Biology subjects

Pucker, B.

Publications and source records attributed to Pucker, B..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Phylogenetic placement of Ceratophyllum submersum based on a complete plastome sequence derived from nanopore long read sequencing data

ObjectiveEutrophication poses a mounting concern in todays world. Ceratophyllum submersum L. is one of many plants capable of living in eutrophic conditions, therefore it could play a critical role in addressing the problem of eutrophication. This study aimed to take a first genomic look at C. submersum. ResultsSequencing of gDNA from C. submersum yielded enough reads to assemble a plastome. Subsequent annotation and phylogenetic analysis validated existing information regarding angiosperm relationships and the positioning of Ceratophylalles in a wider phylogenetic context.

genomics↗

bioRxiv2023

Genomic characterization of a nematode tolerance locus in sugar beet

BackgroundInfection by beet cyst nematodes (BCN, Heterodera schachtii) causes a serious disease of sugar beet, and climatic change is expected to improve the conditions for BCN infection. Yield and yield stability under adverse conditions are among the main breeding objectives. Breeding of BCN tolerant sugar beet cultivars offering high yield in the presence of the pathogen is therefore of high relevance. ResultsTo identify causal genes providing tolerance against BCN infection, we combined several experimental and bioinformatic approaches. Relevant genomic regions were detected through mapping-by-sequencing using a segregating F2 population. DNA sequencing of contrasting F2 pools and analyses of allele frequencies for variant positions identified a single genomic region which confers nematode tolerance. The genomic interval was confirmed and narrowed down by genotyping with newly developed molecular markers. To pinpoint the causal genes within the potential nematode tolerance locus, we generated long read-based genome sequence assemblies of the tolerant parental breeding line Strube U2Bv and the susceptible reference line 2320Bv. We analyzed continuous sequences of the potential locus with regard to functional gene annotation and differential gene expression upon BCN infection. A cluster of genes with similarity to the Arabidopsis thaliana gene encoding nodule inception protein-like protein 7 (NLP7) was identified. Gene expression analyses confirmed transcriptional activity and revealed clear differences between susceptible and tolerant genotypes. ConclusionsOur findings provide new insights into the genomic basis of plant-nematode interactions that can be used to design and accelerate novel management strategies against BCN.

plant biology↗

bioRxiv2023

Genetic factors explaining anthocyanin pigmentation differences

BackgroundAnthocyanins represent one of the most abundant coloration factors found in plants. Biological functions of anthocyanins range from reproduction to protection against biotic and abiotic stressors. Owing to a clearly visible phenotype of mutants, the anthocyanin biosynthesis and its sophisticated regulation have been studied in numerous plant species. Genes encoding the anthocyanin biosynthesis enzymes are regulated by a transcription factor complex comprising MYB, bHLH and WD40 proteins. ResultsA systematic comparison of anthocyanin-pigmented vs. non-pigmented varieties across flowering plant species was performed. Literature was screened for cases in which genetic factors causing anthocyanin loss were reported. Additionally, transcriptomic data sets from previous studies were reanalyzed to determine the genes most likely to be responsible for color variation based on their expression pattern. The contribution of different structural and regulatory genes to the pigmentation differences was quantified. Gene expression differences concerning transcription factors are by far the most frequent explanation for pigmentation differences observed between two varieties of the same species. Among the transcription factors in the analyzed cases, MYB genes are substantially more likely to explain pigmentation differences than bHLH or WD40 genes. ConclusionsThese findings support previous assumptions about the plasticity of transcriptional regulation and its importance for the evolution of novel coloration phenotypes. Our findings underline the particular significance of MYBs and their apparent dominant role in the specificity of the MBW complex.

plant biology↗

bioRxiv2023

Genomic and transcriptomic analysis of camptothecin producing novel fungal endophyte - Alternaria burnsii NCIM 1409

