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1.
Nat Commun ; 15(1): 3895, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38719832

ABSTRACT

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.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Biosensing Techniques , Gene Expression Regulation, Plant , Gibberellins , Meristem , Signal Transduction , Gibberellins/metabolism , Meristem/metabolism , Meristem/growth & development , Arabidopsis/metabolism , Arabidopsis/growth & development , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Plant Growth Regulators/metabolism , Plant Shoots/metabolism , Plant Shoots/growth & development , Plants, Genetically Modified
2.
Nat Plants ; 9(4): 572-587, 2023 04.
Article in English | MEDLINE | ID: mdl-36973414

ABSTRACT

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.


Subject(s)
Arabidopsis , Clustered Regularly Interspaced Short Palindromic Repeats , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Arabidopsis/genetics , Plant Breeding , Plants/genetics , Genome, Plant , CRISPR-Cas Systems , Plants, Genetically Modified/genetics , Gene Editing
3.
Curr Opin Plant Biol ; 65: 102111, 2022 02.
Article in English | MEDLINE | ID: mdl-34543915

ABSTRACT

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.


Subject(s)
Magnoliopsida , Meristem , Plant Development , Plant Growth Regulators , Plant Shoots , Stem Cells
4.
Methods Mol Biol ; 2094: 59-65, 2020.
Article in English | MEDLINE | ID: mdl-31797291

ABSTRACT

Axillary meristems (AMs) are established postembryonically at the leaf axils and can develop into lateral branches. The initiation of AMs establishes new growth axis and is of primary importance for understanding plant development. Understanding plant development requires live imaging of morphogenesis and gene expression. However, AMs are embedded in the leaf axil, making it challenging to perform live imaging. In this chapter, we describe how to prepare and culture Arabidopsis thaliana leaves in vitro, to perform one-time or time-lapse imaging of AM initiation with a confocal microscope.


Subject(s)
Arabidopsis/metabolism , Cell Culture Techniques/methods , Meristem/metabolism , Microscopy, Confocal/methods , Cells, Cultured , Microscopy, Confocal/instrumentation , Plant Leaves/metabolism
5.
Curr Top Dev Biol ; 131: 81-107, 2019.
Article in English | MEDLINE | ID: mdl-30612633

ABSTRACT

The shoot apical meristem (SAM) generates all above-ground organs throughout the life of plants. The development and maintenance of the SAM are crucial for building the plant architecture. The spatiotemporal patterning of lateral organs (leaves and flowers), called phyllotaxis, is one of the best-characterized self-organizing systems and has long been proposed to be driven by inhibitory fields generated by the existing organs and blocking new initiations in their vicinity. Recent years have seen impressive progress in our understanding of the molecular mechanisms controlling SAM function, and on how these mechanisms act in phyllotactic patterning. In this chapter, we first review the regulation of SAM stem cell activity and discuss how feedback signals coming from the differentiated organs affect stem cell homeostasis. Then we highlight experimental and theoretical works that have revealed the chemical and biophysical factors acting in the regulation of phyllotaxis. Finally, we summarize the important roles of SAM geometry in phyllotaxis.


Subject(s)
Flowers/growth & development , Meristem/growth & development , Plant Development , Plant Leaves/growth & development , Plant Shoots/growth & development , Plants
6.
Nat Commun ; 10(1): 141, 2019 01 11.
Article in English | MEDLINE | ID: mdl-30635575

ABSTRACT

Gene regulatory networks control development via domain-specific gene expression. In seed plants, self-renewing stem cells located in the shoot apical meristem (SAM) produce leaves from the SAM peripheral zone. After initiation, leaves develop polarity patterns to form a planar shape. Here we compare translating RNAs among SAM and leaf domains. Using translating ribosome affinity purification and RNA sequencing to quantify gene expression in target domains, we generate a domain-specific translatome map covering representative vegetative stage SAM and leaf domains. We discuss the predicted cellular functions of these domains and provide evidence that dome seemingly unrelated domains, utilize common regulatory modules. Experimental follow up shows that the RABBIT EARS and HANABA TARANU transcription factors have roles in axillary meristem initiation. This dataset provides a community resource for further study of shoot development and response to internal and environmental signals.


