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Light elicits different growth responses in different organs of plants. These organ-specific responses are prominently displayed during de-etiolation. While major light-responsive components and early signaling pathways in this process have been identified, this information has yet to explain how organ-specific light responses are achieved. Here, we report that members of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factor family participate in photomorphogenesis and facilitate light-induced cotyledon opening in Arabidopsis (Arabidopsis thaliana). Chromatin immunoprecipitation sequencing and RNA sequencing analyses indicated that TCP4 targets a number of SMALL AUXIN UPREGULATED RNA (SAUR) genes that have previously been shown to exhibit organ-specific, light-responsive expression. We demonstrate that TCP4-like transcription factors, which are predominantly expressed in the cotyledons of both light- and dark-grown seedlings, activate SAUR16 and SAUR50 expression in response to light. Light regulates the binding of TCP4 to the promoters of SAUR14, SAUR16, and SAUR50 through PHYTOCHROME-INTERACTING FACTORs (PIFs). PIF3, which accumulates in etiolated seedlings and its levels rapidly decline upon light exposure, also binds to the SAUR16 and SAUR50 promoters, while suppressing the binding of TCP4 to these promoters in the dark. Our study reveals that the interplay between light-responsive factors PIFs and the developmental regulator TCP4 determines the cotyledon-specific light regulation of SAUR16 and SAUR50, which contributes to cotyledon closure and opening before and after de-etiolation.

Epigenetic modifications include histone modifications and DNA methylation; such modifications can induce heritable changes in gene expression by altering DNA accessibility and chromatin structure. A number of studies have demonstrated that epigenetic factors regulate plant developmental timing in response to environmental changes. However, we still have an incomplete picture of how epigenetic factors can regulate developmental events such as organogenesis. The small number of cell types and the relatively simple developmental progression required to form the Arabidopsis petal makes it a good model to investigate the molecular mechanisms driving plant organogenesis. In this minireview, we summarize recent studies demonstrating the epigenetic control of gene expression during various developmental transitions, and how such regulatory mechanisms can potentially act in petal growth and differentiation.

Development of leaf margins is an important process in leaf morphogenesis. CIN-clade TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PCF) transcription factors are known to have redundant roles in specifying leaf margins, but the specific mechanisms through which individual TCP genes function remain elusive. In this study, we report that the CIN-TCP gene TCP5 is involved in repressing the initiation and outgrowth of leaf serrations by activating two key regulators of margin development, the Class II KNOX factor KNAT3 and BEL-like SAW1. Specifically, TCP5 directly promotes the transcription of KNAT3 and indirectly activates the expression of SAW1. We also show that TCP5 regulates KNAT3 and SAW1 in a temporal- and spatial- specific manner that is largely in accordance with the progress of formation of serrations. This regulation might serve as a key mechanism in patterning margin morphogenesis and in sculpting the final form of the leaf.

The phytochrome B (phyB) photoreceptor stimulates light responses in plants in part by inactivating repressors of light responses, such as PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Activated phyB inhibits PIF3 by rapid protein degradation and decreased transcription. PIF3 protein degradation is mediated by EIN3-BINDING F-BOX PROTEIN (EBF) and LIGHT-RESPONSE BTB (LRB) E3 ligases, the latter of which simultaneously targets phyB for degradation. In this study, we show that PIF3 levels are additionally regulated by alternative splicing and protein translation in Arabidopsis (Arabidopsis thaliana). Overaccumulation of photo-activated phyB, which occurs in the mutant defective for LRB genes under continuous red light, induces a specific alternative splicing of PIF3 that results in retention of an intron in the 5' untranslated region of PIF3 mRNA. In turn, the upstream open reading frames contained within this intron inhibit PIF3 protein synthesis. The phyB-dependent alternative splicing of PIF3 is diurnally regulated under the short-day light cycle. We hypothesize that this reversible regulatory mechanism may be utilized to fine tune the level of PIF3 protein in light-grown plants and may contribute to the oscillation of PIF3 protein abundance under the short-day environment.

Summary Differential regulation of stem cell activity in shoot meristems contributes to the wide variation in shoot architecture. 1 , 2 , 3 In most Citrus species, a thorn meristem and a dormant axillary meristem co-localize at each leaf base, offset from each other in a spiral phyllotactic pattern. We recently identified THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, as necessary for the termination of meristem proliferation and concomitant thorn production in Citrus. 4 However, how the dormant axillary meristem at the same leaf axil maintains stem cell activity is still unknown. The phosphatidylethanolamine-binding protein (PEBP)-type transcription factors CENTRORADIALIS (CEN) and TERMINAL FLOWER1 (TFL1) maintain inflorescence meristem indeterminacy in many plant species by antagonizing floral meristem identity regulators. 5 , 6 , 7 , 8 , 9 Here, we show that, in Citrus, Citrus CEN (CsCEN) maintains vegetative axillary meristem indeterminacy by antagonizing TI1. CsCEN is expressed in the axillary meristem, but not in the thorn meristem. Disruption of CsCEN function results in termination of the stem cell activity and conversion of dormant axillary meristems into thorns, although ectopic overexpression of CsCEN represses TI1 expression and converts thorns into dormant buds, a phenotype similar to the ti1 mutant. We further show that CsCEN interacts with Citrus FD (CsFD) to repress TI1 expression. CsCEN activity depends on the function of TI1 and TI2, as mutations in TI1 and TI2 rescue the cscen mutant phenotype. We suggest that the antagonistic roles of CsCEN and TI1 define the pattern of axillary meristem determinacy, which shapes vegetative Citrus tree shoot architecture.

