ABSTRACT
How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.
Subject(s)
Arabidopsis/growth & development , Cardamine/growth & development , Plant Leaves/growth & development , Arabidopsis/genetics , Cardamine/genetics , Cell Lineage/genetics , Computational Biology/methods , Gene Expression Regulation, Plant/genetics , Plant Leaves/genetics , Plant Proteins/metabolismABSTRACT
How the interplay between cell- and tissue-level processes produces correctly proportioned organs is a key problem in biology. In plants, the relative size of leaves compared with their lateral appendages, called stipules, varies tremendously throughout development and evolution, yet relevant mechanisms remain unknown. Here we use genetics, live imaging, and modeling to show that in Arabidopsis leaves, the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth. LM1 acts through directly activating the conserved mitosis blocker WEE1, which is sufficient to bypass the LMI1 requirement for leaf proportionality.
Subject(s)
Arabidopsis Proteins/physiology , Endoreduplication , Homeodomain Proteins/physiology , Transcription Factors/physiology , Arabidopsis/anatomy & histology , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Plant Leaves/anatomy & histology , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Two interrelated problems in biology are understanding the regulatory logic and predictability of morphological evolution. Here, we studied these problems by comparing Arabidopsis thaliana, which has simple leaves, and its relative, Cardamine hirsuta, which has dissected leaves comprising leaflets. By transferring genes between the two species, we provide evidence for an inverse relationship between the pleiotropy of SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) homeobox genes and their ability to modify leaf form. We further show that cis-regulatory divergence of BP results in two alternative configurations of the genetic networks controlling leaf development. In C. hirsuta, ChBP is repressed by the microRNA164A (MIR164A)/ChCUP-SHAPED COTYLEDON (ChCUC) module and ChASYMMETRIC LEAVES1 (ChAS1), thus creating cross-talk between MIR164A/CUC and AS1 that does not occur in A. thaliana. These different genetic architectures lead to divergent interactions of network components and growth regulation in each species. We suggest that certain regulatory genes with low pleiotropy are predisposed to readily integrate into or disengage from conserved genetic networks influencing organ geometry, thus rapidly altering their properties and contributing to morphological divergence.
Subject(s)
Arabidopsis/growth & development , Arabidopsis/genetics , Cardamine/growth & development , Cardamine/genetics , Gene Regulatory Networks/genetics , Homeodomain Proteins/genetics , Plant Leaves , Plant Proteins/genetics , Arabidopsis/anatomy & histology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cardamine/anatomy & histology , Gene Expression Regulation, Plant , Homeodomain Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Plant Leaves/anatomy & histology , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Proteins/metabolismABSTRACT
As an essential nutrient, copper (Cu) scarcity causes a decrease in agricultural production. Cu deficiency responses include the induction of several microRNAs, known as Cu-miRNAs, which are responsible for degrading mRNAs from abundant and dispensable cuproproteins to economize copper when scarce. Cu-miRNAs, such as miR398 and miR408 are conserved, as well as the signal transduction pathway to induce them under Cu deficiency. The Arabidopsis thaliana SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family member SPL7 binds to the cis-regulatory motifs present in the promoter regions of genes expressed under Cu deficiency, including Cu-miRNAs. The expression of several other SPL transcription factor family members is regulated by miR156. This regulatory miR156-SPL module plays a crucial role in developmental phase transitions while integrating internal and external cues. Here, we show that Cu deficiency also affects miR156 expression and that SPL3 overexpressing plants, resistant to miR156 regulation, show a severe decrease in SPL7-mediated Cu deficiency responses. These include the expression of Cu-miRNAs and their targets and is probably due to competition between SPL7 and miR156-regulated SPL3 in binding to cis-regulatory elements in Cu-miRNA promoters. Thus, the conserved SPL7-mediated Cu-miRNA pathway could generally be affected by the miR156-SPL module, thereby underscoring the integration of the Cu-miRNA pathway with developmental and environmental stress responses in Arabidopsis thaliana.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Copper/metabolism , DNA-Binding Proteins/metabolism , MicroRNAs/metabolism , Transcription Factors/metabolism , Arabidopsis/growth & development , Plant Development , Stress, PhysiologicalABSTRACT
A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, by using plant leaves as an example, we show that the causes of diversity at these two evolutionary scales can be divergent. Some species like the model plant Arabidopsis thaliana have simple leaves, whereas others like the A. thaliana relative Cardamine hirsuta bear complex leaves comprising leaflets. Previous work has shown that these interspecific differences result mostly from variation in local tissue growth and patterning. Now, by cloning and characterizing a quantitative trait locus (QTL) for C. hirsuta leaf shape, we find that a different process, age-dependent progression of leaf form, underlies variation in this trait within species. This QTL effect is caused by cis-regulatory variation in the floral repressor ChFLC, such that genotypes with low-expressing ChFLC alleles show both early flowering and accelerated age-dependent changes in leaf form, including faster leaflet production. We provide evidence that this mechanism coordinates leaf development with reproductive timing and may help to optimize resource allocation to the next generation.
