Plant leaves show pronounced plasticity of size and form. In the classical, partially dominant mutation Lanceolate (La), the large compound leaves of tomato (Solanum lycopersicum) are converted into small simple ones. We show that LA encodes a transcription factor from the TCP family containing an miR319-binding site. Five independent La isolates are gain-of-function alleles that result from point mutations within the miR319-binding site and confer partial resistance of the La transcripts to microRNA (miRNA)-directed inhibition. The reduced sensitivity to miRNA regulation leads to elevated LA expression in very young La leaf primordia and to precocious differentiation of leaf margins. In contrast, downregulation of several LA-like genes using ectopic expression of miR319 resulted in larger leaflets and continuous growth of leaf margins. Our results imply that regulation of LA by miR319 defines a flexible window of morphogenetic competence along the developing leaf margin that is required for leaf elaboration.

Leaves are formed at the flanks of the shoot apical meristem (SAM) and develop into a variety of forms. In tomato, prolonged leaf patterning enables the elaboration of compound leaves by reiterative initiation of leaflets with lobed margins. In goblet (gob) loss-of-function mutants, primary leaflets are often fused, secondary leaflets and marginal serrations are absent, and SAMs often terminate precociously. We show that GOB encodes a NAC-domain transcription factor expressed in narrow stripes at the leaf margins, flanking the distal side of future leaflet primordia, and at the boundaries between the SAM and leaf primordia. Leaf-specific overexpression of the microRNA miR164, a negative regulator of GOB-like genes, also leads to loss of secondary-leaflet initiation and to smooth leaflet margins. Plants carrying a dominant gob allele with an intact ORF but disrupted miR164 binding site produce more cotyledons and floral organs, have split SAMs and, surprisingly, simpler leaves. Overexpression of a form of GOB with an altered miR164 binding site in leaf primordia leads to delayed leaflet maturation, frequent, improperly timed and spaced initiation events, and a simple mature leaflet form owing to secondary-leaflet fusion. miR164 also affects leaflet separation in Cardamine hirsuta, a Brassicaceae species with complex leaves. Genetic and molecular analyses suggest that GOB expression is intact in the simplified leaves of entire tomato mutants, which have a defect in a putative repressor of auxin responses. Our results show that GOB marks leaflet boundaries and that its accurate spatial, temporal and quantitative activity affects leaf elaboration in a context-dependent manner.

Elaboration of a compound leaf shape depends on extended morphogenetic activity in developing leaves. In tomato (Solanum lycopersicum), the CIN-TCP transcription factor LANCEOLATE (LA) promotes leaf differentiation. LA is negatively regulated by miR319 during the early stages of leaf development, and decreased sensitivity of LA mRNA to miR319 recognition in the semi-dominant mutant La leads to prematurely increased LA expression, precocious leaf differentiation and a simpler and smaller leaf. Increased levels or responses of the plant hormone gibberellin (GA) in tomato leaves also led to a simplified leaf form. Here, we show that LA activity is mediated in part by GA. Expression of the SlGA20 oxidase1 (SlGA20ox1) gene, which encodes an enzyme in the GA biosynthesis pathway, is increased in gain-of-function La mutants and reduced in plants that over-express miR319. Conversely, the transcript levels of the GA deactivation gene SlGA2 oxidase4 (SlGA2ox4) are increased in plants over-expressing miR319. The miR319 over-expression phenotype is suppressed by exogenous GA application and by a mutation in the PROCERA (PRO) gene, which encodes an inhibitor of the GA response. SlGA2ox4 is expressed in initiating leaflets during early leaf development. Its expression expands as a result of miR319 over-expression, and its over-expression leads to increased leaf complexity. These results suggest that LA activity is partly mediated by positive regulation of the GA response, probably by regulation of GA levels.

Elaboration of a complex leaves depends on the morphogenetic activity of a specific tissue at the leaf margin termed marginal-blastozon (MB). In tomato (Solanum lycopersicym), prolonged activity of the MB leads to the development of compound leaves. The activity of the MB is restricted by the TCP transcription factor LANCEOLATE (LA). Plants harboring the dominant LA mutant allele La-2 have simple leaves with a uniform blade. Conversely, leaves of pFIL > > miR319 are compound and grow indeterminately in their margins due to leaf overexpression of miR319, a negative regulator of LA and additional miR319-sensitive genes. We have recently shown that the auxin-response sensor DR5::VENUS marks and precedes leaflet initiation events in the MB. Mutations in ENTIRE (E), an auxin signal inhibitor from the Aux/IAA family, lead to the expansion of the DR5::VENUS signal to throughout the leaf-primordia margin, and to a simplified leaf phenotype. Here, we examined the interaction between auxin, E, and LA in tomato leaf development. In La-2 leaf primordia, the auxin signal is very weak and is diffused to throughout the leaf margin, suggesting that auxin acts within the developmental-context of MB activity, which is controlled by LA. e La-2 double mutants showed an enhanced simple leaf phenotype and e pFIL > > miR319 leaves initiated less leaflets than wild-type, but their margins showed continuous growth. These results suggest that E and auxin affect leaflet initiation within the context of the extended MB activity, but their influence on the extent of indeterminate growth of the leaf is minor.

The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development.