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.

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance.

Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.

A matter of time: auxin signaling dynamics and the regulation of auxin responses during plant development.

As auxin is a major regulator of plant development, studying the signaling mechanisms by which auxin influences cellular activities is of primary importance. In this review, we describe current knowledge on the different modalities of signaling, from the well-characterized canonical nuclear auxin pathway, to the more recently discovered or re-discovered non-canonical modes of auxin signaling. In particular, we discuss how both the modularity of the nuclear auxin pathway and the dynamic regulation of its core components allow specific transcriptomic responses to be triggered. We highlight the fact that the diversity of modes of auxin signaling allows for a wide range of time scales of auxin responses, from second-scale cytoplasmic responses to minute-/hour-scale modifications of gene expression. Finally, we question the extent to which the temporality of auxin signaling and responses contributes to development in both the shoot and the root meristems. We conclude by stressing the fact that future investigations should allow an integrative view to be built not only of the spatial control, but also of the temporality of auxin-mediated regulation of plant development, from the cell to the whole organism.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

Decoding the Auxin Matrix: Auxin Biology Through the Eye of the Computer.

The plant hormone auxin is certainly the most studied developmental regulator in plants. The many functions of auxin during development, from the embryo to the root and shoot construction, are mediated by an ever-growing collection of molecular regulators, with an overwhelming degree of both ubiquity and complexity that we are still far from fully understanding and that biological experiments alone cannot grasp. In this review, we discuss how bioinformatics and computational modeling approaches have helped in recent years to explore this complexity and to push the frontiers of our understanding of auxin biology. We focus on how analysis of massive amounts of genomic data and construction of computational models to simulate auxin-regulated processes at different scales have complemented wet experiments to increase the understanding of how auxin acts in the nucleus to regulate transcription and how auxin movement between cells regulates development at the tissular scale.

bHLH heterodimer complex variations regulate cell proliferation activity in the meristems of Arabidopsis thaliana.

Root, shoot, and lateral meristems are the main regions of cell proliferation in plants. It has been proposed that meristems might have evolved dedicated transcriptional networks to balance cell proliferation. Here, we show that basic helix-loop-helix (bHLH) transcription factor heterodimers formed by members of the TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) subclades are general regulators of cell proliferation in all meristems. Yet, genetics and expression analyses suggest specific functions of these transcription factors in distinct meristems, possibly due to their expression domains determining heterodimer complex variations within meristems, and to a certain extent to the absence of some of them in a given meristem. Target gene specificity analysis for heterodimer complexes focusing on the LONELY GUY gene targets further suggests differences in transcriptional responses through heterodimer diversification that could allow a common bHLH heterodimer complex module to contribute to cell proliferation control in multiple meristems.

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.

Imaging the living plant cell: From probes to quantification.

At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.

Hormonal control of cell identity and growth in the shoot apical meristem.

How cells acquire their identities and grow coordinately within a tissue is a fundamental question to understand plant development. In angiosperms, the shoot apical meristem (SAM) is a multicellular tissue containing a stem cell niche, which activity allows for a dynamic equilibrium between maintenance of stem cells and production of differentiated cells that are incorporated in new aerial tissues and lateral organs produced in the SAM. Plant hormones are small-molecule signals controlling many aspects of plant development and physiology. Several hormones are essential regulators of SAM activities. This review highlights current advances that are starting to decipher the complex mechanisms underlying the hormonal control of cell identity and growth in the SAM.

What shoots can teach about theories of plant form.

Plants generate a large variety of shoot forms with regular geometries. These forms emerge primarily from the activity of a stem cell niche at the shoot tip. Recent efforts have established a theoretical framework of form emergence at the shoot tip, which has empowered the use of modelling in conjunction with biological approaches to begin to disentangle the biochemical and physical mechanisms controlling form development at the shoot tip. Here, we discuss how these advances get us closer to identifying the construction principles of plant shoot tips. Considering the current limits of our knowledge, we propose a roadmap for developing a general theory of form development at the shoot tip.

Auxin Does the SAMba: Auxin Signaling in the Shoot Apical Meristem.

Plants, in contrast to animals, are unique in their capacity to postembryonically develop new organs due to the activity of stem cell populations, located in specialized tissues called meristems. Above ground, the shoot apical meristem generates aerial organs and tissues throughout plant life. It is well established that auxin plays a central role in the functioning of the shoot apical meristem. Auxin distribution in the meristem is not uniform and depends on the interplay between biosynthesis, transport, and degradation. Auxin maxima and minima are created, and result in transcriptional outputs that drive the development of new organs and contribute to meristem maintenance. To uncover and understand complex signaling networks such as the one regulating auxin responses in the shoot apical meristem remains a challenge. Here, we will discuss our current understanding and point to important research directions for the future.

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.

Phyllotaxis from a Single Apical Cell.

Phyllotaxis, the geometry of leaf arrangement around stems, determines plant architecture. Molecular interactions coordinating the formation of phyllotactic patterns have mainly been studied in multicellular shoot apical meristems of flowering plants. Phyllotaxis evolved independently in the major land plant lineages. In mosses, it arises from a single apical cell, raising the question of how asymmetric divisions of a single-celled meristem create phyllotactic patterns and whether associated genetic processes are shared across lineages. We present an overview of the mechanisms governing shoot apical cell specification and activity in the model moss, Physcomitrium patens, and argue that similar molecular regulatory modules have been deployed repeatedly across evolution to operate at different scales and drive apical function in convergent shoot forms.

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.

Low Blue Light Enhances Phototropism by Releasing Cryptochrome1-Mediated Inhibition of PIF4 Expression.

Shade-avoiding plants, including Arabidopsis (Arabidopsis thaliana), display a number of growth responses, such as elongation of stem-like structures and repositioning of leaves, elicited by shade cues, including a reduction in the blue and red portions of the solar spectrum and a low-red to far-red ratio. Shade also promotes phototropism of de-etiolated seedlings through repression of phytochrome B, presumably to enhance capture of unfiltered sunlight. Here we show that both low blue light and a low-red to far-red light ratio are required to rapidly enhance phototropism in Arabidopsis seedlings. However, prolonged low blue light treatments are sufficient to promote phototropism through reduced cryptochrome1 (cry1) activation. The enhanced phototropic response of cry1 mutants in the lab and in response to natural canopies depends on PHYTOCHROME INTERACTING FACTORs (PIFs). In favorable light conditions, cry1 limits the expression of PIF4, while in low blue light, PIF4 expression increases, which contributes to phototropic enhancement. The analysis of quantitative DII-Venus, an auxin signaling reporter, indicates that low blue light leads to enhanced auxin signaling in the hypocotyl and, upon phototropic stimulation, a steeper auxin signaling gradient across the hypocotyl. We conclude that phototropic enhancement by canopy shade results from the combined activities of phytochrome B and cry1 that converge on PIF regulation.

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.

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

Evolution of the Auxin Response Factors from charophyte ancestors.

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most 'ancient' ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.