Laboratoire de GCJtique des Systhnes VegCtaux, G. S. Moulon Gif-sur-YvetteThe level of genetic variation revealed by two-dimensional electrophoresis of proteins from seedlings of two wheat lines strongly depends on the technical procedures. Improvements in extraction and electrophoresis procedures relative to earlier experiments on the same material led to a significant increase in the genetic variation revealed: 15.2 % instead of 6.7 % ofthe spots were genetically variable. The improved procedure is based on (i) precipipation of proteins from wheat seedlings with trichloroacetic acid and acetone, (ii) solubilization of the proteins with a solution containing urea, potassium carbonate and sodium dodecyl sulfate, (iii) isoelectric focusing in an optimized pH gradient, obtained with a mixture of carrier ampholytes (Pharmalyte and Servalyt), and (iv) running electrophoresis in the second dimension on gels with increased surface. Two-dimensional (2-D) gel electrophoresis of denatured proteins [ l l has proven to be an important technique in many areas of biological research. However, its genetic applications are at present limited because this method has often been claimed to reveal lower levels of polymorphism than allozyme electrophoresis [2-61. In addition to technical considerations, genetic reasons have been suggested to explain this limitation [7-101. Nevertheless, we show in this paper that a substantial increase in the amount of genetic variation revealed can be achieved by improving the quality of 2-D gels.Genetic variation between two unrelated wheat lines, "Chinese Spring" (CS) and "Selkirk" (Sk), has been previously studied in our laboratory [11][12], The same plant material was used in this investigation but with the following modified procedures. Two samples per line (each consisting of three dark-germinated seedlings of the same age) were dry-crushed in a liquid nitrogen cooled mortar, and the powder was reCorrespondence: C. Damerval, Laboratoire de G6netique des Systtrmes Vegetaux, G. S. Moulon, la Ferme du Moulon, F-91190 Gif-sur-Yvette, France Abbreviations: IEF, isoelectric focusing; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; UKS, urea-potassium' carbonate-sodium dodecyl sulfate solution suspended in a solution of 10 % trichloroacetic acid (TCA) in acetone with 0.07 % 2-mercaptoethanol. Proteins were allowed to precipitate for 45 min at -18 OC. Then the extract was centrifuged at 35 000 g for 15 min, and the pellet rinsed with acetone containing 0.07 % 2-mercaptoethanol for 1 h at -18 "C. The supernatant was discarded and the pellet dried under vacuum. It was solubilized, in 50 pL per mg of pellet, with the "UKS" solution: 9.5 M urea, 5 mM K2C03, 1.25 % sodium dodecyl sulfate (SDS), 0.5 % dithiothreitol, 2 % LKB Ampholines, pH 3.5 to 10, 6 % Triton X-100. After a 5 min centrifugation at 14 000 g, the supernatants were stored at -80 "C. The isoelectric focusing (IEF) rod gels were 19.5 cm long and had a diameter of 1 mm. The gel mixture was 3.78 % acrylamide, 0.22 % N,N'-methylenebisacr...

Three-week-old plants of two unrelated lines of maize (Zea mays L.) and their hybrid were submitted to progressive water stress for 10 d. Changes induced in leaf proteins were studied by twodimensional electrophoresis and quantitatively analyzed using image analysis. Seventy-eight proteins out of a total of 413 showed a significant quantitative variation (increase or decrease), with 38 of them exhibiting a different expression in the two genotypes. Eleven proteins that increased by a factor of 1.3 to 5 in stressed plants and 8 proteins detected only in stressed plants were selected for internal amino acid microsequencing, and by similarity search 16 were found to be closely related to previously reported proteins. In addition to proteins already known to be involved in the response to water stress (e.g. RAB17 [ Responsive to ABA]), several enzymes involved in basic metabolic cellular pathways such as glycolysis and the Krebs cycle (e.g. enolase and triose phosphate isomerase) were identified, as well as several others, including caffeate O-methyltransferase, the induction of which could be related to lignification.

A cDNA clone encoding a Brassica napus drought-induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought-adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought-adapted plants, the BnD22 transcript was expressed in an organ-specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Künitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.