Camptothecin is an important anticancer alkaloid produced by particular plant species. No suitable synthetic route has been established for camptothecin production yet, imposing a stress on plant-based production systems. Endophytes associated with these camptothecin-producing plants have been reported to also produce camptothecin and other high-value phytochemicals. A previous study identified a fungal endophyte Alternaria burnsii NCIM 1409, isolated from Nothapodytes nimmoniana, to be a sustainable producer of camptothecin. Our study provides key insights on camptothecin biosynthesis in this recently discovered endophyte. The whole genome sequence of Alternaria burnsii NCIM 1409 was assembled and screened for biosynthetic gene clusters. Comparative studies with related fungi supported the identification of candidate genes involved in camptothecin synthesis and also helped to understand some aspects of the endophytes defense against the toxic effects of camptothecin. No evidence for horizontal gene transfer of the camptothecin biosynthetic genes from the host plant to the endophyte was detected suggesting an independent evolution of the camptothecin biosynthesis in this fungus.

genomics↗

bioRxiv2023

Automatic annotation of the bHLH gene family in plants

BackgroundThe bHLH transcription factor family is named after the basic helix-loop-helix (bHLH) domain that is a characteristic element of their members. Understanding the function and characteristics of this family is important for the examination of a wide range of functions. As the availability of genome sequences and transcriptome assemblies has increased significantly, the need for automated solutions that provide reliable functional annotations is emphasised. ResultsA phylogenetic approach was adapted for the automatic identification and functional annotation of the bHLH transcription factor family. The bHLH_annotator for the automated functional annotation of bHLHs was implemented in Python3. Sequences of bHLHs described in literature were collected to represent the full diversity of bHLH sequences. Previously described orthologs form the basis for the functional annotation assignment to candidates which are also screened for bHLH-specific motifs. The pipeline was successfully deployed on the two Arabidopsis thaliana accessions Col-0 and Nd-1, the monocot species Dioscorea dumetorum, and a transcriptome assembly of Croton tiglium. Depending on the applied search parameters for the initial candidates in the pipeline, species-specific candidates or members of the bHLH family which experienced domain loss can be identified. ConclusionsThe bHLH_annotator allows a detailed and systematic investigation of the bHLH family in land plant species and classifies candidates based on bHLH-specific characteristics, which distinguishes the pipeline from other established functional annotation tools. This provides the basis for the functional annotation of the bHLH family in land plants and the systematic examination of a wide range of functions regulated by this transcription factor family.

bioinformatics↗

bioRxiv2023

Identification of annotation artifacts concerning the CHALCONE SYNTHASE (CHS)

ObjectiveChalcone synthase (CHS) catalyzes the initial step of the flavonoid biosynthesis. The CHS encoding gene is well studied in numerous plant species. Rapidly growing sequence databases contain hundreds of CHS entries that are the result of automatic annotation. In this study, we evaluated apparent multiplication of CHS domains in CHS gene models of four plant species. Main findingsCHS genes with an apparent triplication of the CHS domain encoding part were discovered through database searches. Such genes were found in Macadamia integrifolia, Musa balbisiana, Musa troglodytarum, and Nymphaea colorata. A manual inspection of the CHS gene models in these four species with massive RNA-seq data suggests that these gene models are the result of artificial fusions in the annotation process. While there are hundreds of apparently correct CHS records in the databases, it is not clear why these annotation artifacts appeared.

bioinformatics↗

bioRxiv2022

Evolutionary blocks to anthocyanin accumulation and the loss of an anthocyanin carrier protein in betalain-pigmented Caryophyllales

{square} The order Caryophyllales exhibits complex pigment evolution, with mutual exclusion of anthocyanin and betalain pigments. Given recent evidence for multiple shifts to betalain pigmentation, we re-evaluated potential mechanisms underpinning the exclusion of anthocyanins from betalain-pigmented lineages. {square}We examined the evolution of the flavonoid pathway using transcriptomic and genomic datasets covering 309 species in 31 families. Orthologs and paralogs of known flavonoid synthesis genes were identified by sequence similarity, with gene duplication and gene loss inferred by phylogenetic and syntenic analysis. Relative transcript abundances were assessed to reveal broad-scale gene expression changes between betalain- and anthocyanin-pigmented lineages. {square}Most flavonoid genes are retained and transcribed in betalain-pigmented lineages, and many also show evidence of extensive gene duplication within betalain-pigmented lineages. However, expression of several flavonoid genes is reduced in betalain-pigmented lineages, especially the late-stage genes dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). Notably flavonoid 3',5'-hydroxylase (F3'5'H) homologs have been repeatedly lost in belatain-pigmented lineages, and Anthocyanin9 (AN9) homologs are undetectable in any betalain-pigmented lineages. {square}Down-regulation of ANS and DFR homolog expression (limiting synthesis) and reiterative loss of AN9 homologs (limiting transport), coincident with multiple shifts to betalain pigmentation, are likely crucial the loss of anthocyanins in betalain-pigmented Caryophyllales.

