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1.  A Regulatory Network for Coordinated Flower Maturation 
PLoS Genetics  2012;8(2):e1002506.
For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.
Author Summary
Perfect flowers have both male organs that produce and release pollen and female organs that make and harbor seeds. Flowers also often attract pollinators using visual or chemical signals. So that male, female, and pollinator attraction functions occur at the right time, flower organs must grow and mature in a coordinated fashion. In the model self-pollinating plant Arabidopsis, a transcriptional network regulates genes that ensure coordinated growth of different flower organs, as well as pollen release and gynoecium (female) competence to support pollination. This network also regulates nectary development and production of volatile chemicals that may attract or repel insects. We have studied growth, chemical signal levels, and gene expression in mutants affected in components of this network, in order to determine how flower growth is controlled. Several plant hormones act in a cascade that promotes flower maturation. Moreover, regulatory feedback loops affect the timing and extent of developmental steps. Positive feedbacks may ensure that the development of different flower organs is coordinated and rapid, whereas negative feedbacks may allow growth to cease once flowers have opened. Our results provide a framework to understand how flower opening and reproduction are coordinated in Arabidopsis and other flowering plants.
doi:10.1371/journal.pgen.1002506
PMCID: PMC3276552  PMID: 22346763
2.  The auxin signalling network translates dynamic input into robust patterning at the shoot apex 
We provide a comprehensive expression map of the different genes (TIR1/AFBs, ARFs and Aux/IAAs) involved in the signalling pathway regulating gene transcription in response to auxin in the shoot apical meristem (SAM).We demonstrate a relatively simple structure of this pathway using a high-throughput yeast two-hybrid approach to obtain the Aux/IAA-ARF full interactome.The topology of the signalling network was used to construct a model for auxin signalling and to predict a role for the spatial regulation of auxin signalling in patterning of the SAM.We used a new sensor to monitor the input in the auxin signalling pathway and to confirm the model prediction, thus demonstrating that auxin signalling is essential to create robust patterns at the SAM.
The plant hormone auxin is a key morphogenetic signal involved in the control of cell identity throughout development. A striking example of auxin action is at the shoot apical meristem (SAM), a population of stem cells generating the aerial parts of the plant. Organ positioning and patterning depends on local accumulations of auxin in the SAM, generated by polar transport of auxin (Vernoux et al, 2010). However, it is still unclear how auxin is distributed at cell resolution in tissues and how the hormone is sensed in space and time during development. A complex ensemble of 29 Aux/IAAs and 23 ARFs is central to the regulation of gene transcription in response to auxin (for review, see Leyser, 2006; Guilfoyle and Hagen, 2007; Chapman and Estelle, 2009). Protein–protein interactions govern the properties of this transduction pathway (Del Bianco and Kepinski, 2011). Limited interaction studies suggest that, in the absence of auxin, the Aux/IAA repressors form heterodimers with the ARF transcription factors, preventing them from regulating target genes. In the presence of auxin, the Aux/IAA proteins are targeted to the proteasome by an SCF E3 ubiquitin ligase complex (Chapman and Estelle, 2009; Leyser, 2006). In this process, auxin promotes the interaction between Aux/IAA proteins and the TIR1 F-box of the SCF complex (or its AFB homologues) that acts as an auxin co-receptor (Dharmasiri et al, 2005a, 2005b; Kepinski and Leyser, 2005; Tan et al, 2007). The auxin-induced degradation of Aux/IAAs would then release ARFs to regulate transcription of their target genes. This includes activation of most of the Aux/IAA genes themselves, thus establishing a negative feedback loop (Guilfoyle and Hagen, 2007). Although this general scenario provides a framework for understanding gene regulation by auxin, the underlying protein–protein network remains to be fully characterized.
In this paper, we combined experimental and theoretical analyses to understand how this pathway contributes to sensing auxin in space and time (Figure 1). We first analysed the expression patterns of the ARFs, Aux/IAAs and TIR1/AFBs genes in the SAM. Our results demonstrate a general tendency for most of the 25 ARFs and Aux/IAAs detected in the SAM: a differential expression with low levels at the centre of the meristem (where the stem cells are located) and high levels at the periphery of the meristem (where organ initiation takes place). We also observed a similar differential expression for TIR1/AFB co-receptors. To understand the functional significance of the distribution of ARFs and Aux/IAAs in the SAM, we next investigated the global structure of the Aux/IAA-ARF network using a high-throughput yeast two-hybrid approach and uncover a rather simple topology that relies on three basic generic features: (i) Aux/IAA proteins interact with themselves, (ii) Aux/IAA proteins interact with ARF activators and (iii) ARF repressors have no or very limited interactions with other proteins in the network.
The results of our interaction analysis suggest a model for the Aux/IAA-ARF signalling pathway in the SAM, where transcriptional activation by ARF activators would be negatively regulated by two independent systems, one involving the ARF repressors, the other the Aux/IAAs. The presence of auxin would remove the inhibitory action of Aux/IAAs, but leave the ARF repressors to compete with ARF activators for promoter-binding sites. To explore the regulatory properties of this signalling network, we developed a mathematical model to describe the transcriptional output as a function of the signalling input that is the combinatorial effect of auxin concentration and of its perception. We then used the model and a simplified view of the meristem (where the same population of Aux/IAAs and ARFs exhibit a low expression at the centre and a high expression in the peripheral zone) for investigating the role of auxin signalling in SAM function. We show that in the model, for a given ARF activator-to-repressor ratio, the gene induction capacity increases with the absolute levels of ARF proteins. We thus predict that the differential expression of the ARFs generates differences in auxin sensitivities between the centre (low sensitivity) and the periphery (high sensitivity), and that the expression of TIR1/AFB participates to this regulation (prediction 1). We also use the model to analyse the transcriptional response to rapidly changing auxin concentrations. By simulating situations equivalent either to the centre or the periphery of our simplified representation of the SAM, we predict that the signalling pathway buffers its response to the auxin input via the balance between ARF activators and repressors, in turn generated by their differential spatial distributions (prediction 2).
To test the predictions from the model experimentally, we needed to assess both the input (auxin level and/or perception) and the output (target gene induction) of the signalling cascade. For measuring the transcriptional output, the widely used DR5 reporter is perfectly adapted (Figure 5) (Ulmasov et al, 1997; Sabatini et al, 1999; Benkova et al, 2003; Heisler et al, 2005). For assaying pathway input, we designed DII-VENUS, a novel auxin signalling sensor that comprises a constitutively expressed fusion of the auxin-binding domain (termed domain II or DII) (Dreher et al, 2006; Tan et al, 2007) of an IAA to a fast-maturating variant of YFP, VENUS (Figure 5). The degradation patterns from DII-VENUS indicate a high auxin signalling input both in flower primordia and at the centre of the SAM. This is in contrast to the organ-specific expression pattern of DR5::VENUS (Figure 5). These results indicate that the signalling pathway limits gene activation in response to auxin at the meristem centre and confirm the differential sensitivity to auxin between the centre and the periphery (prediction 1). We further confirmed the buffering capacities of the signalling pathway (prediction 2) by carrying out live imaging experiments to monitor DII-VENUS and DR5::VENUS expression in real time (Figure 5). This analysis reveals the presence of important temporal variations of DII-VENUS fluorescence, while DR5::VENUS does not show such global variations. Our approach thus provides evidence that the Aux/IAA-ARF pathway has a key role in patterning in the SAM, alongside the auxin transport system. Our results illustrate how the tight spatio-temporal regulation of both the distribution of a morphogenetic signal and the activity of the downstream signalling pathway provides robustness to a dynamic developmental process.
