MicroRNA1917 targets CTR4 splice variants to regulate ethylene responses in tomato

Abstract

Ethylene perception is regulated by receptors, and the downstream protein CONSTITUTIVE TRIPLE RESPONSE1 is a key suppressor of ethylene signalling. The non-conserved tomato (Solanum lycopersicum) microRNA1917 (Sly-miR1917) mediates degradation of SlCTR4 splice variants (SlCTR4sv) but the molecular details of this pathway remain unknown. Sly-miR1917 and the targeted SlCTR4sv are ubiquitously expressed in all tomato organs. Overexpression of Sly-miR1917 enhances ethylene responses, including the triple response in etiolated seedlings, in the absence of ethylene, as well as epinastic petiole growth, accelerated pedicel abscission, and fruit ripening. Enhanced ethylene signalling in Sly-miR1917-overexpressing plants (1917-OE) is accompanied by up-regulation of ethylene biosynthesis and signalling genes, and increased ethylene emission. These phenotypes were recovered by repressing the positive ethylene regulator EIN2. Moreover, the Sly-miR1917-targeted SlCTR4 splice variant SlCTR4sv3, expressed specifically in the abscission zone, exhibited the opposite expression pattern to Sly-miR1917. Complementation of the Arabidopsis thaliana ctr-1 mutant and yeast two-hybrid and bimolecular fluorescence complementation assays suggested that SlCTR4sv3 functions in ethylene signalling. Co-expression of Sly-miR1917 and SlCTR4sv3 in Nicotiana benthamiana further suggested that Sly-miR1917 cleaves SlCTR4sv3 in vivo. Database homology searching revealed a Solanum tuberosum CTR-like splice variant containing a Sly-miR1917 binding sequence, and a homologue of mature Sly-miR1917 in potato, indicating a conserved function for miR1917 and the regulatory miRNA-mediated ethylene network in solanaceous species.