Plant organ growth requires the proper transition from cell proliferation to cell expansion and differentiation. The CIN-TCP transcription factor gene TCP4 and its post-transcriptional regulator microRNA319 play a pivotal role in this process. In this study, we identified a pathway in which the product of the C2H2 zinc finger gene RABBIT EARS (RBE) regulates the transcription of TCP4 during Arabidopsis (Arabidopsis thaliana) petal development. RBE directly represses TCP4 during the early stages of petal development; this contributes to the role of RBE in controlling the growth of petal primordia. We also found that the rbe-1 mutant strongly enhanced the petal phenotypes of tcp4soj6 and mir319a, two mutants with compromised miR319 regulation of TCP4 Our results show that transcriptional and post-transcriptional regulation function together to pattern the spatial and temporal expression of TCP4 This in turn controls petal size and shape in Arabidopsis.

The WUSCHEL (WUS) gene is necessary for the maintenance of stem cells in the shoot apical meristem. Four recent reports show that cytokinin responsive type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs) directly activate WUS expression, providing a long-awaited explanation for how cytokinin influences the maintenance of the stem cell niche.

Thorns arise from axillary shoot apical meristems that proliferate for a time and then terminally differentiate into a sharp tip. Like other meristems, thorn meristems contain stem cells but, in the case of thorns, these stem cells undergo a programmed cessation of proliferative activity. Using Citrus, we characterize a gene network necessary for thorn development. We identify two Citrus genes, THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TCP transcription factors, as necessary for stem cell quiescence and thorn identity. Disruption of TI1 and TI2 function results in reactivation of stem cells and concomitant conversion of thorns to branches. Expression of WUSCHEL (WUS) defines the shoot stem cell niche in the apical meristems of many angiosperm species; we show that TI1 binds to the Citrus WUS promoter and negatively regulates its expression to terminate stem cell proliferation. We propose that shifts in the timing and function of components of this gene network can account for the evolution of Citrus thorn identity. Modulating this pathway can significantly alter plant architecture and could be leveraged to improve crop yields.

SummaryThe Arabidopsis petal is a simple laminar organ whose development is largely impervious to environmental effects, making it an excellent model for dissecting the regulation of cell-cycle progression and post-mitotic cell expansion that together sculpt organ form [1, 2]. Arabidopsis petals grow via basipetal waves of cell division, followed by a phase of cell expansion [3–5]. RABBIT EARS (RBE) encodes a C2H2 zinc finger transcriptional repressor and is required for petal development [6–9]. During the early phase of petal initiation, RBE regulates a microRNA164-dependent pathway that controls cell proliferation at the petal primordium boundaries [10–12]. The effects of rbe mutations on petal lamina growth suggest that RBE is also required to regulate later developmental events during petal organogenesis [6, 12]. Here, we demonstrate that, early in petal development, RBE represses the transcription of a suite of CIN-TCP genes that in turn act to inhibit the number and duration of cell divisions; the temporal alleviation of that repression results in the transition from cell division to post-mitotic cell expansion and concomitant petal maturation.

Plants consist of many different cell types with specific shapes optimized for their particular functions. For example, most flowering plants have conically shaped epidermal cells on the upper surface of their petals that are important for pollinator attraction. The control of cell morphology in organs such as roots and leaves has been extensively studied, but much less is known about the genes that promote conical expansion of petal epidermal cells. We have developed a technique to rapidly assay the morphology of conical petal epidermal cells, and we employed this method in an unbiased genetic screen to identify mutants that alter the development of these cells. Mutants isolated in this screen affected cell shape, cell size, cuticle synthesis, and cellular chirality. This approach allowed for the identification of novel cellular components that are critical for the morphology of conical petal cells, and demonstrates the usefulness of petal epidermal cells as a model system for studying cellular morphogenesis.

Flowers are organized into concentric whorls of sepals, petals, stamens and carpels, with each of these floral organ types having a unique role in reproduction (Figure 1). Sepals enclose and protect the flower bud, while petals can be large and showy so as to attract pollinators (or people!). Stamens produce pollen grains that contain male gametes, while the carpels contain the ovules that when fertilized will produce the seeds. While the size, shape, number and elaboration of each of these organ types can be quite different, the same general organization of four floral organ types arranged in concentric whorls exists across all flowering plant (angiosperm) species. As I shall explain in this Primer, the 'ABC model' is a simple and satisfying explanation for how this conserved floral architecture is genetically specified.

Summary The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.