A comprehensive expression and interaction map of auxin signalling factors in the Arabidopsis shoot apical meristem is constructed and used to derive a mathematical model of auxin signalling, from which key predictions are experimentally confirmed.
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
doi:10.1038/msb.2011.39
PMCID: PMC3167386  PMID: 21734647
auxin; biosensor; live imaging; ODE; signalling
3.  Emergence of tissue polarization from synergy of intracellular and extracellular auxin signaling 
Here, we provide a novel mechanistic framework for cell polarization during auxin-driven plant development that combines intracellular auxin signaling for regulation of expression of PINFORMED (PIN) auxin efflux transporters and the theoretical assumption of extracellular auxin signaling for regulation of PIN subcellular dynamics.The competitive utilization of auxin signaling component in the apoplast might account for the elusive mechanism for cell-to-cell communication for tissue polarization.Computer model simulations faithfully and robustly recapitulate experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems, and during the competitive regulation of shoot branching by apical dominance.Our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants.
A key question of developmental biology relates to a fundamental issue in cell and tissue polarities, namely, how an individual cell in a polarized tissue senses the polarities of its neighbors and its position within tissue. In plant development, this issue is of pronounced importance, because plants have a remarkable ability to redefine cell and tissue polarities in different developmental programs, such as embryogenesis, postembryonic organogenesis, vascular tissue formation, and tissue regeneration (Kleine-Vehn and Friml, 2008).
A polar, cell-to-cell transport of the small signaling molecule auxin in conjunction with local auxin biosynthesis determines auxin gradients during embryonic and postembryonic development, giving positional cues for primordia formation, organ patterning, and tropistic growth (Friml et al, 2002; Benková et al, 2003; Reinhardt et al, 2003; Heisler et al, 2005; Scarpella et al, 2006; Dubrovsky et al, 2008). Over the past decades, theoretical models proposed that auxin acts as a polarizing cue in the center of a positive feedback mechanisms for auxin transport that has a key role in synchronized polarity rearrangements. However, the mechanistic basis for such a feedback loop between auxin and its own transport remains to a large extent elusive.
The direction of auxin transport largely depends on the polar subcellular localization of PINFORMED (PIN) proteins at the plasma membrane (Petrášek et al, 2006; Wiśniewska et al, 2006). These proteins recycle between the plasma membrane and intracellular endosomal compartments (Geldner et al, 2001; Dhonukshe et al, 2007), and their recycling modulates PIN-dependent auxin efflux rates and enable rapid changes in PIN polarity (Dubrovsky et al, 2008; Kleine-Vehn et al, 2008a). Nevertheless, the molecular basis for PIN polarization in plants remains unknown.
To gain new mechanistic insights in the hypothetical feedback mechanisms governing PIN polarization, several theoretical studies (Mitchison, 1980; Sachs, 1981; Rolland-Lagan and Prusinkiewicz, 2005; Jönsson et al, 2006; Smith et al, 2006; Merks et al, 2007; Bayer et al, 2009; Kramer, 2009) have been carried out. These models suggest that auxin promotes its own transport by modulating the amount of PIN proteins at the plasma membrane by incorporating either not yet identified flux gradient-based component (Mitchison, 1980; Rolland-Lagan and Prusinkiewicz, 2005; Bayer et al, 2009; Kramer, 2009) or an unknown short-range intercellular signal-transmitting auxin concentrations of its direct neighbors (Jönsson et al, 2006; Smith et al, 2006; Merks et al, 2007; Bayer et al, 2009; Sahlin et al, 2009).
Here, we propose a feedback driven, biologically plausible model for PIN polarization and auxin transport that introduces the combination of intracellular and extracellular auxin signaling pathways as a unified approach for tissue polarization in plants. Our computer model is based on chemiosmotic hypothesis (Goldsmith et al, 1981; Figure 1A) and integrates up-to-date experimental data, such as auxin feedback on PIN expression (Peer et al, 2004; Heisler et al, 2005) via a nuclear auxin signaling pathway (Chapman and Estelle, 2009; Figure 1B), auxin carrier recycling auxin (Dubrovsky et al, 2008; Kleine-Vehn et al, 2008a; Figure 1C), and auxin feedback on PIN endocytosis (Paciorek et al, 2005) via novel hypothetical, yet plausible, assumption of extracellular auxin perception (Figure 1D).
The heart of our extracellular receptor-based polarization (ERP) mechanism is the competitive utilization of auxin receptors in the intercellular space that allows a direct and simple cell-to-cell communication scheme. In our model, auxin binds to its extracellular receptor in the concentration-dependent manner and induces signal to modulate PIN protein abundance at the plasma membrane (Figure 1D). The direct mode of the signal transfer involves temporal immobilization of recruited receptors to the plasma membrane, which is reflected by reduced diffusion of receptors involved in auxin signaling (Figure 1D). This competitive utilization mechanism enables cell-to-cell communication in our model, leading to receptor enrichment at the site of higher auxin concentration (Figure 1D). The PIN polarization and polar auxin transport in our model both depend on and contribute to the establishment of differential auxin signaling in the cell wall. This feedback loop leads ultimately to the alignment of PIN polarization within a tissue.
We demonstrated the plausibility of the ERP model for various processes, including de novo vascularization, venation patterning, and tissue regeneration in computer simulations performed with only minimal initial assumptions, a discrete auxin source, and a distal sink. The ERP model reproduces the very detailed PIN polarization events that occur during primary vein initiation (Scarpella et al, 2006), such as basal PIN1 polarity in provascular cells, transient adverse PIN1 polarization in neighboring cells during the alignment of tissue polarization, and inner-lateral polarity displayed by the tissues surrounding a conductive auxin channel (Figure 3). Additionally, the ERP model generates high auxin concentration and high auxin flux simultaneously in emerging veins, revising the classical canalization models (Mitchison, 1980; Rolland-Lagan and Prusinkiewicz, 2005). Importantly, all our model simulations support the claim that the ERP model represents the first single approach that faithfully reproduces PIN polarization, both with the auxin gradient (basal PIN1 polarity in provascular cells) and against the auxin gradient (transient adverse PIN1 polarization in neighboring cells surrounding the provascular bundle), as well as producing the corresponding auxin distribution patterns during auxin canalization.
The proposed model introduces the extracellular auxin signaling pathway, which is crucial to account for coordinated PIN polarization and auxin distribution during venation patterning in plants. The putative candidate for extracellular auxin receptor is auxin-binding protein 1 (ABP1), which resides in the lumen of the endoplasmic reticulum and is secreted to the cell wall (Napier et al, 2002; Tromas et al, 2009) where it is physiologically active (Leblanc et al, 1999; Steffens et al, 2001). Additionally, auxin inhibits clathrin-dependent PIN internalization via binding to ABP1 (Robert et al, 2010). Thus, we speculate that the extracellular fraction of ABP1 (or additionally yet to be identified ABPs) could correspond to the common pool of extracellular auxin receptors in the ERP model. A future challenge will be to test whether the ERP model unifies complex PIN polarization and auxin distribution patterns in embryogenesis, root system maintenance, and de novo organ formation.
