Hervé Thiellement

Numerous and Rapid Nonstochastic Modifications of Gene Products in Newly Synthesized Brassica napus Allotetraploids

Polyploidization is a widespread process that results in the merger of two or more genomes in a common nucleus. To investigate modifications of gene expression occurring during allopolyploid formation, the Brassica napus allotetraploid model was chosen. Large-scale analyses of the proteome were conducted on two organs, the stem and root, so that >1600 polypeptides were screened. Comparative proteomics of synthetic B. napus and its homozygous diploid progenitors B. rapa and B. oleracea showed that very few proteins disappeared or appeared in the amphiploids (<1%), but a strikingly high number (25–38%) of polypeptides displayed quantitative nonadditive pattern. Nonstochastic gene expression repatterning was found since 99% of the detected variations were reproducible in four independently created amphiploids. More than 60% of proteins displayed a nonadditive pattern closer to the paternal parent B. rapa. Interspecific hybridization triggered the majority of the deviations (89%), whereas very few variations (∼3%) were associated with genome doubling and more significant alterations arose from selfing (∼9%). Some nonadditive proteins behaved similarly in both organs, while others exhibited contrasted behavior, showing rapid organ-specific regulation. B. napus formation was therefore correlated with immediate and directed nonadditive changes in gene expression, suggesting that the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms.

Chromosomal localization of structural genes and regulators in wheat by 2D electrophoresis of ditelosomic lines

SummaryAmong the 782 spots observed in two-dimensional gel electrophoresis of denatured proteins from etiolated wheat shoots, 185 were found to be variable between the euploid and 26 ditelosomic lines of ‘Chinese Spring’. Thirty-five structural genes were located on 17 chromosome arms. Numerous intensity changes showing alterations in protein levels were observed and led to the following statements: 1) regulators are frequently found and can be assigned for a same polypeptide to various chromosome arms; 2) for most polypeptides homoeologous arms do not manifest similar effects; 3) nevertheless, when affecting the same polypeptide, homoeologous arms display in most cases identical regulatory effects; 4) gene dosage compensation is observed in only one out of four homoeoallelic situations.

Genetic relationships in the Sitopsis section of Triticum and the origin of the B genome of polyploid wheats

The proteins extracted from etiolated seedlings of 10 accessions, representatives of the species of the Sitopsis section of Triticum were analyzed by two-dimensional (2D) gel electrophoresis. The 2D patterns were compared and similarity indices were computed between all accessions. From the similarity matrix a dendrogram was deduced, that gave interesting information concerning the genetic relationships between the species of the Sitopsis section. The 2D pattern obtained from the Chinese Spring variety of Triticum aestivum was then included in the analysis. From the dendrogram obtained and also from the number of species-specific proteins shared between Chinese Spring and each of the Sitopsis species, it is proposed that the B genome donor of the cultivated wheats originated from a species closer to the T. speltoides-T. aucheri group than to any other diploid Sitopsis species.

Two-dimensional gel electrophoresis of proteins for genetic studies in douglas fir (Pseudotsuga menziesii)

A method of two-dimensional gel electrophoresis of proteins from Douglas fir needles is described. Extraction in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol followed by heating at 100°C produces reliable two-dimensional gels which are convenient for genetic studies. Three genotypes from different geographical origins have been compared: among 225 loci expressed, 22 display regulatory variations and 7 show allelic variations. Thus it is now possible to undertake the genetic study of Douglas fir using this powerful technique.

Comparative proteomics of leaf, stem, and root tissues of synthetic Brassica napus

Comparative proteomics was applied to three vegetative organs of Brassica napus, the leaf, stem, and root using 2‐DE. Among the >1600 analyzed spots, 43% were found to be common to all three organs, suggesting the existence of a “basal” or ubiquitous proteome composed of housekeeping proteins. The green organs, leaf, and stem, were closely related (∼80% common spots) while the root displayed more organ‐specific polypeptides (∼10%). Reference maps were established using MS, allowing the identification of 93, 385, and 266 proteins in leaf, stem, and root proteomes, respectively. Bioinformatic analyses were also performed; in silico functional categorization and cellular localization allow obtaining a precise picture of the cell molecular network within vegetative organs. These proteome maps can be explored using the PROTICdb software at the following address: http://bioinformatique.moulon.inra.fr/proticdb/web_view/.

