Many Kalanchoë species reproduce asexually by forming plantlets in the leaf margins. Plant hormones are involved in meristem and embryogenesis pathways, yet their exact role in Kalanchoë plantlet formation is unknown. Here, we show that auxin and cytokinin-mediated pathways facilitate the establishment of leaf crenulations and, in turn, plantlet primordia in K. pinnata. Antisense transgenic lines with lower expression of the auxin transporter PIN-FORMED1 (PIN1) and the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSFER PROTEIN (AHP) generated fewer plantlets. GREEN FLUORESCENT PROTEIN (GFP) reporter lines visualising the activity of auxin and cytokinin also revealed the accumulation of these hormones in the plantlet primordia. Furthermore, treatment of leaves with gibberellic acid (GA3) prevented plantlet emergence, suggesting that its depletion activates plantlet formation in detached leaves. Our findings highlight the involvement of hormonal regulation in the inducible plantlet formation system, which facilitates the formation of plantlet primordia and exerts precise control over dormancy processes in detached leaves. This study provides insight into the role of hormones in the novel reproductive system in the genus Kalanchoë.

Several Kalanchoë species reproduce asexually by forming plantlets in the leaf crenulations. Some species produce plantlets incessantly via somatic embryogenesis and organogenesis, whereas others exclusively develop plantlets after leaf detachment, presumably through organogenesis. SHOOT MERISTEMLESS (STM), which mediates SAM functions, appears to be involved in Kalanchoë plantlet formation, suggesting that meristem genes may be essential for plantlet formation. However, the genetic regulatory network for establishing and maintaining plantlet primordia in Kalanchoë remains elusive. Here, we showed that meristem genes were differentially expressed in the leaf crenulations of K. pinnata during plantlet development after leaf detachment. The regulatory interactions among these meristem genes are largely conserved in K. pinnata crenulations. Moreover, transgenic antisense (AS) plants with lower expression of these key meristem genes formed significantly fewer plantlets with some morphological defects, suggesting that the meristem genes play an important role in plantlet formation and development. Our research revealed that key meristem genetic pathways were co-opted to the leaf margin to facilitate the unique asexual reproduction mechanism in K. pinnata. This also highlights how evolutionary tinkering invents new structures such as epiphyllous buds and plantlets by rewiring pre-existing genetic pathways.

Few species in the Kalanchoë genus form plantlets on their leaf margins as an asexual reproduction strategy. The limited molecular studies on plantlet formation show that an organogenesis ortholog, SHOOTMERISTEMLESS (STM) and embryogenesis genes, such as LEAFY COTYLEDON1 (LEC1) and FUSCA3 are recruited during plantlet formation. To understand the mechanisms of two Kalanchoë plantlet-forming species with different modes of plantlet formation, RNA-sequencing analysis was performed. Differentially expressed genes between the developmental stages were clustered in K. daigremontiana (Raym.-Hamet and H. Perrier) and K. pinnata (Lam. Pers.), respectively. Of these gene clusters, GO terms that may be involved in plantlet formation of both species, such as signaling, response to wounding, reproduction, regulation of hormone level, and response to karrikin were overrepresented. Compared with the common GO terms, there were more unique GO terms overrepresented during the plantlet formation of each species. A more in-depth investigation is required to understand how these pathways are participating in plantlet formation. Nonetheless, this transcriptome analysis is presented as a reliable basis for future studies on plantlet formation and development in two Kalanchoë plantlet-forming species.

Abstract The unique mechanism by which leaf margin cells regain potency and then form a plantlet in Kalanchoë spp. remains elusive but involves organogenesis and embryogenesis in response to age, day length, nutrient availability, and drought stress. In light of this, we investigated whether TARGET OF RAPAMYCIN (TOR), a conserved protein kinase in eukaryotes that controls cell growth and metabolism in response to nutrient and energy availability, may regulate plantlet formation. Kalanchoë daigremontiana TOR (KdTOR) was expressed in the leaf margin at the site of plantlet initiation, in the early plantlet cotyledons, and in the root tip of the developed plantlet. Both chemical and genetic inhibition of TOR Kinase activity in Kalanchoë daigremontiana leaves disrupted plantlet formation. Furthermore, downregulation of KdTOR in transgenic plants led to wide-ranging transcriptional changes, including decreased K. daigremontiana SHOOTMERISTEMLESS and K. daigremontiana LEAFYCOTYLEDON1 expression, whereas auxin treatments induced KdTOR expression in the plantlet roots. These results suggest that the KdTOR pathway controls plantlet development in cooperation with auxin, organogenesis, and embryogenesis pathways. The ancient and highly conserved TOR Kinase therefore controls diverse and unique developmental pathways, such as asexual reproduction within the land plant lineage.