evolutionary biology↗

bioRxiv2022

Flavonols contrary affect the interconnected glucosinolate and camalexin biosynthesis pathway in Arabidopsis thaliana

Flavonols are structurally and functionally diverse molecules playing roles in plant biotic and abiotic stress tolerance, auxin transport inhibition, pollen development, etc. Despite their ubiquitous occurrence in land plants and multifunctionality, the effect of perturbation of flavonol biosynthesis over global gene expression and pathways other than flavonoid biosynthesis has not been studied in detail. To understand the signaling role of different flavonol metabolites, herein, we used the flavonol deficient Arabidopsis thaliana loss-of-function mutant flavonol synthase1 (fls1-3) as object of study. Comparative transcriptome and metabolic profiling were used to study the effects of genetic flavonol deficiency and exogenous supplementation with flavonol derivatives (kaempferol, quercetin and rutin) on different cellular processes in the seedling. Various flavonol biosynthesis-related regulatory and structural genes were found to be up-regulated in the fls1-3 mutant which could be reversed by exogenous flavonol feeding. Our manifold comparative studies indicated the modulation of various biological processes and metabolic pathways by flavonols. Camalexin biosynthesis was found to be negatively regulated by flavonols. Interestingly, flavonols appeared to promote the accumulation of aliphatic glucosinolate through transcription factor-mediated up-regulation of biosynthesis genes. Overall, this study provides new insights into molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets and adds new knowledge to the expanding plethora of biological activity of flavonols in plants. SignificanceComparative transcriptome and metabolomic profiling of genetic flavonol deficiency and exogenous flavonol supplementation in A. thaliana seedlings, for the first-time revealed the inverse regulation of interconnected specialized metabolite pathways by flavonol aglycones, and -glycosides. Flavonols negatively regulate camalexin biosynthesis, while promoting the accumulation of aliphatic glucosinolates. Our study adds new insights into the expanding plethora of biological activity of flavonols in plants and will help to uncover the molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets.

plant biology↗

bioRxiv2022

Three R2R3-MYB transcription factors from banana (Musa spp.) activate structural anthocyanin biosynthesis genes as part of an MBW complex

Bananas are among the most popular fruits in the world and provide food security and employment opportunities in several developing countries. An increased anthocyanin content could enhance the health promoting properties of banana fruits. The biosynthesis of anthocyanins is largely regulated at the transcriptional level. However, little is known about transcriptional activation of anthocyanin biosynthesis in banana. We analysed the regulatory activity of three MusaMYBs predicted by bioinformatic analysis to transcriptionally regulate anthocyanin biosynthesis in banana. MusaMYBA1, MusaMYBA2 and MusaMYBPA2 did not complement the anthocyanin deficiency phenotype of the A. thaliana pap1/pap2 mutant. However, co-transfection experiments in A. thaliana protoplasts showed that MusaMYBA1, MusaMYBA2 and MusaMYBPA2 function as components of a transcription factor complex with a bHLH and WD40 protein, called MBW complex, resulting in the activation of the anthocyanin synthase and dihydroflavonol 4-reductase promoters from A. thaliana. The activation potential of MusaMYBA1, MusaMYBA2 and MusaMYBPA2 increased when combined with the monocot bHLH ZmR instead of the dicot AtEGL3. This work paves the path towards decoding the MBW complex-mediated transcriptional activation of anthocyanin biosynthesis in banana. Moreover, it facilitates research towards an elevated anthocyanin content in banana and other monocot crops.