Subject(s)
Cardamine/genetics , Plant Leaves/anatomy & histology , Quantitative Trait Loci , Alleles , Arabidopsis , Base Sequence , Biodiversity , Chromosome Mapping , Cloning, Molecular , Flowers , Gene Expression Regulation, Plant , Genotype , Light , Models, Genetic , Molecular Sequence Data , Phenotype , Plants, Genetically Modified , Polymorphism, Genetic , Seeds , Sequence Homology, Nucleic AcidABSTRACT
Copper homeostasis under deficiency is regulated by the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) transcription factor. The daily oscillating expression of two SPL7-dependent copper deficiency markers, COPPER TRANSPORTER (COPT2) and IRON SUPEROXIDE DISMUTASE (FSD1), has been followed by quantitative PCR and in promoter:LUCIFERASE transgenic plants. Both genes showed circadian and diurnal regulation. Under copper deficiency, their expression decreased drastically in continuous darkness. Accordingly, total copper content was slightly reduced in etiolated seedlings under copper deficiency. The expression of SPL7 and its targets COPT2 and FSD1 was differently regulated in various light signalling mutants. On the other hand, increased copper levels reduced the amplitude of nuclear circadian clock components, such as GIGANTEA (GI). The alteration of copper homeostasis in the COPT1 overexpression line and spl7 mutants also modified the amplitude of a classical clock output, namely the circadian oscillation of cotyledon movements. In the spl7 mutant, the period of the oscillation remained constant. These results suggest a feedback of copper transport on the circadian clock and the integration of rhythmic copper homeostasis into the central oscillator of plants.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/physiology , Cation Transport Proteins/genetics , Copper/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant , Superoxide Dismutase/genetics , Transcription Factors/genetics , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Cation Transport Proteins/metabolism , Circadian Rhythm , Copper/deficiency , DNA-Binding Proteins/metabolism , Homeostasis , Plants, Genetically Modified/genetics , Plants, Genetically Modified/physiology , SLC31 Proteins , Superoxide Dismutase/metabolism , Transcription Factors/metabolismABSTRACT
Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.
Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , Chromatin Assembly and Disassembly/physiology , DNA Methylation/physiology , Nucleosomes/metabolism , Arabidopsis/enzymology , Chromatin Assembly and Disassembly/genetics , Chromatin Immunoprecipitation , DNA Methylation/genetics , DNA Polymerase II/analysis , DNA Polymerase II/metabolism , DNA, Plant/genetics , DNA, Plant/metabolism , Exons/genetics , Genes, Plant/genetics , Genome, Plant/genetics , Humans , Micrococcal Nuclease/metabolism , Nucleosomes/genetics , Sequence Analysis, DNAABSTRACT
Following the recognition of pathogen-encoded effectors, plant TIR-NB-LRR immune receptors induce defense signaling by a largely unknown mechanism. We identify a novel and conserved role for the SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-domain transcription factor SPL6 in enabling the activation of the defense transcriptome following its association with a nuclear-localized immune receptor. During an active immune response, the Nicotiana TIR-NB-LRR N immune receptor associates with NbSPL6 within distinct nuclear compartments. NbSPL6 is essential for the N-mediated resistance to Tobacco mosaic virus. Similarly, the presumed Arabidopsis ortholog AtSPL6 is required for the resistance mediated by the TIR-NB-LRR RPS4 against Pseudomonas syringae carrying the avrRps4 effector. Transcriptome analysis indicates that AtSPL6 positively regulates a subset of defense genes. A pathogen-activated nuclear-localized TIR-NB-LRR like N can therefore regulate defense genes through SPL6 in a mechanism analogous to the induction of MHC genes by mammalian immune receptors like CIITA and NLRC5.