Subject(s)
Arabidopsis/embryology , Arabidopsis/genetics , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Plant/genetics , Gene Regulatory Networks/genetics , Meristem/growth & development , Alternative Splicing , Arabidopsis Proteins/genetics , Base Sequence , GATA Transcription Factors/genetics , Gene Expression Profiling , Homeodomain Proteins/genetics , Meristem/genetics , Plant Leaves/growth & development , Repressor Proteins/genetics , Sequence Analysis, RNA , Transcription Factors/genetics
7.
Dev Cell ; 44(2): 204-216.e6, 2018 01 22.
Article in English | MEDLINE | ID: mdl-29401419

ABSTRACT

Stem cells must balance self-renewal and differentiation; thus, their activities are precisely controlled. In plants, the control circuits that underlie division and differentiation within meristems have been well studied, but those that underlie feedback on meristems from lateral organs remain largely unknown. Here we show that long-distance auxin transport mediates this feedback in a non-cell-autonomous manner. A low-auxin zone is associated with the shoot apical meristem (SAM) organization center, and auxin levels negatively affect SAM size. Using computational model simulations, we show that auxin transport from lateral organs can inhibit auxin transport from the SAM through an auxin transport switch and thus maintain SAM auxin homeostasis and SAM size. Genetic and microsurgical analyses confirmed the model's predictions. In addition, the model explains temporary change in SAM size of yabby mutants. Our study suggests that the canalization-based auxin flux can be widely adapted as a feedback control mechanism in plants.


Subject(s)
Indoleacetic Acids/metabolism , Meristem/growth & development , Plant Shoots/growth & development , Biological Transport , Feedback, Physiological , Genes, Plant , Meristem/anatomy & histology , Meristem/metabolism , Mutation , Plant Shoots/anatomy & histology , Plant Shoots/metabolism , Stem Cells/cytology , Stem Cells/metabolism
8.
Plant Cell ; 29(6): 1373-1387, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28576845

ABSTRACT

The homeodomain transcription factor WUSCHEL (WUS) defines the shoot stem cell niche, but the mechanisms underlying the establishment of WUS expression remain unclear. Here, we show that cytokinin signaling precedes WUS expression in leaf axils and activates WUS expression de novo in the leaf axil to promote axillary meristem initiation. Furthermore, type-B Arabidopsis response regulator proteins, which are transcriptional activators in the cytokinin signaling pathway, directly bind to the WUS promoter and activate its expression. Finally, we show that cytokinin activation of WUS in the leaf axil correlates with increased histone acetylation and methylation markers associated with transcriptional activation, supporting the fact that WUS expression requires a permissive epigenetic environment to restrict it to highly defined meristematic tissues. Taken together, these findings explain how cytokinin regulates axillary meristem initiation and establish a mechanistic framework for the postembryonic establishment of the shoot stem cell niche.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cytokinins/metabolism , Meristem/metabolism , Acetylation , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cytokinins/genetics , Gene Expression Regulation, Plant , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Meristem/cytology , Meristem/genetics , Signal Transduction
9.
PLoS Genet ; 12(7): e1006168, 2016 07.
Article in English | MEDLINE | ID: mdl-27398935

ABSTRACT

Shoot branching requires the establishment of new meristems harboring stem cells; this phenomenon raises questions about the precise regulation of meristematic fate. In seed plants, these new meristems initiate in leaf axils to enable lateral shoot branching. Using live-cell imaging of leaf axil cells, we show that the initiation of axillary meristems requires a meristematic cell population continuously expressing the meristem marker SHOOT MERISTEMLESS (STM). The maintenance of STM expression depends on the leaf axil auxin minimum. Ectopic expression of STM is insufficient to activate axillary buds formation from plants that have lost leaf axil STM expressing cells. This suggests that some cells undergo irreversible commitment to a developmental fate. In more mature leaves, REVOLUTA (REV) directly up-regulates STM expression in leaf axil meristematic cells, but not in differentiated cells, to establish axillary meristems. Cell type-specific binding of REV to the STM region correlates with epigenetic modifications. Our data favor a threshold model for axillary meristem initiation, in which low levels of STM maintain meristematic competence and high levels of STM lead to meristem initiation.


