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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.

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.

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.

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.

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.

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.

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.

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.

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.

▪ 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.

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.

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.

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 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.

Flowers come in a variety of colors, shapes and sizes. Despite this variety, flowers have a very stereotypical architecture, consisting of a series of sterile organs surrounding the reproductive structures. Arabidopsis, as the premier model system for molecular and genetic analyses of plant development, has provided a wealth of insights into how this architecture is specified. With the advent of the completion of the Arabidopsis genome sequence a decade ago, in combination with a rich variety of forward and reverse genetic strategies, many of the genes and regulatory pathways controlling flower initiation, patterning, growth and differentiation have been characterized. A central theme that has emerged from these studies is the complexity and abundance of both positive and negative feedback loops that operate to regulate different aspects of flower formation. Presumably, this considerable degree of feedback regulation serves to promote a robust and stable transition to flowering, even in the face of genetic or environmental perturbations. This review will summarize recent advances in defining the genes, the regulatory pathways, and their interactions, that underpin how the Arabidopsis flower is formed.

The Arabidopsis thaliana MADS box transcription factors APETALA3 (AP3) and PISTILLATA (PI) heterodimerize and are required to specify petal identity, yet many details of how this regulatory process is effected are unclear. We have identified three related genes, BHLH136/BANQUO1 (BNQ1), BHLH134/BANQUO2 (BNQ2), and BHLH161/BANQUO3 (BNQ3), as being directly and negatively regulated by AP3 and PI in petals. BNQ1, BNQ2, and BNQ3 encode products belonging to a family of atypical non-DNA binding basic helix-loop-helix (bHLH) proteins that heterodimerize with and negatively regulate bHLH transcription factors. We show that bnq3 mutants have pale-green sepals and carpels and decreased chlorophyll levels, suggesting that BNQ3 has a role in regulating light responses. The ap3 bnq3 double mutant displays pale second-whorl organs, supporting the hypothesis that BNQ3 is downstream of AP3. Consistent with a role in light response, we show that the BNQ gene products regulate the function of HFR1 (for LONG HYPOCOTYL IN FAR-RED1), which encodes a bHLH protein that regulates photomorphogenesis through modulating phytochrome and cryptochrome signaling. The BNQ genes also are required for appropriate regulation of flowering time. Our results suggest that petal identity is specified in part through downregulation of BNQ-dependent photomorphogenic and developmental signaling pathways.

We have mapped the fate of cells in the Arabidopsis embryonic shoot apical meristem by irradiating seed and scoring the resulting clonally derived sectors. 176 white, yellow, pale green or variegated sectors were identified and scored for their position and extent in the resulting plants. Most sectors were confined to a fraction of a leaf, and only occasionally extended into the inflorescence. Sectors that extended into the inflorescence were larger, and usually encompassed about a third to a half of the inflorescence circumference. We also find that axillary buds in Arabidopsis are clonally related to the subtending leaf. Sections through the dry seed embryo indicate that the embryonic shoot apical meristem contains approximately 110 cells in the three meristematic layers prior to the formation of the first two leaf primordia. The histological analysis of cell number in the shoot apical meristem, in combination with the sector analysis have been used to construct a map of the probable fate of cells in the embryonic shoot apical meristem.

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.

Plants flower in response to both environmental and endogenous signals. The Arabidopsis LEAFY (LFY) transcription factor is crucial in integrating these signals, and acts in part by activating the expression of multiple floral homeotic genes. LFY-dependent activation of the homeotic APETALA3 (AP3) gene requires the activity of UNUSUAL FLORAL ORGANS (UFO), an F-box component of an SCF ubiquitin ligase, yet how this regulation is effected has remained unclear. Here, we show that UFO physically interacts with LFY both in vitro and in vivo, and this interaction is necessary to recruit UFO to the AP3 promoter. Furthermore, a transcriptional repressor domain fused to UFO reduces endogenous LFY activity in plants, supporting the idea that UFO acts as part of a transcriptional complex at the AP3 promoter. Moreover, chemical or genetic disruption of proteasome activity compromises LFY-dependent AP3 activation,indicating that protein degradation is required to promote LFY activity. These results define an unexpected role for an F-box protein in functioning as a DNA-associated transcriptional co-factor in regulating floral homeotic gene expression. These results suggest a novel mechanism for promoting flower development via protein degradation and concomitant activation of the LFY transcription factor. This mechanism may be widely conserved, as homologs of UFO and LFY have been identified in a wide array of plant species.

