Assessing duplication and loss of APETALA1/FRUITFULL homologs in Ranunculales.

Gene duplication and loss provide raw material for evolutionary change within organismal lineages as functional diversification of gene copies provide a mechanism for phenotypic variation. Here we focus on the APETALA1/FRUITFULL MADS-box gene lineage evolution. AP1/FUL genes are angiosperm-specific and have undergone several duplications. By far the most significant one is the core-eudicot duplication resulting in the euAP1 and euFUL clades. Functional characterization of several euAP1 and euFUL genes has shown that both function in proper floral meristem identity, and axillary meristem repression. Independently, euAP1 genes function in floral meristem and sepal identity, whereas euFUL genes control phase transition, cauline leaf growth, compound leaf morphogenesis and fruit development. Significant functional variation has been detected in the function of pre-duplication basal-eudicot FUL-like genes, but the underlying mechanisms for change have not been identified. FUL-like genes in the Papaveraceae encode all functions reported for euAP1 and euFUL genes, whereas FUL-like genes in Aquilegia (Ranunculaceae) function in inflorescence development and leaf complexity, but not in flower or fruit development. Here we isolated FUL-like genes across the Ranunculales and used phylogenetic approaches to analyze their evolutionary history. We identified an early duplication resulting in the RanFL1 and RanFL2 clades. RanFL1 genes were present in all the families sampled and are mostly under strong negative selection in the MADS, I and K domains. RanFL2 genes were only identified from Eupteleaceae, Papaveraceae s.l., Menispermaceae and Ranunculaceae and show relaxed purifying selection at the I and K domains. We discuss how asymmetric sequence diversification, new motifs, differences in codon substitutions and likely protein-protein interactions resulting from this Ranunculiid-specific duplication can help explain the functional differences among basal-eudicot FUL-like genes.

Duplicated STM-like KNOX I genes act in floral meristem activity in Eschscholzia californica (Papaveraceae).

In angiosperms, the shoot apical meristem is at the origin of leaves and stems and is eventually transformed into the floral meristem. Class I knotted-like homeobox (KNOX I) genes are known as crucial regulators of shoot meristem formation and maintenance. KNOX I genes maintain the undifferentiated state of the apical meristem and are locally downregulated upon leaf initiation. In Arabidopsis, KNOX I genes, especially SHOOTMERISTEMLESS (STM), have been shown to regulate flower development and the formation of carpels. We investigated the role of STM-like genes in the reproductive development of Eschscholzia californica, to learn more about the evolution of KNOX I gene function in basal eudicots. We identified two orthologs of STM in Eschscholzia, EcSTM1 and EcSTM2, which are predominantly expressed in floral tissues. In contrast, a KNAT1/BP-like and a KNAT2/6-like KNOX I gene are mainly expressed in vegetative organs. Virus-induced gene silencing (VIGS) was used to knockdown gene expression, revealing that both EcSTM genes are required for the formation of reproductive organs. Silencing of EcSTM1 resulted in the loss of the gynoecium and a reduced number of stamens. EcSTM2-VIGS flowers had reduced and defective gynoecia and a stronger reduction in the number of stamen than observed in EcSTM1-VIGS. Co-silencing of both genes led to more pronounced phenotypes. In addition, silencing of EcSTM2 alone or together with EcSTM1 resulted in altered patterns of internodal elongation and sometimes in other floral defects. Our data suggest that some aspects of STM function present in Arabidopsis evolved already before the basal eudicots diverged from core eudicots.

Virus-induced gene silencing (VIGS) in Cysticapnos vesicaria, a zygomorphic-flowered Papaveraceae (Ranunculales, basal eudicots).

BACKGROUND AND AIMS: Studies of evolutionary diversification in the basal eudicot family Papaveraceae, such as the transition from actinomorphy to zygomorphy, are hampered by the lack of comparative functional studies. So far, gene silencing methods are only available in the actinomorphic species Eschscholzia californica and Papaver somniferum. This study addresses the amenability of Cysticapnos vesicaria, a derived fumitory with zygomorphic flowers, to virus-induced gene silencing (VIGS), and describes vegetative and reproductive traits in this species. METHODS: VIGS-mediated downregulation of the C. vesicaria PHYTOENE DESATURASE gene (CvPDS) and of the FLORICAULA gene CvFLO was carried out using Agrobacterium tumefaciens transfer of Tobacco rattle virus (TRV)-based vectors. Wild-type and vector-treated plants were characterized using reverse transcription-PCR (RT-PCR), in situ hybridization, and macroscopic and scanning electron microscopic imaging. KEY RESULTS: Cysticapnos vesicaria germinates rapidly, can be grown at high density, has a short life cycle and is self-compatible. Inoculation of C. vesicaria with a CvPDS-VIGS vector resulted in strong photobleaching of green parts and reduction of endogenous CvPDS transcript levels. Gene silencing persisted during inflorescence development until fruit set. Inoculation of plants with CvFLO-VIGS affected floral phyllotaxis, symmetry and floral organ identities. CONCLUSIONS: The high penetrance, severity and stability of pTRV-mediated silencing, including the induction of meristem-related phenotypes, make C. vesicaria a very promising new focus species for evolutionary-developmental (evo-devo) studies in the Papaveraceae. This now enables comparative studies of flower symmetry, inflorescence determinacy and other traits that diversified in the Papaveraceae.

The bHLH transcription factor SPATULA controls final leaf size in Arabidopsis thaliana.

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.

KNOX overexpression in transgenic Kohleria (Gesneriaceae) prolongs the activity of proximal leaf blastozones and drastically alters segment fate.

