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The phytohormone gibberellic acid (GA) is critical for environmentally sensitive plant development including germination, skotomorphogenesis, and flowering. The Förster resonance energy transfer biosensor GIBBERELLIN PERCEPTION SENSOR1, which permits single-cell GA measurements in vivo, has been used to observe a GA gradient correlated with cell length in dark-grown, but not light-grown, hypocotyls. We sought to understand how light signaling integrates into cellular GA regulation. Here, we show how the E3 ligase CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) play central roles in directing cellular GA distribution in skoto- and photomorphogenic hypocotyls, respectively. We demonstrate that the expression pattern of the GA biosynthetic enzyme gene GA20ox1 is the key determinant of the GA gradient in dark-grown hypocotyls and is a target of COP1 signaling. We engineered a second generation GPS2 biosensor with improved orthogonality and reversibility. GPS2 revealed a previously undetectable cellular pattern of GA depletion during the transition to growth in the light. This GA depletion partly explains the resetting of hypocotyl growth dynamics during photomorphogenesis. Achieving cell-level resolution has revealed how GA distributions link environmental conditions with morphology and morphological plasticity. The GPS2 biosensor is an ideal tool for GA studies in many conditions, organs, and plant species.
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During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically-encoded second generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions and maintaining accumulation in the mature nodule meristem. We show, through mis-expression of GA catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation.
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The nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismoRESUMO
The ABACUS1-2 µ (ABscisic Acid Concentration and Uptake Sensor 1-2 µ) and GPS1 (Gibberellin Perception Sensor 1) are direct Förster resonance energy transfer (FRET) biosensors that can be used to measure hormone levels in planta. We provide detailed protocols to image FRET biosensors under exogenously applied hormones in roots, either as a single time point or for treatment time courses before and after hormone application. A new, free, open-source analysis toolset for Fiji is introduced and used to get full 3D segmentation of images of nuclear localized FRET biosensors and calculate emission ratios on a per nucleus basis allowing in-depth analysis of biosensor data.
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Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Giberelinas , Hormônios , Reguladores de Crescimento de PlantasRESUMO
Control over cell growth by mobile regulators underlies much of eukaryotic morphogenesis. In plant roots, cell division and elongation are separated into distinct longitudinal zones and both division and elongation are influenced by the growth regulatory hormone gibberellin (GA). Previously, a multicellular mathematical model predicted a GA maximum at the border of the meristematic and elongation zones. However, GA in roots was recently measured using a genetically encoded fluorescent biosensor, nlsGPS1, and found to be low in the meristematic zone grading to a maximum at the end of the elongation zone. Furthermore, the accumulation rate of exogenous GA was also found to be higher in the elongation zone. It was still unknown which biochemical activities were responsible for these mobile small molecule gradients and whether the spatiotemporal correlation between GA levels and cell length is important for root cell division and elongation patterns. Using a mathematical modeling approach in combination with high-resolution GA measurements in vivo, we now show how differentials in several biosynthetic enzyme steps contribute to the endogenous GA gradient and how differential cellular permeability contributes to an accumulation gradient of exogenous GA. We also analyzed the effects of altered GA distribution in roots and did not find significant phenotypes resulting from increased GA levels or signaling. We did find a substantial temporal delay between complementation of GA distribution and cell division and elongation phenotypes in a GA deficient mutant. Together, our results provide models of how GA gradients are directed and in turn direct root growth.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Técnicas Biossensoriais/métodos , Regulação da Expressão Gênica de Plantas , Giberelinas/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Transdução de SinaisRESUMO
Arabinogalactan proteins (AGPs) are a family of plant extracellular proteoglycans involved in many physiological events. AGPs are often anchored to the extracellular side of the plasma membrane and are highly glycosylated with arabinogalactan (AG) polysaccharides, but the molecular function of this glycosylation remains largely unknown. The ß-linked glucuronic acid (GlcA) residues in AG polysaccharides have been shown in vitro to bind to calcium in a pH-dependent manner. Here, we used Arabidopsis (Arabidopsis thaliana) mutants in four AG ß-glucuronyltransferases (GlcAT14A, -B, -D, and -E) to understand the role of glucuronidation of AG. AG isolated from glcat14 triple mutants had a strong reduction in glucuronidation. AG from a glcat14a/b/d triple mutant had lower calcium binding capacity in vitro than AG from wild-type plants. Some mutants had multiple developmental defects such as reduced trichome branching. glcat14a/b/e triple mutant plants had severely limited seedling growth and were sterile, and the propagation of calcium waves was perturbed in roots. Several of the developmental phenotypes were suppressed by increasing the calcium concentration in the growth medium. Our results show that AG glucuronidation is crucial for multiple developmental processes in plants and suggest that a function of AGPs might be to bind and release cell-surface apoplastic calcium.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Cálcio/metabolismo , Galactanos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Pleiotropia Genética , Glucuronídeos/metabolismo , Mutação , Filogenia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismoRESUMO
The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first Förster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.
