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International audience; Untangling chromatin interactions The plant nucleus contains myriad subdomains that define and are defined by the composition of their chromatin. On a whole nucleus scale, this includes regions of active euchromatin and inactive heterochromatin, although the boundaries between these states are blurred. Different loci constantly enter and exit nuclear structures including the nucleolus and transient regulatory nuclear bodies. Chromatin is defined by its complex combinations of epigenetic modifications and variation in its constituent histone proteins. Here we preview articles that describe many of the mechanisms that regulate this dynamic chromatin.

In this review, we explore recent advances in knowledge of the structure and dynamics of the plant nuclear envelope. As a paradigm, we focused our attention on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, a structurally conserved bridging complex comprising SUN domain proteins in the inner nuclear membrane and KASH domain proteins in the outer nuclear membrane. Studies have revealed that this bridging complex has multiple functions with structural roles in positioning the nucleus within the cell, conveying signals across the membrane and organizing chromatin in the 3D nuclear space with impact on gene transcription. We also provide an up-to-date survey in nuclear dynamics research achieved so far in the model plant Arabidopsis thaliana that highlights its potential impact on several key plant functions such as growth, seed maturation and germination, reproduction and response to biotic and abiotic stress. Finally, we bring evidences that most of the constituents of the LINC Complex and associated components are, with some specificities, conserved in monocot and dicot crop species and are displaying very similar functions to those described for Arabidopsis. This leads us to suggest that a better knowledge of this system and a better account of its potential applications will in the future enhance the resilience and productivity of crop plants.

International audience; Histone chaperones regulate the flow and dynamics of histone variants and ensure their assembly into nucleosomal structures, thereby contributing to the repertoire of histone variants in specialized cells or tissues. To date, not much is known on the distribution of histone variants and their modifications in the dry seed embryo. Here, we bring evidence that genes encoding the replacement histone variant H3.3 are expressed in Arabidopsis dry seeds and that embryo chromatin is characterized by a low H3.1/H3.3 ratio. Loss of HISTONE REGULATOR A (HIRA), a histone chaperone responsible for H3.3 deposition, reduces cellular H3 levels and increases chromatin accessibility in dry seeds. These molecular differences are accompanied by increased seed dormancy in hira-1 mutant seeds. The loss of HIRA negatively affects seed germination even in the absence of HISTONE MONOUBIQUITINATION 1 or TRANSCRIPTION ELONGATION FACTOR II S, known to be required for seed dormancy. Finally, hira-1 mutant seeds show lower germination efficiency when aged under controlled deterioration conditions or when facing unfavorable environmental conditions such as high salinity. Altogether, our results reveal a dependency of dry seed chromatin organization on the replication-independent histone deposition pathway and show that HIRA contributes to modulating seed dormancy and vigor.

Mathematical modelling to compute ground truth from 3D images is an area of research that can strongly benefit from machine learning methods. Deep neural networks (DNNs) are state-of-the-art methods design for solving these kinds of difficulties. Convolutional neural networks (CNNs), as one class of DNNs, can overcome special requirements of quantitative analysis especially when image segmentation is needed. This article presents a system that uses a cascade of CNNs with symmetric blocks of layers in chain, dedicated to 3D image segmentation from microscopic images of 3D nuclei. The system is designed through eight experiments that differ in following aspects: number of training slices and 3D samples for training, usage of pre-trained CNNs and number of slices and 3D samples for validation. CNNs parameters are optimized using linear, brute force, and random combinatorics, followed by voter and median operations. Data augmentation techniques such as reflection, translation and rotation are used in order to produce sufficient training set for CNNs. Optimal CNN parameters are reached by defining 11 standard and two proposed metrics. Finally, benchmarking demonstrates that CNNs improve segmentation accuracy, reliability and increased annotation accuracy, confirming the relevance of CNNs to generate high-throughput mathematical ground truth 3D images.

