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1.
Mol Plant ; 16(8): 1269-1282, 2023 08 07.
Article in English | MEDLINE | ID: mdl-37415334

ABSTRACT

Survival of living organisms is fully dependent on their maintenance of genome integrity, being permanently threatened by replication stress in proliferating cells. Although the plant DNA damage response (DDR) regulator SOG1 has been demonstrated to cope with replication defects, accumulating evidence points to other pathways functioning independent of SOG1. Here, we report the roles of the Arabidopsis E2FA and EF2B transcription factors, two well-characterized regulators of DNA replication, in plant response to replication stress. Through a combination of reverse genetics and chromatin immunoprecipitation approaches, we show that E2FA and E2FB share many target genes with SOG1, providing evidence for their involvement in the DDR. Analysis of double- and triple-mutant combinations revealed that E2FB, rather than E2FA, plays the most prominent role in sustaining plant growth in the presence of replication defects, either operating antagonistically or synergistically with SOG1. Conversely, SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants. Collectively, our data reveal a complex transcriptional network controlling the replication stress response in which E2Fs and SOG1 act as key regulatory factors.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Transcription Factors/metabolism , E2F Transcription Factors/genetics , E2F Transcription Factors/metabolism , Gene Expression Regulation, Plant/genetics
2.
Plant J ; 106(5): 1197-1207, 2021 06.
Article in English | MEDLINE | ID: mdl-33989439

ABSTRACT

Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants' viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non-replicative polymerase involved in trans-lesion synthesis during DNA replication and double-strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase epsilon, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants' responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , DNA Polymerase II/metabolism , DNA Repair , DNA Replication , DNA, Plant/genetics , DNA-Directed DNA Polymerase/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/genetics , DNA Breaks, Double-Stranded , DNA Damage , DNA Polymerase II/genetics , DNA-Directed DNA Polymerase/genetics , Genomic Instability , Genotype , Meristem/genetics , Meristem/physiology , Models, Biological , Mutation , Phenotype , Plant Roots/genetics , Plant Roots/physiology , Stress, Physiological , DNA Polymerase theta
3.
Sci Rep ; 10(1): 11268, 2020 07 09.
Article in English | MEDLINE | ID: mdl-32647331

ABSTRACT

Programmed cell death (PCD) is essential for several aspects of plant life. We previously identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalysing myo-inositol synthesis, and that displays light-dependent formation of lesions on leaves due to Salicylic Acid (SA) over-accumulation. Rationale of this work was to identify novel regulators of plant PCD using a genetic approach. A screen for secondary mutations that abolish the mips1 PCD phenotype identified a mutation in the BIG gene, encoding a factor of unknown molecular function that was previously shown to play pleiotropic roles in plant development and defence. Physiological analyses showed that BIG is required for lesion formation in mips1 via SA-dependant signalling. big mutations partly rescued transcriptomic and metabolomics perturbations as stress-related phytohormones homeostasis. In addition, since loss of function of the ceramide synthase LOH2 was not able to abolish cell death induction in mips1, we show that PCD induction is not fully dependent of sphingolipid accumulation as previously suggested. Our results provide further insights into the role of the BIG protein in the control of MIPS1-dependent cell death and also into the impact of sphingolipid homeostasis in this pathway.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Calmodulin-Binding Proteins/genetics , Inositol/metabolism , Salicylic Acid/metabolism , Arabidopsis Proteins/metabolism , Calmodulin-Binding Proteins/metabolism , Cluster Analysis , Epistasis, Genetic , Homeostasis , Mutation , Phenotype , Plant Growth Regulators/metabolism , Plant Leaves/metabolism , Signal Transduction , Sphingolipids/metabolism
4.
Plants (Basel) ; 9(8)2020 Jul 23.
Article in English | MEDLINE | ID: mdl-32717805

