Pinnate compound leaves have laminae called leaflets distributed at intervals along an axis, the rachis, whereas simple leaves have a single lamina. In simple-and compound-leaved species, the PHANTASTICA (PHAN) gene is required for lamina formation. Antirrhinum majus mutants lacking a functional gene develop abaxialized, bladeless adult leaves. Transgenic downregulation of PHAN in the compound tomato (Solanum lycopersicum) leaf results in an abaxialized rachis without leaflets. The extent of PHAN gene expression was found to be correlated with leaf morphology in diverse compound-leaved species; pinnate leaves had a complete adaxial domain of PHAN gene expression, and peltate leaves had a diminished domain. These previous studies predict the form of a compound-leaved phan mutant to be either peltate or an abaxialized rachis. Here, we characterize crispa, a phan mutant in pea (Pisum sativum), and find that the compound leaf remains pinnate, with individual leaflets abaxialized, rather than the whole leaf. The mutant develops ectopic stipules on the petiole-rachis axis, which are associated with ectopic class 1 KNOTTED1-like homeobox (KNOX) gene expression, showing that the interaction between CRISPA and the KNOX gene PISUM SATIVUM KNOTTED2 specifies stipule boundaries. KNOX and CRISPA gene expression patterns indicate that the mechanism of pea leaf initiation is more like Arabidopsis thaliana than tomato.

SUMMARYA major goal in biology is to identify the genetic basis for phenotypic diversity. This goal underpins research in areas as diverse as evolutionary biology, plant breeding and human genetics. A limitation for this research is no longer the availability of sequence information but the development of functional genetic tools to understand the link between changes in sequence and phenotype. Here we describe Cardamine hirsuta, a close relative of the reference plant Arabidopsis thaliana, as an experimental system in which genetic and transgenic approaches can be deployed effectively for comparative studies. We present high-resolution genetic and cytogenetic maps for C. hirsuta and show that the genome structure of C. hirsuta closely resembles the eight chromosomes of the ancestral crucifer karyotype and provides a good reference point for comparative genome studies across the Brassicaceae. We compared morphological and physiological traits between C. hirsuta and A. thaliana and analysed natural variation in stamen number in which lateral stamen loss is a species characteristic of C. hirsuta. We constructed a set of recombinant inbred lines and detected eight quantitative trait loci that can explain stamen number variation in this population. We found clear phylogeographic structure to the genetic variation in C. hirsuta, thus providing a context within which to address questions about evolutionary changes that link genotype with phenotype and the environment.

Plant MYB genes can be divided into subgroups on the basis of additional conserved regions of sequence. In some cases, genes within a subgroup share similarities in function, as well as sequence. The functions of three proteins in subgroup 9 have been described, with AmMYBMX regulating the differentiation of conical-papillate petal epidermal cells, PhMYB1 involved in extending the growth of these cells, and AmMYBML1 involved in differentiation of several petal epidermal cell types. Here, the isolation of a gene encoding a new member of MYB subgroup 9, AmMYBML3 (Antirrhinum majus MYB MIXTA-LIKE 3) is described, which contains the defining regions of conserved sequence but is lacking the majority of the C-terminus, including the amphipathic alpha-helix presumed necessary for transcriptional activation. AmMYBML3 is expressed in all aerial organs, but its expression is restricted to outgrowing epidermal cells, including trichomes, stigmatic papillae, and petal conical-papillate cells. Ectopic expression of AmMYBML3 in tobacco results in the formation of conical-papillate cells in the usually flat carpel epidermis. These data suggest that this protein is capable of altering epidermal development, thus resulting in cellular outgrowth, despite the missing C-terminus, and may act in conjunction with other transcriptional activators to enhance cellular outgrowth from the epidermis of all aerial organs.

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.

The genomes of several legume species contain two Phantastica-like genes. Previous studies on leaf development have found that Phantastica confers leaf blade adaxial identity in plant species with simple leaves and leaflet adaxial identity in pea (Pisum sativum L.), a legume with compound leaves. Previous characterisation of the phantastica mutant of pea, crispa, showed it had radialised leaflets, but stipules were not radialised. This suggested either that mutation of a second redundant gene was required for radialisation of stipules, or, that a null mutation was required. Previously characterised crispa mutants may not have exhibited radialised stipules because they were weak alleles. In this work we show that pea has a second Phantastica-like gene, which lies on a different chromosome to Crispa. The second gene was found to be a pseudogene in several genotypes of pea, therefore it would not have a role in conferring stipule adaxial identity. A new deletion mutant, crispa-4 was identified. The mutant has radialised stipules and leaflets, showing that Crispa confers adaxial identity on both these organs in pea. The nucleotide sequence data reported here are in the EMBL and GenBank Nucleotide Databases under the accession numbers DQ486060 (JI 2822), DQ486061 (JI 15), DQ486062 (JI 281) and DQ486063 (JI 399).