Rhamnose is required in Arabidopsis thaliana for synthesizing pectic polysaccharides and glycosylating flavonols. RHAMNOSE BIOSYNTHESIS1 (RHM1) encodes a UDP-l-rhamnose synthase, and rhm1 mutants exhibit many developmental defects, including short root hairs, hyponastic cotyledons, and left-handed helically twisted petals and roots. It has been proposed that the hyponastic cotyledons observed in rhm1 mutants are a consequence of abnormal flavonol glycosylation, while the root hair defect is flavonol-independent. We have recently shown that the helical twisting of rhm1 petals results from decreased levels of rhamnose-containing cell wall polymers. In this study, we found that flavonols indirectly modify the rhm1 helical petal phenotype by altering rhamnose flux to the cell wall. Given this finding, we further investigated the relationship between flavonols and the cell wall in rhm1 cotyledons. We show that decreased flavonol rhamnosylation is not responsible for the cotyledon phenotype of rhm1 mutants. Instead, blocking flavonol synthesis or rhamnosylation can suppress rhm1 defects by diverting UDP-l-rhamnose to the synthesis of cell wall polysaccharides. Therefore, rhamnose is required in the cell wall for normal expansion of cotyledon epidermal cells. Our findings suggest a broad role for rhamnose-containing cell wall polysaccharides in the morphogenesis of epidermal cells.

Although specific organs in some plant species exhibit helical growth patterns of fixed or variable handedness, most plant organs are not helical. Here we report that mutations in Arabidopsis RHAMNOSE BIOSYNTHESIS 1 (RHM1) cause dramatic left-handed helical growth of petal epidermal cells, leading to left-handed twisted petals. rhm1 mutant roots also display left-handed growth. Furthermore, we find that RHM1 is required to promote epidermal cell expansion. RHM1 encodes a UDP-L-rhamnose synthase, and rhm1 mutations affect synthesis of the pectic polysaccharide rhamnogalacturonan-I. Unlike other mutants that exhibit helical growth of fixed handedness, the orientation of cortical microtubule arrays is unaltered in rhm1 mutants. Our findings reveal a novel source of left-handed plant growth caused by changes in cell wall composition that is independent of microtubule orientation. We propose that an important function of rhamnose-containing cell wall polymers is to suppress helical twisting of expanding plant cells.

Inducible gene expression systems, in which the level or timing of activity of a gene of interest can be controlled exogenously, are an effective means to assess gene function. However, such systems have not been widely employed in crop plants. Here we show that the glucocorticoid receptor (GR)-based inducible gene expression system functions in Citrange (Citrus sinensis × C. trifoliata) plants. We generated transgenic Citrange plants containing a two component 35S::LhGR/pOp6::β-glucuronidase (GUS) system (Wielopolska et al. (2005) Plant Biotechnol. J. 3, 583), in which the synthetic transcription factor, LhGR, is glucocorticoid-inducible and can activate the pOp6 promoter driving expression of the β-glucuronidase (GUS) reporter gene. We describe a method for inducing LhGR activity using the synthetic glucocorticoid dexamethasone (DEX) in transgenic Citrange. With the advent of transgenic approaches to engineer new traits in Citrus, control of transgene activity will be essential to realize the full potential of such manipulations. The method we describe here is likely to be of general use in many Citrus cultivars to temporally control the activity of introduced transgenes.

The establishment and maintenance of organ boundaries are vital for animal and plant development. In the Arabidopsis flower, three microRNA164 genes (MIR164a, b and c) regulate the expression of CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, which encode key transcriptional regulators involved in organ boundary specification. These three miR164 genes are expressed in distinct spatial and temporal domains that are crucial for their function. Here, we show that the C2H2 zinc finger transcriptional repressor encoded by RABBIT EARS (RBE) regulates the expression of all three miR164 genes. Furthermore, we demonstrate that RBE directly interacts with the promoter of MIR164c and negatively regulates its expression. We also show that the role of RBE in sepal and petal development is mediated in part through the concomitant regulation of the CUC1 and CUC2 gene products. These results indicate that one role of RBE is to fine-tune miR164 expression to regulate the CUC1 and CUC2 effector genes, which, in turn, regulate developmental events required for sepal and petal organogenesis.

Although many models have been proposed that could lead to the maintenance of gene duplicates, the ways in which interacting gene duplicates influence each other's evolution and function remain poorly understood. Here, we focus on duplication and loss of the B class MADS box transcription factor genes in the euasterids I and the ramifications of such changes on paralog evolution and their encoded functions. In core eudicots, the B class genes belong to two paralogous lineages whose products form obligate heterodimers. Based on comparative genomic and phylogenetic analyses, we show that five stepwise B class MADS box gene gain or loss events occurred during the radiation of the euasterids I within core eudicots. Gene loss in one sublineage was correlated with a deficit of other sublineage genes. We also show that the gain or loss of B class MADS box gene paralogs were associated with altered protein-protein interactions among the remaining copies. These altered protein interactions were correlated with asymmetric patterns of sequence diversification and selection, suggesting that compensatory changes were driving the evolution of such genes. Furthermore, these B class MADS box gene gain or loss events were associated with the evolutionary divergence of floral morphology in the euasterids I. Together, these observations point to a cooperative strategy by which gene networks evolve, with selection maintaining the overall logic of a network despite changes in individual components.

