Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (removal of all preformed shoot apical meristems, leaving a single cotyledon) of greenhouse-grown cotyledon-stage seedlings to test the hypothesis that such abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. To understand why shoot regeneration starts with defective organogenesis, the regeneration response was characterized by morphology and scanning electron and light microscopy in decapitated cotyledon-stage Cucurbita pepo seedlings. Several leaf primordia were observed to regenerate prior to differentiation of a de novo shoot apical meristem from dividing cells on the wound surface. Early regenerating primordia have a greatly distorted structure with dramatically altered dorsoventrality. Aberrant leaf morphogenesis in C. pepo gradually disappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of families (Chenopodiaceae, Compositae, Convolvulaceae, Cucurbitaceae, Cruciferae, Fabaceae, Malvaceae, Papaveraceae, and Solanaceae) of several dicotyledonous clades (Ranunculales, Caryophyllales, Asterids, and Rosids), stems are regenerated bearing abnormal leaves; the normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is manifest in exceptional circumstances such as during recovery from excision of all preformed shoot meristems of a seedling.     

Simulation of organ patterning on the floral meristem using a polar auxin transport model

An intriguing phenomenon in plant development is the timing and positioning of lateral organ initiation, which is a fundamental aspect of plant architecture. Although important progress has been made in elucidating the role of auxin transport in the vegetative shoot to explain the phyllotaxis of leaf formation in a spiral fashion, a model study of the role of auxin transport in whorled organ patterning in the expanding floral meristem is not available yet. We present an initial simulation approach to study the mechanisms that are expected to play an important role. Starting point is a confocal imaging study of Arabidopsis floral meristems at consecutive time points during flower development. These images reveal auxin accumulation patterns at the positions of the organs, which strongly suggests that the role of auxin in the floral meristem is similar to the role it plays in the shoot apical meristem. This is the basis for a simulation study of auxin transport through a growing floral meristem, which may answer the question whether auxin transport can in itself be responsible for the typical whorled floral pattern. We combined a cellular growth model for the meristem with a polar auxin transport model. The model predicts that sepals are initiated by auxin maxima arising early during meristem outgrowth. These form a pre-pattern relative to which a series of smaller auxin maxima are positioned, which partially overlap with the anlagen of petals, stamens, and carpels. We adjusted the model parameters corresponding to properties of floral mutants and found that the model predictions agree with the observed mutant patterns. The predicted timing of the primordia outgrowth and the timing and positioning of the sepal primordia show remarkable similarities with a developing flower in nature.     

SUPERMAN regulates floral whorl boundaries through control of auxin biosynthesis

Proper floral patterning, including the number and position of floral organs in most plant species, is tightly controlled by the precise regulation of the persistence and size of floral meristems (FMs). In Arabidopsis, two known feedback pathways, one composed of WUSCHEL (WUS) and CLAVATA3 (CLV3) and the other composed of AGAMOUS (AG) and WUS, spatially and temporally control floral stem cells, respectively. However, mounting evidence suggests that other factors, including phytohormones, are also involved in floral meristem regulation. Here, we show that the boundary gene SUPERMAN (SUP) bridges floral organogenesis and floral meristem determinacy in another pathway that involves auxin signaling. SUP interacts with components of polycomb repressive complex 2 (PRC2) and fine‐tunes local auxin signaling by negatively regulating the expression of the auxin biosynthesis genes YUCCA1/4 (YUC1/4). In sup mutants, derepressed local YUC1/4 activity elevates auxin levels at the boundary between whorls 3 and 4, which leads to an increase in the number and the prolonged maintenance of floral stem cells, and consequently an increase in the number of reproductive organs. Our work presents a new floral meristem regulatory mechanism, in which SUP, a boundary gene, coordinates floral organogenesis and floral meristem size through fine‐tuning auxin biosynthesis.     

Pattern formation during de novo assembly of the Arabidopsis shoot meristem

Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.     