Developmental phase transitions are often characterized by changes in the chromatin landscape and heterochromatin reorganization. In Arabidopsis, clustering of repetitive heterochromatic loci into so-called chromocenters is an important determinant of chromosome organization in nuclear space. Here, we investigated the molecular mechanisms involved in chromocenter formation during the switch from a heterotrophic to a photosynthetically competent state during early seedling development. We characterized the spatial organization and chromatin features at centromeric and pericentromeric repeats and identified mutant contexts with impaired chromocenter formation. We find that clustering of repetitive DNA loci into chromocenters takes place in a precise temporal window and results in reinforced transcriptional repression. Although repetitive sequences are enriched in H3K9me2 and linker histone H1 before repeat clustering, chromocenter formation involves increasing enrichment in H3.1 as well as H2A.W histone variants, hallmarks of heterochromatin. These processes are severely affected in mutants impaired in replication-coupled histone assembly mediated by CHROMATIN ASSEMBLY FACTOR 1 (CAF-1). We further reveal that histone deposition by CAF-1 is required for efficient H3K9me2 enrichment at repetitive sequences during chromocenter formation. Taken together, we show that chromocenter assembly during post-germination development requires dynamic changes in nucleosome composition and histone post-translational modifications orchestrated by the replication-coupled H3.1 deposition machinery.

International audience; In the interphase cell nucleus, chromosomes adopt a conserved and non-random arrangement in subnuclear domains called chromosome territories (CTs). Whereas chromosome translocation can affect CT organization in tumor cell nuclei, little is known about how aneuploidies can impact CT organization. Here, we performed 3D-FISH on control and trisomic 21 nuclei to track the patterning of chromosome territories, focusing on the radial distribution of trisomic HSA21 as well as 11 disomic chromosomes. We have established an experimental design based on cultured chorionic villus cells which keep their original mesenchymal features including a characteristic ellipsoid nuclear morphology and a radial CT distribution that correlates with chromosome size. Our study suggests that in trisomy 21 nuclei, the extra HSA21 induces a shift of HSA1 and HSA3 CTs out toward a more peripheral position in nuclear space and a higher compaction of HSA1 and HSA17 CTs. We posit that the presence of a supernumerary chromosome 21 alters chromosome compaction and results in displacement of other chromosome territories from their usual nuclear position.

Identification of membrane protein interactomes is a key issue to better understand how these molecules carry out their functions. However, protein-protein interactions using conventional interaction assays are particularly challenging for integral membrane proteins, because of their hydrophobic nature. Here we describe the membrane yeast two-hybrid (MbY2H) system, a powerful tool for identifying the interactors of membrane and membrane-associated proteins.

The analysis of nuclear envelope components and their function has recently been progressed by the use of computational methods of analysis. The methods in this chapter provided by members of the International Plant Nucleus Consortium address the identification of novel nuclear envelope proteins and the study of structure and mobility of the nucleus. DORY2 is an upgrade of the KASH-finder DORY, and NucleusJ is used to characterize the three-dimensional structure of the nucleus in light microscope images. Finally, a method is provided for analysis of the migration of the nucleus, a key technique for exploring the function of plant nuclear proteins.

Image analysis is a classical way to study nuclear organization. While nuclear organization used to be investigated by colorimetric or fluorescent labeling of DNA or specific nuclear compartments, new methods in microscopy imaging now enable qualitative and quantitative analyses of chromatin pattern, and nuclear size and shape. Several procedures have been developed to prepare samples in order to collect 3D images for the analysis of spatial chromatin organization, but only few preserve the positional information of the cell within its tissue context. Here, we describe a whole mount tissue preparation procedure coupled to DNA staining using the PicoGreen® intercalating agent suitable for image analysis of the nucleus in living and fixed tissues. 3D Image analysis is then performed using NucleusJ, an open source ImageJ plugin, which allows for quantifying variations in nuclear morphology such as nuclear volume, sphericity, elongation, and flatness as well as in heterochromatin content and position in respect to the nuclear periphery.