Plant development is exceptionally flexible as manifested by its potential for organogenesis and regeneration, which are processes involving rearrangements of tissue polarities. Fundamental questions concern how individual cells can polarize in a coordinated manner to integrate into the multicellular context. In canalization models, the signaling molecule auxin acts as a polarizing cue, and feedback on the intercellular auxin flow is key for synchronized polarity rearrangements. We provide a novel mechanistic framework for canalization, based on up-to-date experimental data and minimal, biologically plausible assumptions. Our model combines the intracellular auxin signaling for expression of PINFORMED (PIN) auxin transporters and the theoretical postulation of extracellular auxin signaling for modulation of PIN subcellular dynamics. Computer simulations faithfully and robustly recapitulated the experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems and during the competitive regulation of shoot branching by apical dominance. Additionally, our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants.
doi:10.1038/msb.2010.103
PMCID: PMC3018162  PMID: 21179019
auxin; canalization; cell polarity; PIN proteins
4.  Hormones talking 
Plant Signaling & Behavior  2012;7(12):1698-1701.
The proper development of fruits is important for the sexual reproduction and propagation of many plant species. The fruit of Arabidopsis derives from the fertilized gynoecium, which initiates at the center of the flower and obtains its final shape, size, and functional tissues through progressive stages of development. Hormones, specially auxins, play important roles in gynoecium and fruit patterning. Cytokinins, which act as counterparts to auxins in other plant tissues, have been studied more in the context of ovule formation and parthenocarpy. We recently studied the role of cytokinins in gynoecium and fruit patterning and found that they have more than one role during gynoecium and fruit patterning. We also compared the cytokinin response localization to the auxin response localization in these organs, and studied the effects of spraying cytokinins in young flowers of an auxin response line. In this addendum, we discuss further the implications of the observed results in the knowledge about the relationship between cytokinins and auxins at the gynoecium.
doi:10.4161/psb.22422
PMCID: PMC3578912  PMID: 23072997
gynoecium; fruit; patterning and development; auxin; cytokinin
5.  A model for an early role of auxin in Arabidopsis gynoecium morphogenesis 
The female reproductive organ of angiosperms, the gynoecium, often consists of the fusion of multiple ovule-bearing carpels. It serves the important function of producing and protecting ovules as well as mediating pollination. The gynoecium has likely contributed to the tremendous success of angiosperms over their 160 million year history. In addition, being a highly complex plant organ, the gynoecium is well suited to serving as a model system for use in the investigation of plant morphogenesis and development. The longstanding model of gynoecium morphogenesis in Arabidopsis holds that apically localized auxin biosynthesis in the gynoecium results in an apical to basal gradient of auxin that serves to specify along its length the development of style, ovary, and gynophore in a concentration-dependent manner. This model is based primarily on the observed effects of the auxin transport blocker N-1-naphthylphthalamic acid (NPA) as well as analyses of mutants of Auxin Response Factor (ARF) 3/ETTIN (ETT). Both NPA treatment and ett mutation disrupt gynoecium morphological patterns along the apical–basal axis. More than a decade after the model’s initial proposal, however, the auxin gradient on which the model critically depends remains elusive. Furthermore, multiple observations are inconsistent with such an auxin-gradient model. Chiefly, the timing of gynoecium emergence and patterning occurs at a very early stage when the organ has little-to-no apical–basal dimension. Based on these observations and current models of early leaf patterning, we propose an alternate model for gynoecial patterning. Under this model, the action of auxin is necessary for the early establishment of adaxial–abaxial patterning of the carpel primordium. In this case, the observed gynoecial phenotypes caused by NPA and ett are due to the disruption of this early adaxial–abaxial patterning of the carpel primordia. Here we present the case for this model based on recent literature and current models of leaf development.
doi:10.3389/fpls.2014.00327
PMCID: PMC4086399  PMID: 25071809
gynoecium; auxin; ETTIN; abaxial; adaxial
6.  Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato 
Journal of Experimental Botany  2014;65(9):2507-2520.
Summary
Investigations into the role of tomato ARF6 and ARF8 reveal that they are critical components in floral and gynoecium development before anthesis.
Auxin regulates the expression of diverse genes that affect plant growth and development. This regulation requires AUXIN RESPONSE FACTORS (ARFs) that bind to the promoter regions of these genes. ARF6 and ARF8 in Arabidopsis thaliana are required to promote inflorescence stem elongation and late stages of petal, stamen, and gynoecium development. All seed plants studied thus far have ARF6 and ARF8 orthologues as well as the microRNA miR167, which targets ARF6 and ARF8. Whether these genes have broadly conserved roles in flower development is not known. To address this question, the effects of down-regulation of ARF6 and ARF8 were investigated through transgenic expression of Arabidopsis MIR167a in tomato, which diverged from Arabidopsis before the radiation of dicotyledonous plants approximately 90–112 million years ago. The transgenic tomato plants overexpressing MIR167a exhibited reductions in leaf size and internode length as well as shortened petals, stamens, and styles. More significantly, the transgenic plants were female-sterile as a result of failure of wild-type pollen to germinate on the stigma surface and/or to grow through the style. RNA-Seq analysis identified many genes with significantly altered expression patterns, including those encoding products with functions in ‘transcription regulation’, ‘cell wall’ and ‘lipid metabolism’ categories. Putative orthologues of a subset of these genes were also differentially expressed in Arabidopsis arf6 arf8 mutant flowers. These results thus suggest that ARF6 and ARF8 have conserved roles in controlling growth and development of vegetative and flower organs in dicots.
doi:10.1093/jxb/eru141
PMCID: PMC4036516  PMID: 24723401
ARF6; ARF8; expression; female sterility; flower development; tomato.
7.  A Rho Scaffold Integrates the Secretory System with Feedback Mechanisms in Regulation of Auxin Distribution 
PLoS Biology  2010;8(1):e1000282.
In plants, auxin distribution and tissue patterning are coordinated via a feedback loop involving the auxin-regulated cell polarity factor ICR1 and the secretory machinery.
Development in multicellular organisms depends on the ability of individual cells to coordinate their behavior by means of small signaling molecules to form correctly patterned tissues. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells connects cell polarity and tissue patterning and thus is required for many aspects of plant development. Direction of auxin flow is determined by polar subcellular localization of PIN auxin efflux transporters. Dynamic PIN polar localization results from the constitutive endocytic cycling to and from the plasma membrane, but it is not well understood how this mechanism connects to regulators of cell polarity. The Rho family small GTPases ROPs/RACs are master regulators of cell polarity, however their role in regulating polar protein trafficking and polar auxin transport has not been established. Here, by analysis of mutants and transgenic plants, we show that the ROP interactor and polarity regulator scaffold protein ICR1 is required for recruitment of PIN proteins to the polar domains at the plasma membrane. icr1 mutant embryos and plants display an a array of severe developmental aberrations that are caused by compromised differential auxin distribution. ICR1 functions at the plasma membrane where it is required for exocytosis but does not recycle together with PINs. ICR1 expression is quickly induced by auxin but is suppressed at the positions of stable auxin maxima in the hypophysis and later in the embryonic and mature root meristems. Our results imply that ICR1 is part of an auxin regulated positive feedback loop realized by a unique integration of auxin-dependent transcriptional regulation into ROP-mediated modulation of cell polarity. Thus, ICR1 forms an auxin-modulated link between cell polarity, exocytosis, and auxin transport-dependent tissue patterning.