Subject(s)
Arabidopsis/genetics , Gene Expression Regulation, Plant , Meristem/cytology , Plant Shoots/growth & development , Alleles , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Cell Differentiation , Cell Lineage , Chromatin Immunoprecipitation , Genes, Plant , Genotype , Indoleacetic Acids/metabolism , Phenotype , Plant Leaves/growth & development , Plants, Genetically Modified/growth & development , Up-Regulation
10.
Mol Syst Biol ; 10: 755, 2014 Oct 30.
Article in English | MEDLINE | ID: mdl-25358340

ABSTRACT

Gene regulatory networks (GRNs) control development via cell type-specific gene expression and interactions between transcription factors (TFs) and regulatory promoter regions. Plant organ boundaries separate lateral organs from the apical meristem and harbor axillary meristems (AMs). AMs, as stem cell niches, make the shoot a ramifying system. Although AMs have important functions in plant development, our knowledge of organ boundary and AM formation remains rudimentary. Here, we generated a cellular-resolution genomewide gene expression map for low-abundance Arabidopsis thaliana organ boundary cells and constructed a genomewide protein-DNA interaction map focusing on genes affecting boundary and AM formation. The resulting GRN uncovers transcriptional signatures, predicts cellular functions, and identifies promoter hub regions that are bound by many TFs. Importantly, further experimental studies determined the regulatory effects of many TFs on their targets, identifying regulators and regulatory relationships in AM initiation. This systems biology approach thus enhances our understanding of a key developmental process.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Gene Regulatory Networks , Meristem/genetics , Arabidopsis Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation, Plant , Genome, Plant , Models, Genetic , Promoter Regions, Genetic , Transcription Factors/metabolism
11.
Plant J ; 80(4): 629-41, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25187180

ABSTRACT

In Arabidopsis, AUXIN RESPONSE FACTOR 3 (ARF3) belongs to the auxin response factor (ARF) family that regulates the expression of auxin-responsive genes. ARF3 is known to function in leaf polarity specification and gynoecium patterning. In this study, we discovered a previously unknown role for ARF3 in floral meristem (FM) determinacy through the isolation and characterization of a mutant of ARF3 that enhanced the FM determinacy defects of agamous (ag)-10, a weak ag allele. Central players in FM determinacy include WUSCHEL (WUS), a gene critical for FM maintenance, and AG and APETALA2 (AP2), which regulate FM determinacy by repression and promotion of WUS expression, respectively. We showed that ARF3 confers FM determinacy through repression of WUS expression, and associates with the WUS locus in part in an AG-dependent manner. We demonstrated that ARF3 is a direct target of AP2 and partially mediates AP2's function in FM determinacy. ARF3 exhibits dynamic and complex expression patterns in floral organ primordia; altering the patterns spatially compromised FM determinacy. This study uncovered a role for ARF3 in FM determinacy and revealed relationships among genes in the genetic network governing FM determinacy.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Meristem/metabolism , Nuclear Proteins/metabolism , AGAMOUS Protein, Arabidopsis/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , Flowers/genetics , Flowers/metabolism , Gene Expression Regulation, Plant , Gene Regulatory Networks , Homeodomain Proteins/genetics , Meristem/genetics , Mutation , Nuclear Proteins/genetics , Plants, Genetically Modified
12.
Plant Cell ; 26(5): 2055-2067, 2014 May.
Article in English | MEDLINE | ID: mdl-24850849

ABSTRACT

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

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