Functional Annotation,Genome Organization and Phylogeny of the Grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) Terpene Synthase Gene Family Based on Genome Assembly,FLcDNA Cloning,and Enzyme Assays

Background  

Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.     

Structural,Functional, and Evolutionary Analysis of the Unusually Large Stilbene Synthase Gene Family in Grapevine

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.Plants produce a vast array of secondary metabolites, many of them being restricted to specific groups of plant species. This extraordinary chemical diversity is believed to have evolved from a limited number of ubiquitous biosynthetic pathways through gene duplication followed by functional divergence (Pichersky and Gang, 2000). The phenylpropanoid pathway, derived from Phe, illustrates perfectly this phenomenon, as it gives rise to a large diversity of phenolic compounds playing key roles in plants, including participation in structural polymers, defense against herbivores and pathogens, protection from abiotic stress, and important functions in plant-pollinator interactions. Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including dicotyledon angiosperms such as grapevine (Vitis vinifera), peanut (Arachis hypogaea), and Japanese knotweed (Fallopia japonica, formerly Polygonum cuspidatum), monocotyledons like sorghum (Sorghum bicolor), and gymnosperms such as several Pinus and Picea species. In addition to their participation in both constitutive and inducible defense mechanisms in plants, several stilbenes display important pharmacological properties. Since resveratrol (3,5,4′-trihydroxy-trans-stilbene) was postulated to be involved in the health benefits associated with a moderate consumption of red wine (Renaud and de Lorgeril, 1992), plant stilbenes have received considerable interest. Nowadays, resveratrol ranks among the most extensively studied natural products, and hundreds of studies have shown that it can slow the progression of a wide variety of illnesses, including cancer and cardiovascular disease, as well as extend the life spans of various organisms (Baur and Sinclair, 2006). Stilbene synthases (STSs) are characteristic of stilbene-producing plants and catalyze the biosynthesis of the stilbene backbone from three malonyl-CoA and one CoA-ester of a cinnamic acid derivative. STSs are members of the type III polyketide synthases family, chalcone synthases (CHSs), which catalyze the first step of flavonoid biosynthesis, being the most ubiquitous polyketide synthase in plants. Both CHS and STS use p-coumaroyl-CoA and malonyl-CoA as substrates and synthesize the same linear tetraketide intermediate. However, STS uses a specific cyclization mechanism involving a decarboxylation to form the stilbene backbone. STS proteins share extensive amino acid sequence identity with CHS, and phylogenetic analysis of the STS and CHS gene families has shown that STS genes may have evolved from CHS genes several times independently (Tropf et al., 1994). In most stilbene-producing plants, STS genes form small families of closely related paralogs. For example, two STS cDNAs have been cloned from peanut (Schröder et al., 1988), the genome of Scots pine (Pinus sylvestris) has been shown to contain a small family of four STS genes (Preisig-Müller et al., 1999), and three STS genes have been characterized in Japanese red pine (Pinus densiflora; Kodan et al., 2002). Only one STS gene has been isolated from Japanese knotweed to date (Liu et al., 2011), and the sequencing of sorghum genome has shown that SbSTS1 was the only STS gene in this plant species (Yu et al., 2005; Paterson et al., 2009). Grapevine is a noteworthy exception among stilbene-producing plants, as its genome has been shown to contain a large family of putative STS genes. Early Southern-blot experiments suggested that the grapevine genome contained more than 20 STS genes (Sparvoli et al., 1994). Analyses of the first drafts of the grapevine genome sequence confirmed the large size of this multigene family, with an estimated number of STS genes ranging from 21 to 43 (Jaillon et al., 2007; Velasco et al., 2007). However, these relatively low-coverage sequence drafts did not allow a precise analysis of large families of highly similar genes. The more recently released 12× genome sequence of grapevine inbred Pinot Noir cultivar PN40024 offered an improved sequence quality, allowing an accurate analysis of the STS gene family. In this work, we take advantage of the improved 12× sequence of the grapevine ‘PN40024’ genome to analyze the grapevine STS gene family. Furthermore, we combine molecular evolution to structural and functional analyses to gain more insight into the significance of the remarkable amplification of the STS family in grapevine.     

CD1 gene polymorphisms and phenotypic variability in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is characterized by marked phenotypic variation ranging from adrenomyeloneuropathy (AMN) to childhood cerebral ALD (CCALD). X-ALD is caused by mutations in the ABCD1 gene, but no genotype-phenotype correlation has been established so far and modifier gene variants are suspected to modulate phenotypes. Specific classes of lipids, enriched in very long-chain fatty acids that accumulate in plasma and tissues from X-ALD patients are suspected to be involved in the neuroinflammatory process of CCALD. CD1 proteins are lipid- antigen presenting molecules encoded by five CD1 genes in human (CD1A-E). Association studies with 23 tag SNPs covering the CD1 locus was performed in 52 patients with AMN and 87 patients with CCALD. The minor allele of rs973742 located 4-kb downstream from CD1D was significantly more frequent in AMN patients (χ² = 7.6; P = 0.006). However, this association was no longer significant after Bonferroni correction for multiple testing. The other polymorphisms of the CD1 locus did not reveal significant association. Further analysis of other CD1D polymorphisms did not detect stronger association with X-ALD phenotypes. Although the association with rs973742 warrants further investigations, these results indicate that the genetic variants of CD1 genes do not contribute markedly to the phenotypic variance of X-ALD.     