Author Summary
The coordination of different cells during pattern formation is a fundamental process in the development of multicellular organisms. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells demonstrates the importance of cell polarity for tissue patterning. The direction of auxin flow is determined by polar subcellular localization of auxin transport proteins called PINs, which facilitate auxin efflux. At the same time, an auxin-mediated positive feedback mechanism reinforces the polar distribution of PINs. However, the molecular mechanisms that underlie polar PIN localization are not well understood. In eukaryotic cells, the Rho family of small GTPases function as central regulators of cell polarity. We show that a Rho-interacting protein from plants, called ICR1, is required for recruitment via the secretory system of PIN proteins to polar domains in the cell membrane. As a result, ICR1 is required for directional auxin transport and distribution and thereby for proper pattern formation. In addition, both the expression and subcellular localization of ICR1 are regulated by auxin, suggesting that ICR1 could function in a positive feedback loop that reinforces auxin distribution. Thus, ICR1 forms an auxin-modulated link between cell polarity, protein secretion, and auxin-dependent tissue patterning.
doi:10.1371/journal.pbio.1000282
PMCID: PMC2808208  PMID: 20098722
8.  Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID 
eLife  2014;3:e02860.
The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the—in many cells—asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.
DOI: http://dx.doi.org/10.7554/eLife.02860.001
eLife digest
In plants, a hormone called auxin controls the growth of the stems and roots. This chemical is transported from cell to cell, and its flow though the plant is redirected continuously as the plant is developing. Auxin is pumped out of cells by proteins in the cell membrane called ‘auxin efflux carriers’. These proteins are usually found on one side of each cell and this is what gives the direction to auxin transport.
Zourelidou, Absmanner et al. now report that being positioned on the correct side of a plant cell is not enough to enable an efflux carrier to do its job—it must also be turned on by kinases before it can pump auxin out of cells. Kinases are enzymes that add phosphate groups to specific sites on other proteins, and plants without certain kinases are unable to transport auxin.
When Zourelidou, Absmanner et al. produced the efflux carrier and a plant kinase—which turns the efflux carrier on—in immature egg cells from frogs, auxin was rapidly pumped out of the cells. However, cells that contained the efflux carrier but not the kinase could not transport the hormone. Importantly egg cells from frogs do not normally transport auxin, but these cells are commonly used in experiments because they are large, which makes them easier to work with in the lab.
One of at least two kinases must tag a number of sites on the efflux carrier to ensure that it is switched on. It was already known that some of these sites are involved in making sure that the efflux carrier is located on the correct side of the cell. Zourelidou, Absmanner et al. also found that auxin itself encourages the addition of phosphate groups onto the efflux carrier.
Though it was thought that knowing where the auxin transporters are was enough to explain the direction of auxin transport in plants, it is now clear that activation by the kinases needs to be taken into account too. And since these kinases may activate the transporters to different extents, identifying how these proteins are controlled, for example by auxin itself, will be the next challenge in the field.
DOI: http://dx.doi.org/10.7554/eLife.02860.002
doi:10.7554/eLife.02860
PMCID: PMC4091124  PMID: 24948515
AGC kinases; protein kinase; auxin; auxin transport; Arabidopsis
9.  Auxin polar transport in stamen formation and development: how many actors? 
In flowering plants, proper development of stamens, the male reproductive organs, is required for successful sexual reproduction. In Arabidopsis thaliana normally six stamen primordia arise in the third whorl of floral organs and subsequently differentiate into stamen filaments and anthers, where male meiosis occurs, thus ending the early developmental phase. This early phase is followed by a late developmental phase, which consists of a rapid elongation of stamen filaments coordinated with anther dehiscence and pollen maturation, and terminates with mature pollen grain release at anthesis. Increasing evidence suggests that auxin transport is necessary for both early and late phases of stamen development. It has been shown that different members of PIN (PIN-FORMED) family are involved in the early phase, whereas members of both PIN and P-glycoproteins of the ABCB (PGP) transporter families are required during the late developmental phase. In this review we provide an overview of the increasing knowledge on auxin transporters involved in Arabidopsis stamen formation and development and we discuss their role and functional conservation across plant species.
doi:10.3389/fpls.2014.00333
PMCID: PMC4100440  PMID: 25076953
stamen development; auxin transport; Arabidopsis; dicots; monocots
10.  phot1 Inhibition of ABCB19 Primes Lateral Auxin Fluxes in the Shoot Apex Required For Phototropism 
PLoS Biology  2011;9(6):e1001076.
It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19) as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1). Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3). Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.
Author Summary
Plants depend on sunlight for photosynthesis and adapt their growth to optimize light capture. Phototropism, the reorientation of growth towards light, is one important adaptive response. Modern studies of phototropism began with experiments in monocotyledonous grasses by Charles Darwin and led ultimately to the discovery of the plant growth hormone auxin, establishing the concept that light perception at the shoot apex triggers differential bending in the tissues below. In the past two decades, molecular-genetic analysis in the model flowering plant Arabidopsis thaliana has identified the principle photoreceptor for phototropism, phot1, as well as the major auxin transporters. Despite extensive efforts, how the photoreceptor regulates auxin transport so as to establish differential growth is still poorly understood, as is whether this process is conserved between monocots and dicots. Here, we introduce a new approach to the study of Arabidopsis phototropism in the absence of developmental events associated with seedling photomorphogenesis. In doing so, we show that the proximity of light perception and differential growth is conserved between monocots and dicots: in both plant types, differential growth is a consequence of lateral auxin movements across the shoot apex. Moreover, we identify two auxin transporters, PIN3 and ABCB19, that contribute to these movements, the latter serving to prime lateral auxin fluxes in the shoot apex. ABCB19 function is regulated by phot1, identifying it as a substrate for this class of photoreceptor kinase.
doi:10.1371/journal.pbio.1001076
PMCID: PMC3110179  PMID: 21666806
11.  AGAMOUS Controls GIANT KILLER, a Multifunctional Chromatin Modifier in Reproductive Organ Patterning and Differentiation 
PLoS Biology  2009;7(11):e1000251.
The floral, homeotic protein AGAMOUS coordinates multiple downstream genes through direct transcriptional regulation of the nuclear matrix attachment region binding protein GIANT KILLER.
The Arabidopsis homeotic protein AGAMOUS (AG), a MADS domain transcription factor, specifies reproductive organ identity during flower development. Using a binding assay and expression analysis, we identified a direct target of AG, GIANT KILLER (GIK), which fine-tunes the expression of multiple genes downstream of AG. The GIK protein contains an AT-hook DNA binding motif that is widely found in chromosomal proteins and that binds to nuclear matrix attachment regions of DNA elements. Overexpression and loss of function of GIK cause wide-ranging defects in patterning and differentiation of reproductive organs. GIK directly regulates the expression of several key transcriptional regulators, including ETTIN/AUXIN RESPONSE FACTOR 3 (ETT/ARF3) that patterns the gynoecium, by binding to the matrix attachment regions of target promoters. Overexpression of GIK causes a swift and dynamic change in repressive histone modification in the ETT promoter. We propose that GIK acts as a molecular node downstream of the homeotic protein AG, regulating patterning and differentiation of reproductive organs through chromatin organization.
