Transcriptome analysis in pea allows to distinguish chilling and acclimation mechanisms.

In order to distinguish chilling and freezing tolerance mechanisms in pea, responses to cold exposure were compared between the freezing tolerant line Champagne and the sensitive line Terese. Global gene expression was considered in the two lines and associated with morphological, histological and biochemical approaches. The chilling tolerance in both lines was related to responses of the CBF, COR and LEA genes belonging to the CBF regulon, with greater earliness of expression in the Champagne genotype. The freezing tolerance, only observed in Champagne, was associated with acclimation processes such as cellular osmotic stabilization, photosynthesis modifications, antioxidants production, modifications in hormone metabolism, cell wall composition and dynamics.

Partial sequences of nitrogen metabolism genes in hexaploid wheat.

Our objective was to partially sequence genes controlling nitrogen metabolism in wheat species in order to find sequence polymorphism that would enable their mapping. Primers were designed for nitrate reductase, nitrite reductase, glutamate dehydrogenase and glutamate synthase (GOGAT), and gene fragments were amplified on Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii. We obtained more than 8 kb of gene sequences, mainly as coding regions (60%). Polymorphism was quantified by comparing two-by-two the three genomes of the hexaploid cultivar Arche and genomes of diploid wheat species. On average, the polymorphism rate was higher for non-coding regions, where it ranged from 1/60 to 1/23, than for coding regions (range: 1/110-1/40) except when the hexaploid D genome was compared to that of T. tauschii (1/800 and 1/816, respectively). Genome-specific primers were devised for the ferredoxin-dependent (Fd)-GOGAT gene, and they enabled the mapping of this gene on homoeologous chromosomes of group 2 using Chinese Spring deletion lines. A single nucleotide polymorphism (SNP) detected between the two hexaploid wheat cultivars Arche and Recital was used to genetically map Fd-GOGAT on chromosome 2D using a population of dihaploid lines. Fd-GOGAT-specific primers were used to estimate the SNP rate on a set of 11 hexaploid and nine Durum wheat genotypes leading to the estimate of 1 SNP/515 bp. We demonstrate that polymorphism detection enables heterologous, homeologous and even paralogous copies to be assigned, even if the elaboration of specific primer pairs is time-consuming and expensive because of the sequencing.

rym15 from the Japanese cultivar Chikurin Ibaraki 1 is a new barley mild mosaic virus (BaMMV) resistance gene mapped on chromosome 6H.

Breeding for resistant cultivars is the only way to prevent high yield loss in barley caused by the soil-borne barley mild mosaic virus (BaMMV) complex. We have characterized the BaMMV resistance of barley cv. Chikurin Ibaraki 1. Doubled haploid lines were obtained from the F(1) between the susceptible six-rowed winter barley cultivar, Plaisant, and Chikurin Ibaraki 1. Each line was tested for reaction to BaMMV by mechanical inoculation followed by DAS-ELISA. Of 44 microsatellites that covered the genome, 22 polymorphic markers were tested on one susceptible and one resistant bulk, each comprising 30 lines. Differential markers and additional microsatellite markers in the same region were then tested on the whole population. A bootstrap analysis was used to compute confidence intervals of distances and to test the orders of the resistance gene and the closest markers. A segregation of 84 resistant/98 susceptible lines fitted a 1:1 ratio (chi(2)=1.08, P=0.30), which corresponds to a single gene in this DH lines population. The resistance gene was flanked by two markers near the centromeric region of chromosome 6HS-Bmag0173, at 0.6+/-1.2 cM, and EBmac0874, at 5.8 +/- 3.4 cM. We propose to name this new resistance gene rym15. This resistance gene and associated markers will increase the possibilities to breed efficiently for new cultivars resistant to the barley mosaic disease.

Proteomics for genetic and physiological studies in plants.

