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1.
Plant Commun ; 5(6): 100848, 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38379284

ABSTRACT

The phytohormone ethylene is a major regulator of plant adaptive responses to flooding. In flooded plant tissues, ethylene quickly increases to high concentrations owing to its low solubility and diffusion rates in water. Ethylene accumulation in submerged plant tissues makes it a reliable cue for triggering flood acclimation responses, including metabolic adjustments to cope with flood-induced hypoxia. However, persistent ethylene accumulation also accelerates leaf senescence. Stress-induced senescence hampers photosynthetic capacity and stress recovery. In submerged Arabidopsis, senescence follows a strict age-dependent pattern starting with the older leaves. Although mechanisms underlying ethylene-mediated senescence have been uncovered, it is unclear how submerged plants avoid indiscriminate breakdown of leaves despite high systemic ethylene accumulation. We demonstrate that although submergence triggers leaf-age-independent activation of ethylene signaling via EIN3 in Arabidopsis, senescence is initiated only in old leaves. EIN3 stabilization also leads to overall transcript and protein accumulation of the senescence-promoting transcription factor ORESARA1 (ORE1) in both old and young leaves during submergence. However, leaf-age-dependent senescence can be explained by ORE1 protein activation via phosphorylation specifically in old leaves, independent of the previously identified age-dependent control of ORE1 via miR164. A systematic analysis of the roles of the major flooding stress cues and signaling pathways shows that only the combination of ethylene and darkness is sufficient to mimic submergence-induced senescence involving ORE1 accumulation and phosphorylation. Hypoxia, most often associated with flooding stress in plants, appears to have no role in these processes. Our results reveal a mechanism by which plants regulate the speed and pattern of senescence during environmental stresses such as flooding. Age-dependent ORE1 activity ensures that older, expendable leaves are dismantled first, thus prolonging the life of younger leaves and meristematic tissues that are vital to whole-plant survival.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Ethylenes , Floods , Plant Leaves , Signal Transduction , Transcription Factors , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/physiology , Ethylenes/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Plant Leaves/metabolism , Plant Leaves/genetics , Phosphorylation , Transcription Factors/metabolism , Transcription Factors/genetics , Plant Senescence/genetics , Gene Expression Regulation, Plant
2.
Curr Biol ; 33(1): 75-85.e5, 2023 01 09.
Article in English | MEDLINE | ID: mdl-36538931

ABSTRACT

Although plants are immobile, many of their organs are flexible to move in response to environmental cues. In dense vegetation, plants detect neighbors through far-red light perception with their leaf tip. They respond remotely, with asymmetrical growth between the abaxial and adaxial sides of the leafstalk, the petiole. This results in upward movement that brings the leaf blades into better lit zones of the canopy. The plant hormone auxin is required for this response, but it is not understood how non-differential leaf tip-derived auxin can remotely regulate movement. Here, we show that remote signaling of far-red light promotes auxin accumulation in the abaxial petiole. This local auxin accumulation is facilitated by reinforcing an intrinsic directionality of the auxin transport protein PIN3 on the petiole endodermis, as visualized with a PIN3-GFP line. Using an auxin biosensor, we show that auxin accumulates in all cell layers from endodermis to epidermis in the abaxial petiole, upon far-red light signaling in the remote leaf tip. In the petiole, auxin elicits a response to both auxin itself as well as a second growth promoter; gibberellin. We show that this dual regulation is necessary for hyponastic leaf movement in response to light. Our data indicate that gibberellin is required to permit cell growth, whereas differential auxin accumulation determines which cells can grow. Our results reveal how plants can spatially relay information about neighbor proximity from their sensory leaf tips to the petiole base, thus driving adaptive growth.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Indoleacetic Acids/metabolism , Gibberellins/metabolism , Arabidopsis/metabolism , Plant Growth Regulators/metabolism , Light , Plant Leaves , Arabidopsis Proteins/metabolism
3.
New Phytol ; 235(5): 1884-1899, 2022 09.
Article in English | MEDLINE | ID: mdl-35612785

ABSTRACT

Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA-seq data and co-expression analysis were used for SL biosynthetic gene discovery. This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro-orobanchol (DDH) isomers. Virus-induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy-orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers. Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.