Author Summary
Multicellular development depends on proper expression of thousands of genes. Master regulators, such as homeotic proteins, code for transcription factors in both plants and animals and are thought to act by regulating other genes. Recent genomic studies in the plant Arabidopsis have shown that over 1,000 genes are regulated by homeotic proteins that directly control various target genes, including different classes of transcriptional regulators. It is not known, however, how expression of so many genes is coordinated by a single homeotic gene to form functional organs and tissues. Here we identified a transcriptional target of the plant homeotic protein AGAMOUS using bioinformatics analysis and showed that AGAMOUS directly controls GIANT KILLER, a multifunctional chromatin modifier. GIANT KILLER then binds to the upstream regions of multiple genes involved in patterning and differentiation in the AGAMOUS pathway and fine-tunes the expression of these genes. These data therefore provide a possible mechanism by which a homeotic gene coordinates multiple downstream targets in plants.
doi:10.1371/journal.pbio.1000251
PMCID: PMC2774341  PMID: 19956801
12.  Genome-wide identification and transcriptional profiling analysis of auxin response-related gene families in cucumber 
BMC Research Notes  2014;7:218.
Background
Auxin signaling has a vital function in the regulation of plant growth and development, both which are known to be mediated by auxin-responsive genes. So far, significant progress has been made toward the identification and characterization of auxin-response genes in several model plants, while no systematic analysis for these families was reported in cucumber (Cucumis sativus L.), a reference species for Cucurbitaceae crops. The comprehensive analyses will help design experiments for functional validation of their precise roles in plant development and stress responses.
Results
A genome-wide search for auxin-response gene homologues identified 16 auxin-response factors (ARFs), 27 auxin/indole acetic acids (Aux/IAAs), 10 Gretchen Hagen 3 (GH3s), 61 small auxin-up mRNAs (SAURs), and 39 lateral organ boundaries (LBDs) in cucumber. Sequence analysis together with the organization of putative motifs indicated the potential diverse functions of these five auxin-related family members. The distribution and density of auxin response-related genes on chromosomes were not uniform. Evolutionary analysis showed that the chromosomal segment duplications mainly contributed to the expansion of the CsARF, CsIAA, CsGH3, and CsLBD gene families. Quantitative real-time RT-PCR analysis demonstrated that many ARFs, AUX/IAAs, GH3s, SAURs, and LBD genes were expressed in diverse patterns within different organs/tissues and during different development stages. They were also implicated in IAA, methyl jasmonic acid, or salicylic acid response, which is consistent with the finding that a great number of diverse cis-elements are present in their promoter regions involving a variety of signaling transduction pathways.
Conclusion
Genome-wide comparative analysis of auxin response-related family genes and their expression analysis provide new evidence for the potential role of auxin in development and hormone response of plants. Our data imply that the auxin response genes may be involved in various vegetative and reproductive developmental processes. Furthermore, they will be involved in different signal pathways and may mediate the crosstalk between various hormone responses.
doi:10.1186/1756-0500-7-218
PMCID: PMC4108051  PMID: 24708619
13.  DNA Methylation and Histone Modifications Regulate De Novo Shoot Regeneration in Arabidopsis by Modulating WUSCHEL Expression and Auxin Signaling 
PLoS Genetics  2011;7(8):e1002243.
Plants have a profound capacity to regenerate organs from differentiated somatic tissues, based on which propagating plants in vitro was made possible. Beside its use in biotechnology, in vitro shoot regeneration is also an important system to study de novo organogenesis. Phytohormones and transcription factor WUSCHEL (WUS) play critical roles in this process but whether and how epigenetic modifications are involved is unknown. Here, we report that epigenetic marks of DNA methylation and histone modifications regulate de novo shoot regeneration of Arabidopsis through modulating WUS expression and auxin signaling. First, functional loss of key epigenetic genes—including METHYLTRANSFERASE1 (MET1) encoding for DNA methyltransferase, KRYPTONITE (KYP) for the histone 3 lysine 9 (H3K9) methyltransferase, JMJ14 for the histone 3 lysine 4 (H3K4) demethylase, and HAC1 for the histone acetyltransferase—resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Second, we showed that regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications through bisulfite sequencing and chromatin immunoprecipitation. Third, DNA methylation in the regulatory regions of WUS was lost in the met1 mutant, thus leading to increased WUS expression and its localization. Fourth, we did a genome-wide transcriptional analysis and found out that some of differentially expressed genes between wild type and met1 were involved in signal transduction of the phytohormone auxin. We verified that the increased expression of AUXIN RESPONSE FACTOR3 (ARF3) in met1 indeed was due to DNA demethylation, suggesting DNA methylation regulates de novo shoot regeneration by modulating auxin signaling. We propose that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role in this process.
Author Summary
Plants have a strong ability to generate organs from differentiated somatic tissues. Due to this feature, shoot regeneration in vitro has been used as an important way for producing whole plants in agriculture and biotechnology. Phytohormones and the transcription factor WUSCHEL (WUS) are essential for reprogramming during de novo shoot regeneration. Epigenetic modifications are also critical for mammalian cell differentiation and organogenesis. Here, we show that epigenetic modifications mediate the de novo shoot regeneration in Arabidopsis. Mutations of key epigenetic genes resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Bisulfite sequencing and chromatin immunoprecipitation revealed that the regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications. By transcriptome analysis, we identified that some differentially expressed genes between wild type and met1 are involved in signal transduction of the phytohormone auxin. Our results suggest that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role during de novo organogenesis.
doi:10.1371/journal.pgen.1002243
PMCID: PMC3158056  PMID: 21876682
14.  A Division in PIN-Mediated Auxin Patterning during Organ Initiation in Grasses 
PLoS Computational Biology  2014;10(1):e1003447.
The hormone auxin plays a crucial role in plant morphogenesis. In the shoot apical meristem, the PIN-FORMED1 (PIN1) efflux carrier concentrates auxin into local maxima in the epidermis, which position incipient leaf or floral primordia. From these maxima, PIN1 transports auxin into internal tissues along emergent paths that pattern leaf and stem vasculature. In Arabidopsis thaliana, these functions are attributed to a single PIN1 protein. Using phylogenetic and gene synteny analysis we identified an angiosperm PIN clade sister to PIN1, here termed Sister-of-PIN1 (SoPIN1), which is present in all sampled angiosperms except for Brassicaceae, including Arabidopsis. Additionally, we identified a conserved duplication of PIN1 in the grasses: PIN1a and PIN1b. In Brachypodium distachyon, SoPIN1 is highly expressed in the epidermis and is consistently polarized toward regions of high expression of the DR5 auxin-signaling reporter, which suggests that SoPIN1 functions in the localization of new primordia. In contrast, PIN1a and PIN1b are highly expressed in internal tissues, suggesting a role in vascular patterning. PIN1b is expressed in broad regions spanning the space between new primordia and previously formed vasculature, suggesting a role in connecting new organs to auxin sinks in the older tissues. Within these regions, PIN1a forms narrow canals that likely pattern future veins. Using a computer model, we reproduced the observed spatio-temporal expression and localization patterns of these proteins by assuming that SoPIN1 is polarized up the auxin gradient, and PIN1a and PIN1b are polarized to different degrees with the auxin flux. Our results suggest that examination and modeling of PIN dynamics in plants outside of Brassicaceae will offer insights into auxin-driven patterning obscured by the loss of the SoPIN1 clade in Brassicaceae.
