A quantitative gibberellin signaling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem.

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signaling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signaling activity in the SAM. We show that high GA signaling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organization of internodes, thus contributing to internode specification in the SAM.

Transcriptional reprogramming during floral fate acquisition.

Coordinating growth and patterning is essential for eukaryote morphogenesis. In plants, auxin is a key regulator of morphogenesis implicated throughout development. Despite this central role, our understanding of how auxin coordinates cell fate and growth changes is still limited. Here, we addressed this question using a combination of genomic screens to delve into the transcriptional network induced by auxin at the earliest stage of flower development, prior to morphological changes. We identify a shoot-specific network suggesting that auxin initiates growth through an antagonistic regulation of growth-promoting and growth-repressive hormones, quasi-synchronously to floral fate specification. We further identify two DNA-binding One Zinc Finger (DOF) transcription factors acting in an auxin-dependent network that could interface growth and cell fate from the early stages of flower development onward.

Developmental roles of Auxin Binding Protein 1 in Arabidopsis thaliana.

Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy.

A network of transcriptional repressors modulates auxin responses.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

Immediate targets of ETTIN suggest a key role for pectin methylesterase inhibitors in the control of Arabidopsis gynecium development.

The control of gynecium development in Arabidopsis thaliana by the auxin response factor ETTIN (ETT) correlates with a reduction in the methylesterification of cell-wall pectins and a decrease in cell-wall stiffness in the valve tissues of the ovary. Here, we determine the list of genes rapidly regulated following the in-vivo activation of an ETT fusion protein, and show these to be significantly enriched in genes encoding cell-wall proteins, including several pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs). We also perform a genome-wide scan for potential ETT-binding sites, and incorporate the results of this procedure into a comparison of datasets, derived using four distinct methods, to identify genes regulated directly or indirectly by ETT. We conclude from our combined analyses that PMEIs are likely to be key actors that mediate the regulation of gynecium development by ETT, while ETT may simultaneously regulate PMEs to prevent exaggerated developmental effects from the regulation of PMEIs. We also postulate the existence of one or more rapidly-acting intermediate factors in the transcriptional regulation of PMEs and PMEIs by ETT.

Temporal integration of auxin information for the regulation of patterning.

Positional information is essential for coordinating the development of multicellular organisms. In plants, positional information provided by the hormone auxin regulates rhythmic organ production at the shoot apex, but the spatio-temporal dynamics of auxin gradients is unknown. We used quantitative imaging to demonstrate that auxin carries high-definition graded information not only in space but also in time. We show that, during organogenesis, temporal patterns of auxin arise from rhythmic centrifugal waves of high auxin travelling through the tissue faster than growth. We further demonstrate that temporal integration of auxin concentration is required to trigger the auxin-dependent transcription associated with organogenesis. This provides a mechanism to temporally differentiate sites of organ initiation and exemplifies how spatio-temporal positional information can be used to create rhythmicity.

Plants, like animals and many other multicellular organisms, control their body architecture by creating organized patterns of cells. These patterns are generally defined by signal molecules whose levels differ across the tissue and change over time. This tells the cells where they are located in the tissue and therefore helps them know what tasks to perform. A plant hormone called auxin is one such signal molecule and it controls when and where plants produce new leaves and flowers. Over time, this process gives rise to the dashing arrangements of spiraling organs exhibited by many plant species. The leaves and flowers form from a relatively small group of cells at the tip of a growing stem known as the shoot apical meristem. Auxin accumulates at precise locations within the shoot apical meristem before cells activate the genes required to make a new leaf or flower. However, the precise role of auxin in forming these new organs remained unclear because the tools to observe the process in enough detail were lacking. Galvan-Ampudia, Cerutti et al. have now developed new microscopy and computational approaches to observe auxin in a small plant known as Arabidopsis thaliana. This showed that dozens of shoot apical meristems exhibited very similar patterns of auxin. Images taken over a period of several hours showed that the locations where auxin accumulated were not fixed on a group of cells but instead shifted away from the center of the shoot apical meristems faster than the tissue grew. This suggested the cells experience rapidly changing levels of auxin. Further experiments revealed that the cells needed to be exposed to a high level of auxin over time to activate genes required to form an organ. This mechanism sheds a new light on how auxin regulates when and where plants make new leaves and flowers. The tools developed by Galvan-Ampudia, Cerutti et al. could be used to study the role of auxin in other plant tissues, and to investigate how plants regulate the response to other plant hormones.

Methods to Visualize Auxin and Cytokinin Signaling Activity in the Shoot Apical Meristem.

