ABSTRACT
The presence of diffuse morphogen gradients in tissues supports a view in which growth is locally homogenous. Here we challenge this view: we used a high-resolution quantitative approach to reveal significant growth variability among neighboring cells in the shoot apical meristem, the plant stem cell niche. This variability was strongly decreased in a mutant impaired in the microtubule-severing protein katanin. Major shape defects in the mutant could be related to a local decrease in growth heterogeneity. We show that katanin is required for the cell's competence to respond to the mechanical forces generated by growth. This provides the basis for a model in which microtubule dynamics allow the cell to respond efficiently to mechanical forces. This in turn can amplify local growth-rate gradients, yielding more heterogeneous growth and supporting morphogenesis.
Subject(s)
Adenosine Triphosphatases/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , Meristem/cytology , Adenosine Triphosphatases/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Homeostasis , Katanin , Meristem/growth & development , Meristem/metabolism , Microtubules/metabolism , Models, Biological , Morphogenesis , Mutation , Plant Cells/physiology , Plant Shoots/cytology , Plant Shoots/growth & development , Stress, MechanicalABSTRACT
Organ morphogenesis largely relies on cell division and elongation, which need to be both coordinated between cells and orchestrated with cytoskeleton dynamics. However, components that bridge the biological signals and the effectors that define cell shape remain poorly described. We have addressed this issue through the functional characterisation of QUIRKY (QKY), previously isolated as being involved in the STRUBBELIG (SUB) genetic pathway that controls cell-cell communication and organ morphogenesis in Arabidopsis. QKY encodes a protein containing multiple C2 domains and transmembrane regions, and SUB encodes an atypical LRR-receptor-like kinase. We show that twisting of the gynoecium observed in qky results from the abnormal division pattern and anisotropic growth of clustered cells arranged sporadically along the gynoecium. Moreover, the cortical microtubule (CMT) network of these cells is disorganised. A cross to botero, a katanin mutant in which the normal orientation of CMTs and anisotropic cell expansion are impaired, strongly reduces silique deviation, reinforcing the hypothesis of a role for QKY in CMT-mediated cell growth anisotropy. We also show that QKY is localised at the plasma membrane and functions in a multiprotein complex that includes SUB and PAL OF QUIRKY (POQ), a previously uncharacterised PB1-domain-containing protein that localises both at the plasma membrane and in intracellular compartments. Our data indicate that QKY and its interactors play central roles linking together cell-cell communication and cellular growth.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/embryology , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Anisotropy , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Carrier Proteins/genetics , Cell Communication , Cell Membrane/metabolism , Gene Expression Regulation, Plant , Membrane Proteins/genetics , Microtubules , Molecular Sequence Data , Morphogenesis , Receptor Protein-Tyrosine Kinases/genetics , Sequence Alignment , Signal Transduction/geneticsABSTRACT
RNA editing plays an important role in organelle gene expression in various organisms, including flowering plants, changing the nucleotide information at precise sites. Here, we present evidence that the maize (Zea mays) nuclear gene Pentatricopeptide repeat 2263 (PPR2263) encoding a DYW domain-containing PPR protein is required for RNA editing in the mitochondrial NADH dehydrogenase5 (nad5) and cytochrome b (cob) transcripts at the nad5-1550 and cob-908 sites, respectively. Its putative ortholog, MITOCHONDRIAL EDITING FACTOR29, fulfills the same role in Arabidopsis thaliana. Both the maize and the Arabidopsis proteins show preferential localization to mitochondria but are also detected in chloroplasts. In maize, the corresponding ppr2263 mutation causes growth defects in kernels and seedlings. Embryo and endosperm growth are reduced, leading to the production of small but viable kernels. Mutant plants have narrower and shorter leaves, exhibit a strong delay in flowering time, and generally do not reach sexual maturity. Whereas mutant chloroplasts do not have major defects, mutant mitochondria lack complex III and are characterized by a compromised ultrastructure, increased transcript levels, and the induction of alternative oxidase. The results suggest that mitochondrial RNA editing at the cob-908 site is necessary for mitochondrion biogenesis, cell division, and plant growth in maize.
