Characterization of VvSERK1, VvSERK2, VvSERK3 and VvL1L genes and their expression during somatic embryogenesis of grapevine (Vitis vinifera L.)

Little is known about the genes expressed during grapevine somatic embryogenesis. Both groups of Somatic Embryogenesis Receptor Kinase (SERK) and Leafy Cotyledon (LEC and L1L) genes seem to play key roles during somatic embryogenesis in various plant species. Therefore, we identified and analysed the sequences of VvSERK and VvL1L (Leafy cotyledon1-Like) genes. The deduced amino acid sequences of VvSERK1, VvSERK2 and VvSERK3 are very similar to that of registered SERK proteins, with highest homologies for the kinase domain in the C-terminal region. The amino acid sequence of VvL1L presents all the domains that are characteristic for LEC1 and L1L proteins, particularly, the 16 amino acid residues that serve as signature of the B-domain. Phylogenetic analysis distinguishes members of subclass LEC1 and subclass L1L, and VvL1L is closely related to L1L proteins. Using semi-quantitative RT-PCR, we studied gene expression of VvSERK1, VvSERK2, VvSERK3 and VvL1L in calli and somatic embryos obtained from anther culture of Vitis vinifera L. cv Chardonnay. Expression of VvSERK2 is relatively stable during in vitro culture. In contrast, VvSERK1, VvSERK3 and VvL1L are expressed more 4 to 6 weeks after transfer of the calli onto embryo induction medium, before the visible appearance of embryos on the calli as seen by environmental scanning electron microscopy. Later on (8 weeks after transfer) VvSERK1 expression is maintained in the embryogenic calli and VvSERK3 in the embryos, whereas VvL1L expression is very low. All together, these data suggest the involvement of VvSERK and VvL1L genes in grapevine somatic embryogenesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Paul Schellenbaum and Alban Jacques contributed equally to this work.     

Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.

Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.     

Structure-activity relationship of antithrombotic polysaccharide derivatives

Heparin has been the drug of choice in clinical pre-surgical and post-surgical prophylaxis of thrombotic events. However, because of its side-effects, such as bleeding and other disadvantages (i.e. chemical inhomogeneity and variability of its physiological activities), alternatives to heparin are an important field of research. A necessary procedure in the development of new drugs is the evaluation of structure-activity relationships. Genuine neutral polysaccharides were chemically modified and examined for their anticoagulant activities. The linear β-1,3-glucan curdlan, an easily available bacterial polysaccharide, served as the basic polymer. It could be established that the anticoagulant activity was dependent on the degree of sulfation and the molecular weight. For heparin, the sulfation pattern, i.e. the actual location of the sulfate groups along the heparin chain, was of importance in addition to the degree of sulfation. Therefore, we investigated whether there was also a relationship between the substitution pattern of the curdlan sulfates and their anticoagulant activity. For determination of the substitution pattern of the sulfated polysaccharides, a method was developed that is based on synthesis of the partially alkylated alditol acetates of the polymer and examination of these derivatives using combined gas chromatographymass spectrometry. In addition to the analytical data, the structure-activity relationship of anticoagulative curdlan sulfates is presented.     

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H⁺-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae , even after deletion of the enzyme's carboxy terminus. Functional chimerical H⁺-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H⁺-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H⁺-ATPase. The reverse Ala-598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H⁺-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K _m values for MgATP and decreased V _max compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H⁺-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.     

Comparative demographics of a Hawaiian forest bird community

Estimates of demographic parameters such as survival and reproductive success are critical for guiding management efforts focused on species of conservation concern. Unfortunately, reliable demographic parameters are difficult to obtain for any species, but especially for rare or endangered species. Here we derived estimates of adult survival and recruitment in a community of Hawaiian forest birds, including eight native species (of which three are endangered) and two introduced species at Hakalau Forest National Wildlife Refuge, Hawai?i. Integrated population models (IPM) were used to link mark–recapture data (1994–1999) with long‐term population surveys (1987–2008). To our knowledge, this is the first time that IPM have been used to characterize demographic parameters of a whole avian community, and provides important insights into the life history strategies of the community. The demographic data were used to test two hypotheses: 1) arthropod specialists, such as the ‘Akiapōlā‘au Hemignathus munroi, are ‘slower’ species characterized by a greater relative contribution of adult survival to population growth, i.e. lower fecundity and increased adult survival; and 2) a species’ susceptibility to environmental change, as reflected by its conservation status, can be predicted by its life history traits. We found that all species were characterized by a similar population growth rate around one, independently of conservation status, origin (native vs non‐native), feeding guild, or life history strategy (as measured by ‘slowness’), which suggested that the community had reached an equilibrium. However, such stable dynamics were achieved differently across feeding guilds, as demonstrated by a significant increase of adult survival and a significant decrease of recruitment along a gradient of increased insectivory, in support of hypothesis 1. Supporting our second hypothesis, we found that slower species were more vulnerable species at the global scale than faster ones. The possible causes and conservation implications of these patterns are discussed.