MADS-box genes are crucial regulators of floral development, yet how their functions have evolved to control different aspects of floral patterning is unclear. To understand the extent to which MADS-box gene functions are conserved or have diversified in different angiosperm lineages, we have exploited the capability for functional analyses in a new model system, Papaver somniferum (opium poppy). P. somniferum is a member of the order Ranunculales, and so represents a clade that is evolutionarily distant from those containing traditional model systems such as Arabidopsis, Petunia, maize or rice. We have identified and characterized the roles of several candidate MADS-box genes in petal specification in poppy. In Arabidopsis, the APETALA3(AP3) MADS-box gene is required for both petal and stamen identity specification. By contrast, we show that the AP3 lineage has undergone gene duplication and subfunctionalization in poppy, with one gene copy required for petal development and the other responsible for stamen development. These differences in gene function are due to differences both in expression patterns and co-factor interactions. Furthermore, the genetic hierarchy controlling petal development in poppy has diverged as compared with that of Arabidopsis. As these are the first functional analyses of AP3 genes in this evolutionarily divergent clade, our results provide new information on the similarities and differences in petal developmental programs across angiosperms. Based on these observations, we discuss a model for how the petal developmental program has evolved.

Arabidopsis (Arabidopsis thaliana) contains approximately 100 homeobox genes, many of which have been shown to play critical roles in various developmental processes. Here we characterize the zinc finger-homeodomain (ZF-HD) subfamily of homeobox genes, consisting of 14 members in Arabidopsis. We demonstrate that the HDs of the ZF-HD proteins share some similarities with other known HDs in Arabidopsis, but they contain distinct features that cluster them as a unique class of plant HD-containing proteins. We have carried out mutational analyses to show that the noncanonical residues present in the HDs of this family of proteins are important for function. Yeast (Saccharomyces cerevisiae) two-hybrid matrix analyses of the ZF-HD proteins reveal that these proteins both homo- and heterodimerize, which may contribute to greater selectivity in DNA binding. These assays also show that most of these proteins do not contain an intrinsic activation domain, suggesting that interactions with other factors are required for transcriptional activation. We also show that the family members are all expressed predominantly or exclusively in floral tissue, indicating a likely regulatory role during floral development. Furthermore, we have identified loss-of-function mutations for six of these genes that individually show no obvious phenotype, supporting the idea that the encoded proteins have common roles in floral development. Based on these results, we propose the ZF-HD gene family encodes a group of transcriptional regulators with unique biochemical activities that play overlapping regulatory roles in Arabidopsis floral development.

There is considerable morphological diversity among angiosperm flowers. What is the developmental basis of this diversity? Some of this variation appears to be due to changes in the roles of a subset of MADS‐box genes, which have been shown to control various aspects of flower development in several model species, notably Arabidopsis . Here I focus on perianth diversification and the roles that duplication and diversification of MADS‐box genes may play in this process. I review several hypotheses for the types of changes in MADS‐box genes that are responsible for phenotypic variation in perianth form. To critically test such hypotheses, functional analyses in phylogenetically informative taxa are needed. I explore the possibilities for utilizing virus‐induced gene silencing as a tool to functionally assess the roles of MADS‐box genes in nonmodel species. By carrying out functional analyses in a phylogenetic context it will be possible to infer the direction and type of developmental shifts that have occurred. Together these approaches will provide a robust means of inferring the relative contributions of regulatory evolution, protein evolution, and duplication of the MADS‐box genes for building new floral morphologies.

Developmental processes shape plant morphologies, which constitute important adaptive traits selected for during evolution. Identifying the genes that act in developmental pathways and determining how they are modified during evolution is the focus of the field of evolutionary developmental biology, or evo-devo. Knowledge of genetic pathways in the plant model Arabidopsis serves as the starting point for investigating how the toolkit of developmental pathways has been used and reused to form different plant body plans. One productive approach is to identify genes in other species that are orthologous to genes known to control developmental pathways in Arabidopsis and then determine what changes have occurred in the protein coding sequence or in the gene's expression to produce an altered morphology. A second approach relies on natural variation among wild populations or crop plants. Natural variation can be exploited to identify quantitative trait loci that underlie important developmental traits and, thus, define those genes that are responsible for adaptive changes. The possibility of applying comparative genomics approaches to Arabidopsis and related species promises profound new insights into the interplay of evolution and development.