Background Gene duplication has often been invoked as a key mechanism responsible for evolution of new morphologies. The floral homeotic B-group gene family has undergone a number of gene duplication events, and yet the functions of these genes appear to be largely conserved. However, detailed comparative analysis has indicated that such duplicate genes have considerable cryptic variability in their functions. In the Solanaceae, two duplicate B-group gene lineages have been retained in three subfamilies. Comparisons of orthologous genes across members of the Solanaceae have demonstrated that the combined function of all four B-gene members is to establish petal and stamen identity, but that this function was partitioned differently in each species. These observations emphasize both the robustness and the evolvability of the B-system. Scope We provide an overview of how the B-function genes can robustly specify petal and stamen identity and at the same time evolve through changes in protein-protein interaction, gene expression patterns, copy number variation or alterations in the downstream genes they control. By using mathematical models we explore regulatory differences between species and how these impose constraints on downstream gene regulation. Conclusions Evolvability of the B-genes can be understood through the multiple ways in which the B-system can be robust. Quantitative approaches should allow for the incorporation of more biological realism in the representations of these regulatory systems and this should contribute to understanding the constraints under which different B-systems can function and evolve. This, in turn, can provide a better understanding of the ways in which B-genes have contributed to flower diversity.

Basal asterid families, and to a lesser extent the asterids as a whole, are characterized by a high variation in petal and stamen morphology. Moreover, the stamen number, the adnation of stamens to petals, and the degree of sympetaly vary considerably among basal asterid taxa. The B group genes, members of the APETALA3 (AP3) and PISTILLATA (PI) gene lineages, have been shown to specify petal and stamen identities in several core eudicot species. Duplicate genes in these lineages have been shown in some cases to have diversified in their function; for instance in Petunia, a PI paralog is required for the fusion of stamens to the corolla tube, illustrating that such genes belonging to this lineage are not just involved in specifying the identity of the stamens and petals but can also specify novel floral morphologies. This motivated us to study the duplication history of class B genes throughout asterid lineages, which comprise approximately one-third of all flowering plants. The evolutionary history of the PI gene subfamily indicates that the two genes in Petunia result from an ancient duplication event, coinciding with the origin of core asterids. A second duplication event occurred before the speciation of basal asterid Ericales families. These and other duplications in the PI lineage are not correlated with duplications in the AP3 lineage. To understand the molecular evolution of the Ericales PI genes after duplication, we have described their expression patterns using reverse transcription polymerase chain reaction and in situ hybridization, reconstructed how selection shaped their protein sequences and tested their protein interaction specificity with other class B proteins. We find that after duplication, PI paralogs have acquired multiple different expression patterns and negative selective pressure on their codons is relaxed, whereas substitutions in sites putatively involved in protein-protein interactions show positive selection, allowing for a change in the interaction behavior of the PI paralogs after duplication. Together, these observations suggest that the asterids have preferentially recruited PI duplicate genes to diverse and potentially novel roles in asterid flower development.

Petals appear in many angiosperm taxa, yet when and how these attractive organs originated remains unclear. Phylogenetic reconstructions based on morphological data suggest that petals have evolved multiple times during the radiation of the angiosperms. Based on the diversity of petal morphologies, it is likely that the developmental programmes specifying petal identity are distinct in different lineages. On the other hand, molecular genetic analyses have suggested that the specification of petal identity in different lineages utilizes similar genetic pathways. Together, these observations indicate that the evolution of petals has relied on the repeated recruitment of a suite of interacting developmental control genes, albeit in different ways in different lineages. These observations suggest that this gene regulatory network represents a 'deep homology' in plant evolution. A major challenge is to understand how this ancestral developmental pathway has been redeployed in different angiosperm lineages, and how changes in the workings of this pathway have led to the myriad shapes, colours, and sizes of petals.

The origin and evolution of the perianth remains enigmatic. While it seems likely that an undifferentiated perianth consisting of tepals arose early in angiosperm evolution, it is unclear when and how differentiated perianths consisting of distinct organs, such as petals and sepals, arose. Phylogenetic reconstructions of ancestral perianth states across angiosperms have traditionally relied on morphological data from extant species, but these analyses often produce equivocal results. Here we describe the use of devel opmental genetic data as an additional strategy to infer the ancestral perianth character state for different angiosperm clades. By assessing functional data in combination with expression data in a maximum likelihood framework, we provide a novel approach for investigating the evolutionary history of the perianth. Results of this analysis provide new insights into perianth evolution and provide a proof of concept for using this strategy to explore the incorporation of developmental genetic data in character state re constructions. As the assumptions outlined here are tested and more genetic data are generated, we hope that ancestral state recon structions based on multiple lines of evidence will converge. The evolution of flowers is thought to underlie the extensive radiation of angiosperms through enhancement of efficient in teractions with animal pollinators to facilitate reproductive suc cess (Regal, 1977). Flowers are generally composed of a perianth of sterile organs surrounding the reproductive organs? the pollen-bearing stamens and ovule-bearing carpels, with the perianth often differentiated into protective outer organs (se pals) and showy inner organs (petals). Flower morphology has diversified through evolutionary modifications of these floral organs, with some of the most striking changes in form due to modifications in perianth morphology. For example, perianth organization has transitioned from spiral to whorled phyllotaxy, distinct perianth organ types have evolved multiple times, and there have been a number of transitions from radial to bilateral perianth symmetry. Across flowering plants, the perianth may remain undifferentiated as tepals, or may be highly differenti