Author Summary
Computational models and functional studies using the plant Arabidopsis thaliana have led to competing models for how the PIN-FORMED1 (PIN1) auxin transporter polarizes in the cell to create both the maxima required for organ initiation and the narrow streams required for vein patterning. Here we identify a previously uncharacterized PIN protein most closely related to PIN1 that is present in all flowering plants but lost in the Brassicaceae, including Arabidopsis. We localized this protein, here termed Sister-of-PIN1 (SoPIN1), along with duplicate members of PIN1 (PIN1a and PIN1b), in two grass species. Our localization data provide striking evidence for a spatial and temporal split between SoPIN1 and the two PIN1s during organ initiation in grasses. Based on our localization results we created a computational model showing that the observed patterns of expression and polarization of the grass PINs can emerge assuming SoPIN1 polarizes up the gradient of auxin concentration while the PIN1 members polarize with the auxin flux. This model reveals a minimal framework of necessary functions involved in auxin-transport-mediated patterning in the shoot and demonstrates that work outside of Arabidopsis is essential to understanding how auxin-transport mediates patterning in most flowering plants.
doi:10.1371/journal.pcbi.1003447
PMCID: PMC3907294  PMID: 24499933
15.  Evolution and Structural Diversification of PILS Putative Auxin Carriers in Plants 
The phytohormone auxin contributes to virtually every aspect of the plant development. The spatiotemporal distribution of auxin depends on a complex interplay between auxin metabolism and intercellular auxin transport. Intracellular auxin compartmentalization provides another link between auxin transport processes and auxin metabolism. The PIN-LIKES (PILS) putative auxin carriers localize to the endoplasmic reticulum (ER) and contribute to cellular auxin homeostasis. PILS proteins regulate intracellular auxin accumulation, the rate of auxin conjugation and, subsequently, affect nuclear auxin signaling. Here, we investigate sequence diversification of the PILS family in Arabidopsis thaliana and provide insights into the evolution of these novel putative auxin carriers in plants. Our data suggest that PILS proteins are conserved throughout the plant lineage and expanded during higher plant evolution. PILS proteins diversified early during plant evolution into three clades. Besides the ancient Clade I encompassing non-land plant species, PILS proteins evolved into two clades. The diversification of Clade II and Clade III occurred already at the level of non-vascular plant evolution and, hence, both clades contain vascular and non-vascular plant species. Nevertheless, Clade III contains fewer non- and increased numbers of vascular plants, indicating higher importance of Clade III for vascular plant evolution. Notably, PILS proteins are distinct and appear evolutionarily older than the prominent PIN-FORMED auxin carriers. Moreover, we revealed particular PILS sequence divergence in Arabidopsis and assume that these alterations could contribute to distinct gene regulations and protein functions.
doi:10.3389/fpls.2012.00227
PMCID: PMC3470039  PMID: 23091477
PILS proteins; auxin; evolution; phylogeny; auxin metabolism; auxin homeostasis
16.  Tracking the development of the petaloid fertile stamen in Canna indica: insights into the origin of androecial petaloidy in the Zingiberales 
AoB Plants  2013;5:plt009.
The order Zingiberales comprises ∼2500 species of tropical to subtropical plants, including agriculturally (e.g. banana, ginger) and horticulturally (e.g. cannas, heliconias, bird-of-paradise) important plants. Throughout the evolution of this order, the stamens have been modified from the ancestral filamentous structures that produce pollen (seen in Banana flowers) to petal-like structures that no longer bear pollen sacs (seen in Canna flowers). This results in a reduction of pollen, but an effective increase in the overall size of the floral display and perhaps in the efficacy of specialized pollinators by converting stamens into ‘petals’. This study investigates the genetic mechanisms that are involved in making petal-like structures in place of pollen-producing stamens.
Flowers of the order Zingiberales demonstrate a remarkable trend of reduction in the number of fertile stamens; from five or six fertile, filamentous stamens bearing two thecae each in Musaceae and Strelitziaceae to just a single petaloid stamen bearing a single theca in Cannaceae and Marantaceae. As one progresses from ancestral to derived floral forms, 5–6 fertile stamens are replaced by 4–5 petaloid staminodes. In Cannaceae and Costaceae, all members of the androecial whorls exhibit petaloidy, including the fertile stamen. In Costaceae, a single fertile stamen develops two thecae embedded on a broad petaloid appendage, while in Cannaceae the single fertile stamen is further reduced to a single theca with a prominent, expanded petaloid appendage. Whether petaloidy of the fertile stamen is a synapomorphy of the entire ginger clade (including Cannaceae, Costaceae, Zingiberaceae and Marantaceae), or the result of independent convergent evolution in Cannaceae, Costaceae, and some Zingiberaceae, is unclear. We combine a developmental series of the formation of the petaloid fertile stamen in Canna indica with data on the expression of B- and C-class floral organ identity genes to elucidate the organogenetic identity of the petaloid stamen and staminodes. Our data indicate that the single fertile theca in C. indica and its petaloid appendage are derived from one-half of the primordium of a single stamen, with no contribution from the remaining part of the stamen (i.e. the second theca primordium) which aborts early in development. The petaloid appendage expands later, and develops from the position of the filament/connective of the developing theca. Floral identity gene expression shows that petal identity genes (i.e. B-class genes) are expressed in all floral organs studied while C-class gene AG-1 is expressed in an increasing gradient from sepals to gynoecium, and AG-2 is expressed in all floral organs except the petals. The canonical model for molecular specification of floral organ identity is not sufficient to explain petaloidy in the androecial whorl in Canna sp. Further studies understanding the regulation of gene networks are required.
doi:10.1093/aobpla/plt009
PMCID: PMC3608240  PMID: 23539493
Canna; evo-devo; floral development; MADS-box genes; petaloid stamens; petaloidy; Zingiberales
17.  ROP GTPase-Dependent Actin Microfilaments Promote PIN1 Polarization by Localized Inhibition of Clathrin-Dependent Endocytosis 
PLoS Biology  2012;10(4):e1001299.
A study in leaf epidermal pavement cells reveals that auxin activation of a Rho-like GTPase from plants induces inhibition of endocytosis through the clathrin-mediated pathway by regulating the accumulation of cortical F-actin.
Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis.
