The transition to ripening in non-climacteric species is governed by several signals, including hormones that enhance or counteract the abscisic acid (ABA)-promoting effect. The SQUAMOSA Promoter-binding protein-Like (SPL) transcription factors are involved in ripening through the modulation of anthocyanin biosynthesis. In sweet cherry fruits, several miR156-targeted PavSPLs are expressed before and during ripening. Recently, some PavSPLs were found in the transition from development to ripening in cultivars contrasting in maturity time. Additionally, several forms of miR156 were expressed in sweet cherry seeds of an early-season cultivar. In this work, we addressed the relevance of endocarp lignification and PavSPLs expression for the transition to ripening. First, we characterized early- and late-season sweet cherry cultivars, ‘Celeste’ and ‘Regina’, focusing on fruit and seed development, endocarp lignification, and PavSPL expression profile. Fruit growth dynamics revealed an earlier onset of color development and lignification in ‘Celeste’, while ‘Regina’ exhibited a prolonged lag phase and delayed embryo development. Transcript profiling at the light green stage showed a higher expression of PavSPL genes in fruits and identified cultivar-specific expressions, especially between ‘Regina’ and ‘Celeste’ seeds. Co-expression networks linked PavSPLs to genes involved in lignin and anthocyanin biosynthesis. We focused on PavSPL2 and PavSPL9, which were targeted by mtr-miR156a and gma-miR156f. Both PavSPLs and miRNAs were expressed in fruits and seeds at the yellow stage, an advanced point in the transition to ripening in sweet cherry. Exogenous application of auxin-related compounds in the mid-season cultivar ‘Lapins’ modulated endocarp lignification and pigmentation. Notably, p-IBA treatment, which enzymatically targets the lignin pathway, transiently increased anthocyanin accumulation and reduced lignin deposition, effects that correlated with the downregulation of PavSPL gene expression. These findings highlight the interplay between lignification, color evolution, and pigment biosynthesis during the transition from development to ripening in sweet cherry fruits, and suggest a role for PavSPL genes in this transition.

Several signals control fruit ripening, including hormones and metabolites, many of which are produced by the seeds. However, the molecular signatures of seeds and fruits at the transition from development to ripening (t-DtoR) have not yet been elucidated in non-climacteric species. Thus, we aimed to characterize sweet cherry fruits and seeds through directed hormone profiling, untargeted metabolomics, as well as mRNA-seq and small RNA-seq. The early- and late-season cultivars, ‘Celeste’ and ‘Regina’ respectively, were analyzed at the t-DtoR, where ‘Regina’ had delayed ripening evolution, accompanied by a higher content of negative ripening markers in fruits, such as amino acids and chlorophylls, whereas ‘Celeste’ had more positive markers, including abscisic acid (ABA) and D-Fru. PCA analysis revealed that jasmonic acic (JA) and JA-Ile mostly contributed to clustering according to cultivar. Overrepresentation of ‘auxin response’ and ‘inositol catabolic process” categories occurred in ‘Celeste’ fruits (CF), whereas ‘lignin catabolic process’ was overrepresented in ‘Celeste’ seeds (CS). Higher sugar content, enriched ‘disaccharide metabolic process’ category plus increased levels of cytokinins and gibberellins were distinguishable features of CS. Target search and degradome networks showed several squamosa promoter-binding-like (SPL) encoding sequences abundant in fruits, which were predicted targets of miRNA156s. Interactome networks of the four omics datasets revealed alternative molecular landscapes in the contrasting cultivars, where small RNAs had multiple associations. For instance, the small RNAs mes-miR535d/ csi-MIR166f (‘Celeste’) and gra-MIR8771d/ csi-MIR166f (‘Regina’) had associations with several hormones and transcripts. The gene ontology (GO) analysis of these transcripts showed overrepresentation of ‘response to cytokinin in ‘Celeste’ and ‘auxin response’, in ‘Regina’, respectively. This evidences differences between cultivars at the t-DtoR. Finally, in the interactome seed-fruit connections were identified, with a putative inositol oxidase expressed in fruits having several connections with seed transcripts but only in the early-phenotype. This work highlights small RNAs, SPLs, lignin and inositol pathways as novel players in non-climacteric ripening.