A highly mutable GST is essential for bract colouration in Euphorbia pulcherrima Willd. Ex Klotsch.

BACKGROUND: Mutation breeding is an extraordinary tool in plant breeding to increase the genetic variability, where mutations in anthocyanin biosynthesis are targets to generate distinctive phenotypes in ornamental species. In poinsettia, ionizing radiation is routinely applied in breeding programs to obtaining a range of colours, with nearly all pink and white varieties being obtained after γ- or X-ray mutagenesis of red varieties. In the present study we performed a thorough characterization of a potential mutagenesis target gene as the main responsible for the 'white paradox' in poinsettia. RESULTS: We identified a GST gene in poinsettia (Bract1) as an essential factor for the expression of anthocyanin-based red colouration of bracts, which presents a high phylogenetic similarity to known anthocyanin-related GSTs. Red poinsettia varieties and white mutants generated from these varieties by X-ray were analysed for polymorphisms related to the 'white paradox' in the species. A 4 bp mutation in a short repeat within the coding region of Bract1 is most likely responsible for the appearance of white phenotypes upon irradiation treatment. The polymorphism between wild-type and mutant alleles co-segregates with the phenotype in progeny from heterozygous red and white parents. Moreover, overexpression of Bract1 wild-type allele in Arabidopsis tt19 mutants restored the anthocyanin phenotype, while the Bract1 mutated allele showed to be non-functional. CONCLUSIONS: The identified repeat seems to be highly unstable, since mutated plants can be easily detected among fewer than 200 shoots derived from 10 mutated plants. Our data indicate that particular short repeat sequences, similar to microsatellite sequences or so-called dynamic mutations, might be hot spots for genetic variability. Moreover, the identification of the Bract1 mutation fills a gap on the understanding on the molecular mechanism of colour formation in poinsettia.

Hybrid de novo transcriptome assembly of poinsettia (Euphorbia pulcherrima Willd. Ex Klotsch) bracts.

BACKGROUND: Poinsettia is a popular and important ornamental crop, mostly during the Christmas season. Its bract coloration ranges from pink/red to creamy/white shades. Despite its ornamental value, there is a lack of knowledge about the genetics and molecular biology of poinsettia, especially on the mechanisms of color formation. We performed an RNA-Seq analysis in order to shed light on the transcriptome of poinsettia bracts. Moreover, we analyzed the transcriptome differences of red- and white-bracted poinsettia varieties during bract development and coloration. For the assembly of a bract transcriptome, two paired-end cDNA libraries from a red and white poinsettia pair were sequenced with the Illumina technology, and one library from a red-bracted variety was used for PacBio sequencing. Both short and long reads were assembled using a hybrid de novo strategy. Samples of red- and white-bracted poinsettias were sequenced and comparatively analyzed in three color developmental stages in order to understand the mechanisms of color formation and accumulation in the species. RESULTS: The final transcriptome contains 288,524 contigs, with 33% showing confident protein annotation against the TAIR10 database. The BUSCO pipeline, which is based on near-universal orthologous gene groups, was applied to assess the transcriptome completeness. From a total of 1440 BUSCO groups searched, 77% were categorized as complete (41% as single-copy and 36% as duplicated), 10% as fragmented and 13% as missing BUSCOs. The gene expression comparison between red and white varieties of poinsettia showed a differential regulation of the flavonoid biosynthesis pathway only at particular stages of bract development. An initial impairment of the flavonoid pathway early in the color accumulation process for the white poinsettia variety was observed, but these differences were no longer present in the subsequent stages of bract development. Nonetheless, GSTF11 and UGT79B10 showed a lower expression in the last stage of bract development for the white variety and, therefore, are potential candidates for further studies on poinsettia coloration. CONCLUSIONS: In summary, this transcriptome analysis provides a valuable foundation for further studies on poinsettia, such as plant breeding and genetics, and highlights crucial information on the molecular mechanism of color formation.

The rare orange-red colored Euphorbia pulcherrima cultivar 'Harvest Orange' shows a nonsense mutation in a flavonoid 3'-hydroxylase allele expressed in the bracts.