Author Summary
Formation of cell polarity is a process of distributing cellular structures or molecules in an asymmetric manner. This process plays an important role in the generation of diverse cell forms and types. In plants, the quintessential hormone auxin is important for diverse physiological functions, including growth and development of cells and organs. To perform these functions, auxin must be transported and localized to specific regions within the plant. This is partially mediated by polar distribution of the PIN-FORMED (PIN) auxin efflux transporters, which transport auxin outside of the cell and allow for the directional short- and long-distance transport of auxin throughout plant tissues and organs. Although auxin itself has been implicated as a signal to regulate PIN polar distribution, how auxin does so remains to be elucidated. We previously showed that auxin promotes the generation of “puzzle-piece” polarity in leaf epidermal pavement cells, which contain interdigitated lobes and indentations, by activating the ROP (Rho-like GTPases from plants) members of the conserved Rho family of small GTPases. Here, we find that auxin-dependent local activation of ROP2 in the lobe region inhibits PIN1 internalization into the endosomal compartments (or endocytosis), leaving higher levels of PIN1 polar distribution in the lobe region. PIN1 internalization is inhibited by altering the actin cytoskeleton through the ROP2 effector protein RIC4, a protein involved in cytoskeletal remodeling. On the basis of our findings, we propose that the Rho GTPase-mediated inhibition of endocytosis of PIN1 provides a self-organizing mechanism for the polar PIN1 distribution. Rho GTPase-based inhibition of endocytosis is also important for the formation of cell polarity in animal cells. Thus, we conclude that Rho GTPase signaling to inhibit endocytosis is a common mechanism for cell polarization in multicellular organisms.
doi:10.1371/journal.pbio.1001299
PMCID: PMC3317906  PMID: 22509133
18.  Simulation of Organ Patterning on the Floral Meristem Using a Polar Auxin Transport Model 
PLoS ONE  2012;7(1):e28762.
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.
doi:10.1371/journal.pone.0028762
PMCID: PMC3264561  PMID: 22291882
19.  The Arabidopsis IDD14, IDD15, and IDD16 Cooperatively Regulate Lateral Organ Morphogenesis and Gravitropism by Promoting Auxin Biosynthesis and Transport 
PLoS Genetics  2013;9(9):e1003759.
The plant hormone auxin plays a critical role in regulating various aspects of plant growth and development, and the spatial accumulation of auxin within organs, which is primarily attributable to local auxin biosynthesis and polar transport, is largely responsible for lateral organ morphogenesis and the establishment of plant architecture. Here, we show that three Arabidopsis INDETERMINATE DOMAIN (IDD) transcription factors, IDD14, IDD15, and IDD16, cooperatively regulate auxin biosynthesis and transport and thus aerial organ morphogenesis and gravitropic responses. Gain-of-function of each IDD gene in Arabidopsis results in small and transversally down-curled leaves, whereas loss-of-function of these IDD genes causes pleiotropic phenotypes in aerial organs and defects in gravitropic responses, including altered leaf shape, flower development, fertility, and plant architecture. Further analyses indicate that these IDD genes regulate spatial auxin accumulation by directly targeting YUCCA5 (YUC5), TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS1 (TAA1), and PIN-FORMED1 (PIN1) to promote auxin biosynthesis and transport. Moreover, mutation or ectopic expression of YUC suppresses the organ morphogenic phenotype and partially restores the gravitropic responses in gain- or loss-of-function idd mutants, respectively. Taken together, our results reveal that a subfamily of IDD transcription factors plays a critical role in the regulation of spatial auxin accumulation, thereby controlling organ morphogenesis and gravitropic responses in plants.
Author Summary
Auxin is a key plant hormone and the spatial accumulation of auxin is essential for lateral organ morphogenesis and gravitropic responses in higher plants. However, the various mechanisms through which spatial auxin accumulation is regulated remain to be fully elucidated. Here, we identify a gain-of-function mutant of Arabidopsis IDD14 that exhibits small and transversally down-curled leaves. Further characterization of both gain- and loss-of-function mutants in IDD14 and its close homologs, IDD15 and IDD16, reveals that these three IDD transcription factors function redundantly and cooperatively in the regulation of multiple aspects of lateral organ morphogenesis and gravitropic responses. We further demonstrate that these IDD transcription factors influence the spatial accumulation of auxin by directly targeting auxin biosynthetic and transport genes to activate their expression. These findings identify a subfamily of IDD transcription factors that coordinates spatial auxin gradients and thus directs lateral organ morphogenesis and gravitropic responses in plants.
doi:10.1371/journal.pgen.1003759
PMCID: PMC3764202  PMID: 24039602
20.  The role of auxin transporters in monocots development 
Auxin is a key regulator of plant growth and development, orchestrating cell division, elongation and differentiation, embryonic development, root and stem tropisms, apical dominance, and transition to flowering. Auxin levels are higher in undifferentiated cell populations and decrease following organ initiation and tissue differentiation. This differential auxin distribution is achieved by polar auxin transport (PAT) mediated by auxin transport proteins. There are four major families of auxin transporters in plants: PIN-FORMED (PIN), ATP-binding cassette family B (ABCB), AUXIN1/LIKE-AUX1s, and PIN-LIKES. These families include proteins located at the plasma membrane or at the endoplasmic reticulum (ER), which participate in auxin influx, efflux or both, from the apoplast into the cell or from the cytosol into the ER compartment. Auxin transporters have been largely studied in the dicotyledon model species Arabidopsis, but there is increasing evidence of their role in auxin regulated development in monocotyledon species. In monocots, families of auxin transporters are enlarged and often include duplicated genes and proteins with high sequence similarity. Some of these proteins underwent sub- and neo-functionalization with substantial modification to their structure and expression in organs such as adventitious roots, panicles, tassels, and ears. Most of the present information on monocot auxin transporters function derives from studies conducted in rice, maize, sorghum, and Brachypodium, using pharmacological applications (PAT inhibitors) or down-/up-regulation (over-expression and RNA interference) of candidate genes. Gene expression studies and comparison of predicted protein structures have also increased our knowledge of the role of PAT in monocots. However, knockout mutants and functional characterization of single genes are still scarce and the future availability of such resources will prove crucial to elucidate the role of auxin transporters in monocots development.
doi:10.3389/fpls.2014.00393
PMCID: PMC4133927  PMID: 25177324
IAA; PIN; ABCB; AUX/LAX; PILS; PAT
21.  Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape 
BMC Plant Biology  2009;9:49.
Background
Universally accepted landmark stages are necessary to highlight key events in plant reproductive development and to facilitate comparisons among species. Domestication and selection of tomato resulted in many varieties that differ in fruit shape and size. This diversity is useful to unravel underlying molecular and developmental mechanisms that control organ morphology and patterning. The tomato fruit shape gene SUN controls fruit elongation. The most dramatic effect of SUN on fruit shape occurs after pollination and fertilization although a detailed investigation into the timing of the fruit shape change as well as gene expression profiles during critical developmental stages has not been conducted.
Results
We provide a description of floral and fruit development in a red-fruited closely related wild relative of tomato, Solanum pimpinellifolium accession LA1589. We use established and propose new floral and fruit landmarks to present a framework for tomato developmental studies. In addition, gene expression profiles of three key stages in floral and fruit development are presented, namely floral buds 10 days before anthesis (floral landmark 7), anthesis-stage flowers (floral landmark 10 and fruit landmark 1), and 5 days post anthesis fruit (fruit landmark 3). To demonstrate the utility of the landmarks, we characterize the tomato shape gene SUN in fruit development. SUN controls fruit shape predominantly after fertilization and its effect reaches a maximum at 8 days post-anthesis coinciding with fruit landmark 4 representing the globular embryo stage of seed development. The expression profiles of the NILs that differ at sun show that only 34 genes were differentially expressed and most of them at a less than 2-fold difference.