International audience; Histones are essential components of the nucleosome, the major chromatin subunit that structures linear DNA molecules and regulates access of other proteins to DNA. Specific histone chaperone complexes control the correct deposition of canonical histones and their variants to modulate nucleosome structure and stability. In this study, we characterize the Arabidopsis thaliana Alpha Thalassemia-mental Retardation X-linked (ATRX) ortholog and show that ATRX is involved in histone H3 deposition. Arabidopsis ATRX mutant alleles are viable, but show developmental defects and reduced fertility. Their combination with mutants of the histone H3.3 chaperone HIRA (Histone Regulator A) results in impaired plant survival, suggesting that HIRA and ATRX function in complementary histone deposition pathways. Indeed, ATRX loss of function alters cellular histone H3.3 pools and in consequence modulates the H3.1/H3.3 balance in the cell. H3.3 levels are affected especially at genes characterized by elevated H3.3 occupancy, including the 45S ribosomal DNA (45S rDNA) loci, where loss of ATRX results in altered expression of specific 45S rDNA sequence variants. At the genome-wide scale, our data indicate that ATRX modifies gene expression concomitantly to H3.3 deposition at a set of genes characterized both by elevated H3.3 occupancy and high expression. Together, our results show that ATRX is involved in H3.3 deposition and emphasize the role of histone chaperones in adjusting genome expression.

International audience; The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences.

International audience; In this study, we explore the plasticity during evolution of proteins of the higher plant nuclear envelope (NE) from the most ancestral plant species to advanced angiosperms. The higher plant NE contains a functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex based on conserved Sad1-Unc84 (SUN) domain proteins and plant specific Klarsicht/Anc1/Syne homology (KASH) domain proteins. Recent evidence suggests the presence of a plant lamina underneath the inner membrane and various coiled-coil proteins have been hypothesized to be associated with it including Crowded Nuclei (CRWN; also termed LINC and NMCP), Nuclear Envelope Associated Protein (NEAP) protein families as well as the CRWN binding protein KAKU4. SUN domain proteins appear throughout with a key role for mid-SUN proteins suggested. Evolution of KASH domain proteins has resulted in increasing complexity, with some appearing in all species considered, while other KASH proteins are progressively gained during evolution. Failure to identify CRWN homologs in unicellular organisms included in the study and their presence in plants leads us to speculate that convergent evolution may have occurred in the formation of the lamina with each kingdom having new proteins such as the Lamin B receptor (LBR) and Lamin-Emerin-Man1 (LEM) domain proteins (animals) or NEAPs and KAKU4 (plants). Our data support a model in which increasing complexity at the nuclear envelope occurred through the plant lineage and suggest a key role for mid-SUN proteins as an early and essential component of the nuclear envelope.

International audience; This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

The precise location of chromatin domains within the cell nucleus has\ud seen growing recognition in the past decade as an additional\ud mechanism of controlling gene expression in both plants and\ud animals (Dekker et al., 2017). Consequently, international efforts are\ud devoted to understanding the organising principle of this organelle in\ud plants, and notably the nature and the role of functional compartments\ud on gene expression (Graumann et al., 2013; Sotelo-Silveira et al.,\ud 2018). The European cooperation ‘Impact of Nuclear Domains on\ud Gene Expression and Plant Traits’ (INDEPTH) brings together\ud molecular cell biologists, plant physiologists, bioinformaticians, image\ud analysts and computer scientists. They aim to address the question of\ud how nuclear architecture, chromatin organisation and gene expression\ud are connected in plants, particularly in relation to traits of interest such\ud as biomass, reproduction and resistance to pathogens (https://\ud www.brookes.ac.uk/indepth/). The kick-off meeting of the INDEPTH\ud consortium took place in Clermont-Ferrand, France, on 12–14thMarch\ud 2018, where more than 80 researchers set the agenda for the coming\ud four years of research and collaboration.

International audience; The centromere is a specific chromosomal region where the kinetochore assembles to ensure the faithful segregation of sister chromatids during mitosis and meiosis. Centromeres are defined by a local enrichment of the specific histone variant CenH3 mostly at repetitive satellite sequences. A larger pericentromeric region containing repetitive sequences and transposable elements surrounds the centromere that adopts a particular chromatin state characterized by specific histone variants and post-translational modifications and forms a transcriptionally repressive chromosomal environment. In the model organism Arabidopsis thaliana centromeric and pericentromeric domains form conspicuous heterochromatin clusters called chromocenters in interphase. Here we discuss, using Arabidopsis as example, recent insight into mechanisms involved in maintenance and establishment of centromeric and pericentromeric chromatin signatures as well as in chromocenter formation.

