Marker gene tethering by nucleoporins affects gene expression in plants

In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localise at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants

A novel family of plant nuclear envelope associated proteins

This paper describes the characterisation of a new family of higher plant nuclear envelope associated proteins (NEAPs) that interact with 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 knock out mutant showed reduced root growth and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as INM anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure

The plant LINC complex at the nuclear envelope

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

Extensive conservation of sequences and chromatin structures in the bxd Polycomb Response Element among Drosophilid species

The Polycomb Response Element (PRE) is the nucleation site for the Polycomb silencing complexes. The sequences responsible for the recruitment of the components of the Polycomb complex are not well understood. A comparison of the bxd PRE sequences from several different Drosophila species shows that some changes have occurred during phylogeny but large blocks of sequence are conserved after a divergence of some 60 million years. We compare the PRE sequences, the sites of some known PRE binding proteins, the conservation of DNasel hypersensitive sites and relate them to the sequence of the Ultrabithorax promoter which these PREs regulate

S1 SINE retroposons are methylated at symmetrical and non-symmetrical positions in Brassica napus: identification of a preferred target site for asymmetrical methylation.

International audienceDNA methylation has been often proposed to operate as a genome defence system against parasitic mobile elements. To test this possibility, the methylation status of a class of plant mobile elements, the S1Bn SINEs, was analysed in detail using the bisulfite modification method. We observed that S1Bn SINE retroposons are methylated at symmetrical and asymmetrical positions. Methylated cytosines are not limited to transcriptionally important regions but are well distributed along the sequence. S1Bn SINE retroposons are two-fold more methylated than the average methylation level of the Brassica napus nuclear DNA. By in situ hybridization, we showed that this high level of methylation does not result from the association of S1Bn elements to genomic regions known to be highly methylated suggesting that S1Bn elements were specifically methylated. A detailed analysis of the methylation context showed that S1Bn cytosines in symmetrical CpG and CpNpG sites are methylated at a level of 87% and 44% respectively. We observed that 5.3% of S1Bn cytosines in non-symmetrical positions were also methylated. Of this asymmetrical methylation, 57% occurred at a precise motif (Cp(A/T)pA) that only represented 12% of the asymmetrical sites in S1Bn sequences suggesting that it represents a preferred asymmetrical methylation site. This motif is methylated in S1Bn elements at only half the level observed for the Cp(A/T)pG sites. We show that non-S1Bn CpTpA sites can also be methylated in DNA from B. napus and from other plant species

S1 SINE retroposons are methylated at symmetrical and non-symmetrical positions in Brassica napus: identification of a preferred target site for asymmetrical methylation.

International audienceDNA methylation has been often proposed to operate as a genome defence system against parasitic mobile elements. To test this possibility, the methylation status of a class of plant mobile elements, the S1Bn SINEs, was analysed in detail using the bisulfite modification method. We observed that S1Bn SINE retroposons are methylated at symmetrical and asymmetrical positions. Methylated cytosines are not limited to transcriptionally important regions but are well distributed along the sequence. S1Bn SINE retroposons are two-fold more methylated than the average methylation level of the Brassica napus nuclear DNA. By in situ hybridization, we showed that this high level of methylation does not result from the association of S1Bn elements to genomic regions known to be highly methylated suggesting that S1Bn elements were specifically methylated. A detailed analysis of the methylation context showed that S1Bn cytosines in symmetrical CpG and CpNpG sites are methylated at a level of 87% and 44% respectively. We observed that 5.3% of S1Bn cytosines in non-symmetrical positions were also methylated. Of this asymmetrical methylation, 57% occurred at a precise motif (Cp(A/T)pA) that only represented 12% of the asymmetrical sites in S1Bn sequences suggesting that it represents a preferred asymmetrical methylation site. This motif is methylated in S1Bn elements at only half the level observed for the Cp(A/T)pG sites. We show that non-S1Bn CpTpA sites can also be methylated in DNA from B. napus and from other plant species

Utilization of the IR hybrid dysgenesis system in Drosophila to test in vivo mobilization of synthetic SINEs sharing 3' homology with the I factor.

