Nardjis Amiour

Arabidopsis TONNEAU1 Proteins Are Essential for Preprophase Band Formation and Interact with Centrin

Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein–TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.

Allelic diversity of HMW and LMW glutenin subunits and omega-gliadins in French bread wheat (Triticum aestivum L.)

Wheat endosperm storage proteins, namely gliadins and glutenins, are the major components of gluten. They play an important role in dough properties and in bread making quality in various wheat varieties. In the present study, the different alleles encoded at the 6 glutenin loci and at 3 ω-gliadin loci were identified from a set of 200 hexaploid wheat cultivars grown primarily in France using SDS PAGE. At Glu-A1, Glu-B1 and Glu-D1, encoding high molecular weight glutenin subunits (HMW-GS), 3, 8 and 5 alleles were observed respectively. Low molecular weight glutenin subunits (LMW-GS) displayed similar polymorphism, as 5 and 11 alleles were identified at loci Glu-A3 and Glu-B3 respectively. Four alleles were observed at Glu-D3 loci. Omega-gliadin diversity was also very high, as 7, 13 and 9 alleles were found at Gli-A1, Gli-B1 and Gli-D1, respectively. A total of 147 (or 149) patterns resulted from the genetic combination of the alleles encoding at the six glutenin loci (or Glu-1 and Gli-1 loci). Although Glu-1 and Glu-3 loci were located on different chromosome arms and were theoretically independent, some associations were revealed due to pedigree relatedness between some French wheat cultivars. The usefulness of allelic identification of LMW-GS together with HMW-GS and gliadins for future genetic and technological wheat improvement is discussed.

Proteomic analysis of amphiphilic proteins of hexaploid wheat kernels

Wheat proteins and specially gluten proteins have been well studied and are closely associated with baking products. Amphiphilic proteins (proteins that are soluble using nonionic detergent Triton X‐114 ) also play an important role in wheat quality. Some of them, like puroindolines, are lipid binding proteins, and are strongly linked to dough foaming properties and to fine crumb texture. However many amphiphilic proteins are still unknown and both their physiological and technological functions remain to be analysed. In order to explore these proteins, proteomic analysis was carried out using 81 F9 lines, progeny obtained from an interspecific cross “W7984”דOpata”, and already used to built a map of more than 2000 molecular markers (International Triticeae Mapping Initiative, ITMImap). Two‐dimensional electrophoresis (immobilized pH gradient (pH 6–11)×sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) was performed on amphiphilic proteins with three to five replicates for each line. Silver stained gels were analysed using Melanie 3 software. Genetic determinism was carried out on 170 spots segregating between the two parental hexaploïd wheats. Many of these spots were mapped on different chromosomes of the ITMImap. Spots of interest were identified using matrix‐assisted laser desorption/ionization‐time of flight and some of them were partly sequenced using electrospray ionization‐tandem mass spectrometry. This proteomic approach provided some very useful information about some proteic components linked to bread wheat quality and particularly to kernel hardness.

Assessing the metabolic impact of nitrogen availability using a compartmentalized maize leaf genome-scale model.

A cell type- and leaf tissue-specific model provides new insights into nitrogen metabolism in the maize leaf. Maize (Zea mays) is an important C4 plant due to its widespread use as a cereal and energy crop. A second-generation genome-scale metabolic model for the maize leaf was created to capture C4 carbon fixation and investigate nitrogen (N) assimilation by modeling the interactions between the bundle sheath and mesophyll cells. The model contains gene-protein-reaction relationships, elemental and charge-balanced reactions, and incorporates experimental evidence pertaining to the biomass composition, compartmentalization, and flux constraints. Condition-specific biomass descriptions were introduced that account for amino acids, fatty acids, soluble sugars, proteins, chlorophyll, lignocellulose, and nucleic acids as experimentally measured biomass constituents. Compartmentalization of the model is based on proteomic/transcriptomic data and literature evidence. With the incorporation of information from the MetaCrop and MaizeCyc databases, this updated model spans 5,824 genes, 8,525 reactions, and 9,153 metabolites, an increase of approximately 4 times the size of the earlier iRS1563 model. Transcriptomic and proteomic data have also been used to introduce regulatory constraints in the model to simulate an N-limited condition and mutants deficient in glutamine synthetase, gln1-3 and gln1-4. Model-predicted results achieved 90% accuracy when comparing the wild type grown under an N-complete condition with the wild type grown under an N-deficient condition.

Mutations in DMI3 and SUNN Modify the Appressorium-Responsive Root Proteome in Arbuscular Mycorrhiza

Modification of the Medicago truncatula root proteome during the early stage of arbuscular mycorrhizal symbiosis was investigated by comparing, using two-dimensional electrophoresis, the protein patterns obtained from non-inoculated roots and roots synchronized for Glomus intraradices appressorium formation. This approach was conducted in wild-type (J5), mycorrhiza-defective (TRV25, dmi3), and autoregulation-defective (TR122, sunn) M. truncatula genotypes. The groups of proteins that responded to appressorium formation were further compared between wild-type and mutant genotypes; few overlaps and major differences were recorded, demonstrating that mutations in DMI3 and SUNN modified the appressorium-responsive root proteome. Except for a chalcone reductase, none of the differentially displayed proteins that could be identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry previously was known as appressorium responsive. A DMI3-dependent increased accumulation of signal transduction-related proteins (dehydroascorbate reductase, cyclophilin, and actin depolymerization factor) was found to precede mycorrhiza establishment. Differences in the accumulation of proteins related to plant defense reactions, cytoskeleton rearrangements, and auxin signaling upon symbiont contact were recorded between wild-type and hypermycorrhizal genotypes, pointing to some putative pathways by which SUNN may regulate very early arbuscule formation.