Visualizing the distribution of hormone signaling activity such as auxin and cytokinins is of key importance for understanding regulation of plant development and physiology. Live imaging and genetically encoded hormone biosensors and reporters allow monitoring the spatial and temporal distribution of these phytohormones. Here, we describe how to cultivate live shoot apical meristems after dissection for observation under the confocal microscope for up to 4 days. The shoot apical meristems are maintained on an appropriate medium allowing them to grow and initiate new organs at a frequency similar to plants grown on soil. Meristems expressing hormone biosensors and reporters allows following hormone signaling activity distribution at high spatiotemporal resolution without chemical fixation, an approach that that can also be applied to follow the dynamics of expression in vivo of any fluorescent marker.

Dissecting the pathways coordinating patterning and growth by plant boundary domains.

Boundary domains play important roles during morphogenesis in plants and animals, but how they contribute to patterning and growth coordination in plants is not understood. The CUC genes determine the boundary domains in the aerial part of the plants and, in particular, they have a conserved role in regulating leaf complexity across Angiosperms. Here, we used tooth formation at the Arabidopsis leaf margin controlled by the CUC2 transcription factor to untangle intertwined events during boundary-controlled morphogenesis in plants. Combining conditional restoration of CUC2 function with morphometrics as well as quantification of gene expression and hormone signaling, we first established that tooth morphogenesis involves a patterning phase and a growth phase. These phases can be separated, as patterning requires CUC2 while growth can occur independently of CUC2. Next, we show that CUC2 acts as a trigger to promote growth through the activation of three functional relays. In particular, we show that KLUH acts downstream of CUC2 to modulate auxin response and that expressing KLUH can compensate for deficient CUC2 expression during tooth growth. Together, we reveal a genetic and molecular network that allows coordination of patterning and growth by CUC2-defined boundaries during morphogenesis at the leaf margin.

Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression.

Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions.

A stochastic multicellular model identifies biological watermarks from disorders in self-organized patterns of phyllotaxis.

Exploration of developmental mechanisms classically relies on analysis of pattern regularities. Whether disorders induced by biological noise may carry information on building principles of developmental systems is an important debated question. Here, we addressed theoretically this question using phyllotaxis, the geometric arrangement of plant aerial organs, as a model system. Phyllotaxis arises from reiterative organogenesis driven by lateral inhibitions at the shoot apex. Motivated by recurrent observations of disorders in phyllotaxis patterns, we revisited in depth the classical deterministic view of phyllotaxis. We developed a stochastic model of primordia initiation at the shoot apex, integrating locality and stochasticity in the patterning system. This stochastic model recapitulates phyllotactic patterns, both regular and irregular, and makes quantitative predictions on the nature of disorders arising from noise. We further show that disorders in phyllotaxis instruct us on the parameters governing phyllotaxis dynamics, thus that disorders can reveal biological watermarks of developmental systems.

Reproductive failure in Arabidopsis thaliana under transient carbohydrate limitation: flowers and very young siliques are jettisoned and the meristem is maintained to allow successful resumption of reproductive growth.

The impact of transient carbon depletion on reproductive growth in Arabidopsis was investigated by transferring long-photoperiod-grown plants to continuous darkness and returning them to a light-dark cycle. After 2 days of darkness, carbon reserves were depleted in reproductive sinks, and RNA in situ hybridization of marker transcripts showed that carbon starvation responses had been initiated in the meristem, anthers and ovules. Dark treatments of 2 or more days resulted in a bare-segment phenotype on the floral stem, with 23-27 aborted siliques. These resulted from impaired growth of immature siliques and abortion of mature and immature flowers. Depolarization of PIN1 protein and increased DII-VENUS expression pointed to rapid collapse of auxin gradients in the meristem and inhibition of primordia initiation. After transfer back to a light-dark cycle, flowers appeared and formed viable siliques and seeds. A similar phenotype was seen after transfer to sub-compensation point irradiance or CO2 . It also appeared in a milder form after a moderate decrease in irradiance and developed spontaneously in short photoperiods. We conclude that Arabidopsis inhibits primordia initiation and aborts flowers and very young siliques in C-limited conditions. This curtails demand, safeguarding meristem function and allowing renewal of reproductive growth when carbon becomes available again.

Overexpression of the Arabidopsis thaliana signalling peptide TAXIMIN1 affects lateral organ development.