Subject(s)
Arabidopsis/immunology , Gene Expression Regulation, Plant , Nicotiana/immunology , Plant Diseases/immunology , Plant Immunity , Plant Proteins/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/immunology , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , Gene Expression Profiling , Immunity, Innate , Mutation , Oligonucleotide Array Sequence Analysis , Plant Diseases/microbiology , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/immunology , Plants, Genetically Modified , Protein Structure, Tertiary , Pseudomonas syringae/physiology , Signal Transduction , Nicotiana/cytology , Nicotiana/genetics , Nicotiana/virology , Tobacco Mosaic Virus/physiology , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolism , TranscriptomeABSTRACT
Proper copper (Cu) homeostasis is required by living organisms to maintain essential cellular functions. In the model plant Arabidopsis (Arabidopsis thaliana), the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7) transcription factor participates in reprogramming global gene expression during Cu insufficiency in order to improve the metal uptake and prioritize its distribution to Cu proteins of major importance. As a consequence, spl7 null mutants show morphological and physiological disorders during Cu-limited growth, resulting in lower fresh weight, reduced root elongation, and chlorosis. On the other hand, the Arabidopsis KIN17 homolog belongs to a well-conserved family of essential eukaryotic nuclear proteins known to be stress activated and involved in DNA and possibly RNA metabolism in mammals. In the study presented here, we uncovered that Arabidopsis KIN17 participates in promoting the Cu deficiency response by means of a direct interaction with SPL7. Moreover, the double mutant kin17-1 spl7-2 displays an enhanced Cu-dependent phenotype involving growth arrest, oxidative stress, floral bud abortion, and pollen inviability. Taken together, the data presented here provide evidence for SPL7 and KIN17 protein interaction as a point of convergence in response to both Cu deficiency and oxidative stress.
Subject(s)
Adaptation, Physiological/drug effects , Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Conserved Sequence , Copper/toxicity , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Arabidopsis/drug effects , Arabidopsis/physiology , Base Sequence , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Models, Biological , Molecular Sequence Data , Mutation/genetics , Nuclear Proteins/metabolism , Oxidative Stress/drug effects , Phenotype , Plant Development/drug effects , Plant Leaves/drug effects , Plant Leaves/metabolism , Pollen/drug effects , Pollen/metabolism , Protein Binding/drug effects , Nicotiana/drug effects , Nicotiana/metabolismABSTRACT
The transition metal copper (Cu) is essential for all living organisms but is toxic when present in excess. To identify Cu deficiency responses comprehensively, we conducted genome-wide sequencing-based transcript profiling of Arabidopsis thaliana wild-type plants and of a mutant defective in the gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), which acts as a transcriptional regulator of Cu deficiency responses. In response to Cu deficiency, FERRIC REDUCTASE OXIDASE5 (FRO5) and FRO4 transcript levels increased strongly, in an SPL7-dependent manner. Biochemical assays and confocal imaging of a Cu-specific fluorophore showed that high-affinity root Cu uptake requires prior FRO5/FRO4-dependent Cu(II)-specific reduction to Cu(I) and SPL7 function. Plant iron (Fe) deficiency markers were activated in Cu-deficient media, in which reduced growth of the spl7 mutant was partially rescued by Fe supplementation. Cultivation in Cu-deficient media caused a defect in root-to-shoot Fe translocation, which was exacerbated in spl7 and associated with a lack of ferroxidase activity. This is consistent with a possible role for a multicopper oxidase in Arabidopsis Fe homeostasis, as previously described in yeast, humans, and green algae. These insights into root Cu uptake and the interaction between Cu and Fe homeostasis will advance plant nutrition, crop breeding, and biogeochemical research.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Copper/metabolism , DNA-Binding Proteins/metabolism , FMN Reductase/genetics , Iron/metabolism , Transcription Factors/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant , Gene Knockdown Techniques , High-Throughput Nucleotide Sequencing , Homeostasis , Plant Roots/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , RNA, Plant/genetics , Transcription Factors/genetics , TranscriptomeABSTRACT