Changes in homeotic gene expression patterns or in the functions of the encoded proteins are thought to play a prominent role in the evolution of new morphologies. The floral homeotic APETALA3 ( AP3 ) and PISTILLATA ( PI ) genes encode MADS domain-containing transcription factors required to specify petal and stamen identities in Arabidopsis . We have previously shown that perianth expression of AP3 and PI homologs varies in different groups of angiosperms with diverse floral structures, suggesting that changes in expression may contribute to changing morphology. We have investigated the possibility that changes in the functions of the encoded gene products may also have played a role in the evolution of different floral morphologies. AP3 and PI are members of paralogous gene lineages and share extensive similarity along the length of the protein products. Genes within these lineages encode products with characteristic C-terminal motifs that we show are critical for functional specificity. In particular, the C terminus of AP3 is sufficient to confer AP3 functionality on the heterologous PI protein. Furthermore, we have shown that the evolution of the divergent AP3 C-terminal domain in the core eudicots is correlated with the acquisition of a role in specifying perianth structures. These results suggest that divergence in these sequence motifs has contributed to the evolution of distinct functions for these floral homeotic gene products.

The COP9 signalosome (CSN) is involved in multiple developmental processes. It interacts with SCF ubiquitin ligases and deconjugates Nedd8/Rub1 from cullins (deneddylation). CSN is highly expressed in Arabidopsis floral tissues. To investigate the role of CSN in flower development, we examined the expression pattern of CSN in developing flowers. We report here that two csn1 partially deficient Arabidopsis strains exhibit aberrant development of floral organs, decline of APETALA3 (AP3) expression, and low fertility in addition to defects in shoot and inflorescence meristems. We show that UNUSUAL FLORAL ORGANS (UFO) forms a SCF(UFO) complex, which is associated with CSN in vivo. Genetic interaction analysis indicates that CSN is necessary for the gain-of-function activity of the F-box protein UFO in AP3 activation and in floral organ transformation. Compared with the previously reported csn5 antisense and csn1 null mutants, partial deficiency of CSN1 causes a reduction in the level of CUL1 in the mutant flowers without an obvious defect in CUL1 deneddylation. We conclude that CSN is an essential regulator of Arabidopsis flower development and suggest that CSN regulates Arabidopsis flower development in part by modulating SCF(UFO)-mediated AP3 activation.

The Arabidopsis APETALA3 (AP3) floral homeotic gene is required for specifying petal and stamen identities, and is expressed in a spatially limited domain of cells in the floral meristem that will give rise to these organs. Here we show that the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) are required for the activation of AP3. The LFY transcription factor binds to a sequence, with dyad symmetry, that lies within a region of the AP3 promoter required for early expression of AP3. Mutation of this region abolishes LFY binding in vitro and in yeast one hybrid assays, but has no obvious effect on AP3 expression in planta. Experiments using a steroid-inducible form of LFY show that, in contrast to its direct transcriptional activation of other floral homeotic genes, LFY acts in both a direct and an indirect manner to regulate AP3 expression. This LFY-induced expression of AP3 depends in part on the function of the APETALA1 (AP1) floral homeotic gene, since mutations in AP1 reduce LFY-dependent induction of AP3 expression. LFY therefore appears to act through several pathways, one of which is dependent on AP1 activity, to regulate AP3 expression.

The shoot apical meristem of Arabidopsis thaliana consists of three cell layers that proliferate to give rise to the aerial organs of the plant. By labeling cells in each layer using an Ac-based transposable element system, we mapped their contributions to the floral organs, as well as determined the degree of plasticity in this developmental process. We found that each cell layer proliferates to give rise to predictable derivatives: the L1 contributes to the epidermis, the stigma, part of the transmitting tract and the integument of the ovules, while the L2 and L3 contribute, to different degrees, to the mesophyll and other internal tissues. In order to test the roles of the floral homeotic genes in regulating these patterns of cell proliferation, we carried out similar clonal analyses in apetala3-3 and agamous-1 mutant plants. Our results suggest that cell division patterns are regulated differently at different stages of floral development. In early floral stages, the pattern of cell divisions is dependent on position in the floral meristem, and not on future organ identity. Later, during organogenesis, the layer contributions to the organs are controlled by the homeotic genes. We also show that AGAMOUS is required to maintain the layered structure of the meristem prior to organ initiation, as well as having a non-autonomous role in the regulation of the layer contributions to the petals.

The APETALA3 floral homeotic gene is required for petal and stamen development in Arabidopsis. APETALA3 transcripts first detected in a meristematic region that will give rise to the petal and stamen primordia, and expression is maintained in this region during subsequent development of these organs. To dissect how the APETALA3 gene is expressed in this spatially and temporally restricted domain, various APETALA3 promoter fragments were fused to the uidA reporter gene encoding β -glucuronidase and assayed for the resulting patterns of expression in transgenic Arabidopsis plants. Based on these promoter analyses, we defined cis-acting elements required for distinct phases of APETALA3 expression, as well as for petal-specific and stamen-specific expression. By crossing the petal-specific construct into different mutant backgrounds, we have shown that several floral genes, including APETALA3, PISTILLATA, UNUSUAL are FLORAL ORGANS, and APETALA1, encode trans-acting factors required for second-whorl-specific APETALA3 expression. We have also shown that the products of the APETALA1, APETALA3, PISTILLATA and AGAMOUS genes bind to several conserved sequence motifs within the APETALA3 promoter. We present a model whereby spatially and temporally restricted APETALA3 transcription is controlled via interactions between proteins binding to different domains of the APETALA3 promoter.