molecular biology↗

bioRxiv2022

KIPEs3: Automatic annotation of biosynthesis pathways

Flavonoids and carotenoids are pigments involved in stress mitigation and numerous other processes. Both pigment classes can contribute to flower and fruit coloration. Flavonoid aglycones and carotenoids are produced by a pathway that is largely conserved across land plants. Glycosylations, acylations, and methylations of the flavonoid aglycones can be species-specific and lead to a plethora of biochemically diverse flavonoids. We previously developed KIPEs for the automatic annotation of biosynthesis pathways and presented an application on the flavonoid aglycone biosynthesis. KIPEs3 is an improved version with additional features and the potential to identify not just the core biosynthesis players, but also candidates involved in the decoration steps and in the transport of flavonoids. Functionality of KIPEs3 is demonstrated through the analysis of the flavonoid biosynthesis in Arabidopsis thaliana Nd-1, Capsella grandiflora, and Dioscorea dumetorum. We demonstrate the applicability of KIPEs to other pathways by adding the carotenoid biosynthesis to the repertoire. As a technical proof of concept, the carotenoid biosynthesis was analyzed in the same species and Daucus carota. KIPEs3 is available as an online service to enable access without prior bioinformatics experience. KIPEs3 facilitates the automatic annotation and analysis of biosynthesis pathways with a consistent and high quality in a large number of plant species. Numerous genome sequencing projects are generating a huge amount of data sets that can be analyzed to identify evolutionary patterns and promising candidate genes for biotechnological and breeding applications.

bioinformatics↗

bioRxiv2022

Mapping-by-sequencing reveals genomic regions associated with seed quality parameters in Brassica napus

Rapeseed (Brassica napus L.) is an important oil crop and harbours the potential to serve as a highly productive source of protein. This protein exhibits an excellent amino acid composition and has a high nutritional value for humans. Seed protein content (SPC) and seed oil content (SOC) are two complex quantitative and polygenic traits which are negatively correlated and assumed to be controlled by additive and epistatic effects. A reduction of seed glucosinolate (GSL) content is desired as GSLs cause a stringent and bitter taste. The goal here was the identification of genomic intervals relevant for seed GSL content and SPC/SOC. Mapping-by-sequencing (MBS) revealed 30 and 15 new and known genomic intervals associated with seed GSL content and SPC/SOC, respectively. Within these intervals we identified known but also so far unknown putatively causal genes and sequence variants. A 4 bp insertion in the MYB28 homolog on C09 shows a significant correlation with a reduction in seed GSL content. This study provides insights into the genetic architecture and potential mechanisms underlying seed quality traits, which will enhance future breeding approaches in B. napus.

genomics↗

bioRxiv2022

Apiaceae FNS I originated from F3H through tandem gene duplication

BackgroundFlavonoids are specialized metabolites with numerous biological functions in stress response and reproduction of plants. Flavones are one subgroup that is produced by the flavone synthase (FNS). Two distinct enzyme families evolved that can catalyze the biosynthesis of flavones. While the membrane-bound FNS II is widely distributed in seed plants, one lineage of soluble FNS I appeared to be unique to Apiaceae species. ResultsWe show through phylogenetic and comparative genomic analyses that Apiaceae FNS I evolved through tandem gene duplication of flavanone 3-hydroxylase (F3H) followed by neofunctionalization. Currently available datasets suggest that this event happened within the Apiaceae in a common ancestor of Daucus carota and Apium graveolens. The results also support previous findings that FNS I in the Apiaceae evolved independent of FNS I in other plant species. ConclusionWe validated a long standing hypothesis about the evolution of Apiaceae FNS I and predicted the phylogenetic position of this event. Our results explain how an Apiaceae-specific FNS I lineage evolved and confirm independence from other FNS I lineages reported in non-Apiaceae species.

plant biology↗

bioRxiv2021

The evidence for anthocyanins in the betalain-pigmented genus Hylocereus is weak

Here we respond to Zhou et al., 2020 "Combined Transcriptome and Metabolome analysis of Pitaya fruit unveiled the mechanisms underlying Peel and pulp color formation" published in BMC Genomics. Given the evolutionary conserved anthocyanin biosynthesis pathway in betalain-pigmented species, we are open to the idea that species with both anthocyanins and betalains might exist. However, in absence of LC-MS/MS spectra, apparent lack of biological replicates, and no comparison to authentic standards, the findings of Zhou et al., 2020 are not a strong basis to propose the presence of anthocyanins in betalain-pigmented pitaya. In addition, our re-analysis of the datasets indicates the misidentification of important genes and the omission of key anthocyanin synthesis genes ANS and DFR. Finally, our re-analysis of the RNA-Seq dataset reveals no correlation between anthocyanin biosynthesis gene expression and pigment status.