Subject(s)
Solanum lycopersicum , Catalysis , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Heterocyclic Compounds, 3-Ring , Lactones/metabolism , Solanum lycopersicum/genetics , Solanum lycopersicum/metabolism , Phylogeny , Plant Growth Regulators/metabolism , Rhizosphere , Saccharomyces cerevisiae/metabolism , Nicotiana/genetics , Nicotiana/metabolism
4.
Curr Biol ; 31(22): 4946-4955.e4, 2021 11 22.
Article in English | MEDLINE | ID: mdl-34610273

ABSTRACT

Premitotic control of cell division orientation is critical for plant development, as cell walls prevent extensive cell remodeling or migration. While many divisions are proliferative and add cells to existing tissues, some divisions are formative and generate new tissue layers or growth axes. Such formative divisions are often asymmetric in nature, producing daughters with different fates. We have previously shown that, in the Arabidopsis thaliana embryo, developmental asymmetry is correlated with geometric asymmetry, creating daughter cells of unequal volume. Such divisions are generated by division planes that deviate from a default "minimal surface area" rule. Inhibition of auxin response leads to reversal to this default, yet the mechanisms underlying division plane choice in the embryo have been unclear. Here, we show that auxin-dependent division plane control involves alterations in cell geometry, but not in cell polarity axis or nuclear position. Through transcriptome profiling, we find that auxin regulates genes controlling cell wall and cytoskeleton properties. We confirm the involvement of microtubule (MT)-binding proteins in embryo division control. Organization of both MT and actin cytoskeleton depends on auxin response, and genetically controlled MT or actin depolymerization in embryos leads to disruption of asymmetric divisions, including reversion to the default. Our work shows how auxin-dependent control of MT and actin cytoskeleton properties interacts with cell geometry to generate asymmetric divisions during the earliest steps in plant development.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Shape/physiology , Cytoskeleton/metabolism , Indoleacetic Acids/metabolism , Microtubules/metabolism
5.
Cell ; 180(3): 427-439.e12, 2020 02 06.
Article in English | MEDLINE | ID: mdl-32004461

ABSTRACT

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.


Subject(s)
Arabidopsis/chemistry , Arabidopsis/cytology , Cell Polarity/physiology , Plant Cells/physiology , Polymerization , Protein Domains , Animals , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Axin Protein/chemistry , Axin Protein/metabolism , Bryopsida/chemistry , Bryopsida/cytology , Bryopsida/genetics , Bryopsida/growth & development , COS Cells , Chlorocebus aethiops , Dishevelled Proteins/metabolism , HEK293 Cells , Humans , Marchantia/chemistry , Marchantia/cytology , Marchantia/genetics , Marchantia/growth & development , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Plants, Genetically Modified , Repressor Proteins/metabolism , Wnt Signaling Pathway
6.
Methods Mol Biol ; 2122: 49-61, 2020.
Article in English | MEDLINE | ID: mdl-31975295

ABSTRACT

Virtually all growth, developmental, physiological, and defense responses in plants are accompanied by reorganization of subcellular structures to enable altered cellular growth, differentiation or function. Visualizing cellular reorganization is therefore critical to understand plant biology at the cellular scale. Fluorescently labeled markers for organelles, or for cellular components are widely used in combination with confocal microscopy to visualize cellular reorganization. Early during plant embryogenesis, the precursors for all major tissues of the seedling are established, and in Arabidopsis, this entails a set of nearly invariant switches in cell division orientation and directional cell expansion. Given that these cellular reorganization events are genetically regulated and coupled to formative events in plant development, they offer a good model to understand the genetic control of cellular reorganization in plant development. Until recently, it has been challenging to visualize subcellular structures in the early Arabidopsis embryo for two reasons: embryos are deeply embedded in seed coat and fruit, and in addition, no dedicated fluorescent markers, expressed in the embryo, were available. We recently established both an imaging approach and a set of markers for the early Arabidopsis embryo. Here, we describe a detailed protocol to use these new tools in imaging cellular reorganization.


Subject(s)
Arabidopsis/embryology , Fluorescent Dyes/analysis , Microscopy, Confocal/methods , Seeds/embryology , Arabidopsis/cytology , Arabidopsis/ultrastructure , Microscopy, Confocal/instrumentation , Seeds/cytology , Seeds/ultrastructure
7.
Plant Cell ; 31(1): 52-67, 2019 01.
Article in English | MEDLINE | ID: mdl-30573473

ABSTRACT

Land plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how this process is modified during nonzygotic embryogenesis. The Arabidopsis (Arabidopsis thaliana) zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We found that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to create resilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.