Flux-based transport enhancement as a plausible unifying mechanism for auxin transport in meristem development

Plants continuously generate new organs through the activity of populations of stem cells called meristems. The shoot apical meristem initiates leaves, flowers, and lateral meristems in highly ordered, spiralled, or whorled patterns via a process called phyllotaxis. It is commonly accepted that the active transport of the plant hormone auxin plays a major role in this process. Current hypotheses propose that cellular hormone transporters of the PIN family would create local auxin maxima at precise positions, which in turn would lead to organ initiation. To explain how auxin transporters could create hormone fluxes to distinct regions within the plant, different concepts have been proposed. A major hypothesis, canalization, proposes that the auxin transporters act by amplifying and stabilizing existing fluxes, which could be initiated, for example, by local diffusion. This convincingly explains the organised auxin fluxes during vein formation, but for the shoot apical meristem a second hypothesis was proposed, where the hormone would be systematically transported towards the areas with the highest concentrations. This implies the coexistence of two radically different mechanisms for PIN allocation in the membrane, one based on flux sensing and the other on local concentration sensing. Because these patterning processes require the interaction of hundreds of cells, it is impossible to estimate on a purely intuitive basis if a particular scenario is plausible or not. Therefore, computational modelling provides a powerful means to test this type of complex hypothesis. Here, using a dedicated computer simulation tool, we show that a flux-based polarization hypothesis is able to explain auxin transport at the shoot meristem as well, thus providing a unifying concept for the control of auxin distribution in the plant. Further experiments are now required to distinguish between flux-based polarization and other hypotheses.     

Multiple MONOPTEROS-dependent pathways are involved in leaf initiation

Initiation of leaves at the flanks of the shoot apical meristem occurs at sites of auxin accumulation and pronounced expression of auxin-inducible PIN-FORMED1 (PIN) genes, suggesting a feedback loop to progressively focus auxin in concrete spots. Because PIN expression is regulated by auxin response factor activity, including MONOPTEROS (MP), it appeared possible that MP affects leaf formation as a positive regulator of PIN genes and auxin transport. Here, we analyze a novel, completely leafless phenotype arising from simultaneous interference with both auxin signaling and auxin transport. We show that mp pin1 double mutants, as well as mp mutants treated with auxin-efflux inhibitors, display synergistic abnormalities not seen in wild type regardless of how strongly auxin transport was reduced. The synergism of abnormalities indicates that the role of MP in shoot meristem organization is not limited to auxin transport regulation. In the mp mutant background, auxin transport inhibition completely abolishes leaf formation. Instead of forming leaves, the abnormal shoot meristems dramatically increase in size, harboring correspondingly enlarged expression domains of CLAVATA3 and SHOOTMERISTEMLESS, molecular markers for the central stem cell zone and the complete meristem, respectively. The observed synergism under conditions of auxin efflux inhibition was further supported by an unrestricted PIN1 expression in mp meristems, as compared to a partial restriction in wild-type meristems. Auxin transport-inhibited mp meristems also lacked detectable auxin maxima. We conclude that MP promotes the focusing of auxin and leaf initiation in part through pathways not affected by auxin efflux inhibitors.     

Thidiazuron-induced morphogenesis in tamarind seedlings

Summary Germination of tamarind seeds in medium containing thidiazuron (TDZ) resulted in induction of nodular protrusions in and around the cotyledonary node meristem. The structures developed radially in well-defined circles and subsequently spread towards the cotyledonary bridge and also in the proximal part of the hypocotyl. The structures developed into shoots on transfer to medium devoid of growth regulators. Histological studies revealed that the protrusions initiated from the nodal meristem and extended to the non-meristematic region between the two meristems and also in the proximal part of the hypocotyl in seedlings germinated in 9.08 μM TDZ. Newly formed cell layers and less-differentiated meristematic protrusions were also seen. With the increase in the distance from the meristem, the buds were less differentiated; in the proximal part of the hypocotyl only the multiple layers of meristematic cells were noted. With extension of the period of incubation, the TDZ-induced meristematic activity extended laterally in circles towards the neighboring region. The radial spread of the meristematic activity from the center of the nodal meristem was also evident at 18.16 μM TDZ. From the pattern of the morphogenic development and the histological studies it may be hypothesized that in tamarind, TDZ influences the existing meristems specifically. Subsequently de novo organogenesis is triggered in the neighboring cells.     

AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis

In contrast to animals, little is known about pattern formation in plants. Physiological and genetic data suggest the involvement of the phytohormone auxin in this process. Here, we characterize a novel member of the PIN family of putative auxin efflux carriers, Arabidopsis PIN4, that is localized in developing and mature root meristems. Atpin4 mutants are defective in establishment and maintenance of endogenous auxin gradients, fail to canalize externally applied auxin, and display various patterning defects in both embryonic and seedling roots. We propose a role for AtPIN4 in generating a sink for auxin below the quiescent center of the root meristem that is essential for auxin distribution and patterning.     

NO VEIN Mediates Auxin-Dependent Specification and Patterning in the Arabidopsis Embryo,Shoot, and Root

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type–specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type–specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.     

Hormonal and cell division analyses in Watsonia lepida seedlings

The regeneration ability, cell division activity, auxin and cytokinin content of seedling regions and hypocotyl subsections of Watsonia lepida were studied. A total of 21 different cytokinins or conjugates were found in seedlings, with the highest cytokinin content in meristematic regions (root and shoot apical meristems). The greatest contribution to the cytokinin pool came from the biologically inactive cZRMP, suggesting that significant de novo synthesis was occurring. Five different auxins or conjugates were detected, being concentrated largely in the shoot apical meristem and leaves, IAA being the most abundant. Analysis of hypocotyl subsections (C1–C4) revealed that cell division was highest in subsection C2, although regeneration in vitro was significantly lower than in subsection C1. Anatomically, subsection C1 contains the apical meristem, and hence has meristematic cells that are developmentally plastic. In contrast, subsection C2 has cells that have recently exited the meristem and are differentiating. Despite high rates of cell division, cells in subsection C2 appear no longer able to respond to cues that promote proliferation in vitro. Auxin and cytokinin analyses of these subsections were conducted. Possibly, a lower overall cytokinin content, and in particular the free-base cytokinins, could account for this observed difference.     

The Relationship between auxin transport and maize branching

Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.     

BRANCHED SILKLESS mediates the transition from spikelet to floral meristem during Zea mays ear development

The molecular and genetic control of inflorescence and flower development has been studied in great detail in model dicotyledonous plants such as Arabidopsis and Antirrhinum . In contrast, little is known about these important developmental steps in monocotyledonous species. Here we report the analysis of the Zea mays mutant branched silkless1–2 (bd1–2) , allelic to bd1 , which we have used as a tool to study the transition from spikelet to floret development in maize. Floret development is blocked in the female inflorescence (the ear) of bd1–2 plants, whereas florets develop almost normally in the male inflorescence (the tassel). Detailed phenotypic analyses indicate that in bd1–2 mutants ear inflorescence formation initiates normally, however, the spikelet meristems do not proceed to form floret meristems. The ear spikelets, at anthesis, contain various numbers of spikelet-like meristems and glume-like structures. Furthermore, growth of branches from the base of the ear is often observed. Expression analyses show that the floral-specific MADS box genes Zea mays AGAMOUS1 ( ZAG1 ), ZAG2 and Zea mays MADS 2 ( ZMM2 ) are not expressed in ear florets in bd1–2 mutants, whereas their expression in tassel florets is similar to that of wild type. Taken together, these data indicate that the development from spikelet to floret meristem is differentially controlled in the ear and tassel in the monoecious grass species Zea mays , and that BRANCHED SILKLESS plays an important role in regulating the transition from spikelet meristem to floral meristem during the development of the female inflorescence of maize.     

Root Meristem Establishment and Maintenance: The Role of Auxin

Auxin has a central role in the establishment and elaboration of pattern in root meristems. Regulation of root development by auxin begins early in embryogenesis, perhaps even as early as the establishment of polarity in the zygote, and persists throughout the lifetime of a root. Auxin-regulated development depends on a balance of synthesis/import and metabolism/export/sequestration. The overall result of these processes is to establish a state of auxin homeostasis which we hypothesize is required for normal root meristem patterning and development.     