BACKGROUND: Commercially available poinsettia (Euphorbia pulcherrima) varieties prevalently accumulate cyanidin derivatives and show intense red coloration. Orange-red bract color is less common. We investigated four cultivars displaying four different red hues with respect to selected enzymes and genes of the anthocyanin pathway, putatively determining the color hue. RESULTS: Red hues correlated with anthocyanin composition and concentration and showed common dark red coloration in cultivars 'Christmas Beauty' and 'Christmas Feeling' where cyanidin derivatives were prevalent. In contrast, orange-red bract color is based on the prevalent presence of pelargonidin derivatives that comprised 85% of the total anthocyanin content in cv. 'Premium Red' and 96% in cv. 'Harvest Orange' (synonym: 'Orange Spice'). cDNA clones of flavonoid 3'-hydroxylase (F3'H) and dihydroflavonol 4-reductase (DFR) were isolated from the four varieties, and functional activity and substrate specificity of the corresponding recombinant enzymes were studied. Kinetic studies demonstrated that poinsettia DFRs prefer dihydromyricetin and dihydroquercetin over dihydrokaempferol, and thus, favor the formation of cyanidin over pelargonidin. Whereas the F3'H cDNA clones of cultivars 'Christmas Beauty', 'Christmas Feeling', and 'Premium Red' encoded functionally active enzymes, the F3'H cDNA clone of cv. 'Harvest Orange' contained an insertion of 28 bases, which is partly a duplication of 20 bases found close to the insertion site. This causes a frameshift mutation with a premature stop codon after nucleotide 132 and, therefore, a non-functional enzyme. Heterozygosity of the F3'H was demonstrated in this cultivar, but only the mutated allele was expressed in the bracts. No correlation between F3'H-expression and the color hue could be observed in the four species. CONCLUSIONS: Rare orange-red poinsettia hues caused by pelargonidin based anthocyanins can be achieved by different mechanisms. F3'H is a critical step in the establishment of orange red poinsettia color. Although poinsettia DFR shows a low substrate specificity for dihydrokaempferol, sufficient precursor for pelargonidin formation is available in planta, in the absence of F3'H activity.

Effect of stacking insecticidal cry and herbicide tolerance epsps transgenes on transgenic maize proteome.

BACKGROUND: The safe use of stacked transgenic crops in agriculture requires their environmental and health risk assessment, through which unintended adverse effects are examined prior to their release in the environment. Molecular profiling techniques can be considered useful tools to address emerging biosafety gaps. Here we report the first results of a proteomic profiling coupled to transgene transcript expression analysis of a stacked commercial maize hybrid containing insecticidal and herbicide tolerant traits in comparison to the single event hybrids in the same genetic background. RESULTS: Our results show that stacked genetically modified (GM) genotypes were clustered together and distant from other genotypes analyzed by PCA. Twenty-two proteins were shown to be differentially modulated in stacked and single GM events versus non-GM isogenic maize and a landrace variety with Brazilian genetic background. Enrichment analysis of these proteins provided insight into two major metabolic pathway alterations: energy/carbohydrate and detoxification metabolism. Furthermore, stacked transgene transcript levels had a significant reduction of about 34% when compared to single event hybrid varieties. CONCLUSIONS: Stacking two transgenic inserts into the genome of one GM maize hybrid variety may impact the overall expression of endogenous genes. Observed protein changes differ significantly from those of single event lines and a conventional counterpart. Some of the protein modulation did not fall within the range of the natural variability for the landrace used in this study. Higher expression levels of proteins related to the energy/carbohydrate metabolism suggest that the energetic homeostasis in stacked versus single event hybrid varieties also differ. Upcoming global databases on outputs from "omics" analyses could provide a highly desirable benchmark for the safety assessment of stacked transgenic crop events. Accordingly, further studies should be conducted in order to address the biological relevance and implications of such changes.

Author Correction: An integrated multi-omics analysis of the NK603 Roundup-tolerant GM maize reveals metabolism disturbances caused by the transformation process.

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Systematic miRNome profiling reveals differential microRNAs in transgenic maize metabolism.

BACKGROUND: While some genetically modified organisms (GMOs) are created to produce new double-stranded RNA molecules (dsRNA), in others, such molecules may occur as an unintended effect of the genetic engineering process. Furthermore, GMOs might produce naturally occurring dsRNA molecules in higher or lower quantities than its non-transgenic counterpart. This study is the first to use high-throughput technology to characterize the miRNome of commercialized GM maize events and to investigate potential alterations in miRNA regulatory networks. RESULTS: Thirteen different conserved miRNAs were found to be dys-regulated in GM samples. The insecticide Bt GM variety had the most distinct miRNome. These miRNAs target a range of endogenous transcripts, such as transcription factors and nucleic acid binding domains, which play key molecular functions in basic genetic regulation. In addition, we have identified 20 potential novel miRNAs with target transcripts involved in lipid metabolism in maize. isomiRs were also found in 96 conserved miRNAs sequences, as well as potential transgenic miRNA sequences, which both can be a source of potential off-target effects in the plant genome. We have also provided information on technical limitations and when to carry on additional in vivo experimental testing. CONCLUSIONS: These findings do not reveal hazards per se but show that robust and reproducible miRNA profiling technique can strengthen the assessment of risk by detecting any new intended and unintended dsRNA molecules, regardless of the outcome, at any stage of GMO development.

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses.