TARGET OF RAPAMYCIN (TOR) kinase has been recognised as a key developmental regulator in both plants and animals. Despite their distinct developmental programmes, all eukaryotes studied possess a functional TOR kinase, which integrates environmental and nutrient signals to direct growth and development. This is particularly important in plants, as they are sessile and must sense and respond to external signals to coordinate multicellular growth appropriately. Thus, the investigation of TOR is essential for plant developmental studies in the context of the resources available for growth. Recently, links have been shown between TOR and plant development from embryogenesis through to senescence, however more investigation is crucial to fully elucidate TOR function in each developmental process.

Nature often creates complex structures by rearranging pre-existing units. One such example is the flower head (capitulum) in daisies, where a group of flowers (florets) and phyllaries (modified bracts) are arranged to superficially mimic a single flower. The capitulum is a key taxonomical innovation that defines the daisy family (Asteraceae), the largest flowering plant group. However, patterning mechanisms underlying its structure remain elusive. Here, we show that auxin, a plant hormone, provides a developmental patterning cue for the capitulum. During capitulum development, a temporal auxin gradient occurs, regulating the successive and centripetal formation of distinct florets and phyllaries. Disruption of the endogenous auxin gradient led to homeotic conversions of florets and phyllaries in the capitulum. Furthermore, auxin regulates floral meristem identity genes, such as Matricaria inodora RAY2 and M. inodora LEAFY, which determine floret and phyllary identity. This study reveals the mechanism of capitulum patterning and highlights how common developmental tools, such as hormone gradients, have independently evolved in plants and animals.

While the role of proteins in determining cell identity has been extensively studied, the contribution of small noncoding RNA molecules such as miRNAs and siRNAs has been also recognised. miRNAs bind to complementary sites in target mRNA molecules to trigger the degradation or translational inhibition of those targets. Recent studies have revealed that miRNAs play pivotal roles in key developmental processes such as patterning of the embryo, meristem, leaf, and flower. Furthermore, these miRNAs have been recruited throughout plant evolution into pathways that create diverse plant organ forms and shapes. This review focuses on the roles of miRNAs in establishing plant cell identity during key plant development processes and creating morphological diversity during plant evolution.

Flower forms are both highly diverse and multifaceted. As well as varying in colour, size, organ number, and much more, flowers show different types of symmetry. Floral symmetry can be grouped into three main categories: asymmetry, bilateral symmetry and radial symmetry, characterised by zero, one, and multiple planes of symmetry, respectively. This review will first explore floral symmetry from a classical morphological view, then from a modern molecular perspective. The recent molecular work on symmetry in monocots and eudicots will be discussed, followed by an in-depth discussion into the evolution of CYC genes, particularly in the capitulum of the sunflower family (Asteraceae). Whilst recent studies on non-model species are helping to bring new light to this field, more species coverage is required to understand how traits such as bilateral symmetry have evolved so many times, and whether the same molecular regulators were recruited for this function.

All members of Asteraceae, the largest flowering family, have a unique compressed inflorescence known as a capitulum, which resembles a solitary flower. The capitulum often consists of bilateral (zygomorphic) ray florets and radial (actinomorphic) disc florets. In Antirrhinum majus, floral zygomorphy is established by the interplay between dorsal petal identity genes, CYCLOIDEA (CYC) and RADIALIS (RAD), and a ventral gene DIVARICATA (DIV). To investigate the role of CYC, RAD, and DIV in the development of ray and disc florets within a capitulum, we isolated homologs of these genes from an Asteraceae species, Senecio vulgaris (common groundsel). After initial uniform expression of RAY3 (CYC), SvRAD, and SvDIV1B in ray florets only, RAY3 and SvRAD were exclusively expressed in the ventral petals of the ray florets. Our functional analysis further showed that RAY3 promotes and SvDIV1B represses petal growth, confirming their roles in floral zygomorphy. Our results highlight that while floral symmetry genes such as RAY3 and SvDIV1B appear to have a conserved role in petal growth in both Senecio and Antirrhinum, the regulatory relationships and expression domains are divergent, allowing ventral petal elongation in Senecio versus dorsal petal elongation in Antirrhinum. In S. vulgaris, diversification of CYC genes has led to novel interactions; SvDIV1B inhibits RAY3 and SvRAD, and may activate RAY2. This highlights how recruitment of floral symmetry regulators into dynamic networks was crucial for creating a complex and elaborate structure such as the capitulum.