Lateral organ boundary formation is highly regulated by transcription factors and hormones such as auxins and brassinosteroids. However, in contrast to many other developmental processes in plants, no role for signalling peptides in the regulation of this process has been reported yet. The first characterization of the secreted cysteine-rich TAXIMIN (TAX) signalling peptides in Arabidopsis is presented here. TAX1 overexpression resulted in minor alterations in the primary shoot and root metabolome, abnormal fruit morphology, and fusion of the base of cauline leaves to stems forming a decurrent leaf attachment. The phenotypes at the paraclade junction match TAX1 promoter activity in this region and are similar to loss of LATERAL ORGAN FUSION (LOF) transcription factor function. Nevertheless, TAX1 expression was unchanged in lof1lof2 paraclade junctions and, conversely, LOF gene expression was unchanged in TAX1 overexpressing plants, suggesting TAX1 may act independently. This study identifies TAX1 as the first plant signalling peptide influencing lateral organ separation and implicates the existence of a peptide signal cascade regulating this process in Arabidopsis.

Reporters for sensitive and quantitative measurement of auxin response.

The visualization of hormonal signaling input and output is key to understanding how multicellular development is regulated. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack the sensitivity or precision to visualize these. We developed a set of fluorescent reporters that allow sensitive and semiquantitative readout of auxin responses at cellular resolution in Arabidopsis thaliana. These generic tools are suitable for any transformable plant species.

A fluorescent hormone biosensor reveals the dynamics of jasmonate signalling in plants.

Activated forms of jasmonic acid (JA) are central signals coordinating plant responses to stresses, yet tools to analyse their spatial and temporal distribution are lacking. Here we describe a JA perception biosensor termed Jas9-VENUS that allows the quantification of dynamic changes in JA distribution in response to stress with high spatiotemporal sensitivity. We show that Jas9-VENUS abundance is dependent on bioactive JA isoforms, the COI1 co-receptor, a functional Jas motif and proteasome activity. We demonstrate the utility of Jas9-VENUS to analyse responses to JA in planta at a cellular scale, both quantitatively and dynamically. This included using Jas9-VENUS to determine the cotyledon-to-root JA signal velocities on wounding, revealing two distinct phases of JA activity in the root. Our results demonstrate the value of developing quantitative sensors such as Jas9-VENUS to provide high-resolution spatiotemporal data about hormone distribution in response to plant abiotic and biotic stresses.

Structural basis for oligomerization of auxin transcriptional regulators.

The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.

Cytokinin signalling inhibitory fields provide robustness to phyllotaxis.

How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.

Halotropism is a response of plant roots to avoid a saline environment.

Tropisms represent fascinating examples of how plants respond to environmental signals by adapting their growth and development. Here, a novel tropism is reported, halotropism, allowing plant seedlings to reduce their exposure to salinity by circumventing a saline environment. In response to a salt gradient, Arabidopsis, tomato, and sorghum roots were found to actively prioritize growth away from salinity above following the gravity axis. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by the PIN-FORMED 2 (PIN2) auxin efflux carrier. We show that salt-induced phospholipase D activity stimulates clathrin-mediated endocytosis of PIN2 at the side of the root facing the higher salt concentration. The intracellular relocalization of PIN2 allows for auxin redistribution and for the directional bending of the root away from the higher salt concentration. Our results thus identify a cellular pathway essential for the integration of environmental cues with auxin-regulated root growth that likely plays a key role in plant adaptative responses to salt stress.

A light-regulated genetic module was recruited to carpel development in Arabidopsis following a structural change to SPATULA.

A key innovation of flowering plants is the female reproductive organ, the carpel. Here, we show that a mechanism that regulates carpel margin development in the model flowering plant Arabidopsis thaliana was recruited from light-regulated processes. This recruitment followed the loss from the basic helix-loop-helix transcription factor SPATULA (SPT) of a domain previously responsible for its negative regulation by phytochrome. We propose that the loss of this domain was a prerequisite for the light-independent expression in female reproductive tissues of a genetic module that also promotes shade avoidance responses in vegetative organs. Striking evidence for this proposition is provided by the restoration of wild-type carpel development to spt mutants by low red/far-red light ratios, simulating vegetation shade, which we show to occur via phytochrome B, PHYTOCHROME INTERACTING FACTOR4 (PIF4), and PIF5. Our data illustrate the potential of modular evolutionary events to generate rapid morphological change and thereby provide a molecular basis for neo-Darwinian theories that describe this nongradualist phenomenon. Furthermore, the effects shown here of light quality perception on carpel development lead us to speculate on the potential role of light-regulated mechanisms in plant organs that, like the carpel, form within the shade of surrounding tissues.