Subject(s)
Arabidopsis Proteins/metabolism , Indoleacetic Acids/metabolism , Plants, Genetically Modified/metabolism , Seeds/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Plants, Genetically Modified/genetics , Seeds/genetics , Signal Transduction/genetics , Signal Transduction/physiology
8.
Curr Biol ; 28(19): 3031-3043.e2, 2018 10 08.
Article in English | MEDLINE | ID: mdl-30245102

ABSTRACT

Oriented cell divisions are significant in plant morphogenesis because plant cells are embedded in cell walls and cannot relocate. Cell divisions follow various regular orientations, but the underlying mechanisms have not been clarified. We propose that cell-shape-dependent self-organization of cortical microtubule arrays is able to provide a mechanism for determining planes of early tissue-generating divisions and may form the basis for robust control of cell division orientation in the embryo. To show this, we simulate microtubules on actual cell surface shapes, from which we derive a minimal set of three rules for proper array orientation. The first rule captures the effects of cell shape alone on microtubule organization, the second rule describes the regulation of microtubule stability at cell edges, and the third rule includes the differential effect of auxin on local microtubule stability. These rules generate early embryonic division plane orientations and potentially offer a framework for understanding patterned cell divisions in plant morphogenesis.


Subject(s)
Cell Division/physiology , Microtubules/physiology , Seeds/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cell Shape/physiology , Computer Simulation , Embryonic Development , Indoleacetic Acids/metabolism , Meristem/metabolism , Orientation, Spatial , Plant Cells/physiology , Plant Development , Plant Roots/metabolism
9.
Plant J ; 93(6): 963-976, 2018 03.
Article in English | MEDLINE | ID: mdl-29383853

ABSTRACT

Considerable progress has been made in understanding the influence of physical and genetic factors on the patterns of cell division in various model systems. However, how each of these factors directs changes in subcellular structures has remained unclear. Generic machineries for the execution of cell expansion and division have been characterized, but how these are influenced by genetic regulators and physical cell properties remains an open question. To a large degree, the complexity of growing post-embryonic tissues and a lack of precise predictability have prevented the extraction of rigid correlations between subcellular structures and future orientation of cell division. The Arabidopsis embryo offers an exquisitely predictable and simple model for studying such correlations, but so far the tools and methodology for studying subcellular structures in the early embryo have been lacking. Here, we describe a set of markers to visualize a range of subcellular structures in the early Arabidopsis embryo. We have designed a series of fluorescent cellular reporters optimized for embryos, and demonstrate the effectiveness of using these 'ACE' reporters with simple three-dimensional imaging procedures that preserve delicate cellular structures. We describe the ontogeny of subcellular structures in the early embryo and find that central/peripheral cell polarity is established much earlier than suspected. In addition, we show that the actin and microtubule cytoskeleton has distinct topologies in the embryo. These tools and methods will allow detailed analysis of the events of cellular reorganization that underlie morphogenesis in the Arabidopsis embryo.


Subject(s)
Arabidopsis/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Seeds/genetics , Arabidopsis/cytology , Arabidopsis/embryology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Division/genetics , Microscopy, Confocal , Microtubules/metabolism , Morphogenesis/genetics , Organelles/metabolism , Plants, Genetically Modified , Seeds/cytology , Seeds/embryology
10.
Sci Rep ; 7: 42664, 2017 03 03.
Article in English | MEDLINE | ID: mdl-28256503

ABSTRACT

Root and shoot branches are major determinants of plant form and critical for the effective capture of resources below and above ground. These branches are often maintained at specific angles with respect to gravity, known as gravitropic set point angles (GSAs). We have previously shown that the mechanism permitting the maintenance of non-vertical GSAs is highly auxin-dependent and here we investigate the developmental and environmental regulation of root and shoot branch GSA. We show that nitrogen and phosphorous deficiency have opposing, auxin signalling-dependent effects on lateral root GSA in Arabidopsis: while low nitrate induces less vertical lateral root GSA, phosphate deficiency results in a more vertical lateral root growth angle, a finding that contrasts with the previously reported growth angle response of bean adventitious roots. We find that this root-class-specific discrepancy in GSA response to low phosphorus is mirrored by similar differences in growth angle response to auxin treatment between these root types. Finally we show that both shaded, low red/far-red light conditions and high temperature induce more vertical growth in Arabidopsis shoot branches. We discuss the significance of these findings in the context of efforts to improve crop performance via the manipulation of root and shoot branch growth angle.