ABSTRACT

Thymidine kinase 1 (TK1) phosphorylates thymidine nucleosides to generate thymidine monophosphate. This reaction belongs to the pyrimidine salvage route that is phylogenetically conserved. In the model plant Arabidopsis thaliana, TK activity contributes to maintain nuclear and organellar genome integrity by providing deoxythymidine-triphosphate (dTTP) for DNA synthesis. Arabidopsis has two TK1 genes (TK1a and TK1b) and double mutants show an albino phenotype and develop poorly. In contrast, maize (Zea mays L.) has a single TK1 (ZmTK1) gene and mutant plants are albino and display reduced genome copy number in chloroplasts. We studied the role of ZmTK1 during development and genotoxic stress response by assessing its activity at different developmental stages and by complementing Arabidopsis tk1 mutants. We found that ZmTK1 transcripts and activity are present during germination and throughout maize development. We show that ZmTK1 translocation to chloroplasts depends on a 72-amino-acid N-signal and its plastid localization is consistent with its ability to complement Arabidopsis tk1b mutants which are hypersensitive to ciprofloxacin (CIP), a genotoxic agent to organellar DNA. Also, ZmTK1 partly complemented the Arabidopsis double mutant plants during development. Our results contribute to the understanding of TK1 function in monocot species as an organellar enzyme for genome replication and repair.

5.
Plant J ; 97(3): 430-446, 2019 02.
Article in English | MEDLINE | ID: mdl-30317699

ABSTRACT

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.


Subject(s)
Arabidopsis/genetics , Genome, Plant/genetics , Pyrimidines/metabolism , Thymidine Kinase/metabolism , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , Chloroplasts/metabolism , DNA Damage , Nucleotides/metabolism , Thymidine/metabolism , Thymidine Kinase/genetics
6.
Plant Physiol ; 173(3): 1735-1749, 2017 03.
Article in English | MEDLINE | ID: mdl-28153919

ABSTRACT

Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , DNA Polymerase II/genetics , DNA Replication , Arabidopsis/enzymology , Arabidopsis Proteins/metabolism , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , DNA Polymerase II/metabolism , DNA, Plant/genetics , DNA, Plant/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Plant , Gene Ontology , Hydroxyurea/pharmacology , Microscopy, Fluorescence , Models, Genetic , Mutation , Nucleic Acid Synthesis Inhibitors/pharmacology , Plants, Genetically Modified , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction
7.
Nucleic Acids Res ; 44(15): 7251-66, 2016 09 06.
Article in English | MEDLINE | ID: mdl-27193996

ABSTRACT

Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/enzymology , Cell Cycle/physiology , DNA Damage , DNA Polymerase II/chemistry , DNA Polymerase II/metabolism , DNA Repair , DNA Replication , DNA-Binding Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , DNA Polymerase II/genetics , DNA-Binding Proteins/genetics
8.
Plant Physiol ; 170(3): 1745-56, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26747283

ABSTRACT

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3'-phosphoadenosine 5'-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5'-3' exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.


Subject(s)
Adenosine Diphosphate/metabolism , Apoptosis/physiology , Arabidopsis/metabolism , Chloroplasts/metabolism , Signal Transduction/physiology , Apoptosis/genetics , Arabidopsis/genetics , Arabidopsis/microbiology , Chlorophyll/metabolism , Chloroplasts/genetics , Disease Resistance/genetics , Mutation , Myo-Inositol-1-Phosphate Synthase/genetics , Myo-Inositol-1-Phosphate Synthase/metabolism , Oxidation-Reduction , Photosynthesis/genetics , Photosynthesis/physiology , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Immunity/genetics , Pseudomonas syringae/physiology , Signal Transduction/genetics
9.
Plant Cell ; 27(6): 1801-14, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26048869

ABSTRACT

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/physiology , Cell Death/physiology , Hexokinase/physiology , Inositol/physiology , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Gas Chromatography-Mass Spectrometry , Genes, Plant/genetics , Genes, Plant/physiology , Hexokinase/genetics , Inositol/metabolism
10.
Plant Physiol ; 166(1): 152-67, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25037213

ABSTRACT

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/physiology , Cell Cycle , Chloroplasts/physiology , Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Proliferation , Cyclins/metabolism , Gene Expression Regulation, Plant
11.
Plant Physiol ; 165(2): 732-746, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24706550