International audienceThe 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

EMP4 GENE

The present invention relates to altering plant development and more particularly to altering the development of the plant endosperm. It concerns in particular nucleic acid molecules which alter the endosperm development

Different mutations in the ZmCAD2 gene underlie the maize brown-midrib1 (bm1) phenotype with similar effects on lignin characteristics and have potential interest for bioenergy production

International audienceThe maize ZmCAD2 gene has been fully sequenced in several normal and bm1 maize lines, highlighting a large diversity of mutations underlying the bm1 phenotype. Mutations in three bm1 lines (F2bm1, A619bm1, and 511Jbm1) were found corresponding to short InDels inducing premature stop codons and truncated proteins. In two lines (511Kbm1 and 5803Cbm1), mutations were limited to an only SNP or to a few SNP, modifying the catalytic sites, and likely inactivating the proteins. Results also established that the 5803Ibm7 mutant was in fact a bm1 mutant, with a sequence fully identical to the 5803Cbm1 sequence. The two new F7803bm1 (natural mutant) and Ev2210bm1 (transposon tagging Mutator investigations) both had a transposon insertion in the ZmCAD2 DNA, resulting in a truncated protein, even if the mRNA was produced. The biochemical characteristics of the Ev2210bm1 lignins corroborated the signature of CAD2 deficiency in plants, with the presence of aldehydes and atypical compounds and linkages. Considering lignin structure and content, CAD2 is likely a good target for the improvement of energy production based on maize and grass lignocellulosic biomass, including a greater susceptibility to environmentally friendly pretreatments, as it was shown in bmr sorghum. The interest in maize bm1 hybrids for cattle feeding also should be considered because there seem to be little or limited negative effects of CAD2 mutations on other agronomical traits

Toward the discovery of maize cell wall genes involved in silage quality and capacity to biofuel production

Silage maize is Currently, with grazing, the basis of cattle feeding. In the near future, maize stover and cereal straws will also be a major Source of carbohydrates for sustainable biofuel production. The embedding of cell wall in lignins and the linkages between lignins, p-hydroxycinnamic acids, and arabinoxylans greatly influences cell wall properties, including the enzymatic degradability of structural polysaccharides in animal rumen or industrial fermenters. Breeding for higher silage quality and biofuel production will thus be based on the discovery of genetic traits involved in each component biosynthesis and deposition in each lignified tissue. Genes involved or putatively involved in the biosynthesis of the grass cell wall were searched for, including phenolic Compounds, cell wall carbohydrates and regulation factors. While most cytosolic steps of monolignol biosynthesis have been identified, most of lignin pathway genes belong to small multigene families which were all identified based on data available in the Maize Genomics Sequencing Project (MGSP) database. Cell wall carbohydrate genes were identified based on their Arabidopsis orthologs and previous research in C3 grasses by MITCHELL et al. (2007). Transcription and regulation factors of cell wall genes were similarly identified based their orthologs described both in Arabidopsis and woody species. All these genes were mapped in silico considering their physical position in the MGSP database. Physical positions of previously described QTL for cell wall degradability, lignin and p-hydroxycinnamic acid contents were also searched for based on the position of the flanking market in the MGSP database and distances between QTL and flanking markets. While only a few lignin pathway genes mapped to QTL positions, several colocalizations were shown between QTL and transcription factor physical positions. This last result is in agreement with expression Studies which highlighted that several genes in the lignin pathway are simultaneously under-expressed in lines with higher cell wall degradability, likely corroborating an upstream regulation rather than co-regulation phenomena. However, none of these possible candidate genes have yet been validated and many QTL still do not have relevant candidates. A lot of relevant candidate genes are still to be discovered among those involved in lignin pathway gene regulation, in regulation of lignified tissue assembly, and in cell wall carbohydrate biosynthesis and deposition. In addition, in spite of their critical involvement in maize cell wall assembly and degradability, genes involved in ferulic acid biosynthesis and linkages with other cell wall components are little known