KNOX (knotted1-like homeobox) genes have a widely conserved role in the generation of dissected leaves. Ectopic KNOX activity in leaves in various angiosperm lineages causes leaf form changes that can elucidate how the configuration of leaf development evolved. We present an analysis of leaf morphology and morphogenesis in transgenic Kohleria lines overexpressing a heterologous KNOX gene. Kohleria, like many members of Gesneriaceae, has simple-serrated leaves with pinnate venation. KNOX overexpression causes prolonged segment proliferation in proximal, but not distal, parts of leaf blades. Elaborate dissected segments reiterate the zonation of the whole leaf, with organogenic activity persisting between a distal maturation zone and a proximal intercalary elongation zone. The architecture of vascular bundles is severely altered, with a reduced midvein and a more palmate venation. The initial establishment of organogenically competent primordial margins (marginal blastozones) and the onset of tissue differentiation in early stages of leaf development were similar in wild-type and KNOX overexpressing lines. However, leaves overexpressing KNOX often failed to fully mature, and persistent marginal blastozones were found at the base of blades in mature portions of the shoot. We conclude that KNOX-mediated perpetuation of marginal blastozones in Kohleria is sufficient to induce a set of processes that result in highly dissected leaflets, which are unusual in this plant family. Spatial confinement of blastozones between an early maturing tip and a late elongating petiole zone reflects the presence of distinct maturation processes that limit the ability of the leaf margins to respond to ectopic KNOX gene expression.

Highly efficient virus-induced gene silencing (VIGS) in California poppy (Eschscholzia californica): an evaluation of VIGS as a strategy to obtain functional data from non-model plants.

BACKGROUND AND AIMS: Eschscholzia californica (California poppy) is an emerging model plant for 'evo-devo' studies from the basal eudicot clade of Papaveraceae. California poppy has a relatively small genome, a short life cycle and, most importantly, it is amenable for transformation. However, since this transformation protocol is time consuming, virus-induced gene silencing (VIGS) was evaluated as a fast method to obtain functional data for California poppy genes. METHODS: Commercially available California poppy plants were infiltrated with Agrobacterium tumefaciens carrying the tobacco rattle virus plasmids pTRV1 and pTRV2. pTRV2 contained part of the eschscholzia Phytoene Desaturase (EcPDS) gene whose loss of function results in photobleaching of the green parts of the plant and in a lack of floral coloration. The degree and duration of these symptoms was evaluated for vegetative rosettes and plants in flower. KEY RESULTS: It is shown that VIGS is able to effectively down-regulate the EcPDS gene in eschscholzia. Various degrees of silencing were observed starting <2 weeks after infiltration with Agrobacterium tumefaciens in 92 % of the plants. Tissue with silencing symptoms also showed complete or strong reduction of EcPDS transcripts. Strong silencing resulted in almost completely white petals, fruits, shoots and leaves. Plants with a strong degree of silencing will eventually die off; however, others are able to produce EcPDS gene product even after a strong initial silencing and will recover. Silencing was found to be not always systemic, but was often restricted to certain organs or parts of organs. CONCLUSIONS: 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.

Expression patterns of STM-like KNOX and Histone H4 genes in shoot development of the dissected-leaved basal eudicot plants Chelidonium majus and Eschscholzia californica (Papaveraceae).

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.

Developmental events leading to peltate leaf structure in Tropaeolum majus (Tropaeolaceae) are associated with expression domain changes of a YABBY gene.

Peltate leaf architecture has evolved from conventional bifacial leaves many times in flowering plant evolution. Characteristics of peltate leaves, such as the differentiation of a cross zone and of a radially symmetric, margin-less petiole, have also been observed in mutants of genes responsible for adaxial-abaxial polarity establishment. This suggests that altered regulation of such genes provided a mechanism for the evolution of peltate leaf structure. Here, we show that evolution of leaf peltation in Tropaeolum majus, a species distantly related to Arabidopsis thaliana, was associated with altered expression of Tropaeolum majus FILAMENTOUS FLOWER (TmFIL), a gene conferring abaxial identity. In situ hybridization indicates that adaxial and abaxial domains are established in early leaf primordia as in species with bifacial leaves. Upon initiation of the cross zone by fusion of the blade margins, localized expansion of TmFIL to the upper leaf side could be seen, indicating a local loss of adaxial leaf identity. The observed changes in expression are consistent with a role of TmFIL in radialization of the petiole and circularization of the leaf blade margin by the cross zone. In addition, expression was observed in segment primordia and during expansion of the bifacial blade, suggesting additional roles for TmFIL in leaf development.

Comparative analysis of leaf shape development in Eschscholzia californica and other Papaveraceae-Eschscholzioideae.

Dissected leaves in Papaveraceae-Eschscholzioideae have an architecture frequently encountered in the basal eudicot clade Ranunculales that could represent an ancestral condition for eudicots. Developmental morphology of foliage leaves was investigated using scanning electron microscopy and focusing on primordium formation activity (primary morphogenesis) at the leaf margin. Eschscholzia californica, E. lobii, and Hunnemannia fumariaefolia had a polyternate-acropetal mode of leaf dissection. Segment formation continued around the whole leaf blade periphery. Differences in mature leaf architecture was traced to variations in regional blastozone activity and duration. Epidermal cell size measurements in E. californica indicated that the leaf tip tissue starts to differentiate already at the onset of organogenic activity and that tip cells remain larger than epidermal cells at the basal margins during further growth. It is argued that early differentiation of the tip does not set up a general basipetal differentiation gradient, but is a local effect that allows acropetal pinna initiation to occur in subapical blastozones. In Dendromecon, secondarily entire leaves have evolved through the loss of primordium formation activity. Marginal corrugations found in Dendromecon form late in development and are not reminiscent of lateral primordia.

EcFLO, a FLORICAULA-like gene from Eschscholzia californica is expressed during organogenesis at the vegetative shoot apex.

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.