Christelle Mazubert

Characterization of the expression of a wheat cystatin gene during caryopsis development

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.

Multiple functions of Kip-related protein5 connect endoreduplication and cell elongation.

The cell cycle inhibitor KRP5 binds chromatin to coordinately control endoreduplication and chromatin structure and to allow the expression of genes required for cell elongation. 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.

The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis

A mutation in the Arabidopsis mRNA polyadenylation factor suppresses the cell death associated with the immunity response mediated by salicylic acid and defective myoinositol biosynthesis. 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.

Identification of novel drought-related mRNAs in common bean roots by differential display RT-PCR

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.

Dual function of MIPS1 as a metabolic enzyme and transcriptional regulator.

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.

Involvement of Arabidopsis Hexokinase1 in Cell Death Mediated by Myo-Inositol Accumulation

A novel pathway of HXK1-mediated cell death in which two MIPS enzymes act cooperatively highlights a novel checkpoint of inositol homeostasis in plants. 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.

Chloroplasts activity and PAP-signaling regulate programmed cell death in Arabidopsis

Chloroplasts modulate programmed cell death via at least two different pathways: photosynthetic activity is required for cell death while a retrograde signal negatively regulates cell death and plant innate immunity. 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.

Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant

The constitutive stress response induced by chloroplast dysfunction causes early differentiation via the activation of cell cycle inhibitors. 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.

The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

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.

Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis

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.