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia

Background

Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.

Results

Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30 % of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.

Conclusions

Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20 %.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1698-7) contains supplementary material, which is available to authorized users.     

A putative physiological role of hypothalamic CNTF in the control of energy homeostasis

Administration of CNTF durably reduces food intake and body weight in obese humans and rodent models. However, the involvement of endogenous CNTF in the central regulation of energy homeostasis needs to be elucidated. Here, we demonstrate that CNTF and its receptor are expressed in the arcuate nucleus, a key hypothalamic region controlling food intake, and that CNTF levels are inversely correlated to body weight in rats fed a high-sucrose diet. Thus endogenous CNTF may act, in some individuals, as a protective factor against weight gain during hypercaloric diet and could account for individual differences in the susceptibility to obesity.     

Endocytic prolactin routes to the secretory pathway in lactating mammary epithelial cells

Plasma-borne prolactin is carried from blood to milk by transcytosis across the mammary epithelial cell through the endocytic and secretory pathways. To determine the precise route of prolactin endocytosis, intracellular transport of biotinylated prolactin was monitored, in parallel with endocytosis of fluorescein isothiocyanate-conjugated dextran and IgG, by using pulse-chase experiments in lactating mammary fragments and in enzymatically dissociated acini. Biotinylated prolactin was sorted to vesiculo-tubular organelles whereas dextran was very rapidly carried to the lumen and IgG remained accumulated in the basal region of cells. To determine whether prolactin uses routes into and across the Golgi and trans-Golgi network, localisation of biotinylated prolactin was combined with the immunofluorescence detection of caseins and, respectively, p58 and TGN38. Biotinylated prolactin strongly colocalised with caseins during a chase but not all or only very little with p58 and TGN38. To characterise the organelles involved in transcytosis, gold-labelled prolactin, experimentally accumulated in late endosomes and which recovered a normal transport, was localised by electron microscopy. In mammary fragments incubated at low temperature, and in mammary fragments from rats fed with a lipid-deprived diet, transport of gold-labelled prolactin was restored by increasing the temperature and by adding arachidonic acid, respectively. These data demonstrate that a sorting occurs very rapidly between prolactin, dextran and IgG. They suggest that prolactin may reach the biosynthetic pathway after direct fusion between multivesicular bodies and secretory vesicles.     

Mouse liver PMP70 and ALDP: homomeric interactions prevail in vivo

ALDP, ALDPR, PMP70 and PMP70R are half ATP-binding cassette (ABC) transporters of the mammalian peroxisomal membrane. By analogy with other members of this family, it is assumed that peroxisomal ABC transporters must dimerize to become functional units. However, not much is known regarding the type of dimers (i.e., homodimers and/or heterodimers) that are formed in vivo under normal expression conditions. In this work, we have characterized the quaternary structure of mouse liver PMP70 and ALDP. The PMP70 protein complex was purified to apparent homogeneity using a two-step purification protocol. The ALDP-containing protein complex was characterized by preparative immunoprecipitation experiments. In both cases, no evidence for the existence of heteromeric interactions or for the presence of accessory proteins in these ABC transporter protein complexes could be obtained. Our data indicate that the majority (if not all) of mouse liver PMP70 and ALDP are homomeric proteins.     

Contrasted Microcolinearity and Gene Evolution Within a Homoeologous Region of Wheat and Barley Species

We study here the evolution of genes located in the same physical locus using the recently sequenced Ha locus in seven wheat genomes in diploid, tetraploid, and hexaploid species and compared them with barley and rice orthologous regions. We investigated both the conservation of microcolinearity and the molecular evolution of genes, including coding and noncoding sequences. Microcolinearity is restricted to two groups of genes (Unknown gene-2, VAMP, BGGP, Gsp-1, and Unknown gene-8 surrounded by several copies of ATPase), almost conserved in rice and barley, but in a different relative position. Highly conserved genes between wheat and rice run along with genes harboring different copy numbers and highly variable sequences between close wheat genomes. The coding sequence evolution appeared to be submitted to heterogeneous selective pressure and intronic sequences analysis revealed that the molecular clock hypothesis is violated in most cases.     

Direct quantification of mitochondrial DNA and its 4.9-kb common deletion without DNA purification

We studied the extraction and analysis of integral membrane proteins possessing hydrophobic and hydrophilic domains and found that a nonionic detergent called MEGA-10, used in lysis buffers, had a superior extraction effect compared to most conventional detergents. A sodium dodecyl sulfate (SDS) concentration of >0.4% (w/v) in the sample buffer was crucial for those proteins to be clearly analyzed by electrophoresis and Western blotting. Furthermore, MEGA-10 had the tendency to maximally extract proteins around its critical micelle concentration (CMC) of 0.24% (w/v). These solutions can greatly assist functional investigations of membrane proteins in the proteomics era.