ABSTRACT

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Indeed, incompatible plant-pathogen interactions are well known to induce the hypersensitive response, a localized cell death. Mutational analyses have identified several key PCD components, and we recently identified the mips1 mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for the key enzyme catalyzing the limiting step of myoinositol synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD, revealing roles for myoinositol or inositol derivatives in the regulation of PCD. Here, we identified a regulator of plant PCD by screening for mutants that display transcriptomic profiles opposing that of the mips1 mutant. Our screen identified the oxt6 mutant, which has been described previously as being tolerant to oxidative stress. In the oxt6 mutant, a transfer DNA is inserted in the CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) gene, which encodes a polyadenylation factor subunit homolog. We show that CPSF30 is required for lesion formation in mips1 via SA-dependent signaling, that the prodeath function of CPSF30 is not mediated by changes in the glutathione status, and that CPSF30 activity is required for Pseudomonas syringae resistance. We also show that the oxt6 mutation suppresses cell death in other lesion-mimic mutants, including lesion-simulating disease1, mitogen-activated protein kinase4, constitutive expressor of pathogenesis-related genes5, and catalase2, suggesting that CPSF30 and, thus, the control of messenger RNA 3' end processing, through the regulation of SA production, is a key component of plant immune responses.

12.
Plant Physiol ; 161(4): 1694-705, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23426196

ABSTRACT

Despite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclin-dependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis (Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication, indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization. Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithorax-related protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required for cell elongation.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , Endoreduplication , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cyclin-Dependent Kinase Inhibitor Proteins/genetics , Cyclins/metabolism , Genes, Plant/genetics , Heterochromatin/metabolism , Models, Biological , Mutation/genetics , Protein Binding/genetics , Protein Transport , Seedlings/metabolism , Transcriptional Activation/genetics
13.
Nucleic Acids Res ; 41(5): 2907-17, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23341037

ABSTRACT

Because regulation of its activity is instrumental either to support cell proliferation and growth or to promote cell death, the universal myo-inositol phosphate synthase (MIPS), responsible for myo-inositol biosynthesis, is a critical enzyme of primary metabolism. Surprisingly, we found this enzyme to be imported in the nucleus and to interact with the histone methyltransferases ATXR5 and ATXR6, raising the question of whether MIPS1 has a function in transcriptional regulation. Here, we demonstrate that MIPS1 binds directly to its promoter to stimulate its own expression by locally inhibiting the spreading of ATXR5/6-dependent heterochromatin marks coming from a transposable element. Furthermore, on activation of pathogen response, MIPS1 expression is reduced epigenetically, providing evidence for a complex regulatory mechanism acting at the transcriptional level. Thus, in plants, MIPS1 appears to have evolved as a protein that connects cellular metabolism, pathogen response and chromatin remodeling.


Subject(s)
Arabidopsis/genetics , Gene Expression Regulation, Plant , Meristem/genetics , Myo-Inositol-1-Phosphate Synthase/physiology , Apoptosis , Arabidopsis/cytology , Arabidopsis/enzymology , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Cell Nucleus/enzymology , Chromatin Assembly and Disassembly , Cytoplasm/enzymology , DNA Methylation , Epigenesis, Genetic , Flagellin/immunology , Gene Expression , Histones/metabolism , Meristem/cytology , Meristem/enzymology , Methylation , Methyltransferases/metabolism , Methyltransferases/physiology , Myo-Inositol-1-Phosphate Synthase/genetics , Myo-Inositol-1-Phosphate Synthase/metabolism , Plant Immunity/genetics , Promoter Regions, Genetic , Protein Binding , Protein Processing, Post-Translational , Protein Transport , Nicotiana
14.
Plant Mol Biol ; 78(4-5): 323-36, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22170036

ABSTRACT

The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.