Homoeology and phylogeny of the A, S, and D genomes of the triticinae

SummaryTwo-dimensional gel electrophoresis of seedling proteins has been undertaken on diploid representatives of the A, S, and DTriticum genomes. The 457 reproducible spots of the hexaploid Chinese Spring were taken into account in the comparisons. More than 82% of the hexaploid spots were found among the representatives of each of the three genomes. Thus, it was proposed that the homoeologous structural loci, on the three homoeologous genomes, can be present in the same allelic forms and that from 72% to 86% of the spots of the hexaploid pattern are in two or three doses. The dendrogram, deduced from the similarity indices computed between the 16 diploid accessions, was very consistent with our present knowledge of the genetic relationships in theTriticum genus. This result, in addition to previous ones, shows that the comparison of two-dimensional protein patterns represents a novel experimental approach for studying the phylogenetic relationships in theTriticinae.

Parental genome expression in synthetic wheats (Triticum turgidum sp. × T. tauschii sp.) revealed by two-dimensional electrophoresis of seedling proteins.

SummaryTwo-dimensional gel electrophoresis was conducted on etiolated seedling proteins from two distinct amphiploids (ABD1, ABD2) and their parental lines (AB1, D1 and AB2, D2), AB1 and AB2 being used as female. On the amphiploid patterns were found all the parental spots except 8 D spots of which 3 are cytoplasmically encoded. One exceptional polypeptide observed in ABD1 was present neither in AB1 nor D1. The patterns fromt the amphiploids very closely resemble the co-electrophoresis done with 1/3 D protein extract and 2/3 AB protein extract. Thus it is very likely that for most gene products revealed the genomes act independently of each other.

Regulatory effects of homoeologous chromosome arms on wheat proteins at two developmental stages

SummaryTwo-dimensional gel electrophoresis was conducted on denatured proteins of the 10-day-old first leaf (1F stage) of 18 homoeologous ditelosomic (DT) lines of wheat cultivar ‘Chinese Spring’. The observations, compared to the euploid control and relative to previous data found on 7-day-old etiolated seedlings (G7 stage) of the same lines lead to the following statements: 1) the structural genes of 24 spots can be assigned to 12 chromosome arms; 2) regulatory effects are completely different between the 1F and the G7 stages which may indicate that the regulation of protein amounts is often stage-specific; 3) no case of complete gene dosage compensation is observed among 4 groups of hypothesized homoeoallelic products; 4) homoeologous DT lines do not manifest similar effects which suggest the absence of homoeology for the detected regulatory effects.

Two-dimensional gel electrophoresis of proteins as a tool in wheat genetics.

In this minireview are reported several genetic investigations undertaken on wheat with the use of two-dimensional gel electrophoresis of total proteins extracted mainly from etiolated seedlings or from green leaves. Differences between developmental stages or organs of one genotype and nuclear and cytoplasmic genetic variations between genotypes are revealed by this method. We have also localized on the chromosomes structural genes coding for the proteins revealed and assigned their subcellular location to many polypeptides. We obtained new information concerning the regulation of protein amounts as well as the phylogenetic and homeology relationships between the A, B and D genomes.

Study of Gene Expression During in Vitro Culture of Tobacco thin Cell Layers by two-Dimensional Electrophoresis of Proteins

Compared to animal systems, plant systems have tremendous potential for organ regeneration, embryogenesis from somatic cells, pollen grains, and ovules, for early sexual reproduction in in vivo conditions and in vitro conditions. Advanced progress already made in genetic manipulation technology would have led to important plant improvement if its feasibility was not limited to a small number of species. This limit is due to the difficulties in controlling in vitro plant regeneration via protoplasts, cells, tissues and/or organs culture of recalcitrant species such as monocotyledons, leguminous, and woody species which also have great economic importance. It is generally observed in recalcitrant species that regeneration of organs can be induced only during the juvenile phase. For example, mature tissues of woody plants lose their ability to differentiate organs in vitro. It is important to understand the phenomenon of maturation in recalcitrant species as it is important to understand the mechanisms of organ regeneration in recalcitrant, and nonrecalcitrant species. Progress is slow, because of the lack of regeneration /maturation mutants and because of the complexity of the eucaryotic genome. In species such as Arabidopsis for which the genome size is relatively reduced and for which several mutants are available, it still lacks developmental and regeneration mutants. There exist mutants with altered flower shape; however, these mutations are not suitable for studying the control of organogenesis.