Shaping up: miR319 and LANCEOLATE control tomato fruit morphology

Abstract

The domestication of tomato (Solanum lycopersicum) has given rise to a wide range of cultivars with distinct fruit shapes and sizes. These traits are not only relevant to consumer preferences, often indicating culinary applications, but also bear importance for mechanical harvesting. Thus, understanding the molecular basis of fruit formation is a priority for scientists and breeders alike. Yet, few genes have been identified that regulate tomato fruit morphology. Among these, the transcriptional repressor OVATE is one of the best characterised. Loss of OVATE function causes increased cell division along the proximal-distal axis and reduced cell proliferation along the medial-lateral axis, resulting in elongated, pear-shaped fruits (Snouffer et al., 2020). How OVATE itself is regulated, however, is unclear.

Small RNAs, especially microRNAs (miRNAs) and phased secondary small interfering RNAs (phasiRNAs), are critical modulators of fruit development (Huang et al., 2022). In the model plant Arabidopsis thaliana, miR319a affects the development of petals, stamen and siliques by targeting the mRNA encoding transcription factors TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) 3 and TCP4 (Cao et al., 2022; Nag et al., 2009). In tomato, miR160, miR166 and miR396 have been implicated in the regulation of fruit size and shape (Huang et al., 2022). Tomato miR319 and its target SlTCP2/LANCEOLATE (LA), on the other hand, act as important regulators of leaf morphology (Ori et al., 2007); their role in fruit formation had not been explored.

Fabio Nogueira developed a passion for miRNAs and their role in plant development during his postdoctoral studies with Marja Timmermans at Cold Spring Harbor Laboratory. After returning to Brazil to establish his own research group, he focused on the control of reproductive development, contrasting miRNA function in Arabidopsis with that in crops such as tomato. The highlighted study began as a Master's project of co-first author Airton Carvalho Jr, who investigated the links between tomato fruit shape, LA and miR319.

Carvalho et al. characterised La-1 mutants, which harbour a mutation in LA's miR319 recognition site, which results in LA de-repression and ectopic expression (Ori et al., 2007). While homozygous mutants were rarely viable, heterozygous La-1/+ plants produced elongated fruits with a malformed septum, a rudimental placenta and, in some genetic backgrounds, a reduced number of seeds (Figure 1a). These phenotypes can be traced back to changes in the morphology of La-1/+ flowers, which have shorter sepals as well as carpels with a medial constriction in the ovary (Figure 1b). These phenotypes were associated with deregulated cell division in the developing flower and fruit: La-1/+ plants displayed an increased number of cell layers in the ovary wall and a reduced number in the pericarp. Using in situ hybridisation, the authors found that LA transcripts were nearly undetectable during early flower development in the wild type but accumulated ectopically in the early stages of gynoecium development in La-1/+, likely driving the observed morphological changes. The effects of LA on fruit development were further investigated by expressing another miR319-resistant LA allele, La-2, in the abaxial side of floral organs in wild-type plants. These plants produced elongated ovaries and fruits resembling those of La-1/+, confirming that ectopic accumulation of LA is sufficient to alter fruit shape and size.

As the project expanded, Carvalho collaborated with co-first authors Mateus Vicente and Leticia Ferigolo, who contributed expertise in gene expression and protein-DNA interaction analyses. In a search for putative downstream targets of LA, the team identified two TCP-like binding sites in the OVATE gene and, using chromatin immunoprecipitation and yeast-1-hybrid assays, demonstrated that one of them is directly bound by LA. OVATE transcript levels were reduced in La-1/+, suggesting that LA directly represses OVATE expression. Analysis of La-1/+ ovate double mutants, however, showed that the La-1 mutation is epistatic to ovate, suggesting that LA also regulates fruit development via an OVATE-independent pathway.

The plant hormone auxin also plays a major role in shaping tomato fruit morphology: knock-down of the Aux/IAA repressor gene ENTIRE (E) results in precocious fruit set and the formation of seedless fruits (Wang et al., 2005) while application of auxin causes fruit elongation (Wang et al., 2019). Therefore, the authors explored a potential link between auxin and the La-1/+ phenotype. They observed reduced auxin responses in La-1/+ floral buds based on expression of a pDR5::GUS reporter, which correlated with reduced expression of several auxin biosynthesis and response genes, most notably the biosynthesis gene SlYUCCA4 (SlYUC4). The SlYUC4 promoter was directly bound by LA, suggesting that SlYUC4 represents another key LA target.

Taken together, Carvalho et al. identified the miR319-LA module, previously known as a critical regulator of leaf shape (Ori et al., 2007), as a novel determinant of tomato fruit morphology, acting through the transcriptional repressor OVATE and the auxin biosynthesis gene SlYUC4 to regulate cell division in the gynoecium. Timing of miR319 accumulation is thus crucial for proper tomato fruit development. Key aspects of miR319 regulation, however, remain unknown, including how miR319 accumulation is controlled during fruit development and whether it responds to environmental cues. These questions represent exciting avenues for future research in Nogueira's lab.