plant biology↗

bioRxiv2021

Automatic identification and annotation of MYB gene family members in plants

BackgroundMYBs are among the largest transcription factor families in plants. Consequently, members of this family are involved in a plethora of processes including development and specialized metabolism. The MYB families of many plant species were investigated in the last two decades since the first investigation looked at Arabidopsis thaliana. This body of knowledge and characterized sequences provide the basis for the identification, classification, and functional annotation of candidate sequences in new genome and transcriptome assemblies. ResultsA pipeline for the automatic identification and functional annotation of MYBs in a given sequence data set was implemented in Python. MYB candidates are identified, screened for the presence of a MYB domain and other motifs, and finally placed in a phylogenetic context with well characterized sequences. In addition to technical benchmarking based on existing annotation, the transcriptome assembly of Croton tiglium and the annotated genome sequence of Castanea crenata were screened for MYBs. Results of both analyses are presented in this study to illustrate the potential of this application. The analysis of one species takes only a few minutes depending on the number of predicted sequences and the size of the MYB gene family. This pipeline, the required bait sequences, and reference sequences for a classification are freely available on github: https://github.com/bpucker/MYB_annotator. ConclusionsThis automatic annotation of the MYB gene family in novel assemblies makes genome-wide investigations consistent and paves the way for comparative studies in the future. Candidate genes for in-depth analyses are presented based on their orthology to previously characterized sequences which allows the functional annotation of the newly identified MYBs with high confidence. The identification of orthologs can also be harnessed to detect duplication and deletion events.

plant biology↗

bioRxiv2021

Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives

BackgroundAs the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution. ResultsWe sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provided high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species. Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated. ConclusionIn conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa.

genomics↗

bioRxiv2021

Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis

BackgroundExperimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. ResultsWe sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10% of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. ConclusionLong read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.

plant biology↗

bioRxiv2020

RNA-Seq Time Series of Vitis vinifera Bud Development Reveals Correlation of Expression Patterns with the Local Temperature Profile

Plants display sophisticated mechanisms to tolerate challenging environmental conditions and need to manage their ontogenesis in parallel. Here, we set out to generate an RNA-Seq time series dataset throughout grapevine (Vitis vinifera) early bud development. The expression of the developmental regulator VviAP1 served as an indicator for progress of development. We investigated the impact of changing temperatures on gene expression levels during the time series and detected a correlation between increased temperatures and a high expression level of genes encoding heat-shock proteins. The data set also allowed the exemplary investigation of expression patterns of genes from three transcription factor (TF) gene families, namely MADS-box, WRKY, and R2R3-MYB genes. Inspection of the expression profiles from all three TF gene families indicated that a switch in the developmental program takes place in July which coincides with increased expression of the bud dormancy marker gene VviDRM1.

plant biology↗

bioRxiv2020

Automatic identification of players in the flavonoid biosynthesis with application on the biomedicinal plant Croton tiglium

The flavonoid biosynthesis is a well characterised model system for specialised metabolism and transcriptional regulation in plants. Flavonoids have numerous biological functions like UV protection and pollinator attraction, but also biotechnological potential. Here, we present Knowledge-based Identification of Pathway Enzymes (KIPEs) as an automatic approach for the identification of players in the flavonoid biosynthesis. KIPEs combines comprehensive sequence similarity analyses with the inspection of functionally relevant amino acid residues and domains in subjected peptide sequences. Comprehensive sequence sets of flavonoid biosynthesis enzymes and knowledge about functionally relevant amino acids were collected. As a proof of concept, KIPEs was applied to investigate the flavonoid biosynthesis of the medicinal plant Croton tiglium based on a transcriptome assembly. Enzyme candidates for all steps in the biosynthesis network were identified and matched to previous reports of corresponding metabolites in Croton species.

plant biology↗