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Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Giberelinas/metabolismo , Arabidopsis/metabolismo , Perfusão , Raízes de Plantas/metabolismo , Transdução de SinaisRESUMO
The gibberellin phytohormones regulate growth and development throughout the plant lifecycle. Upstream regulation and downstream responses to gibberellins vary across cells and tissues, developmental stages, environmental conditions, and plant species. The spatiotemporal distribution of gibberellins is the result of an ensemble of biosynthetic, catabolic and transport activities, each of which can be targeted to influence gibberellin levels in space and time. Understanding gibberellin distributions has recently benefited from discovery of transport proteins capable of importing gibberellins as well as novel methods for detecting gibberellins with high spatiotemporal resolution. For example, a genetically-encoded fluorescent biosensor for gibberellins was deployed in Arabidopsis and revealed gibberellin gradients in rapidly elongating tissues. Although cellular accumulations of gibberellins are hypothesized to regulate cell growth in developing embryos, germinating seeds, elongating stems and roots, and developing floral organs, understanding the quantitative relationship between cellular gibberellin levels and cellular growth awaits further investigation. It is also unclear how spatiotemporal gibberellin distributions result from myriad endogenous and environmental factors directing an ensemble of known gibberellin enzymatic and transport steps.
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Giberelinas/metabolismo , Desenvolvimento Vegetal , Transporte Biológico , Giberelinas/biossíntese , Modelos Biológicos , Células Vegetais/metabolismo , Fatores de TempoRESUMO
The phytohormone gibberellin (GA) is a key regulator of plant growth and development. Although the upstream regulation and downstream responses to GA vary across cells and tissues, developmental stages and environmental conditions, the spatiotemporal distribution of GA in vivo remains unclear. Using a combinatorial screen in yeast, we engineered an optogenetic biosensor, GIBBERELLIN PERCEPTION SENSOR 1 (GPS1), that senses nanomolar levels of bioactive GAs. Arabidopsis thaliana plants expressing a nuclear localized GPS1 report on GAs at the cellular level. GA gradients were correlated with gradients of cell length in rapidly elongating roots and dark-grown hypocotyls. In roots, accumulation of exogenously applied GA also correlated with cell length, intimating that a root GA gradient can be established independently of GA biosynthesis. In hypocotyls, GA levels were reduced in a phytochrome interacting factor (pif) quadruple mutant in the dark and increased in a phytochrome double mutant in the light, indicating that PIFs elevate GA in the dark and that phytochrome inhibition of PIFs could lower GA in the light. As GA signalling directs hypocotyl elongation largely through promoting PIF activity, PIF promotion of GA accumulation represents a positive feedback loop within the molecular framework driving rapid hypocotyl growth.
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Arabidopsis/metabolismo , Técnicas Biossensoriais , Giberelinas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células Vegetais/metabolismo , Receptores de Superfície Celular/metabolismo , Distribuição TecidualRESUMO
Light regulates Arabidopsis seed germination through the phyB/PIL5 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 5) transduction pathway, and we have previously shown that the Dof transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a component of this pathway. By means of microarray analysis of dag1 and wild type developing siliques, we identified the EARLY LIGHT-INDUCED PROTEIN1 and 2 (ELIP1 and ELIP2) genes among those deregulated in the loss-of-function dag1 mutant. We analysed seed germination of elip single and double mutants, of elip dag1 double mutants as well as of elip1 elip2 dag1 triple mutant under different environmental conditions. We show that ELIP1 and ELIP2 are involved in opposite ways in the control of this developmental process, in particular under abiotic (light, temperature, salt) stress conditions.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Germinação/efeitos da radiação , Sementes/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Luz , Análise em Microsséries , Mutação , Sementes/metabolismo , Sementes/efeitos da radiação , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We have previously shown that inactivation of the gene encoding the Arabidopsis thaliana transcription factor DOF AFFECTING GERMINATION 1 (DAG1) renders seed germination more sensitive to both phytochrome B (phyB) and gibberellins (GA). dag1 mutant seeds require less red (R) light fluence and a lower GA concentration than WT to germinate. Here, we show that inactivation of the gene PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIL5) results in down-regulation of DAG1. Inactivation of PIL5 in the dag1 mutant background further increased the germination potential of dag1 mutant seeds, supporting the suggestion that DAG1 is under the positive control of PIL5. Germination of dag1phyB seeds showed a reduced requirement of gibberellins as compared with phyB mutant seeds, both in the presence and in the absence of GA biosynthesis. Furthermore, the GA biosynthetic gene AtGA3ox1 is upregulated in dag1 seeds as compared with the WT, and DAG1 actually binds to the AtGA3ox1 promoter, as shown by chromatin immunoprecipitation experiments. Expression analysis at different time points confirms that AtGA3ox1 is directly regulated by DAG1, while suggesting that DAG1 is not a direct regulatory target of PIL5. Our data indicate that in the phyB pathway leading to seed germination, DAG1 negatively regulates GA biosynthesis and suggest that DAG1 acts downstream of PIL5. In addition, the analysis of hypocotyls of dag1 and phyB mutant plantlets, of plantlets overexpressing phyB in the dag1 mutant, as well as of dag1phyB double mutant suggests that DAG1 may act as a negative regulatory element downstream of phyB also in hypocotyl elongation.