Floral symmetry regulators play antagonistic roles in establishing two distinct floral symmetries within a capitulum in Asteraceae. All members of Asteraceae, the largest flowering family, have a unique compressed inflorescence known as a capitulum, which resembles a solitary flower. The capitulum often consists of bilateral (zygomorphic) ray florets and radial (actinomorphic) disc florets. In Antirrhinum majus, floral zygomorphy is established by the interplay between dorsal petal identity genes, CYCLOIDEA (CYC) and RADIALIS (RAD), and a ventral gene DIVARICATA (DIV). To investigate the role of CYC, RAD, and DIV in the development of ray and disc florets within a capitulum, we isolated homologs of these genes from an Asteraceae species, Senecio vulgaris (common groundsel). After initial uniform expression of RAY3 (CYC), SvRAD, and SvDIV1B in ray florets only, RAY3 and SvRAD were exclusively expressed in the ventral petals of the ray florets. Our functional analysis further showed that RAY3 promotes and SvDIV1B represses petal growth, confirming their roles in floral zygomorphy. Our results highlight that while floral symmetry genes such as RAY3 and SvDIV1B appear to have a conserved role in petal growth in both Senecio and Antirrhinum, the regulatory relationships and expression domains are divergent, allowing ventral petal elongation in Senecio versus dorsal petal elongation in Antirrhinum. In S. vulgaris, diversification of CYC genes has led to novel interactions; SvDIV1B inhibits RAY3 and SvRAD, and may activate RAY2. This highlights how recruitment of floral symmetry regulators into dynamic networks was crucial for creating a complex and elaborate structure such as the capitulum.

A mutated master regulator of zygotic embryogenesis is essential for creating somatic embryos and enhancing asexual propagation in Kalanchoë. Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

BACKGROUND AND AIMS Diverse leaf forms in nature can be categorized into two groups: simple and compound. A simple leaf has a single blade unit, whilst a compound leaf is dissected into leaflets. For both simple and compound leaves, a MYB domain transcription factor PHANTASTICA (PHAN) plays an important role in establishing the adaxial domain in the leaf. Absence of PHAN in arabidopsis and antirrhinum leaves supresses blade development, and in tomato suppresses leaflet development. However, in the rachis and petiole regions of tomato leaves where PHAN and the adaxial domain coexist, it has been unclear why leaf blade and leaflets are not formed. We hypothesized that PHAN regulates the medio-lateral extent of the adaxial domain, thereby determining compound leaf architecture. METHODS To test this hypothesis, we generated and analysed transgenic tomato plants expressing tomato PHAN (SlPHAN) under the Cauliflower mosaic virus (CaMV) 35S promoter in both sense and antisense orientations, and tobacco plants that over-express tomato SlPHAN. KEY RESULTS Modulations in SlPHAN resulted in a variety of leaf morphologies such as simple, ternate and compound in either a peltate or non-peltate arrangement. Measurements of the extent of the adaxial domain along the wild-type tomato leaf axis showed that the adaxial domain is narrowed in the rachis and petiole in comparison with regions where laminar tissue arises. In antiSlPHAN transgenic leaves, no blade or leaflet was formed where the adaxial domain was medio-laterally narrowed, and KNOX gene expression was correlatively upregulated. CaMV35S::SlPHAN expression led to widening of the adaxial domain and ectopic blade outgrowth in the rachis of tomato and in the petiole of tobacco. Taken together, these results suggest that SlPHAN plays a role in medio-lateral extension of the adaxial domain and contributes to final leaf morphology in tomato. CONCLUSIONS This study provides a novel insight into leaf architecture in tomato and highlights how changes in the expression domain of a master regulator gene such as SlPHAN can be translated into diverse final leaf morphologies.

