Genetic, molecular and cellular approaches to the analysis of maize embryo development

The development of embryo structures in plants is essential for the formation of the adult plant organs. In cereals, this process has distinct features which have attracted attention from different points of view. Differential gene expression analyses have been used in order to identify genes useful as molecular markers of certain physiological, molecular or developmental processes. Several maize mutants affected in embryo development have been isolated, but only a fraction of them have been characterized at the molecular level. Molecular markers can be useful in the characterization of embryo defective mutants. Here, we describe the different techniques used in the identification of molecular marker genes for embryo development. We describe in more detail some groups of genes coding for cell wall proteins. We also describe the application of these molecular markers in the characterization of some embryo mutants

Cellular localization of the embryo-specific Hybrid PRP from Zea mays, and characterization of promoter regulatory elements of its gene

The expression, regulation and cellular localization of ZmHyPRP, a gene marker of embryo differentiation whose expression declines after ABA induction, was studied. ZmHyPRP is a proline-rich protein with a C-terminal domain having eight cysteines in a CM8 pattern. Transient expression in onion epidermal cells, transformed with a 2x35S::ZmHyPRP-GFP construction, indicated the protein is present in vesicles lining the membrane of the cell. The ZmHyPRP gene expression is under the control of classic promoter seed-specific regulatory elements such as Sph/RY and G-boxes, suggesting regulation by B3 and b-ZIP transcription factors. Promoter deletion analysis, by particle-bombardment transient transformation of maize immature embryos with serial deletions of the promoter fused to GUS, showed the presence of two negative regulatory elements, NE1 (−2070 to −1280) and NE2 (−232 to −178), in the ZmHyPRP promoter. By selective deletion or mutation of ZmHyPRP regulatory promoter elements we conclude that the promoter expression is attenuated by the NE2 element as well as by the G-box2 and the Sph1-2 box together with the G-box2.This work was supported by grants BI02004-01577, BI02007-64791. Consolider CSD2007-00036 Centro de Agrigenómica y Orientación Alimentaria (MICINN). SGR2009 703 Grup de Recerca Consolidat “Genetica Molecular Vegetal” (AGAUR). Xarxa de referencia en Biotecnologia (DIUE).Peer reviewe

Expression of the promoter of HyPRP, an embryo-specific gene from Zea mays in maize and tobacco transgenic plants

zmHyPRP is a gene specifically expressed in maize immature embryos where its transcripts are mainly observed in the scutellum. It has been shown that zmHyPRP expression in the embryo is arrested when ABA levels increase at the beginning of the maturation stage. Here we report the ability of 2 Kb zmHyPRP promoter to reproduce the zmHyPRP gene specific expression pattern in the maize embryo and its repression by ABA at the end of the morphogenetic process. Three different approaches have been used, transient particle bombardment of maize immature excised embryos and stable transformation of maize and tobacco plants with a construct containing 2 Kb of zmHyPRP promoter fused to the GUS gene. This construct has shown to confer specific expression to maize and tobacco embryos but in tobacco expression in the embryo was very low. The same construct was also negatively regulated by ABA in embryos of both species. This suggests that 2 Kb of the zmHyPRP promoter contain all regulatory elements sufficient to confer the developmental expression patterns of the gene characterized to date.This work was supported by the Bio 2001-1721 and MAZE (QLK3-CT-2000-00196) projects and it is under the framework of Centre de Referencia en Biotecnologia de la Generalitat de Catalunya (CERBA).Peer reviewe

Hydroxyproline-rich glycoprotein mRNA accumulation in maize root cells colonized by an arbuscular mycorrhizal fungus as revealed by in situ hybridization

To determine whether the expression of cell wall related genes changes during the establishment of an arbuscular mycorrhizal symbiosis (AM), we studied the expression of a maize hydroxyproline-rich glycoprotein (HRGP) gene. In situ hybridization showed that, in differentiated cells of maize roots, mRNA accumulation corresponding to the gene encoding for HRGP was only found when the cells were colonized by the endomycorrhizal fungusGlomus versiforme.The research was supported by an international project between Spain and Italy (Azioni Integrate Italia-Spagna) and by Italian Ministero Università Ricerca Scientifica e Tecnologica (40%).Peer reviewe

Differential stability of the higher order structure of chromatin associated with genes having different transcriptional activity