Root Meristem Establishment and Maintenance: The Role of Auxin

Auxin has a central role in the establishment and elaboration of pattern in root meristems. Regulation of root development by auxin begins early in embryogenesis, perhaps even as early as the establishment of polarity in the zygote, and persists throughout the lifetime of a root. Auxin-regulated development depends on a balance of synthesis/import and metabolism/export/sequestration. The overall result of these processes is to establish a state of auxin homeostasis which we hypothesize is required for normal root meristem patterning and development.     

REGENERATION OF THE SUNFLOWER CAPITULUM AFTER CYLINDRICAL WOUNDING OF THE RECEPTACLE

Using the young capitulum of Helianthus annuus L., a cylindrical plug of undifferentiated receptacle tissue, 1 mm in diameter, was isolated from lateral communication with the rest of the receptacle surface by a vertical circular wound cut, while retaining continuity with the subapical meristem. Within 24 hr, active cell division was induced at the inner and outer surfaces of the wound and in the receptacle epidermis bordering the wound edges, creating a rounded rim at the top of the wound. Within 3–6 days, floral initials, spaced 133–166 μm apart appeared on the flanks of both rims and later on the top of the plug and surrounding receptacle surface. The first formed initials developed into involucral bracts or ray florets and the later ones into disc florets which were organized into contact parastichies, the number of which did not conform with the Fibonacci series. The base of the plug developed into a stem-like structure completing the regeneration of a fully formed functional capitulum. This operation was demonstrated for two sunflower cultivars and occurred in both long and short daylengths.     

Developmental consequences of the <Emphasis Type="Italic">tumorous shoot development1</Emphasis> mutation,a novel allele of the cellulose-synthesizing <Emphasis Type="Italic">KORRIGAN1</Emphasis> gene

This work describes the further characterization of the tumorous shoot development1 (tsd1) mutant of Arabidopsis thaliana, which develops disorganized tumorous-like shoot tissue instead of organized leaves and stems. Map-based cloning revealed that tsd1 is a novel strong allele of the KOR1 gene, encoding a membrane-bound endo-1,4-β-d-glucanase involved in cellulose synthesis. To study developmental changes accompanying the aberrant growth of the tsd1 mutant, patterning in the meristems and the hormonal status were analysed by marker genes. Expression of key regulators of meristem maintenance, the CLV3 and STM genes, indicated the presence of numerous meristems in the tsd1 shoot callus. Expression of the LFY::GUS marker supported the ability of the tsd1 callus to form organ primordia, which however failed to develop further. An epidermal marker showed that the L1 layer was maintained only in distinct areas of the tsd1 callus, which could be a reason of disorganized shoot growth. In the tsd1 root meristem, quiescent center activity was lost early after germination, which caused differentiation of the root meristem. The spatial expression of genes reporting the auxin and cytokinin status was altered in the tsd1 mutant. Modifying the endogenous levels of these hormones partially rescued shoot and root development of the tsd1 mutant. Together, the work shows that TSD1/KOR1 is required for maintaining a correct meristematic pattern and organ growth as well as for a normal hormonal response.     

The Stem Cell Niche in Leaf Axils Is Established by Auxin and Cytokinin in Arabidopsis

Plants differ from most animals in their ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems harboring new stem cell niches. In seed plants, this is achieved by axillary meristems, which are established in the axil of each leaf base and develop into lateral branches. Here, we describe the initial processes of Arabidopsis thaliana axillary meristem initiation. Using reporter gene expression analysis, we find that axillary meristems initiate from leaf axil cells with low auxin through stereotypical stages. Consistent with this, ectopic overproduction of auxin in the leaf axil efficiently inhibits axillary meristem initiation. Furthermore, our results demonstrate that auxin efflux is required for the leaf axil auxin minimum and axillary meristem initiation. After lowering of auxin levels, a subsequent cytokinin signaling pulse is observed prior to axillary meristem initiation. Genetic analysis suggests that cytokinin perception and signaling are both required for axillary meristem initiation. Finally, we show that cytokinin overproduction in the leaf axil partially rescue axillary meristem initiation-deficient mutants. These results define a mechanistic framework for understanding axillary meristem initiation.