Summary: NucleusJ is a simple and user-friendly ImageJ plugin dedicated to the characterization of nuclear morphology and chromatin organization in 3D. Starting from image stacks, the nuclear boundary is delimited by combining the Otsu segmentation method with optimization of nuclear sphericity. Chromatin domains are segmented by partitioning the nucleus using a 3D watershed algorithm and by thresholding a contrast measure over the resulting regions. As output, NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with different nuclear organization. Availability and implementation: Dataset of nuclei is available at https://www.gred-clermont.fr/media/WorkDirectory.zip. NucleusJ is available at http://imagejdocu.tudor.lu/doku.php?id=plugin:stacks:nuclear_analysis_plugin:start. Contact: philippe.andrey@versailles.inra.fr and christophe.tatout@univ-bpclermont.fr Supplementary information: Supplementary data are available at Bioinformatics online.

International audience; SUN-domain proteins belong to a gene family including classical Cter-SUN and mid-SUN subfamilies differentiated by the position of the SUN domain within the protein. Although present in animal and plant species, mid-SUN proteins have so far remained poorly described. Here, we used a combination of genetics, yeast two-hybrid and in planta transient expression methods to better characterize the SUN family in Arabidopsis thaliana. First, we validated the mid-SUN protein subfamily as a monophyletic group conserved from yeast to plant. Arabidopsis Cter-SUN (AtSUN1 and AtSUN2) and mid-SUN (AtSUN3 and AtSUN4) proteins expressed as fluorescent protein fusions are membrane-associated and localize to the nuclear envelope (NE) and endoplasmic reticulum. However, only the Cter-SUN subfamily is enriched at the NE. We investigated interactions in and between members of the two subfamilies and identified the coiled-coil domain as necessary for mediating interactions. The functional significance of the mid-SUN subfamily was further confirmed in mutant plants as essential for early seed development and involved in nuclear morphology. Finally, we demonstrated that both subfamilies interact with the KASH domain of AtWIP1 and identified a new root-specific KASH-domain protein, AtTIK. AtTIK localizes to the NE and affects nuclear morphology. Our study indicates that Arabidopsis Cter-SUN and mid-SUN proteins are involved in a complex protein network at the nuclear membranes, reminiscent of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex found in other kingdoms.

International audience; Significant advances in understanding the plant nuclear envelope have been made over the past few years; indeed, knowledge of the protein network at the nuclear envelope is rapidly growing. One such network, the linker of nucleoskeleton and cytoskeleton (LINC) complex, is known in animals to connect chromatin to the cytoskeleton through the nuclear envelope. The LINC complex is made of Sad1/Unc84 (SUN) and Klarsicht/Anc1/Syne1 homology (KASH) proteins which have been recently characterized in plants. SUN proteins are located within the inner nuclear membrane, while the KASH proteins are included into the outer nuclear membrane. SUN and KASH domains interact and bridge the two nuclear membranes. In Arabidopsis, KASH proteins also interact with the tryptophan-proline-proline (WPP) domain-interacting tail-anchored protein 1 (WIT1), associated with the nuclear pore complex and with myosin XI-i which directly interacts with the actin cytoskeleton. Although evidence for a plant LINC complex connecting the nucleus to the cytoskeleton is growing, its interaction with chromatin is still unknown, but knowledge gained from animal models strongly suggests its existence in plants. Possible functions of the plant LINC complex in cell division, nuclear shape, and chromatin organization are discussed.

The characterization of a simple bacterial system using commercially available plasmids and strains, developed to assess the effectiveness of trans-acting (antisense RNA and ribozymes) RNA in Escherichia coli, is reported. This system was used to test various trans-acting RNA molecules against the expression of the I factor, a functional transposable element from Drosophila melanogaster. For this target, results indicate that antisense RNA efficiency depends on the hybridization length between sense and antisense RNA. The introduction of a single hammerhead ribozyme within the antisense RNAs does not increase its inhibitory activity. These predictions were subsequently confirmed in Drosophila melanogaster.

