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...

CUP-SHAPED COTYLEDON2 (CUC2) and the interacting microRNA miR164 regulate leaf margin dissection. Here, we further investigate the evolution and the specific roles of the CUC1 to CUC3 genes during Arabidopsis thaliana leaf serration. We show that CUC2 is essential for dissecting the leaves of a wide range of lobed/serrated Arabidopsis lines. Inactivation of CUC3 leads to a partial suppression of the serrations, indicating a role for this gene in leaf shaping. Morphometric analysis of leaf development and genetic analysis provide evidence for different temporal contributions of CUC2 and CUC3. Chimeric constructs mixing CUC regulatory sequences with different coding sequences reveal both redundant and specific roles for the three CUC genes that could be traced back to changes in their expression pattern or protein activity. In particular, we show that CUC1 triggers the formation of leaflets when ectopically expressed instead of CUC2 in the developing leaves. These divergent fates of the CUC1 and CUC2 genes after their formation by the duplication of a common ancestor is consistent with the signature of positive selection detected on the ancestral branch to CUC1. Combining experimental observations with the retraced origin of the CUC genes in the Brassicales, we propose an evolutionary scenario for the CUC genes.

The TFL1 and FT genes, which are key genes in the control of flowering time in Arabidopsis thaliana, belong to a small multigene family characterized by a specific phosphatidylethanolamine-binding protein domain, termed the PEBP gene family. Several PEBP genes are found in dicots and monocots, and act on the control of flowering time. We investigated the evolution of the PEBP gene family in cereals. First, taking advantage of the complete rice genome sequence and EST databases, we found 19 PEBP genes in this species, 6 of which were not previously described. Ten genes correspond to five pairs of paralogs mapped on known duplicated regions of the rice genome. Phylogenetic analysis of Arabidopsis and rice genes indicates that the PEBP gene family consists of three main homology classes (the so-called TFL1-LIKE, MFT-LIKE, and FT-LIKE subfamilies), in which gene duplication and/or loss occurred independently in Arabidopsis and rice. Second, phylogenetic analyses of genomic and EST sequences from five cereal species indicate that the three subfamilies of PEBP genes have been conserved in cereals. The tree structure suggests that the ancestral grass genome had at least two MFT-like genes, two TFL1-like genes, and eight FT-like genes. A phylogenomic approach leads to some hypotheses about conservation of gene function within the subfamilies.