SPL8 and miR156-targeted SPL genes are known to play an essential role in Arabidopsis anther development. Here we show that these SPL genes are also expressed within the developing gynoecium, where they redundantly control development of the female reproductive tract. Whereas the gynoecium morphology in the spl8 single mutant is largely normal, additional down-regulation of miR156-targeted SPL genes results in a shortened style and an apically swollen ovary narrowing onto an elongated gynophore. In particular, the septum does not form properly and lacks a transmitting tract. Loss of SPL8 function enhances the mutant phenotypes of ett, crc and spt, indicating a functional overlap between SPL8 and these genes in regulating gynoecium development. Furthermore, gynoecium development of 35S:MIR156b spl8-1 double mutants shows enhanced sensitivity to a polar auxin transport inhibitor, and the expression pattern of the auxin biosynthesis gene YUCCA4 is altered compared to wild-type. Our observations imply that SPL8 and miR156-targeted SPL genes control gynoecium patterning through interference with auxin homeostasis and signalling.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Flowers/genetics , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , MicroRNAs/genetics , Arabidopsis/cytology , Arabidopsis/drug effects , Arabidopsis/growth & development , Arabidopsis Proteins/metabolism , Body Patterning , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , Flowers/cytology , Flowers/drug effects , Flowers/growth & development , Homeostasis , MicroRNAs/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Multigene Family , Mutation , Organ Specificity , Phenotype , Phthalimides/pharmacology , Plant Leaves/cytology , Plant Leaves/drug effects , Plant Leaves/genetics , Plant Leaves/growth & development , Plants, Genetically Modified , Seedlings/cytology , Seedlings/drug effects , Seedlings/genetics , Seedlings/growth & development , Signal TransductionABSTRACT
Plant development progresses through distinct phases: vegetative growth, followed by a reproductive phase and eventually seed set and senescence. The transitions between these phases are controlled by distinct genetic circuits that integrate endogenous and environmental cues. In recent years, however, it has become evident that the genetic networks that underlie these phase transitions share some common factors. Here, we review recent advances in the field of plant phase transitions, highlighting the role of two microRNAs - miR156 and miR172 - and their respective targets during these transitions. In addition, we discuss the evolutionary conservation of the functions of these miRNAs in regulating the control of plant developmental phase transitions.
Subject(s)
Arabidopsis/genetics , Gene Expression Regulation, Plant , Plants/embryology , Plants/metabolism , Developmental Biology/methods , Flowers , Genes, Plant , MicroRNAs/metabolism , Models, Biological , Models, Genetic , Plant Physiological Phenomena , Pollen , Transcription Factors/metabolismABSTRACT
BACKGROUND: The Arabidopsis SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor SPL7 reprograms cellular gene expression to adapt plant growth and cellular metabolism to copper (Cu) limited culture conditions. Plant cells require Cu to maintain essential processes, such as photosynthesis, scavenging reactive oxygen species, cell wall lignification and hormone sensing. More specifically, SPL7 activity promotes a high-affinity Cu-uptake system and optimizes Cu (re-)distribution to essential Cu-proteins by means of specific miRNAs targeting mRNA transcripts for those dispensable. However, the functional mechanism underlying SPL7 activation is still to be elucidated. As SPL7 transcript levels are largely non-responsive to Cu availability, post-translational modification seems an obvious possibility. Previously, it was reported that the SPL7 SBP domain does not bind to DNA in vitro in the presence of Cu ions and that SPL7 interacts with a kin17 domain protein to raise SPL7-target gene expression upon Cu deprivation. Here we report how additional conserved SPL7 protein domains may contribute to the Cu deficiency response in Arabidopsis. RESULTS: Cytological and biochemical approaches confirmed an operative transmembrane domain (TMD) and uncovered a dual localisation of SPL7 between the nucleus and an endomembrane system, most likely the endoplasmic reticulum (ER). This new perspective unveiled a possible link between Cu deficit and ER stress, a metabolic dysfunction found capable of inducing SPL7 targets in an SPL7-dependent manner. Moreover, in vivo protein-protein interaction assays revealed that SPL7 is able to homodimerize, probably mediated by the IRPGC domain. These observations, in combination with the constitutive activation of SPL7 targets, when ectopically expressing the N-terminal part of SPL7 including the SBP domain, shed some light on the mechanisms governing SPL7 function. CONCLUSIONS: Here, we propose a revised model of SPL7 activation and regulation. According to our results, SPL7 would be initially located to endomembranes and activated during ER stress as a result of Cu deficiency. Furthermore, we added the SPL7 dimerization in the presence of Cu ions as an additional regulatory mechanism to modulate the Cu deficiency response.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Copper , DNA-Binding Proteins , Gene Expression Regulation, Plant , Transcription Factors , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , Copper/deficiency , Copper/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum Stress/physiology , Protein Structure, Tertiary , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Biological shape diversity is often manifested in modulation of organ symmetry and modification of the patterned elaboration of repeated shape elements.1,2,3,4,5 Whether and how these two aspects of shape determination are coordinately regulated is unclear.5,6,7 Plant leaves provide an attractive system to investigate this problem, because they often show asymmetries along the proximodistal (PD) axis of their blades, along which they can also produce repeated marginal outgrowths such as serrations or leaflets.1 One aspect of leaf shape diversity is heteroblasty, where the leaf form in a single genotype is modified with progressive plant age.8,9,10,11 In Arabidopsis thaliana, a plant with simple leaves, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) controls heteroblasty by activating CyclinD3 expression, thereby sustaining proliferative growth and retarding differentiation in adult leaves.12,13 However, the precise significance of SPL9 action for leaf symmetry and marginal patterning is unknown. By combining genetics, quantitative shape analyses, and time-lapse imaging, we show that PD symmetry of the leaf blade in A. thaliana decreases in response to an age-dependent SPL9 expression gradient, and that SPL9 action coordinately regulates the distribution and shape of marginal serrations and overall leaf form. Using comparative analyses, we demonstrate that heteroblastic growth reprogramming in Cardamine hirsuta, a complex-leafed relative of A. thaliana, also involves prolonging the duration of cell proliferation and delaying differentiation. We further provide evidence that SPL9 enables species-specific action of homeobox genes that promote leaf complexity. In conclusion, we identified an age-dependent layer of organ PD growth regulation that modulates leaf symmetry and has enabled leaf shape diversification.
ABSTRACT
How is time encoded into organ growth and morphogenesis? We address this question by investigating heteroblasty, where leaf development and form are modified with progressing plant age. By combining morphometric analyses, fate-mapping through live-imaging, computational analyses, and genetics, we identify age-dependent changes in cell-cycle-associated growth and histogenesis that underpin leaf heteroblasty. We show that in juvenile leaves, cell proliferation competence is rapidly released in a "proliferation burst" coupled with fast growth, whereas in adult leaves, proliferative growth is sustained for longer and at a slower rate. These effects are mediated by the SPL9 transcription factor in response to inputs from both shoot age and individual leaf maturation along the proximodistal axis. SPL9 acts by activating CyclinD3 family genes, which are sufficient to bypass the requirement for SPL9 in the control of leaf shape and in heteroblastic reprogramming of cellular growth. In conclusion, we have identified a mechanism that bridges across cell, tissue, and whole-organism scales by linking cell-cycle-associated growth control to age-dependent changes in organ geometry.