Organogenesis entails the regulation of cell division, cell expansion, cell and tissue type differentiation, and patterning of the organ as a whole. Petals are ideally suited to dissecting these processes. Petals are dispensable for growth and reproduction, enabling varied manipulations to be carried out with ease. In Arabidopsis, petals have a simple laminar structure with a small number of cell types, facilitating the analysis of organogenesis. This review summarizes recent studies that have illuminated some of the complex interplay between the genetic pathways controlling petal specification, growth and differentiation in Arabidopsis. These advances, coupled with the advantages of using petals as a model experimental system, provide an excellent platform to investigate the underlying mechanisms driving plant organogenesis.

Floral organogenesis is dependent on the combinatorial action of MADS-box transcription factors, which in turn control the expression of suites of genes required for growth, patterning, and differentiation. In Arabidopsis (Arabidopsis thaliana), the specification of petal and stamen identity depends on the action of two MADS-box gene products, APETALA3 (AP3) and PISTILLATA (PI). In a screen for genes whose expression was altered in response to the induction of AP3 activity, we identified GNC (GATA, nitrate-inducible, carbon-metabolism-involved) as being negatively regulated by AP3 and PI. The GNC gene encodes a member of the Arabidopsis GATA transcription factor family and has been implicated in the regulation of chlorophyll biosynthesis as well as carbon and nitrogen metabolism. In addition, we found that the GNC paralog, GNL (GNC-like), is also negatively regulated by AP3 and PI. Using chromatin immunoprecipitation, we showed that promoter sequences of both GNC and GNL are bound by PI protein, suggesting a direct regulatory interaction. Analyses of single and double gnc and gnl mutants indicated that the two genes share redundant roles in promoting chlorophyll biosynthesis, suggesting that in repressing GNC and GNL, AP3/PI have roles in negatively regulating this biosynthetic pathway in flowers. In addition, coexpression analyses of genes regulated by AP3, PI, GNC, and GNL indicate a complex regulatory interplay between these transcription factors in regulating a variety of light and nutrient responsive genes. Together, these results provide new insights into the transcriptional cascades controlling the specification of floral organ identities.

One of the biggest unanswered questions in developmental biology is how growth is controlled. Petals are an excellent organ system for investigating growth control in plants: petals are dispensable, have a simple structure, and are largely refractory to environmental perturbations that can alter their size and shape. In recent studies, a number of genes controlling petal growth have been identified. The overall picture of how such genes function in petal organogenesis is beginning to be elucidated. This review will focus on studies using petals as a model system to explore the underlying gene networks that control organ initiation, growth, and final organ morphology.

MIKCc-type MADS-box genes encode key transcriptional regulators of a variety of developmental processes in Arabidopsis thaliana. However, there has been relatively little effort to systematically carry out comparative genomic or functional analyses of these genes across flowering plants. Here we describe a strategy to identify members of the MIKCc-type MADS-box gene family from any angiosperm species of interest. Using this approach, we have identified 24 MIKCc-type MADS-box genes in tomato, including 17 that have not previously been characterized. Using these sequences, we have performed phylogenetic analyses that indicate that there have been a number of gene duplication and loss events in tomato relative to Arabidopsis. We also describe the expression domains of these genes and compare these results with their cognates in Arabidopsis. These analyses demonstrate the utility of this approach for characterizing a large number of MIKCc-type MADS-box genes from any flowering plant species of interest and provide a framework for evolutionary comparisons of this important gene family across angiosperms.

The floral homeotic APETALA3 (AP3) gene in Arabidopsis thaliana encodes a MADS box transcription factor required for specifying petal and stamen identities. AP3 is a member of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. Although Arabidopsis lacks genes in the paralogous Tomato MADS box gene 6 (TM6) lineage, tomato (Solanum lycopersicum) possesses both euAP3 and TM6 genes, which have functionally diversified. A loss-of-function mutation in Tomato AP3 (TAP3) resulted in homeotic transformations of both petals and stamens, whereas RNA interference–induced reduction in TM6 function resulted in flowers with homeotic defects primarily in stamens. The functional differences between these genes can be ascribed partly to different expression domains. When overexpressed in an equivalent domain, both genes can partially rescue the tap3 mutant, indicating that relative levels as well as spatial patterns of expression contribute to functional differences. Our results also indicate that the two proteins have differing biochemical capabilities. Together, these results suggest that TM6 and TAP3 play qualitatively different roles in floral development; they also support the ideas that the ancestral role of AP3 lineage genes was in specifying stamen development and that duplication and divergence in the AP3 lineage allowed for the acquisition of a role in petal specification in the core eudicots.