The structure of compound leaves provides flexibility for morphological change by variation in the shapes, sizes and arrangement of leaflets. Here, we conduct a multilevel analysis of shape variation in compound leaves to explore the developmental plasticity and evolutionary potential that are the basis of diversification in leaf shape. We use the methods of geometric morphometrics to study the shapes of individual leaflets and whole leaves in 20 taxa of Potentilla (sensu lato). A newly developed test based on the bootstrap approach suggests that uncertainty in the molecular phylogeny precludes firm conclusions whether there is a phylogenetic signal in the data on leaf shape. For variation among taxa, variation within taxa, as well as fluctuating asymmetry, there is evidence of strong morphological integration. The patterns of variation are similar across all three levels, suggesting that integration within taxa may act as a constraint on evolutionary change.

Orchids comprise one of the largest and most diverse angiosperm families. Currently, about 24,500 orchid species have been reported, and there are many more to be discovered ([Dressler, 2005][1]). Due to their biological complexity, orchids have been proposed as an attractive system with which to

Hybridization between species can lead to introgression of genes from one species to another, providing a potential mechanism for preserving and recombining key traits during evolution. To determine the molecular basis of such transfers, we analyzed a natural polymorphism for flower-head development in Senecio. We show that the polymorphism arose by introgression of a cluster of regulatory genes, the RAY locus, from the diploid species S. squalidus into the tetraploid S. vulgaris. The RAY genes are expressed in the peripheral regions of the inflorescence meristem, where they promote flower asymmetry and lead to an increase in the rate of outcrossing. Our results highlight how key morphological and ecological traits controlled by regulatory genes may be gained, lost, and regained during evolution.

Stomata, epidermal structures that modulate gas exchange between plants and the atmosphere, play critical roles in primary productivity and the global climate. Positively acting transcription factors and negatively acting mitogen-activated protein kinase (MAPK) signaling control stomatal development in Arabidopsis; however, it is not known how the opposing activities of these regulators are integrated. We found that a unique domain in a basic helix-loop-helix (bHLH) stomatal initiating factor, SPEECHLESS, renders it a MAPK phosphorylation target in vitro and modulates its function in vivo. MAPK cascades modulate a diverse set of activities including development, cell proliferation, and response to external stresses. The coupling of MAPK signaling to SPEECHLESS activity provides cell type specificity for MAPK output while allowing the integration of multiple developmental and environmental signals into the production and spacing of stomata.

Recent work on species with simple leaves suggests that the juxtaposition of abaxial (lower) and adaxial (upper) cell fates (dorsiventrality) in leaf primordia is necessary for lamina outgrowth. However, how leaf dorsiventral symmetry affects leaflet formation in species with compound leaves is largely unknown. In four non-allelic dorsiventrality-defective mutants in tomato, wiry, wiry3, wiry4 and wiry6, partial or complete loss of ab-adaxiality was observed in leaves as well as in lateral organs in the flower, and the number of leaflets in leaves was reduced significantly. Morphological analyses and expression patterns of molecular markers for ab-adaxiality [LePHANTASTICA (LePHAN) and LeYABBY B (LeYAB B)] indicated that ab-adaxial cell fates were altered in mutant leaves. Reduction in expression of both LeT6 (a tomato KNOX gene) and LePHAN during post-primordial leaf development was correlated with a reduction in leaflet formation in the wiry mutants. LePHAN expression in LeT6 overexpression mutants suggests that LeT6 is a negative regulator of LePHAN. KNOX expression is known to be correlated with leaflet formation and we show that LeT6 requires LePHAN activity to form leaflets. These phenotypes and gene expression patterns suggest that the abaxial and adaxial domains of leaf primordia are important for leaflet primordia formation, and thus also important for compound leaf development. Furthermore, the regulatory relationship between LePHAN and KNOX genes is different from that proposed for simple-leafed species. We propose that this change in the regulatory relationship between KNOX genes and LePHAN plays a role in compound leaf development and is an important feature that distinguishes simple leaves from compound leaves.

Long-distance movement of RNA through the phloem is known to occur, but the functional importance of these transported RNAs has remained unclear. Grafting experiments with a naturally occurring dominant gain-of-function leaf mutation in tomato were used to demonstrate long-distance movement of mutant messenger RNA (mRNA) into wild-type scions. The stock-specific pattern of mRNA expression was graft transmissible, indicating that the mRNA accumulation pattern is inherent to the transcript and not attributable to the promoter. The translocated mRNA caused changes in leaf morphology of the wild-type scions, suggesting that the translocated RNA is functional.