In vitro plant regeneration and Agrobacterium tumefaciens-mediated transformation of Datura stramonium (Solanaceae).

Premise of the Study:Datura stramonium is a pharmacologically and evolutionarily important plant species in the family Solanaceae. Stable transformation methodology of this species would be advantageous for future genetic studies. Methods:In vitro plant regeneration and Agrobacterium tumefaciens-mediated transformation techniques were developed for D. stramonium based on methods reported for tomato. A binary vector containing pAtUBQ10::erGFP was used for transformation. Results:We recovered primary transformants harboring the green fluorescent protein (GFP) transgene that resulted in expression of fluorescence in all tissues analyzed. Transformants were allowed to self-pollinate, and two of five progeny contained the GFP transgene and displayed fluorescence identical to the primary transformants. Discussion:We have demonstrated the first stable transformation in the genus Datura. This is a key first step to study the genetic basis of traits in this evolutionarily interesting species

Gene Evolution in Solanaceae

Among flowering plants there have been frequent evolutionary transitions from an ancestral dry fruit to a derived fleshy fruit. These transitions have dramatic consequences since fleshy fruits often attract animals, including humans, that disperse these fruits and their seeds over large distances. Rarely, however, these transitions occur in reverse, shifting from fleshy back to dry fruits. We set out to better understand these transitions and their genetic basis. To enable more detailed studies, we developed a transformation protocol for Datura stramonium, a species whose dry fruit exemplifies this evolutionary reversion. We then complemented this protocol with a draft genome assembly of this plant and used this to show an increased mutation rate following transformation but negligible impact on gene expression. We also found evidence in D. stramonium for lineage-specific gene duplications in a pathway that synthesizes medicinally important tropane alkaloids. Next, we analyzed gene expression patterns over time in the pericarps of five species with differing fruit types. This revealed a core set of 121 genes with conserved patterns of expression among all species. These core fruit development genes contained a number of known developmental regulators but also implicated unexpected developmental pathways potentially involving brassinosteroids or small RNAs

Gene Evolution in Solanaceae

Among flowering plants there have been frequent evolutionary transitions from an ancestral dry fruit to a derived fleshy fruit. These transitions have dramatic consequences since fleshy fruits often attract animals, including humans, that disperse these fruits and their seeds over large distances. Rarely, however, these transitions occur in reverse, shifting from fleshy back to dry fruits. We set out to better understand these transitions and their genetic basis. To enable more detailed studies, we developed a transformation protocol for Datura stramonium, a species whose dry fruit exemplifies this evolutionary reversion. We then complemented this protocol with a draft genome assembly of this plant and used this to show an increased mutation rate following transformation but negligible impact on gene expression. We also found evidence in D. stramonium for lineage-specific gene duplications in a pathway that synthesizes medicinally important tropane alkaloids. Next, we analyzed gene expression patterns over time in the pericarps of five species with differing fruit types. This revealed a core set of 121 genes with conserved patterns of expression among all species. These core fruit development genes contained a number of known developmental regulators but also implicated unexpected developmental pathways potentially involving brassinosteroids or small RNAs

In vitro plant regeneration and Agrobacterium tumefaciens-mediated transformation of Datura stramonium (Solanaceae).

Premise of the studyDatura stramonium is a pharmacologically and evolutionarily important plant species in the family Solanaceae. Stable transformation methodology of this species would be advantageous for future genetic studies.MethodsIn vitro plant regeneration and Agrobacterium tumefaciens-mediated transformation techniques were developed for D. stramonium based on methods reported for tomato. A binary vector containing pAtUBQ10::erGFP was used for transformation.ResultsWe recovered primary transformants harboring the green fluorescent protein (GFP) transgene that resulted in expression of fluorescence in all tissues analyzed. Transformants were allowed to self-pollinate, and two of five progeny contained the GFP transgene and displayed fluorescence identical to the primary transformants.DiscussionWe have demonstrated the first stable transformation in the genus Datura. This is a key first step to study the genetic basis of traits in this evolutionarily interesting species

Evolution and Diversification of FRUITFULL Genes in Solanaceae.