Some genetically modified (GM) plants have transgenes that confer tolerance to abiotic stressors. Meanwhile, other transgenes may interact with abiotic stressors, causing pleiotropic effects that will affect the plant physiology. Thus, physiological alteration might have an impact on the product safety. However, routine risk assessment (RA) analyses do not evaluate the response of GM plants exposed to different environmental conditions. Therefore, we here present a proteome profile of herbicide-tolerant maize, including the levels of phytohormones and related compounds, compared to its near-isogenic non-GM variety under drought and herbicide stresses. Twenty differentially abundant proteins were detected between GM and non-GM hybrids under different water deficiency conditions and herbicide sprays. Pathway enrichment analysis showed that most of these proteins are assigned to energetic/carbohydrate metabolic processes. Among phytohormones and related compounds, different levels of ABA, CA, JA, MeJA and SA were detected in the maize varieties and stress conditions analysed. In pathway and proteome analyses, environment was found to be the major source of variation followed by the genetic transformation factor. Nonetheless, differences were detected in the levels of JA, MeJA and CA and in the abundance of 11 proteins when comparing the GM plant and its non-GM near-isogenic variety under the same environmental conditions. Thus, these findings do support molecular studies in GM plants Risk Assessment analyses.

Levels of DNA methylation and transcript accumulation in leaves of transgenic maize varieties.

BACKGROUND: Prior to their release in the environment, transgenic crops are examined for their health and environmental safety. In addition, transgene expression needs to be consistent in order to express the introduced trait (e.g. insecticidal and/or herbicide tolerance). Moreover, data on expression levels for GM events are usually required for approval, but these are rarely disclosed or they are considered insufficient. On the other hand, biosafety regulators do not consider epigenetic regulation (e.g. DNA methylation, ncRNAs and histone modifications), which are broadly known to affect gene expression, within their risk assessment analyses. Here we report the results of a DNA methylation (bisulfite sequencing) and transgene transcript accumulation (RT-qPCR) analysis of four Bt-expressing single transgenic maize hybrids, under different genetic backgrounds, and a stacked transgenic hybrid expressing both insecticidal and herbicide tolerance traits. RESULTS: Our results showed differences in cytosine methylation levels in the FMV promoter and cry2Ab2 transgene of the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids under the same genetic background showed differences in the 35S promoter sequence. The results of transgene transcript accumulation levels showed differences in both cry1A.105 and cry2Ab2 transgenes among the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids showed difference for the cry2Ab2 transgene only. CONCLUSIONS: Overall, our results show differences in DNA methylation patterns in all varieties, as well as in transgene transcript accumulation levels. Although the detection of changes in DNA methylation and transgenic accumulation levels does not present a safety issue per se, it demonstrates the need for additional studies that focus on detecting possible safety implications of such changes.

An integrated multi-omics analysis of the NK603 Roundup-tolerant GM maize reveals metabolism disturbances caused by the transformation process.

Glyphosate tolerant genetically modified (GM) maize NK603 was assessed as 'substantially equivalent' to its isogenic counterpart by a nutrient composition analysis in order to be granted market approval. We have applied contemporary in depth molecular profiling methods of NK603 maize kernels (sprayed or unsprayed with Roundup) and the isogenic corn to reassess its substantial equivalence status. Proteome profiles of the maize kernels revealed alterations in the levels of enzymes of glycolysis and TCA cycle pathways, which were reflective of an imbalance in energy metabolism. Changes in proteins and metabolites of glutathione metabolism were indicative of increased oxidative stress. The most pronounced metabolome differences between NK603 and its isogenic counterpart consisted of an increase in polyamines including N-acetyl-cadaverine (2.9-fold), N-acetylputrescine (1.8-fold), putrescine (2.7-fold) and cadaverine (28-fold), which depending on context can be either protective or a cause of toxicity. Our molecular profiling results show that NK603 and its isogenic control are not substantially equivalent.

Characterization of 10 new nuclear microsatellite markers in Acca sellowiana (Myrtaceae).

PREMISE OF THE STUDY: Microsatellite primers were identified and characterized in Acca sellowiana in order to expand the limited number of pre-existing polymorphic markers for use in population genetic studies for conservation, phylogeography, breeding, and domestication. • METHODS AND RESULTS: A total of 10 polymorphic microsatellite primers were designed from clones obtained from a simple sequence repeat (SSR)-enriched genomic library. The primers amplified di- and trinucleotide repeats with four to 27 alleles per locus. In all tested populations, the observed heterozygosity ranged from 0.269 to 1.0. • CONCLUSIONS: These new polymorphic SSR markers will allow future genetic studies to be denser, either for genetic structure characterization of natural populations or for studies involving genetic breeding and domestication process in A. sellowiana.