Gilles Vachon

AtGA3ox2, a Key Gene Responsible for Bioactive Gibberellin Biosynthesis, Is Regulated during Embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both β-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.

The Immunophilin-Interacting Protein AtFIP37 from Arabidopsis Is Essential for Plant Development and Is Involved in Trichome Endoreduplication

The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::β-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.

Trichome specific expression of the tobacco (Nicotiana sylvestris) cembratrien-ol synthase genes is controlled by both activating and repressing cis-regions

Tobacco (Nicotiana sylvestris) glandular trichomes make an attractive target for isoprenoid metabolic engineering because they produce large amounts of one type of diterpenoids, α- and β-cembratrien-diols. This article describes the establishment of tools for metabolic engineering of tobacco trichomes, namely a transgenic line with strongly reduced levels of diterpenoids in the exudate and the characterization of a trichome specific promoter. The diterpene-free tobacco line was generated by silencing the major tobacco diterpene synthases, which were found to be encoded by a family of four highly similar genes (NsCBTS-2a, NsCBTS-2b, NsCBTS-3 and NsCBTS-4), one of which is a pseudogene. The promoter regions of all four CBTS genes were sequenced and found to share over 95% identity between them. Transgenic plants expressing uidA under the control of the NsCBTS-2a promoter displayed a specific pattern of GUS expression restricted exclusively to the glandular cells of the tall secretory trichomes. A series of sequential and internal deletions of the NsCBTS-2a promoter led to the identification of two cis-acting regions. The first, located between positions -589 to -479 from the transcription initiation site, conferred a broad transcriptional activation, not only in the glandular cells, but also in cells of the trichome stalk, as well as in the leaf epidermis and the root. The second region, located between positions -279 to -119, had broad repressor activity except in trichome glandular cells and is mainly responsible for the specific expression pattern of the NsCBTS-2a gene. These results establish the basis for the identification of trans-regulators required for the expression of the CBTS genes restricted to the secretory cells of the glandular trichomes.

Both forms of translational initiation factor IF2(α and β) are required for maximal growth of Escherichia coli evidence for two translational initiation codons for IF2β

Abstract The gene infB codes for two forms of translational initiation factor IF2; IF2α (97,300 Da) and IF2β (79,700 Da). IF2β arises from an independent translational event on a GUG codon located 471 bases downstream from IF2α start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2β synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2β results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2β have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2β exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2β start codons could we construct plasmids that express only IF2α. A plasmid expressing only IF2β was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic λ phage, we could cure the λ phage when the plasmids expressing only one form of IF2 were supplied in trans . We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli , but that growth is retarded at 37 °C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

A variant of LEAFY reveals its capacity to stimulate meristem development by inducing RAX1

In indeterminate inflorescences, floral meristems develop on the flanks of the shoot apical meristem, at positions determined by auxin maxima. The floral identity of these meristems is conferred by a handful of genes called floral meristem identity genes, among which the LEAFY (LFY) transcription factor plays a prominent role. However, the molecular mechanism controlling the early emergence of floral meristems remains unknown. A body of evidence indicates that LFY may contribute to this developmental shift, but a direct effect of LFY on meristem emergence has not been demonstrated. We have generated a LFY allele with reduced floral function and revealed its ability to stimulate axillary meristem growth. This role is barely detectable in the lfy single mutant but becomes obvious in several double mutant backgrounds and plants ectopically expressing LFY. We show that this role requires the ability of LFY to bind DNA, and is mediated by direct induction of REGULATOR OF AXILLARY MERISTEMS1 (RAX1) by LFY. We propose that this function unifies the diverse roles described for LFY in multiple angiosperm species, ranging from monocot inflorescence identity to legume leaf development, and that it probably pre-dates the origin of angiosperms.

GeBP and GeBP-Like Proteins Are Noncanonical Leucine-Zipper Transcription Factors That Regulate Cytokinin Response in Arabidopsis