Summary Orchids serve as useful model plants for the discovery and study of genes involved in novel processes in floral development because of their highly modified flowers. In this study three different FRUITFULL ( FUL )-like MADS-box genes, DthyrFL1 , -2 , and -3 have been isolated from the orchid Dendrobium thyrsiflorum . Sequence alignment indicates that the entire sequence of exon 6 is missing in DthyrFL 3 and that a frame shift could explain the missing FUL -like motif found in the sequence of both DthyrFL1 and -2 . Phylogenetic analysis of the APETALA1/FRUITFULL lineage shows that FUL -like sequences from monocots all fall within one major clade and that subsequent gene duplication within this group is specific to monocots. At least two major duplication events have occurred: one before the split of the Poaceae and another within the Poaceae. Quantitative real-time RT-PCR analysis of the DthyrFL genes shows that they are expressed at different levels during inflorescence development but also transcribed in ovules and at very low levels in roots and leaves. These results suggest that the three FUL -like members are involved in floral development in D. thyrsiflorum .

Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are resistant to conventional genetic approaches. However, VIGS has been shown to be effective in only a few, closely related plant species. Tobacco rattle virus (TRV), a bipartite RNA virus, has a wide host range and so in principle could serve as an efficient vector for VIGS in a diverse array of plant species. Here we show that a vector based on TRV sequences is effective at silencing the endogenous phytoene desaturase (PapsPDS) gene in Papaver somniferum (opium poppy). We show that this vector does not compromise the growth or reproduction of poppy and the plants did not display viral symptoms. The silencing of PapsPDS resulted in a significant reduction in PapsPDS mRNA and a concomitant photobleached phenotype. The ability to rapidly assay gene function in P. somniferum will be valuable in manipulation of the opiate pathway in this pharmaceutically important species. We suggest that our vacuum infiltration method used to deliver TRV-based vectors into poppy is a promising approach for expanding VIGS to diverse angiosperm species in which traditional delivery methods fail to induce VIGS. Furthermore, these studies demonstrate the utility of TRV-VIGS for probing gene function in a basal eudicot species that is phylogenetically distant from model plant species.

To identify genes involved in Arabidopsis thaliana petal and stamen organogenesis, we used a gene trap approach to examine the patterns of reporter expression at each stage of flower development of 1765 gene trap lines. In 80 lines, the reporter gene showed petal- and/or stamen-specific expression or lack of expression, or expression in distinct patterns within the petals and/or the stamens, including distinct suborgan domains of expression, such as tissue-specific lines marking epidermis and vasculature, as well as lines demarcating the proximodistal or abaxial/adaxial axes of the organs. Interestingly, reporter gene expression was typically restricted along the proximodistal axis of petals and stamens, indicating the importance of this developmental axis in patterning of gene expression domains in these organs. We identified novel domains of gene expression along the axis marking the midregion of the petals and apical and basal parts of the anthers. Most of the genes tagged in these 80 lines were identified, and their possible functions in petal and/or stamen differentiation are discussed. We also scored the floral phenotypes of the 1765 gene trap lines and recovered two mutants affecting previously uncharacterized genes. In addition to revealing common domains of gene expression, the gene trap lines reported here provide both useful markers and valuable starting points for reverse genetic analyses of the differentiation pathways in petal and stamen development.

Traditionally, developmental studies in plant biology have suffered from the lack of a convenient means to study gene function in non-model plant species. Here we show that virus-induced gene silencing (VIGS) is an effective new tool to study the function of orthologs of floral homeotic genes such as DEFICIENS (DEF) in non-model systems. We used a tobacco rattle virus (TRV)-based VIGS approach to study the function of the Nicotiana benthamiana DEF ortholog (NbDEF). Silencing of NbDEF in N. benthamiana using TRV-VIGS was similar to that of Antirrhinum def and Arabidopsis ap3 mutants and caused transformation of petals into sepals and stamens into carpels. Molecular analysis of the NbDEF -silenced plants revealed a dramatic reduction of the levels of NbDEF mRNA and protein in flowers. NbDEF silencing was specific and has no effect on the mRNA levels of NbTM6, the closest paralog of NbDEF. A dramatic reduction of the levels of N. benthamiana GLOBOSA (NbGLO) mRNA and protein was also observed in flowers of NbDEF-silenced plants, suggesting that cross-regulation of this GLO-like gene by NbDEF. Taken together, our results suggest that NbDEF is a functional homolog of Antirrhinum DEF. Our results are significant in that they show that TRV efficiently induces gene silencing in young and differentiating flowers and that VIGS is a promising new tool for analyses of developmental gene function in non-model organisms.

▪ Abstract Flowering is one of the most intensively studied processes in plant development. Despite the wide diversity in floral forms, flowers have a simple stereotypical architecture. Flowers develop from florally determined meristems. These small populations of cells proliferate to form the floral organs, including the sterile outer organs, the sepals and petals, and the inner reproductive organs, the stamens and carpels. In the past decade, analyses of key flowering genes have been carried out primarily in Arabidopsis and have provided a foundation for understanding the underlying molecular genetic mechanisms controlling different aspects of floral development. Such studies have illuminated the transcriptional cascades responsible for the regulation of these key genes, as well as how these genes effect their functions. In turn, these studies have resulted in the refinement of the original ideas of how flowers develop and have indicated the gaps in our knowledge that need to be addressed.