International audience; The reverse transcription of RNA in DNA is responsible for the generation of large families of repetitive sequences called retroposons or non-LTR retrotransposons. Recent reports established that the integration of mammalian SINE and LINE retroposons occurs at nonrandom staggered breaks, probably resulting from the action of a LINE-encoded endonuclease (Feng et al. 1996; Jurka 1997; Jurka et al. 1998). We report here that plant SINE S1 retroposons also integrate at nonrandom staggered breaks. One of the two nicks involved in S1 integration is associated mainly with the 5'-Y/AAANNNG-3' motif. The other nick at opposite DNA strand occurs preferably within 14-16 bp, a situation also observed for mammalian retroposons, but is not associated with any specific motif. Further studies on the distribution of dinucleotides surrounding the two nicking sites showed that, as for mammalian retroposons, S1 retroposons integrate at sites rich in TA, CA, and TG dinucleotides. These dinucleotides were reported as specific DNA sites where special DNA structures called "kinks" may occur under bending constraints. Nicking sites are preceded by peaks in frequency of di-pyrimidine followed by peaks of di-purine. These results suggest that the general A/T richness of a given DNA region and the presence of short runs of pyrimidines followed by short runs of purines could represent a favorable context for the integration of retroposons. In such a context, an endonuclease upon fixation could be able to generate the kink at the pyrimidine/purine transition and to nick the DNA. The similarities in target site selection observed for plant and mammalian retroposons suggest that retroposition is a surprisingly well conserved process.

Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1(-). In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1(-) exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors' knowledge, this is the first functional characterization of CCR1 in a grass species.

The pentatricopeptide repeat (PPR) family represents one of the largest gene families in plants, with >440 members annotated in Arabidopsis thaliana. PPR proteins are thought to have a major role in the regulation of posttranscriptional processes in organelles. Recent studies have shown that Arabidopsis PPR proteins play an essential, nonredundant role during embryogenesis. Here, we demonstrate that mutations in empty pericarp4 (emp4), a maize (Zea mays) PPR-encoding gene, confer a seed-lethal phenotype. Mutant endosperms are severely impaired, with highly irregular differentiation of transfer cells in the nutrient-importing basal endosperm. Analysis of homozygous mutant plants generated from embryo-rescue experiments indicated that emp4 also affects general plant growth. The emp4-1 mutation was identified in an active Mutator (Mu) population, and cosegregation analysis revealed that it arose from a Mu3 element insertion. Evidence of emp4 molecular cloning was provided by the isolation of four additional emp4 alleles obtained by a reverse genetics approach. emp4 encodes a novel type of PPR protein of 614 amino acids. EMP4 contains nine 35–amino acid PPR motifs and an N-terminal mitochondrion-targeted sequence peptide, which was confirmed by a translational EMP4–green fluorescent protein fusion that localized to mitochondria. Molecular analyses further suggest that EMP4 is necessary to regulate the correct expression of a small subset of mitochondrial transcripts in the endosperm.

The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.

International audience; UDPGDH (UDP-D-glucose dehydrogenase) oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-D-glucuronate), the precursor of UDP-D-xylose and UDP-L-arabinose, major cell wall polysaccharide precursors. Maize (Zea mays L.) has at least two putative UDPGDH genes (A and B), according to sequence similarity to a soya bean UDPGDH gene. The predicted maize amino acid sequences have 95% similarity to that of soya bean. Maize mutants with a Mu-element insertion in UDPGDH-A or UDPGDH-B were isolated (udpgdh-A1 and udpgdh-B1 respectively) and studied for changes in wall polysaccharide biosynthesis. The udpgdh-A1 and udpgdh-B1 homozygotes showed no visible phenotype but exhibited 90 and 60-70% less UDPGDH activity respectively than wild-types in a radiochemical assay with 30 microM UDP-glucose. Ethanol dehydrogenase (ADH) activity varied independently of UDPGDH activity, supporting the hypothesis that ADH and UDPGDH activities are due to different enzymes in maize. When extracts from wild-types and udpgdh-A1 homozygotes were assayed with increasing concentrations of UDP-Glc, at least two isoforms of UDPGDH were detected, having K(m) values of approx. 380 and 950 microM for UDP-Glc. Leaf and stem non-cellulosic polysaccharides had lower Ara/Gal and Xyl/Gal ratios in udpgdh-A1 homozygotes than in wild-types, whereas udpgdh-B1 homozygotes exhibited more variability among individual plants, suggesting that UDPGDH-A activity has a more important role than UDPGDH-B in UDP-GlcA synthesis. The fact that mutation of a UDPGDH gene interferes with polysaccharide synthesis suggests a greater importance for the sugar nucleotide oxidation pathway than for the myo-inositol pathway in UDP-GlcA biosynthesis during post-germinative growth of maize.