This article is dedicated to the memory of Dr. Josep M. Sala-Trepat who died while the work was being completed.We have investigated the stability of the higher order structure of chromatin associated to genes with display a different transcriptional activity in adult rat liver. Nuclei were digested with micrococcal nuclease and chromatin was fractionated by sedimentation in sucrose gradients. Specific DNA sequences were revealed by dot-blotting. In conditions of physiological ionic strength the distribution of the inactive γ-casein gene sequences is similar than the bulk of chromatin. In the same conditions the relative content of the albumin gene, highly expressed in adult rat liver, revealed an enhanced instability of the chromatin superstructure. The distribution of the potentially active but silent α-fetoprotein sequences in adult liver showed an intermediate unfolding of its chromatin superstructure. These distinct behaviour was not observed in non-physiological ionic strength conditions. Our results suggest that distinct folding of the local higher order structure of chromatin actually occurs in the region of active, potentially active and inactive genes.The authors acknowledge the support of CSIC and CAICYT and of an Action Thematique Programmee from CNRS.Peer reviewe

Involvement of a maize proline-rich protein in secondary cell wall formation as deduced from its specific mRNA localization

A clone encoding a proline-rich protein (ZmPRP) has been obtained from maize root by differential screening of a maturing elongation root cDNA library. The amino acid sequence deduced from the full-length cDNA contains a putative signal peptide and a highly repetitive sequence containing the PEPK motif, indicating that the ZmPRP mRNA may code for a cell wall protein. The PEPK repeat is also found in a previously reported wheat sequence but differs from the repeated sequences found in hydroxyproline-rich glycoproteins (HRGP) and in dicot proline-rich proteins (PRP). In the maize genome, the ZmPRP protein is encoded by a single gene that is expressed in maturing regions of the root, in the hypocotyl and in the pericarp. In these organs, the ZmPRP mRNA accumulates in the xylem and surrounding cells, and in the epidermis. No ZmPRP mRNA was found in the phloem. The pattern of mRNA accumulation is very similar to the one observed for genes coding for proteins involved in lignin biosynthesis and, like most cell wall proteins, ZmPRP synthesis is also induced by wounding. These data support the hypothesis that ZmPRP is a member of a new class of fibrous proteins involved in the secondary cell wall formation in monocot species.This work has been financed by Plan Nacional de Investigación Científica y Técnica (grant BIO97-0729). The work has been carried out within the framework of Centre de Referència de Biotecnologia de la Generalitat de Catalunya. F.V. was the recipient of a E.C. fellowship (Grant ERBCHBI930757). The authors are indebted to Limagrain Genetics for support and collaboration.Peer reviewe

The eight-cysteine motif, a versatile structure in plant proteins

A number of protein sequences deduced from the molecular analysis of plant cDNA or genomic libraries can be grouped in relation to a defined number of cysteine residues located in distinct positions of their sequences. This is the case for a group of around 500 polypeptides from different species that contain a small domain (less than 100 amino acids residues) displaying a pattern of eight-cysteines in a specific order. The plant sequences containing this motif belong to proteins having different functions, ranging from storage, protection, enzyme inhibition and lipid transfer, to cell wall structure. The eight-cysteine motif (8CM) appears to be a structural scaffold of conserved helical regions connected by variable loops, as observed by three-dimensional structure analysis. It is proposed that the cysteine residues would form a network of disulfide bridges necessary, for the maintenance of the tertiary structure of the molecule together with the central helical core, while the variable loops would provide the sequences required for the specific functions of the proteins. © 2004 Elsevier SAS. All rights reserved.The work was supported by grant Bio1997-0729 and Bio2003-00132 from Plan Nacional de Investigación Científica y Técnica and within the framework of Center de Referència de Biotecnologia de la Generalitat de CatalunyaPeer Reviewe

Genetic, molecular and cellular approaches to the analysis of maize embryo development

The development of embryo structures in plants is essential for the formation of the adult plant organs. In cereals, this process has distinct features which have attracted attention from different points of view. Differential gene expression analyses have been used in order to identify genes useful as molecular markers of certain physiological, molecular or developmental processes. Several maize mutants affected in embryo development have been isolated, but only a fraction of them have been characterized at the molecular level. Molecular markers can be useful in the characterization of embryo defective mutants. Here, we describe the different techniques used in the identification of molecular marker genes for embryo development. We describe in more detail some groups of genes coding for cell wall proteins. We also describe the application of these molecular markers in the characterization of some embryo mutants