Phylogenetic analyses of angiosperm MADS-box genes suggest that this gene family has undergone multiple duplication events followed by sequence divergence. To determine when such events have taken place and to understand the relationships of particular MADS-box gene lineages, we have identified APETALA1/FRUITFULL-like MADS-box genes from a variety of angiosperm species. Our phylogenetic analyses show two gene clades within the core eudicots, euAP1 (including Arabidopsis APETALA1 and Antirrhinum SQUAMOSA) and euFUL (including Arabidopsis FRUITFULL). Non-core eudicot species have only sequences similar to euFUL genes (FUL-like). The predicted protein products of euFUL and FUL-like genes share a conserved C-terminal motif. In contrast, predicted products of members of the euAP1 gene clade contain a different C terminus that includes an acidic transcription activation domain and a farnesylation signal. Sequence analyses indicate that the euAP1 amino acid motifs may have arisen via a translational frameshift from the euFUL/FUL-like motif. The euAP1 gene clade includes key regulators of floral development that have been implicated in the specification of perianth identity. However, the presence of euAP1 genes only in core eudicots suggests that there may have been changes in mechanisms of floral development that are correlated with the fixation of floral structure seen in this clade.

Plant MADS-box genes encode transcriptional regulators that are critical for a number of developmental processes. In the angiosperms (the flowering plants), these include the specification of floral organ identities, flowering time and fruit development. It appears that the MADS box gene family has undergone considerable gene duplication and sequence divergence within the angiosperms. Here I discuss the possibility that these events have allowed the recruitment of these genes to new developmental pathways in particular angiosperm lineages. Recent analyses of sequence changes, expression patterns and, in a few cases, gene function are beginning to provide tantalizing evidence for deciphering when and how such genetic diversification has led to particular morphological innovations. In the future, comparative studies of large numbers of species will be required to assess the extent of such variation as well as to fully understand the mechanisms by which evolution of these developmental regulators has played a role in shaping new morphologies.

Identifying the genes regulated by the floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI) is crucial for understanding the molecular mechanisms that lead to petal and stamen formation. We have used microarray analysis to conduct a broad survey of genes whose expression is affected by AP3 and PI activity. DNA microarrays consisting of 9216 Arabidopsis ESTs were screened with probes corresponding to mRNAs from different mutant and transgenic lines that misexpress AP3 and/or PI. The microarray results were further confirmed by RNA gel blot analyses. Our results suggest that AP3 and PI regulate a relatively small number of genes, implying that many genes used in petal and stamen development are not tissue specific and likely have roles in other processes as well. We recovered genes similar to previously identified petal- and stamen-expressed genes as well as genes that were not implicated previously in petal and stamen development. A very low percentage of the genes recovered encoded transcription factors. This finding suggests that AP3 and PI act relatively directly to regulate the genes required for the basic cellular processes responsible for petal and stamen morphogenesis.

The genomic approach to understanding how evolution has generated the extraordinary diversity of flowers is to assemble a floral EST database for several missing-link taxa and then use gene phylogenies and expression data to identify genes that are important in flower evolution. However, such a genomic approach is likely to miss important genes that are not members of gene families that control flower development in Arabidopsis , and can overlook genes that are not expressed, or are weakly expressed, at their site of action. Therefore we propose complementary genetic approaches in which a few phylogenetically well distributed species are developed as model systems and floral differentiation among closely related species is studied using functional approaches.

It is clear that cell-cell signaling is critical for the development of both root and shoot structures. Recently, several of the key gene products required for intercellular signaling have been defined, and the developmental processes regulated by cell-cell interactions are beginning to be elucidated. Surprisingly, these results suggest that the mechanisms by which plant cells communicate with each other may be quite distinct from those used in animal systems.

Cell-cell signaling is crucial for the coordination of cell division and differentiation during plant organogenesis. We have developed a novel mosaic analysis method for Arabidopsis, based on the maize Ac/Ds transposable element system, to assess the requirements of individual genes in intercellular signaling. Using this strategy, we have shown that the floral homeotic APETALA3 (AP3) gene has distinct roles in regulating intercellular signaling in different tissues. In petals, AP3 acts primarily in a cell-autonomous fashion to regulate cell type differentiation, but its function is also required in a non-cell-autonomous fashion to regulate organ shape. In contrast, AP3-regulated intercellular interactions are required for conferring both cell type identity and organ shape and size in the stamens. Using antibodies raised against AP3, we have shown that the AP3 protein does not traffic between cells. These observations imply that AP3 acts by differentially regulating the production of intercellular signals in a whorl-specific manner.

