Leaf development has been monitored chiefly by following anatomical markers. Analysis of transcriptome dynamics during leaf maturation revealed multiple expression patterns that rise or fall with age or that display age-specific peaks. These were used to formulate a digital differentiation index (DDI) based on a set of selected markers with informative expression during leaf ontogeny. The leaf-based DDI reliably predicted the developmental state of leaf samples from diverse sources and was independent of mitotic cell division transcripts or propensity of specific cell types. When calibrated by informative root markers, the same algorithm accurately diagnosed dissected root samples. We used the DDI to characterize plants with reduced activities of multiple CINCINNATA (CIN)-TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) growth regulators. These plants had giant curled leaves made up of small cells with abnormal shape, low DDI scores, and low expression of mitosis markers, depicting the primary role of CIN-TCPs as promoters of differentiation. Delayed activity of several CIN-TCPs resulted in abnormally large but flat leaves with regular cells. The application of DDI has therefore portrayed the CIN-TCPs as heterochronic regulators that permit the development of a flexible and robust leaf form through an ordered and protracted maturation schedule.

In seed plants, leaves are born on radial shoots, but unlike shoots, they are determinate dorsiventral organs made of flat lamina. YABBY genes are found only in seed plants and in all cases studied are expressed primarily in lateral organs and in a polar manner. Despite their simple expression, Arabidopsis thaliana plants lacking all YABBY gene activities have a wide range of morphological defects in all lateral organs as well as the shoot apical meristem (SAM). Here, we show that leaves lacking all YABBY activities are initiated as dorsiventral appendages but fail to properly activate lamina programs. In particular, the activation of most CINCINNATA-class TCP genes does not commence, SAM-specific programs are reactivated, and a marginal leaf domain is not established. Altered distribution of auxin signaling and the auxin efflux carrier PIN1, highly reduced venation, initiation of multiple cotyledons, and gradual loss of the SAM accompany these defects. We suggest that YABBY functions were recruited to mold modified shoot systems into flat plant appendages by translating organ polarity into lamina-specific programs that include marginal auxin flow and activation of a maturation schedule directing determinate growth.

The leaves of seed plants evolved from a primitive shoot system and are generated as determinate dorsiventral appendages at the flanks of radial indeterminate shoots. The remarkable variation of leaves has remained a constant source of fascination, and their developmental versatility has provided an advantageous platform to study genetic regulation of subtle, and sometimes transient, morphological changes. Here, we describe how eudicot plants recruited conserved shoot meristematic factors to regulate growth of the basic simple leaf blade and how subsets of these factors are subsequently re-employed to promote and maintain further organogenic potential. By comparing tractable genetic programs of species with different leaf types and evaluating the pros and cons of phylogenetic experimental procedures, we suggest that simple and compound leaves, and, by the same token, leaflets and serrations, are regulated by distinct ontogenetic programs. Finally, florigen, in its capacity as a general growth regulator, is presented as a new upper-tier systemic modulator in the patterning of compound leaves.