Subject(s)
Cytochromes b/genetics , Mitochondrial Proteins/genetics , NADH Dehydrogenase/genetics , Plant Proteins/metabolism , RNA Editing , Zea mays/growth & development , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/growth & development , Chloroplasts/enzymology , Gene Expression Regulation, Plant , Microscopy, Electron, Transmission , Mitochondria/enzymology , Mitochondria/ultrastructure , Mitochondrial Proteins/metabolism , Molecular Sequence Data , Mutagenesis, Insertional , Oxidoreductases/metabolism , Phenotype , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , RNA, Plant/genetics , Seeds/growth & development , Zea mays/genetics , Zea mays/metabolismABSTRACT
Plant and animals have evolved different strategies for their development. Whether this is linked to major differences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.
Subject(s)
Arabidopsis/cytology , Mechanical Phenomena , Animals , Biomechanical Phenomena , Cell Line , Mice , Microtubules/metabolism , Single-Cell Analysis , Species SpecificityABSTRACT
The retromer complex localizes to endosomal membranes and is involved in protein trafficking. In mammals, it is composed of a dimer of sorting nexins and of the core retromer consisting of vacuolar protein sorting (VPS)26, VPS29, and VPS35. Although homologs of these proteins have been identified in plants, how the plant retromer functions remains elusive. To better understand the role of VPS components in the assembly and function of the core retromer, we characterize here Arabidopsis vps26-null mutants. We show that impaired VPS26 function has a dramatic effect on VPS35 levels and causes severe phenotypic defects similar to those observed in vps29-null mutants. This implies that functions of plant VPS26, VPS29, and VPS35 are tightly linked. Then, by combining live-cell imaging with immunochemical and genetic approaches, we report that VPS35 alone is able to bind to endosomal membranes and plays an essential role in VPS26 and VPS29 membrane recruitment. We also show that the Arabidopsis Rab7 homolog RABG3f participates in the recruitment of the core retromer to the endosomal membrane by interacting with VPS35. Altogether our data provide original information on the molecular interactions that mediate assembly of the core retromer in plants.
Subject(s)
Arabidopsis/metabolism , rab GTP-Binding Proteins/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cytosol/metabolism , Endosomes/metabolism , Genotype , Immunochemistry/methods , Microscopy, Confocal/methods , Mutagenesis, Site-Directed , Mutation , Phenotype , Plant Physiological Phenomena , Plant Roots/metabolism , Plants, Genetically Modified/metabolism , Plasmids/metabolism , Subcellular Fractions/metabolism , Two-Hybrid System Techniques , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/geneticsABSTRACT
Auxin response factors (ARF) are key players in plant development. They mediate the cellular response to the plant hormone auxin by activating or repressing the expression of downstream developmental genes. The pivotal activation function of ARF proteins is enabled by their four-domain architecture, which includes both DNA-binding and protein dimerization motifs. To determine the evolutionary origin of this characteristic architecture, we built a comprehensive data set of 224 ARF-related protein sequences that represents all major living divisions of land plants, except hornworts. We found that ARFs are split into three subfamilies that could be traced back to the origin of the land plants. We also show that repeated events of extensive gene duplication contributed to the expansion of those three original subfamilies. Further examination of our data set uncovered a broad diversity in the structure of ARF transcripts and allowed us to identify an additional conserved motif in ARF proteins. We found that additional structural diversity in ARF proteins is mainly generated by two mechanisms: genomic truncation and alternative splicing. We propose that the loss of domains from the canonical, four-domain ARF structure has promoted functional shifts within the ARF family by disrupting either dimerization or DNA-binding capabilities. For instance, the loss of dimerization domains in some ARFs from moss and spikemoss genomes leads to proteins that are reminiscent of Aux/IAA proteins, possibly providing a clue on the evolution of these modulators of ARF function. We also assessed the functional impact of alternative splicing in the case of ARF4, for which we have identified a novel isoform in Arabidopsis thaliana. Genetic analysis showed that these two transcripts exhibit markedly different developmental roles in A. thaliana. Gene duplications, domain rearrangement, and post-transcriptional regulation have thus enabled a subtle control of auxin signaling through ARF proteins that may have contributed to the critical importance of these regulators in plant development and evolution.
Subject(s)
Arabidopsis/genetics , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Multigene Family , Plant Growth Regulators/metabolism , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Alternative Splicing , Bryophyta/genetics , Cloning, Molecular , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Evolution, Molecular , Gene Duplication , Gene Rearrangement , Genes, Plant , Membrane Proteins/genetics , Membrane Proteins/metabolism , Open Reading Frames , Phylogeny , Plants, Genetically Modified , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.