Understanding the role of transcription factors (TFs) is essential in reconstructing developmental regulatory networks. The plant-specific GeBP TF family of Arabidopsis thaliana (Arabidopsis) comprises 21 members, all of unknown function. A subset of four members, the founding member GeBP and GeBP-like proteins (GPL) 1, 2, and 3, shares a conserved C-terminal domain. Here we report that GeBP/GPL genes represent a newly defined class of leucine-zipper (Leu-zipper) TFs and that they play a redundant role in cytokinin hormone pathway regulation. Specifically, we demonstrate using yeast, in vitro, and split-yellow fluorescent protein in planta assays that GeBP/GPL proteins form homo- and heterodimers through a noncanonical Leu-zipper motif located in the C-terminal domain. A triple loss-of-function mutant of the three most closely related genes gebp gpl1 gpl2 shows a reduced sensitivity to exogenous cytokinins in a subset of cytokinin responses such as senescence and growth, whereas root inhibition is not affected. We find that transcript levels of type-A cytokinin response genes, which are involved in the negative feedback regulation of cytokinin signaling, are higher in the triple mutant. Using a GPL version that acts as a constitutive transcriptional activator, we show that the regulation of Arabidopsis response regulators (ARRs) is mediated by at least one additional, as yet unknown, repressor acting genetically downstream in the GeBP/GPL pathway. Our results indicate that GeBP/GPL genes encode a new class of unconventional Leu-zipper TF proteins and suggest that their role in the cytokinin pathway is to antagonize the negative feedback regulation on ARR genes to trigger the cytokinin response.

GeBP/GPL Transcription Factors Regulate a Subset of CPR5-Dependent Processes

The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical function and displays highly pleiotropic functions, particularly in pathogen responses, cell proliferation, cell expansion, and cell death. Here, we demonstrate a link between CPR5 and the GLABRA1 ENHANCER BINDING PROTEIN (GeBP) family of transcription factors. We investigated the primary role of the GeBP/GeBP-like (GPL) genes using transcriptomic analysis of the quadruple gebp gpl1,2,3 mutant and one overexpressing line that displays several cpr5-like phenotypes including dwarfism, spontaneous necrotic lesions, and increased pathogen resistance. We found that GeBP/GPLs regulate a set of genes that represents a subset of the CPR5 pathway. This subset includes genes involved in response to stress as well as cell wall metabolism. Analysis of the quintuple gebp gpl1,2,3 cpr5 mutant indicates that GeBP/GPLs are involved in the control of cell expansion in a CPR5-dependent manner but not in the control of cell proliferation. In addition, to our knowledge, we provide the first evidence that the CPR5 protein is localized in the nucleus of plant cells and that a truncated version of the protein with no transmembrane domain can trigger cpr5-like processes when fused to the VP16 constitutive transcriptional activation domain. Our results provide clues on how CPR5 and GeBP/GPLs play opposite roles in the control of cell expansion and suggest that the CPR5 protein is involved in transcription.

A chymosin-like extracellular acidic endoprotease from Myxococcus xanthus DK101: A potential new tool for protein engineering

SDS‐PAGE and N‐tenninal sequence analysis of hydrolysis products from 3 substrates containing a unique sensitive bond usually recognized by chymosin (k‐casein, a synthetic hexapeptide and a recombinant tripartite protein) revealed that a 45 kDa endoprotease of Myxococcus xanthus DK101 cleaved the same characteristic Phe‐Met bond with high specificity. Such an enzyme, easy to obtain from culture supernatant and to use in acidic conditions, could be a new tool for protein engineering.

APETALA3-nuclease hybrid protein : a potential tool for APETALA3 target gene mutagenesis

Direct target genes of homeotic genes are largely unknown in plants. The class B homeotic gene APETALA3 (AP3) is required for petal and stamen identities. The AP3 gene encodes a MADS-domain containing protein which forms heterodimers in vitro with the second class B homeotic protein PISTILLATA (PI). Here, we describe a new strategy that can be used to isolate mutants of genes that are immediate targets of AP3 or AP3:PI. The strategy is based on providing a nuclease activity to AP3 by translationally fusing the FN nuclease domain of the FokI restriction enzyme. In electro-mobility shift assays, AP3-FN:PI heterodimers display the same binding specificity for CArG-box elements as AP3:PI heterodimers, although with a lower affinity. Transgenic lines carrying the AP3-FN fusion gene under control of the 35S promoter were obtained. The 35S::AP3-FN construct is able to partially suppress the ap3-1 mutant phenotype showing that the AP3 part of the hybrid protein is functional in vivo. When crossed with the DNA-break repair deficient mutant u6h1, offspring were obtained that showed, to various degrees, a lack of fertility consistent with the role of AP3 in gamete development. The mutant phenotypes are inherited to the next generation. This is the first report of a strategy designed to create mutants of genes directly regulated by a homeotic gene.

LEAFY as a master regulator of flower development

Flowers represent a powerful reproductive structure, considered to be largely responsible for the ev…