International audience; The current model of short interspersed nuclear element (SINE) mobility suggests that these non-coding retroposons are able to recruit for their own benefits the enzymatic machinery encoded by autonomous long interspersed nuclear elements (LINEs). The recent characterization of potential SINE-LINE partner pairs that share common 3' end sequences concurs with this model and has led to a potent picture of tRNA-derived SINEs consisting of a tripartite functional structure (Mol. Cell. Biol. 16 (1996) 3756; Mol. Biol. Evol. 16 (1999) 1238; Proc. Natl. Acad. Sci. USA 96 (1999) 2869). This structure consist of a 5' polIII tRNA-related promoter region, a central conserved domain and a variable 3' region with homology to the 3' end of LINEs, believed to be essential to direct recognition by the LINE proteins. To test this model in vivo, we have designed synthetic SINEs possessing this 'canonical' structure, including 3' homology to the 3' UTR of the LINE I factor from Drosophila. These synthetic elements were introduced in a Drosophila reactive strain, and SINE retroposition was assessed following dysgenic crosses that are known to induce high levels of I factor germinal transposition. In the progeny from the dysgenic crosses 3400-4000 flies were analyzed but no retroposed copy of the chimeric SINEs was detected, indicating that what is assumed to be a typical SINE structure is not sufficient per se to allow efficient trans-mobilization of our synthetic SINEs by an actively amplifying partner LINE. Alternatively, the apparent absence of natural fly SINEs may underline intrinsic properties of fly biology that are incompatible with the genesis and/or propagation of SINE-like elements.

The I factor (IF) is a LINE-like transposable element from Drosophila melanogaster. IF is silenced in most strains, but under special circumstances its transposition can be induced and correlates with the appearance of a syndrome of female sterility called hybrid dysgenesis. To elucidate the relationship between IF expression and female sterility, different transgenic antisense and/or sense RNAs homologous to the IF ORF1 have been expressed. Increasing the transgene copy number decreases both the expression of an IF-lacZ fusion and the intensity of the female sterile phenotype, demonstrating that IF expression is correlated with sterility. Some transgenes, however, exert their repressive abilities not only through a copy number-dependent zygotic effect, but also through additional maternal and paternal effects that may be induced at the DNA and/or RNA level. Properties of the maternal effect have been detailed: (1) it represses hybrid dysgenesis more efficiently than does the paternal effect; (2) its efficacy increases with both the transgene copy number and the aging of sterile females; (3) it accumulates slowly over generations after the transgene has been established; and (4) it is maintained for at least two generations after transgene removal. Conversely, the paternal effect increases only with female aging. The last two properties of the maternal effect and the genuine existence of a paternal effect argue for the occurrence, in the IF regulation pathway, of a cellular memory transmitted through mitosis, as well as through male and female meiosis, and akin to epigenetic phenomena.

International audience; In Drosophila melanogaster, several factors have been suggested to influence the rates of P-element transposition and excision, including position effects, size and structure of the elements and differences in transposase source. We have investigated the effect of the size of the starting P-element on the rates of excision and transposition. Four transgenes localized at the same insertion site on the X chromosome and which differ by the number of copies of an internal repeated sequence, were studied. Transgenes with sizes ranging from 11 kb to 22 kb excise at similar rates, and size does not correlate with the differences in transposition rate between them. We also studied the behavior of double P-elements, located at the same site and arranged in various configurations: nested, contiguous or separated by a few base pairs, in the same or reverse orientation. These double P-elements display different mobilities depending on the arrangement of the two transgenes. Transposition and excision rates were also studied for an insertion bearing four transgenes in very close proximity. Our results suggest that several neighboring elements could excise together. We also propose a new model to explain the formation of all the double P-elements we describe.