Our recently acquired understanding of the ABC program, which controls floral organ identity in model plant species such as Arabidopsis thaliana and Antirrhinum majus, has provided a new set of characters with which to evaluate floral evolution. What is still lacking, however, is a clear assessment of the actual degree of conservation of this genetic program across the angiosperms. To this end, we have begun to investigate the evolution of members of the B class gene lineages, which are known to control petal and stamen identity in the higher eudicots, and to analyze their expression patterns in selected species from the lower eudicots and basal angiosperms. The B class genes comprise the homologues of the A. thaliana genes APETALA3 (AP3) and PISTILLATA (PI), which are closely related paralogues encoding MADS box‐containing DNA-binding proteins. This study has uncovered many examples of gene duplication and divergence in both the AP3 and PI lineages as well as complex and variable patterns of gene expression. These findings indicate that although some aspects of the ABC program are conserved, others display a high degree of plasticity and may not have become fixed until later in angiosperm evolution.

The normal development of shoot structures depends on controlling the growth, proliferation and differentiation of cells derived from the shoot apical meristem. We have identified the CYP78A5 gene encoding a putative cytochrome P450 monooxygenase that is the first member of the CYP78 family from Arabidopsis. This gene is strongly expressed in the peripheral regions of the vegetative and reproductive shoot apical meristems, defining a boundary between the central meristematic zone and the developing organ primordia. In addition, CYP78A5 shows a dynamic pattern of expression during floral development. Overexpression of CYP78A5 affects multiple cell types, causing twisting and kinking of the stem and defects in floral development. To define the relationship of CYP78A5 to genes controlling meristem function, we examined CYP78A5 expression in plants mutant for SHOOT MERISTEMLESS, ZWILLE and ARGONAUTE, and have found that CYP78A5 expression is altered in these mutant backgrounds. We propose that CYP78A5 has a role in regulating directional growth in the peripheral region of the shoot apical meristem in response to cues established by genes regulating meristem function.

The angiosperms, or flowering plants, are the most abundant plant group on the earth today. They have evolved sophisticated reproductive structures, the flowers, which have long attracted interest from gardeners, poets, and artists from around the world. Scholars, too, have focused on studying flowers, and floral morphology is one of the most common characters used in the classification of the angiosperms. This is largely because differences in floral forms are so dramatic, and yet flowers from a particular species are remarkably consistent in their development and final morphology. Flowers arise from florally determined shoot apical meristems. Meristems are small populations of stem cells, which divide in a stereotypical fashion to produce both daughter cells which go on to form organs, as well as daughters to replenish the meristematic population. During vegetative development, cells on the flanks of the shoot apical meristem proliferate to form the vegetative structures, the leaves with their associated axillary buds. Once a plant has been induced to flower, some or all of the meristems can become committed to forming a flower. The floral organs arise sequentially on the flanks of the floral meristem: first the sepals, followed by the petals, stamens, and finally carpels (Fig. 1). The sepals protect and enclose the bud, while the petals in many species serve to attract pollinators. The sepals and petals surround the stamens, which produce the pollen, and the carpels, which contain the ovules. This review will focus on two related issues: p

The specification of floral organ identity in the higher dicots depends on the function of a limited set of homeotic genes, many of them members of the MADS-box gene family. Two such genes, APETALA3 (AP3) and PISTILLATA (PI), are required for petal and stamen identity in Arabidopsis; their orthologs in Antirrhinum exhibit similar functions. To understand how changes in these genes may have influenced the morphological evolution of petals and stamens, we have cloned twenty-six homologs of the AP3 and PI genes from two higher eudicot and eleven lower eudicot and magnolid dicot species. The sequences of these genes reveal the presence of characteristic PI- and AP3-specific motifs. While the PI-specific motif is found in all of the PI genes characterized to date, the lower eudicot and magnolid dicot AP3 homologs contain distinctly different motifs from those seen in the higher eudicots. An analysis of all the available AP3 and PI sequences uncovers multiple duplication events within each of the two gene lineages. A major duplication event in the AP3 lineage coincides with the base of the higher eudicot radiation and may reflect the evolution of a petal-specific AP3 function in the higher eudicot lineage.

The Arabidopsis flower is composed of four concentric whorls of organs: the sepals, petals, stamens and carpels. The development of this pattern depends on two general processes: that of establishing a florally determined meristem, and that of establishing particular organ identities. Mutational analyses have been instrumental in defining classes of genes that participate in each of these processes. The floral meristem identity genes are required to specify a florally determined meristem, and also appear to be required in part to activate the floral homeotic genes. The floral homeotic genes function in discrete regions of the florally determined meristem to specify the different organ types. The molecular analysis of these genes is beginning to shed light on the details of their regulation and function. The challenge that remains is to understand how the action of these genes in the dividing meristematic cell population can be coordinated and maintained to result in the differentiation of the complex array of cell and tissue types that make up the Arabidopsis flower.

The controlled ablation of cells is an effective technique with which to study and manipulate plant development. Traditional microsurgical techniques have been used to remove plant cells with varying degrees of success, but modern methods using either lasers or genetic ablation are proving to be easier to control. Lasers can be used to remove accessible single cells or small groups of cells, and genetic ablation — employing a tissue-specific promoter to control the expression of a cytotoxic gene — is effective for killing a specific set of cells. Ablation techniques have been instrumental in investigating cell—cell interactions, as well as for more applied research, such as on the control of fertility and disease resistance.