Subject(s)
Plant Leaves , Transcription Factors , Transcription Factors/metabolism , Cell Proliferation , Cell Division , Morphogenesis , Gene Expression Regulation, PlantABSTRACT
Copper (Cu), an essential redox active cofactor, participates in fundamental biological processes, but it becomes highly cytotoxic when present in excess. Therefore, living organisms have established suitable mechanisms to balance cellular and systemic Cu levels. An important strategy to maintain Cu homeostasis consists of regulating uptake and mobilization via the conserved family of CTR/COPT Cu transport proteins. In the model plant Arabidopsis thaliana, COPT1 protein mediates root Cu acquisition, whereas COPT5 protein functions in Cu mobilization from intracellular storage organelles. The function of these transporters becomes critical when environmental Cu bioavailability diminishes. However, little is know about the mechanisms that mediate plant Cu distribution. In this report, we present evidence supporting an important role for COPT6 in Arabidopsis Cu distribution. Similarly to COPT1 and COPT2, COPT6 fully complements yeast mutants defective in high-affinity Cu uptake and localizes to the plasma membrane of Arabidopsis cells. Whereas COPT2 mRNA is only up-regulated upon severe Cu deficiency, COPT6 transcript is expressed under Cu excess conditions and displays a more gradual increase in response to decreases in environmental Cu levels. Consistent with COPT6 expression in aerial vascular tissues and reproductive organs, copt6 mutant plants exhibit altered Cu distribution under Cu-deficient conditions, including increased Cu in rosette leaves but reduced Cu levels in seeds. This altered Cu distribution is fully rescued when the wild-type COPT6 gene is reintroduced into the copt6 mutant line. Taken together, these findings highlight the relevance of COPT6 in shoot Cu redistribution when environmental Cu is limited.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Copper/metabolism , Gene Expression Regulation, Plant , Membrane Transport Proteins/metabolism , Amino Acid Sequence , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Biological Transport , Cell Membrane/metabolism , Copper/deficiency , Genetic Complementation Test , Homeostasis , Membrane Transport Proteins/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Plant Leaves/cytology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Roots/cytology , Plant Roots/genetics , Plant Roots/metabolism , Plant Shoots/cytology , Plant Shoots/genetics , Plant Shoots/metabolism , Plant Vascular Bundle/cytology , Plant Vascular Bundle/genetics , Plant Vascular Bundle/metabolism , Plants, Genetically Modified , SLC31 Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Seeds/cytology , Seeds/genetics , Seeds/metabolism , Sequence Alignment , Up-RegulationABSTRACT
The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(-)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(-). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , DNA-Binding Proteins/metabolism , Flowers/physiology , MicroRNAs/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Blotting, Western , Chromatin Immunoprecipitation , Cold Temperature , DNA-Binding Proteins/genetics , Flowers/genetics , Flowers/metabolism , Gene Expression Regulation, Plant , MicroRNAs/genetics , Plant Leaves/genetics , Plant Leaves/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/physiology , Protein Binding , Proteolysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , Time Factors , Transcription Factors/geneticsABSTRACT
The SBP-box transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE8 (SPL8) is required for proper development of sporogenic tissues in Arabidopsis thaliana. Here, we show that the semisterile phenotype of SPL8 loss-of-function mutants is due to partial functional redundancy with several other members of the Arabidopsis SPL gene family. In contrast with SPL8, the transcripts of these latter SPL genes are all targeted by miR156/7. Whereas the introduction of single miR156/7-resistant SPL transgenes could only partially restore spl8 mutant fertility, constitutive overexpression of miR156 in an spl8 mutant background resulted in fully sterile plants. Histological analysis of the anthers of such sterile plants revealed an almost complete absence of sporogenous and anther wall tissue differentiation, a phenotype similar to that reported for sporocyteless/nozzle (spl/nzz) mutant anthers. Expression studies indicated a functional requirement for miR156/7-targeted SPL genes limited to early anther development. Accordingly, several miR156/7-encoding loci were found expressed in anther tissues at later stages of development. We conclude that fully fertile Arabidopsis flowers require the action of multiple miR156/7-targeted SPL genes in concert with SPL8. Either together with SPL/NZZ or independently, these SPL genes act to regulate genes mediating cell division, differentiation, and specification early in anther development. Furthermore, SPL8 in particular may be required to secure fertility of the very first flowers when floral transition-related miR156/7 levels might not have sufficiently declined.