Subject(s)
Bryopsida/growth & development , Plant Proteins/metabolism , Plant Shoots/growth & development , RNA-Binding Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Molecular Sequence Data , Mutation , Phenotype , Phylogeny , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Shoots/metabolism , Poaceae/genetics , RNA-Binding Proteins/genetics
15.
Planta ; 231(3): 525-35, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19943172

ABSTRACT

TERMINAL EAR1-like (TEL) genes encode putative RNA-binding proteins only found in land plants. Previous studies suggested that they may regulate tissue and organ initiation in Poaceae. Two TEL genes were identified in both Populus trichocarpa and the hybrid aspen Populus tremula x P. alba, named, respectively, PoptrTEL1-2 and PtaTEL1-2. The analysis of the organisation around the PoptrTEL genes in the P. trichocarpa genome and the estimation of the synonymous substitution rate for PtaTEL1-2 genes indicate that the paralogous link between these two Populus TEL genes probably results from the Salicoid large-scale gene-duplication event. Phylogenetic analyses confirmed their orthology link with the other TEL genes. The expression pattern of both PtaTEL genes appeared to be restricted to the mother cells of the plant body: leaf founder cells, leaf primordia, axillary buds and root differentiating tissues, as well as to mother cells of vascular tissues. Most interestingly, PtaTEL1-2 transcripts were found in differentiating cells of secondary xylem and phloem, but probably not in the cambium itself. Taken together, these results indicate specific expression of the TEL genes in differentiating cells controlling tissue and organ development in Populus (and other Angiosperm species).


Subject(s)
Plant Proteins/genetics , Populus/genetics , Amino Acid Sequence , Cell Differentiation , Evolution, Molecular , Gene Duplication , Genome, Plant , In Situ Hybridization , Molecular Sequence Data , Phylogeny , Plant Proteins/chemistry , Plant Proteins/metabolism , Populus/growth & development , Populus/metabolism , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, Protein , Transcription, Genetic
16.
Plant Sci ; 171(3): 300-7, 2006 Sep.
Article in English | MEDLINE | ID: mdl-22980199

ABSTRACT

Drought is a major constraint for the production of common bean (Phaseolus vulgaris L.). To identify molecular responses to water deficit, we performed a differential display RT-PCR (DDRT) analysis using roots of bean plants grown aeroponically and submitted to dehydration. This allowed us to visualise 1200 DDRT bands, 8.7% of which showed a clear regulation by dehydration, and to clone 42 cDNAs, called PvD1 to PvD42. Among them, 20 early-dehydration-responsive cDNAs were selected by reverse northern that were induced or repressed before detectable water status changes and induction of ABA-regulated genes. Northern analysis for 16 PvD clones confirmed these early regulations and allowed us to identify four late dehydration-responsive genes. Their putative involvement in signalling, protein turn-over and translocation, chaperones as well as root growth modulations in response to water stress is discussed.

17.
Plant Mol Biol ; 51(3): 341-9, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12602865

ABSTRACT

A cDNA coding for a putative organic cation transporter (OCT) was isolated from Phaseolus vulgaris roots by differential display RT-PCR and the corresponding full-length cDNA (named PvOCT1) was subsequently obtained by RACE-PCR. Hydropathy profiles of the deduced amino acid sequence (547 residues) predicted the existence of twelve membrane-spanning domains, which are highly conserved in the major facilitator superfamily (MFS). Three specific domains, which characterize organic ion transporters in animals, can also be observed in the predicted protein. In the non-stressed plants, northern analysis showed that PvOCT1 is strongly expressed in roots and stems, while in situ hybridization revealed the presence of PvOCT1 transcripts in phloem cells. In roots PvOCT1 transcript levels transitorily increased after one hour of dehydration and then dramatically decreased. This decrease was associated with enhanced abundance of PvNeED1 mRNA encoding the enzyme thought to catalyze the limiting step of abscisic acid biosynthesis.


Subject(s)
Organic Cation Transporter 1/genetics , Phaseolus/genetics , Plant Proteins/genetics , Plant Structures/metabolism , Water/pharmacology , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation, Plant/drug effects , Molecular Sequence Data , Organic Cation Transporter 1/metabolism , Plant Proteins/metabolism , Plant Structures/genetics , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid
18.
Plant Mol Biol ; 50(4-5): 687-98, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12374300

ABSTRACT

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.


Subject(s)
Cystatins/genetics , Seeds/genetics , Triticum/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cystatins/metabolism , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Molecular Sequence Data , Peptide Hydrolases/metabolism , Seeds/growth & development , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Time Factors
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