and AQP expression to affect water transport, and both act cooperatively to control the loss of leaf water in tomato. Therefore, this study uncovers a previously unrecognized leaf water loss regulatory factor and a network for coordinating auxin and ABA signalling in this important process. In an evolutionary context, miR160 regulates ARF10 to maintain the water balance in the leaf, thus ensuring normal plant development and environmental adaptation.