An integrated statistical analysis of the genetic variability of nitrogen metabolism in the ear of three maize inbred lines (Zea mays L.)

During the grain-filling period of maize, the changes in metabolite content, enzyme activities, and transcript abundance of marker genes of amino acid synthesis and interconversion and carbon metabolism in three lines F2, Io, and B73 have been monitored in the cob and in the kernels. An integrative statistical approach using principal component analysis (PCA) and hierarchical clustering of physiological and transcript abundance data in the three maize lines was performed to determine if it was possible to link the expression of a physiological trait and a molecular biomarker to grain yield and its components. In this study, it was confirmed that, in maize, there was a genetic and organ-specific control of the main steps of nitrogen (N) and carbon metabolism in reproductive sink organs during the grain-filling period. PCA analysis allowed the identification of groups of physiological and molecular markers linked to either a genotype, an organ or to both biological parameters. A hierarchical clustering analysis was then performed to identify correlative relationships existing between these markers and agronomic traits related to yield. Such a clustering approach provided new information on putative marker traits that could be used to improve yield in a given genetic background. This can be achieved using either genetic manipulation or breeding to increase transcript abundance for the genes encoding the enzymes glutamine synthetase (GS), alanine amino transferase (AlaAT), aspartate amino transferase (AspAT), and Δ1-pyrroline-5-carboxylate synthetase (P5CS).

Allelic variation of HMW and LMW glutenin subunits, HMW secalin subunits and 75K gamma-secalins of hexaploid triticale

Although the endosperm storage protein of hexaploid triticale have alreadybeen analysed, the allelic diversity of glutenins and secalins remains to bedescribed. Analysis, by SDS-PAGE, of about one thousand F2 seeds fromten triticale crosses allowed the inheritance of these storage proteins to bestudied in order to determine their allelic forms and to assign them toparticular chromosomes. Two new alleles encoding HMW subunits ofglutenin and five new alleles encoding HMW subunits of secalin weredetermined at Glu-B1 and Glu-R1 loci respectively. In additionto the three allelic forms of 75K gamma-secalins encoded at Gli-R2and previously reported, a null form was found. A nomenclature for theseproteins and their corresponding alleles was suggested. The composition ofB-LMW glutenin subunits of hexaploid triticale was described and allelicforms were determined: Five alleles were encoded at Glu-A3 andseven at Glu-B3 including one and two new allelic formsrespectively.

An integrated "omics" approach to the characterization of maize (Zea mays L.) mutants deficient in the expression of two genes encoding cytosolic glutamine synthetase.

BackgroundTo identify the key elements controlling grain production in maize, it is essential to have an integrated view of the responses to alterations in the main steps of nitrogen assimilation by modification of gene expression. Two maize mutant lines (gln1.3 and gln1.4), deficient in two genes encoding cytosolic glutamine synthetase, a key enzyme involved in nitrogen assimilation, were previously characterized by a reduction of kernel size in the gln1.4 mutant and by a reduction of kernel number in the gln1.3 mutant. In this work, the differences in leaf gene transcripts, proteins and metabolite accumulation in gln1.3 and gln1.4 mutants were studied at two key stages of plant development, in order to identify putative candidate genes, proteins and metabolic pathways contributing on one hand to the control of plant development and on the other to grain production.ResultsThe most interesting finding in this study is that a number of key plant processes were altered in the gln1.3 and gln1.4 mutants, including a number of major biological processes such as carbon metabolism and transport, cell wall metabolism, and several metabolic pathways and stress responsive and regulatory elements. We also found that the two mutants share common or specific characteristics across at least two or even three of the “omics” considered at the vegetative stage of plant development, or during the grain filling period.ConclusionsThis is the first comprehensive molecular and physiological characterization of two cytosolic glutamine synthetase maize mutants using a combined transcriptomic, proteomic and metabolomic approach. We find that the integration of the three “omics” procedures is not straight forward, since developmental and mutant-specific levels of regulation seem to occur from gene expression to metabolite accumulation. However, their potential use is discussed with a view to improving our understanding of nitrogen assimilation and partitioning and its impact on grain production.

Functional Genomic of Arbuscular Mycorrhizal Symbiosis: Why and How Using Proteomics

This chapter aims to provide a general technical overview of the contribution of proteomics to the identification of arbuscular mycorrhizal symbiosis-related proteins. Key features with regard to biological material handling; equipment, protein separation and detection protocols are first highlighted, before a case study of combined transcriptomic and proteomic investigations targeted at the early stages of this plant–mycorrhiza interaction is exposed.