Subject(s)
Arabidopsis/physiology , Environment , Gravitropism , Plant Development , Indoleacetic Acids/metabolism , Indoleacetic Acids/pharmacology , Light , Nitrates/metabolism , Phosphates/metabolism , Plant Roots/physiology , Temperature
11.
Methods Mol Biol ; 1284: 421-31, 2015.
Article in English | MEDLINE | ID: mdl-25757785

ABSTRACT

Molecular cloning is a vital step in much of today's plant biological research. Particularly, when a species is amenable to transgenic manipulation, cloning enables detailed study of gene and protein function in vivo. Therefore, accurate, consistent, and efficient cloning methods have the potential to accelerate biological research. Traditional restriction-enzyme/ligase-based strategies are often inefficient, while novel alternative methods can be less economical. We have recently optimized a method for Ligation-Independent Cloning (LIC) that is both efficient and economical. We have developed a large set of LIC-compatible plasmids for application in plant research. These include dedicated vectors for gene expression analysis, protein localization studies, and protein misexpression. We describe a detailed protocol that allows the reliable generation of plant transformation-ready constructs from PCR fragments in 2-3 days.


Subject(s)
Cloning, Molecular/methods , Plants/genetics , Genetic Vectors/genetics , Plasmids/genetics , Polymerase Chain Reaction/methods
12.
Nat Methods ; 12(3): 207-10, 2 p following 210, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25643149

ABSTRACT

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.


Subject(s)
Arabidopsis/genetics , Genes, Reporter , Indoleacetic Acids/metabolism , Response Elements/genetics , Arabidopsis/drug effects , Arabidopsis/growth & development , Gene Expression Regulation, Plant , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Indoleacetic Acids/pharmacology , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Imaging/methods , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified , Promoter Regions, Genetic , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Signal Transduction/genetics
13.
Curr Opin Plant Biol ; 23: 25-30, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25449723

ABSTRACT

Multicellular plant development requires strict control of cell division orientation. A key unanswered question is how developmental regulators interact with the generic cell division machinery to trigger oriented divisions. We discuss the Arabidopsis embryo as a model for addressing this question. Recent progress in 3D imaging and computation now allows sketching of a framework for the developmental control of division orientation in which the signaling molecule auxin controls oriented division by preventing a geometrically defined default plane. We expect that the identification of auxin effectors, together with the identification of novel regulators of cell division will help to link developmental regulators to the division machinery.


Subject(s)
Arabidopsis/cytology , Arabidopsis/embryology , Cell Division , Seeds/cytology , Cell Shape , Models, Biological , Plant Development
14.
Plant Physiol ; 156(3): 1292-9, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21562332

ABSTRACT

With plant molecular biology entering the omics era, there is a need for simple cloning strategies that allow high throughput to systematically study the expression and function of large numbers of genes. Such strategies would facilitate the analysis of gene (sub)families and/or sets of coexpressed genes identified by transcriptomics. Here, we provide a set of 34 ligation-independent cloning (LIC) binary vectors for expression analysis, protein localization studies, and misexpression that will be made freely available. This set of plant LIC vectors offers a fast alternative to standard cloning strategies involving ligase or recombination enzyme technology. We demonstrate the use of this strategy and our new vectors by analyzing the expression domains of genes belonging to two subclades of the basic helix-loop-helix transcription factor family. We show that neither the closest homologs of TARGET OF MONOPTEROS7 (TMO7/ATBS1) nor the members of the ATBS1 INTERACTING FACTOR subclade of putative TMO7 interactors are expressed in the embryo and that there is very limited coexpression in the primary root meristem. This suggests that these basic helix-loop-helix transcription factors are most likely not involved in TMO7-dependent root meristem initiation.


Subject(s)
Arabidopsis/genetics , Cloning, Molecular/methods , Genetic Vectors/genetics , Base Sequence , Gene Expression Regulation, Plant , Molecular Sequence Data , Promoter Regions, Genetic/genetics
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