Subject(s)
Conjugation, Genetic , Escherichia coli , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Plasmids/genetics , DNA , DNA, Single-Stranded/genetics , Gene Transfer, HorizontalABSTRACT
The pentatricopeptide repeat (PPR) domain is an RNA binding domain allowing members of the PPR superfamily to participate in post-transcriptional processing of organellar RNA. Loss of PPR8522 from maize (Zea mays) confers an embryo-specific (emb) phenotype. The emb8522 mutation was isolated in an active Mutator (Mu) population and co-segregation analysis revealed that it was tightly linked to a MuDR insertion in the first exon of PPR8522. Independent evidence that disruption of PPR8522 caused the emb phenotype was provided by fine mapping to a region of 116kb containing no other gene than PPR8522 and complementation of the emb8522 mutant by a PPR8522 cDNA. The deduced PPR8522 amino acid sequence of 832 amino acids contains 10 PPR repeats and a chloroplast target peptide, the function of which was experimentally demonstrated by transient expression in Nicotiana benthamiana. Whereas mutant endosperm is apparently normal, mutant embryos deviate from normal development as early as 3 days after pollination, are reduced in size, exhibit more or less severe morphological aberrations depending on the genetic background, and generally do not germinate. The emb8522 mutation is the first to associate the loss of a PPR gene with an embryo-lethal phenotype in maize. Analyses of mutant plantlets generated by embryo-rescue experiments indicate that emb8522 also affects vegetative plant growth and chloroplast development. The loss of chloroplast transcription dependent on plastid-encoded RNA polymerase is the likely cause for the lack of an organized thylakoid network and an albino, seedling-lethal phenotype.
Subject(s)
Chloroplasts/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Zea mays/growth & development , Zea mays/metabolism , Amino Acid Sequence , Chloroplasts/chemistry , Chloroplasts/genetics , Gene Expression Regulation, Plant , Molecular Sequence Data , Plant Proteins/genetics , Protein Structure, Tertiary , Protein Transport , Sequence Alignment , Zea mays/embryology , Zea mays/geneticsABSTRACT
Here we analyze the structural evolution of the paralogous transcription factors ETTIN (ETT/ARF3) and AUXIN RESPONSE FACTOR 4 (ARF4), which control the development of floral organs and leaves in the model angiosperm Arabidopsis. ETT is truncated at its C terminus, and consequently lacks two regulatory domains present in most other ARFs, including ARF4. Our analysis indicates ETT and ARF4 to have been generated by the duplication of a non-truncated ARF gene prior to the radiation of the extant angiosperms. We furthermore show that either ETT or ARF4 orthologs have become modified to encode truncated ARF proteins, lacking C-terminal regulatory domains, in representatives of three groups that separated early in angiosperm evolution: Amborellales, Nymphaeales and the remaining angiosperm clade. Interestingly, the production of truncated ARF4 transcripts in Amborellales occurs through an alternative splicing mechanism, rather than through a permanent truncation, as in the other groups studied. To gain insight into the potential functional significance of truncations to ETT and ARF4, we tested the capacity of native, truncated and chimeric coding sequences of these genes to restore a wild-type phenotype to Arabidopsis ett mutants. We discuss the results of this analysis in the context of the structural evolution of ARF genes in the angiosperms.
Subject(s)
Evolution, Molecular , Magnoliopsida/classification , Magnoliopsida/metabolism , Plant Proteins/classification , Plant Proteins/metabolism , Transcription Factors/classification , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Ephedra/genetics , Ephedra/metabolism , Magnoliopsida/genetics , Nuclear Proteins/classification , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phylogeny , Plant Proteins/genetics , Plants, Genetically Modified/classification , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Transcription Factors/geneticsABSTRACT
Drug-efflux by pump proteins is one of the major mechanisms of antibiotic resistance in bacteria. Here, we use quantitative fluorescence microscopy to investigate the real-time dynamics of drug accumulation and efflux in live E. coli cells. We visualize simultaneously the intrinsically fluorescent protein-synthesis inhibitor tetracycline (Tc) and the fluorescently labelled Tc-specific efflux pump, TetA. We show that Tc penetrates the cells within minutes and accumulates to stable intracellular concentration after â¼20 min. The final level of drug accumulation reflects the balance between Tc-uptake by the cells and Tc-efflux by pump proteins. In wild-type Tc-sensitive cells, drug accumulation is significantly limited by the activity of the multidrug efflux pump, AcrAB-TolC. Tc-resistance wild-type cells carrying a plasmid-borne Tn10 transposon contain variable amounts of TetA protein, produced under steady-state repression by the TetR repressor. TetA content heterogeneity determines the cells' initial ability to efflux Tc. Yet, efflux remains partial until the synthesis of additional TetA pumps allows for Tc-efflux activity to surpass Tc-uptake. Cells overproducing TetA no longer accumulate Tc and become resistant to high concentrations of the drug. This work uncovers the dynamic balance between drug entry, protein-synthesis inhibition, efflux-pump production, drug-efflux activity and drug-resistance levels.