Natural variation in organ morphologies can have adaptive significance and contribute to speciation. However, the underlying allelic differences responsible for variation in organ size and shape remain poorly understood. We have utilized natural phenotypic variation in three Arabidopsis thaliana ecotypes to examine the genetic basis for quantitative variation in petal length, width, area, and shape. We identified 23 loci responsible for such variation, many of which appear to correspond to genes not previously implicated in controlling organ morphology. These analyses also demonstrated that allelic differences at distinct loci can independently affect petal length, width, area or shape, suggesting that these traits behave as independent modules. We also showed that ERECTA (ER), encoding a leucine-rich repeat (LRR) receptor-like serine-threonine kinase, is a major effect locus determining petal shape. Allelic variation at the ER locus was associated with differences in petal cell proliferation and concomitant effects on petal shape. ER has been previously shown to be required for regulating cell division and expansion in other contexts; the ER receptor-like kinase functioning to also control organ-specific proliferation patterns suggests that allelic variation in common signaling components may nonetheless have been a key factor in morphological diversification.

We have characterized the floral phenotypes produced by the recessive homeotic apetala 1-1 (ap1-1) mutation in Arabidopsis. Plants homozygous for this mutation display a homeotic conversion of sepsis into brachts and the concomitant formation of floral buds in the axil of each transformed sepal. In addition, these flowers lack petals. We show that the loss of petal phenotype is due to the failure of petal primordia to be initiated. We have also constructed double mutant combinations with ap1 and other mutations affecting floral development. Based on these results, we suggest that the AP1 and the apetala 2 (AP2) genes may encode similar functions that are required to define the pattern of where floral organs arise, as well as for determinate development of the floral meristem. We propose that the AP1 and AP2 gene products act in concert with the product of the agamous (AG) locus to establish a determinate floral meristem, whereas other homeotic gene products are required for cells to differentiate correctly according to their position. These results extend the proposed role of the homeotic genes in floral development and suggest new models for the establishment of floral pattern.

This year's meeting on the model plant Arabidopsis highlighted the progress that has been made in many areas of its biology by the use of a variety of genetic and genomic tools. One recurring theme was how multiple layers of transcriptional and posttranscriptional regulation are integrated to produce a biological response.

A recent study, comparing the maize SILKY1 gene to its well-characterized homolog APETALA3 from Arabidopsis, has provided some of the first evidence pointing to conservation of homeotic gene function between monocots and dicots.

Several homeotic genes controlling floral development have been isolated in both Antirrhinum and Arabidopsis. Based on the similarities in sequence and in the phenotypes elicited by mutations in some of these genes, it has been proposed that the regulatory hierarchy controlling floral development is comparable in these two species. We have performed a direct experimental test of this hypothesis by introducing a chimeric Antirrhinum Deficiens (DefA)/Arabidopsis APETALA3 (AP3) gene, under the control of the Arabidopsis AP3 promoter, into Arabidopsis. We demonstrated that this transgene is sufficient to partially complement severe mutations at the AP3 locus. In combination with a weak ap3 mutation, this transgene is capable of completely rescuing the mutant phenotype to a fully functional wild-type flower. These observations indicate that despite differences in DNA sequence and expression, DefA coding sequences can compensate for the loss of AP3 gene function. We discuss the implications of these results for the evolution of homeotic gene function in flowering plants.

Flowers develop from the coordinated division and differentiation of cells derived from the shoot apical meristem. By inducing chromosomal deletions in individual shoot apical meristem cells, we have generated Arabidopsis plants that are genetically mosaic for the homeotic PISTILLATA gene. Flowers bearing wild-type PISTILLATA epidermal tissue and mutant pistillata internal tissues are phenotypically normal. Based on this non-cell-autonomy, we suggest that PISTILLATA controls the production of a substance involved in cell-cell communication between the outer and inner tissue layers of the flower. These mosaic flowers were also used to assess the relative contributions of meristematic cells to the developing floral organs. These observations indicate that meristematic cells have discrete but somewhat variable contributions to the Arabidopsis flower. We have used these results to construct a fate map of the Arabidopsis floral primordium.