Ecologically and economically important fleshy edible fruits have evolved from dry fruit numerous times during angiosperm diversification. However, the molecular mechanisms that underlie these shifts are unknown. In the Solanaceae there has been a major shift to fleshy fruits in the subfamily Solanoideae. Evidence suggests that an ortholog of FRUITFULL (FUL), a transcription factor that regulates cell proliferation and limits the dehiscence zone in the silique of Arabidopsis, plays a similar role in dry-fruited Solanaceae. However, studies have shown that FUL orthologs have taken on new functions in fleshy fruit development, including regulating elements of tomato ripening such as pigment accumulation. FUL belongs to the core eudicot euFUL clade of the angiosperm AP1/FUL gene lineage. The euFUL genes fall into two paralogous clades, euFULI and euFULII. While most core eudicots have one gene in each clade, Solanaceae have two: FUL1 and FUL2 in the former, and MBP10 and MBP20 in the latter. We characterized the evolution of the euFUL genes to identify changes that might be correlated with the origin of fleshy fruit in Solanaceae. Our analyses revealed that the Solanaceae FUL1 and FUL2 clades probably originated through an early whole genome multiplication event. By contrast, the data suggest that the MBP10 and MBP20 clades are the result of a later tandem duplication event. MBP10 is expressed at weak to moderate levels, and its atypical short first intron lacks putative transcription factor binding sites, indicating possible pseudogenization. Consistent with this, our analyses show that MBP10 is evolving at a faster rate compared to MBP20. Our analyses found that Solanaceae euFUL gene duplications, evolutionary rates, and changes in protein residues and expression patterns are not correlated with the shift in fruit type. This suggests deeper analyses are needed to identify the mechanism underlying the change in FUL ortholog function

Evolution and Diversification of FRUITFULL Genes in Solanaceae

Ecologically and economically important fleshy edible fruits have evolved from dry fruit numerous times during angiosperm diversification. However, the molecular mechanisms that underlie these shifts are unknown. In the Solanaceae there has been a major shift to fleshy fruits in the subfamily Solanoideae. Evidence suggests that an ortholog of FRUITFULL (FUL), a transcription factor that regulates cell proliferation and limits the dehiscence zone in the silique of Arabidopsis, plays a similar role in dry-fruited Solanaceae. However, studies have shown that FUL orthologs have taken on new functions in fleshy fruit development, including regulating elements of tomato ripening such as pigment accumulation. FUL belongs to the core eudicot euFUL clade of the angiosperm AP1/FUL gene lineage. The euFUL genes fall into two paralogous clades, euFULI and euFULII. While most core eudicots have one gene in each clade, Solanaceae have two: FUL1 and FUL2 in the former, and MBP10 and MBP20 in the latter. We characterized the evolution of the euFUL genes to identify changes that might be correlated with the origin of fleshy fruit in Solanaceae. Our analyses revealed that the Solanaceae FUL1 and FUL2 clades probably originated through an early whole genome multiplication event. By contrast, the data suggest that the MBP10 and MBP20 clades are the result of a later tandem duplication event. MBP10 is expressed at weak to moderate levels, and its atypical short first intron lacks putative transcription factor binding sites, indicating possible pseudogenization. Consistent with this, our analyses show that MBP10 is evolving at a faster rate compared to MBP20. Our analyses found that Solanaceae euFUL gene duplications, evolutionary rates, and changes in protein residues and expression patterns are not correlated with the shift in fruit type. This suggests deeper analyses are needed to identify the mechanism underlying the change in FUL ortholog function

Evidence for three genetic loci involved in both anorexia nervosa risk and variation of body mass index

The maintenance of normal body weight is disrupted in patients with anorexia nervosa (AN) for prolonged periods of time. Prior to the onset of AN, premorbid body mass index (BMI) spans the entire range from underweight to obese. After recovery, patients have reduced rates of overweight and obesity. As such, loci involved in body weight regulation may also be relevant for AN and vice versa. Our primary analysis comprised a cross-trait analysis of the 1000 single-nucleotide polymorphisms (SNPs) with the lowest P-values in a genome-wide association meta-analysis (GWAMA) of AN (GCAN) for evidence of association in the largest published GWAMA for BMI (GIANT). Subsequently we performed sex-stratified analyses for these 1000 SNPs. Functional ex vivo studies on four genes ensued. Lastly, a look-up of GWAMA-derived BMI-related loci was performed in the AN GWAMA. We detected significant associations (P-values <5 × 10-5, Bonferroni-corrected P<0.05) for nine SNP alleles at three independent loci. Interestingly, all AN susceptibility alleles were consistently associated with increased BMI. None of the genes (chr. 10: CTBP2, chr. 19: CCNE1, chr. 2: CARF and NBEAL1; the latter is a region with high linkage disequilibrium) nearest to these SNPs has previously been associated with AN or obesity. Sex-stratified analyses revealed that the strongest BMI signal originated predominantly from females (chr. 10 rs1561589; Poverall: 2.47 × 10-06/Pfemales: 3.45 × 10-07/Pmales: 0.043). Functional ex vivo studies in mice revealed reduced hypothalamic expression of Ctbp2 and Nbeal1 after fasting. Hypothalamic expression of Ctbp2 was increased in diet-induced obese (DIO) mice as compared with age-matched lean controls. We observed no evidence for associations for the look-up of BMI-related loci in the AN GWAMA. A cross-trait analysis of AN and BMI loci revealed variants at three chromosomal loci with potential joint impact. The chromosome 10 locus is particularly promising given that the association with obesity was primarily driven by females. In addition, the detected altered hypothalamic expression patterns of Ctbp2 and Nbeal1 as a result of fasting and DIO implicate these genes in weight regulation