Subject(s)
Antiporters/metabolism , Bacterial Proteins/metabolism , Escherichia coli/cytology , Escherichia coli/metabolism , Tetracycline/metabolism , Microscopy, Fluorescence , Tetracycline Resistance/geneticsABSTRACT
Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor-conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action.
Subject(s)
Carrier Proteins/physiology , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli Proteins/physiology , Escherichia coli/physiology , F Factor/physiology , Anti-Bacterial Agents/pharmacology , Antiporters/antagonists & inhibitors , Antiporters/biosynthesis , Antiporters/genetics , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Carrier Proteins/genetics , Conjugation, Genetic , DNA, Single-Stranded , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , F Factor/genetics , Microscopy , Protein Biosynthesis/drug effects , Tetracycline/pharmacologyABSTRACT
In Arabidopsis, the population of stem cells present in young flower buds is lost after the production of a fixed number of floral organs. The precisely timed repression of the stem cell identity gene WUSCHEL (WUS) by the floral homeotic protein AGAMOUS (AG) is a key part of this process. In this study, we report on the identification of a novel input into the process of floral stem cell regulation. We use genetics and chromatin immunoprecipitation assays to demonstrate that the bZIP transcription factor PERIANTHIA (PAN) plays a role in regulating stem cell fate by directly controlling AG expression and suggest that this activity is spatially restricted to the centermost region of the AG expression domain. These results suggest that the termination of floral stem cell fate is a multiply redundant process involving loci with unrelated floral patterning functions.
Subject(s)
AGAMOUS Protein, Arabidopsis/metabolism , Arabidopsis Proteins/physiology , Arabidopsis/cytology , DNA-Binding Proteins/physiology , Flowers/cytology , Stem Cells/cytology , Transcription Factors/physiology , AGAMOUS Protein, Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Body Patterning/physiology , Cell Differentiation/physiology , DNA-Binding Proteins/genetics , Flowers/physiology , Gene Expression Regulation, Plant , Homeodomain Proteins/metabolism , Mutation , Stem Cells/physiology , Transcription Factors/geneticsABSTRACT
BACKGROUND AND AIMS: CRABS CLAW (CRC) encodes a transcription factor of the YABBY family that plays important roles in carpel and nectary development in Arabidopsis thaliana. Combined evolutionary and developmental studies suggest an ancestor of the CRC gene to have controlled carpel development in the last common ancestor of the angiosperms. Roles for CRC orthologues in leaf development and carpel specification in rice, and in nectary development in core eudicots, have accordingly been interpreted as derived. The aim of this study was to assess the capacity of CRC orthologues from a basal angiosperm and from rice to complement CRC mutants of arabidopsis. These experiments were designed to test the hypothesized ancestral role of CRC in the angiosperms, and to indicate whether putatively novel roles of various CRC orthologues resulted from changes to their encoded proteins, or from other molecular evolutionary events. METHODS: The crc-1 mutant of arabidopsis was genetically transformed with the coding sequences of various CRC orthologues, and with paralogous YABBY coding sequences, under the control of the arabidopsis CRC promoter. The phenotypes of transformed plants were assessed to determine the degree of complementation of the crc-1 mutant phenotype in carpel fusion, carpel form and nectary development. KEY RESULTS: The CRC orthologue from the basal angiosperm Amborella trichopoda partially complemented the crc-1 mutant phenotype in carpels, but not in nectaries. The CRC orthologue from rice partially complemented all aspects of the crc-1 mutant phenotype. Though most non-CRC YABBY coding sequences did not complement crc-1 mutant phenotypes, YABBY2 (YAB2) proved to be an exception. CONCLUSIONS: The data support a hypothesized ancestral role for CRC in carpel development and suggest that novel roles for CRC orthologues in monocots and in core eudicots resulted principally from molecular changes other than those affecting their coding sequences.