No abstract

YABBY genes are seed plant-specific transcriptional regulators that are involved in diverse aspects of leaf, shoot and flower development. A series of duplications gave rise to five gene groups found throughout flowering plants. In Arabidopsis and other species, expression of two gene groups, CRABS CLAW and INNER NO OUTER, is restricted to floral organs. In contrast, members of the FILAMENTOUS FLOWER, YABBY2 and YABBY5 gene groups are also expressed in leaves and have been termed 'vegetative YABBYs'. How the five paralogue groups evolved and how their expression and function diversified have remained largely unresolved, precluding a reconstruction of the natural history of this gene family. Here, we report new genes from Eschscholzia californica (Ranunculales, Papaveraceae) that we use together with currently available database sequences in a comprehensive phylogenetic re-evaluation of the YABBY gene family. Multilayered Bayesian analysis covering seed plants allowed us to locate Eschscholzia YABBY sequences within the gene family phylogeny. We established that vegetative YABBYs do not form a monophyletic clade, and that CRABS CLAW and FILAMENTOUS FLOWER arose from a common ancestor gene. INNER NO OUTER genes are sister to that ancestral gene. We identified several conserved motifs outside of known amino acid domains that define all five angiosperm YABBY gene clades. Further, we inferred the evolution of gene expression and provide evidence for release of purifying constraint in certain branches of the gene family tree. Finally, we report expression patterns for five Eschscholzia YABBY genes consistent with functional conservation between early-diverged and core eudicots.

Leaves possess intrinsic information about their final size, but the developmental mechanisms setting the limits of growth are not well characterized. By screening enhancer trap lines that show a specific expression pattern in leaf primordia, we isolated one line, 576. This line contains a T-DNA insertion upstream of the basic helix-loop-helix (bHLH) transcription factor SPATULA (SPT) gene, and shows expression in the basal region of young leaves, where cell proliferation is active. An spt loss-of-function mutation increased leaf size and total cell number within a leaf, while SPT overexpression decreased leaf size and total cell number within a leaf. Although spt mutations did not affect cell size, SPT overexpression decreased the cell size in fully expanded leaves. Genetic analysis suggested that SPT acts independently from another set of cell proliferation-dependent organ size regulators ANGUSTIFOLIA3 (AN3) and GROWTH REGULATING FACTOR5 (AtGRF5). Detailed analysis of spt leaf development showed that the spt mutation enlarged the size of the meristematic region in leaf primordia, while overexpression of AtGRF5 promoted cell proliferation without affecting the enlargement of the meristematic region. These results suggest that SPT functions as a repressor of leaf growth and that meristematic region size in young leaf primordia, in terms of proliferative cell number within leaf primordia, is another target of leaf size determination, which previously had not been identified.

No abstract

VIGS is an effective, fast and transient method to down-regulate gene expression in eschscholzia. It serves well to detect prominent phenotypes which may become obvious even if some target gene transcript remains in the plant tissue. However, subtle phenotypes will be more difficult to detect, as extremely strong silencing effects occur in <10 % of all flowers from infected plants.

Knotted-like homeobox (KNOX) genes encode important regulators of shoot development in flowering plants. In Arabidopsis, class I KNOX genes are part of a regulatory system that contributes to indeterminacy of shoot development, delimitation of leaf primordia and internode development. In other species, class I KNOX genes have also been recruited in the control of marginal blastozone fractionation during dissected leaf development. Here we report the isolation of class I KNOX genes from two species of the basal eudicot family Papaveraceae, Chelidonium majus and Eschscholzia californica. Sequence comparisons and expression patterns indicate that these genes are orthologs of SHOOTMERISTEMLESS (STM), a class I KNOX gene from Arabidopsis. Both genes are expressed in the center of vegetative and floral shoot apical meristems (SAM), but downregulated at leaf or floral organ initiating sites. While Eschscholzia californica STM (EcSTM) is again upregulated during acropetal pinna formation, in situ hybridization could not detect Chelidonium majus STM (CmSTM) transcripts at any stage of basipetal leaf development, indicating divergent evolution of STM gene function in leaves within Papaveraceae. Immunolocalization of KNOX proteins indicate that other gene family members may control leaf dissection in both species. The contrasting direction of pinna initiation in the two species was also investigated using Histone H4 expression. Leaves at early stages of development did not reveal notable differences in cell division activity of the elongating leaf axis, suggesting that differential meristematic growth may not play a role in determining the observed dissection patterns.

FLORICAULA/ LEAFY-like genes were initially characterized as flower meristem identity genes. In a range of angiosperms, expression occurs also in vegetative shoot apices and developing leaves, and in some species with dissected leaves expression is perpetuated during organogenesis at the leaf marginal blastozone. The evolution of these expression patterns and associated functions is not well understood. We have isolated and characterized a FLORICAULA-like gene from California Poppy, Eschscholzia californica Cham. (Papaveraceae), a species belonging to the basal eudicot clade Ranunculales. EcFLO encodes a putative 416-amino-acid protein with highest similarity to homologous genes from Trochodendron and Platanus. We show that EcFLO mRNA is expressed during the vegetative phase of the shoot apical meristem and in developing dissected leaves in a characteristic manner. This pattern is compared to that of other eudicots and discussed in terms of evolution of FLORICAULA expression and function.

CYCLOIDEA-like genes belong to the TCP family of transcriptional regulators and have been shown to control different aspects of shoot development in various angiosperm lineages, including flower monosymmetry in asterids and axillary meristem growth in monocots. Genes related to the CYC gene from ANTIRRHINUM show independent duplications in both asterids and rosids. However, it remains unclear to what extent this affected the evolution of flower symmetry and shoot branching in these and other eudicot lineages. Here, we show that CYC-like genes have also undergone duplications in two related Ranunculales families, Fumariaceae and Papaveraceae s.str. These families exhibit morphological diversity in flower symmetry and inflorescence architecture that is potentially related to functions of CYC-like genes. We present sequences of 14 CYC-related genes covering 9 genera. Phylogenetic analyses indicate the presence of three clades of CYC-like genes. Shared motifs in the region between the TCP and R domains of CYC-like genes between Fumariaceae, Papaveraceae s.str., and AQUILEGIA (Ranunculaceae) indicate that the observed duplications originated from a single CYC gene present in all Ranunculales. RT-PCR expression data suggest that gene duplication and diversification in Fumariaceae and Papaveraceae s.str. was accompanied by divergence in expression patterns.