Proteomics is becoming a necessity in plant biology, as it is in medicine, zoology and microbiology, for deciphering the function and role of the genes that are or will be sequenced. In this review we focus on the various, mainly genetic, applications of the proteomic tools that have been developed in recent years: characterization of individuals or lines, estimation of genetic variability within and between populations, establishment of genetic distances that can be used in phylogenetic studies, characterization of mutants and localization of the genes encoding the revealed proteins. Improvements in specifically devoted software have permitted precise quantification of the variation in amounts of proteins, leading to the concept of "protein quantity loci" which, combined with the "quantitative trait loci" approach, results in testable hypotheses regarding the role of "candidate proteins" in the metabolism or phenotype under study. This new development is exemplified by the reaction of plants to drought, a trait of major agronomic interest. The accumulation of data regarding genomic and cDNA sequencing will be connected to the protein databases currently developed in plants.

Genetic diversity of old french six-rowed winter barley varieties assessed with molecular, biochemical and morphological markers and its relation to BaMMV resistance

Twenty-six old French six-rowed winter barley (Hordeum vulgare L.) varieties were characterized for their reaction against barley mild mosaic virus (BaMMV). The genetic diversity of these varieties and two recent barley varieties was assessed using molecular, biochemical and morphological data. Seven old varieties were fully resistant to BaMMV. A higher differentiation level between varieties was observed by using DNA molecular markers compared to biochemical and morphological ones. Correspondence analysis using all markers showed that DNA molecular data could fully discriminate between all varieties, whereas biochemical and morphological markers were not able to achieve a complete discrimination. The dendrogram clustering computed with the DNA marker dissimilarity index showed two main groups. The first group included the seven varieties resistant to the BaMMV, whereas the second contained susceptible varieties. The relationships between these varieties, their diversity level, and their characterization are discussed. We infer that the seven BaMMV-resistant varieties have a common origin.

Water-deficit-responsive proteins in maritime pine.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify drought-responsive proteins during progressive water deprivation of two-year old maritime pine seedlings. Stress was applied by withholding water during vegetative growth. Needles were sampled before, during and after the stress. Out of about 1000 spots that were quantified by computer analysis, 38 responded during stress. Some proteins were accumulated while others were suppressed. One to three internal microsequences were obtained for 11 proteins, 10 of which were identified on the basis of sequence homologies. These proteins are quite diverse and are involved in photosynthesis, cell elongation, antioxidant metabolism and lignification.

Organisation of the variability of abundant proteins in seven geographical origins of maritime pine (Pinus pinaster Ait.).

The comparison of 42 two-dimensional protein patterns from megagametophytes of maritime pine from seven geographical origins enabled the analysis of the genetic variability of abundant proteins. More than 84% of the polypeptides were variable. The intra- and inter-origin variability levels were of a similar magnitude. Correspondence analysis and a dendrogram computed using a dissimilarity index between individuals showed three main groups. The first group included the individuals from Landes (France), Portugal, eastern Spain, and Corsica, without individualising the provenances. The second group was composed of accessions from Italy and Sardinia, and the individuals of each location were separated. The third group included all of the individuals of Moroccan origin. This clustering was in agreement with the Atlantic, Mediterranean and North African structuration of maritime pine established from terpene data.

Seed-protein variation in maritime pine (Pinus pinaster Ait.) revealed by two-dimensional electrophoresis: genetic determinism and construction of a linkage map.

Proteins from haploid megagametophytes from 18 trees were studied by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A total of 222 seeds, an average of 12 per tree, were analysed individually. 150 protein spots appeared to segregate on the polyacrylamide gels in at least tree. Genetic interpretations were made to define the number of loci responsible for the presence versus absence, staining differences or position variation of the segregating spots. The complete covariation observed between some spots could be the result of either the separation of a single gene product into two or more constituents, very close linkage, or the action of a pleiotropic gene. Human genetics techniques were used to map the 84 putative loci detected. Sixty-five loci were organised in 17 linkage groups, whereas 19 remained unlinked.

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.

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

Two-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.

Two-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 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 degrees 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.