Conclusion
The landmarks for flower and fruit development in tomato were outlined and integrated with the effect of SUN on fruit shape. Although we did not identify many genes differentially expressed in the NILs that differ at the sun locus, higher or lower transcript levels for many genes involved in phytohormone biosynthesis or signaling as well as organ identity and patterning of tomato fruit were found between developmental time points.
doi:10.1186/1471-2229-9-49
PMCID: PMC2685393  PMID: 19422692
22.  Inducible knock-down of GNOM during root formation reveals tissue-specific response to auxin transport and its modulation of local auxin biosynthesis 
Journal of Experimental Botany  2014;65(4):1165-1179.
Summary
Using DEX-inducible expression of GNOM antisense RNA, it was shown that GNOM-dependent auxin transport could affect local auxin biosynthesis, and this may also contribute to the establishment of GNOM-dependent auxin gradients.
In plants, active transport of auxin plays an essential role in root development. Localization of the PIN1 auxin transporters to the basal membrane of cells directs auxin flow and depends on the trafficking mediator GNOM. GNOM-dependent auxin transport is vital for root development and thus offers a useful tool for the investigation of a possible tissue-specific response to dynamic auxin transport. To avoid pleiotropic effects, DEX-inducible expression of GNOM antisense RNA was used to disrupt GNOM expression transiently or persistently during embryonic root development. It was found that the elongation zone and the pericycle layer are the most sensitive to GNOM-dependent auxin transport variations, which is shown by the phenotypes in cell elongation and the initiation of lateral root primordia, respectively. This suggests that auxin dynamics is critical to cell differentiation and cell fate transition, but not to cell division. The results also reveal that GNOM-dependent auxin transport could affect local auxin biosynthesis. This suggests that local auxin biosynthesis may also contribute to the establishment of GNOM-dependent auxin gradients in specific tissues, and that auxin transport and local auxin biosynthesis may function together in the regulatory network for initiation and development of lateral root primordia. Thus, the data reveal a tissue-specific response to auxin transport and modulation of local auxin biosynthesis by auxin transport.
doi:10.1093/jxb/ert475
PMCID: PMC3935571  PMID: 24453227
Arabidopsis; auxin biosynthesis; auxin transport; GNOM; inducible gene expression; root.
23.  Expression of gibberellin 20-oxidase1 (AtGA20ox1) in Arabidopsis seedlings with altered auxin status is regulated at multiple levels 
Journal of Experimental Botany  2008;59(8):2057-2070.
Bioactive gibberellins (GAs) affect many biological processes including germination, stem growth, transition to flowering, and fruit development. The location, timing, and level of bioactive GA are finely tuned to ensure that optimal growth and development occur. The balance between GA biosynthesis and deactivation is controlled by external factors such as light and by internal factors that include auxin. The role of auxin transport inhibitors (ATIs) and auxins on GA homeostasis in intact light-grown Arabidopsis thaliana (L.) Heynh. seedlings was investigated. Two ATIs, 1-N-naphthylthalamic acid (NPA) and 1-naphthoxyacetic acid (NOA) caused elevated expression of the GA biosynthetic enzyme AtGA20-oxidase1 (AtGA20ox1) in shoot but not in root tissues, and only at certain developmental stages. It was investigated whether enhanced AtGA20ox1 gene expression was a consequence of altered flow through the GA biosynthetic pathway, or was due to impaired GA signalling that can lead to enhanced AtGA20ox1 expression and accumulation of a DELLA protein, Repressor of ga1-3 (RGA). Both ATIs promoted accumulation of GFP-fused RGA in shoots and roots, and this increase was counteracted by the application of GA4. These results suggest that in ATI-treated seedlings the impediment to DELLA protein degradation may be a deficiency of bioactive GA at sites of GA response. It is proposed that the four different levels of AtGA20ox1 regulation observed here are imposed in a strict hierarchy: spatial (organ-, tissue-, cell-specific) > developmental > metabolic > auxin regulation. Thus results show that, in intact auxin- and auxin transport inhibitor-treated light-grown Arabidopsis seedlings, three other levels of regulation supersede the effects of auxin on AtGA20ox1.
doi:10.1093/jxb/ern063
PMCID: PMC2413289  PMID: 18440929
Auxin; auxin transport inhibitors; DELLA proteins; gibberellin 20-oxidase; gibberellin biosynthesis; RGA
24.  The role of auxin in style development and apical-basal patterning of the Arabidopsis thaliana gynoecium 
Plant Signaling & Behavior  2009;4(2):83-85.
In angiosperms, the gynoecium constitutes the female reproductive organ that after fertilization develops into a fruit and in Arabidopsis thaliana the gynoecium is formed by the congenital fusion of two carpels. In the last few years many genes involved in female organ development have been identified and there have been several reports on the involvement of the plant hormone auxin in gynoecium patterning. An auxin gradient has been suggested to establish the apical-basal patterning of the gynoecium and recently it has been shown that elevated apical auxin levels can compensate for the loss of several style-promoting factors but that auxin is dependent on their action in apical-basal patterning. Here we discuss the role of auxin and different upstream, downstream or parallel factors in the apical-basal patterning of the gynoecium. We focus specifically on the development of style and stigma and discuss the most recent findings.
PMCID: PMC2637486  PMID: 19649177
auxin; fruit; gynoecium; style; STYLISH1; PAT; NPA
25.  A soybean MADS-box protein modulates floral organ numbers, petal identity and sterility 
BMC Plant Biology  2014;14:89.
Background
The MADS-box transcription factors play fundamental roles in reproductive developmental control. Although the roles of many plant MADS-box proteins have been extensively studied, there are almost no functional studies of them in soybean, an important protein and oil crop in the world. In addition, the MADS-box protein orthologs may have species-specific functions. Controlling male fertility is an important goal in plant hybrid breeding but is difficult in some crops like soybean. The morphological structure of soybean flowers prevents the cross-pollination. Understanding the molecular mechanisms for floral development will aid in engineering new sterile materials that could be applied in hybrid breeding programs in soybean.
Result
Through microarray analysis, a flower-enriched gene in soybean was selected and designated as GmMADS28. GmMADS28 belongs to AGL9/SEP subfamily of MADS-box proteins, localized in nucleus and showed specific expression patterns in floral meristems as well as stamen and petal primordia. Expression of GmMADS28 in the stamens and petals of a soybean mutant NJS-10Hfs whose stamens are converted into petals was higher than in those of wild-type plants. Constitutive expression of GmMADS28 in tobacco promoted early flowering and converted stamens and sepals to petals. Interestingly, transgenic plants increased the numbers of sepal, petal and stamen from five to six and exhibited male sterility due to the shortened and curly filaments and the failure of pollen release from the anthers. The ectopic expression of GmMADS28 was found to be sufficient to activate expression of tobacco homologs of SOC1, LEAFY, AGL8/FUL, and DEF. In addition, we observed the interactions of GmMADS28 with soybean homologs of SOC1, AP1, and AGL8/FUL proteins.
Conclusion
In this study, we observed the roles of GmMADS28 in the regulation of floral organ number and petal identity. Compared to other plant AGL9/SEP proteins, GmMADS28 specifically regulates floral organ number, filament length and pollen release. The sterility caused by the ectopic expression of GmMADS28 offers a promising way to genetically produce new sterile material that could potentially be applied in the hybrid breeding of crops like soybean.
doi:10.1186/1471-2229-14-89
PMCID: PMC4021551  PMID: 24693922
Fertility; Floral organ number; Petal identity; Glycine max; MADS-box transcription factors

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