Biology subjects

Weisshaar, B.

Publications and source records attributed to Weisshaar, B..

At least 19 recordsLinked to original sources

bioRxiv2025

A sublethal drought and rewatering time course reveals intricate patterning of responses in the annual Arabidopsis thaliana

Drought is a major factor of yield loss in annual crops. It triggers a wide range of physiological and molecular changes which are mediated by a suite of transcription factors from at least four different protein families. In the literature, the observed phenotypic changes and the number of transcriptome changes upon drought in Arabidopsis thaliana varies widely. To resolve the apparent variation, we conducted a phenotyping and transcriptomics experiment with progressive drought which is initially mild and escalates to strong but still sublethal drought followed by rewatering. Phenotypic data is analyzed with machine learning methods and connected to transcriptome data. The phenotypic data show that drought stress manifests in distinct stages. The transcriptional analysis shows one threshold program and gradual expression programs among typical drought responsive regulons over time and during recovery. Plant aging prior to senescence and the drought response overlap to a large degree and drought stressed plants rejuvenate transcriptionally before returning to the control aging program. The phenotypic traits are associated with different transcript abundances again reflecting multiple overlaying programs. Transcripts with high explanatory power of phenotypes are biomarkers and not causal for the phenotype. Significance statementLarge scale phenotyping and transcriptomics during progressive drought demonstrate that the drought response in the annual Arabidopsis thaliana is controlled by multiple transcriptional programs, mostly with gradual onset, that overlap with the plant aging program. Transcripts with high explanatory power for phenotypes are biomarkers and not causal.

plant biology↗

bioRxiv2024

Many transcription factor families have evolutionarily conserved binding motifs in plants

Transcription factors control gene expression during development and in response to a broad range of internal and external stimuli. They regulate promoter activity by directly binding cis- regulatory elements in DNA. The angiosperm Arabidopsis thaliana contains more than 1,500 annotated transcription factors, each containing a DNA-binding domain that is used to define transcription factor families. Analyzing binding motifs of 686 and the binding sites of 335 A. thaliana transcription factors as well as motifs of 92 transcription factors from other plants, we identified a constrained vocabulary of 74 conserved motifs spanning 50 families in plants. Among 21 transcription factor families, we found one core motif for all analyzed members and between 2 and 72% overlapping binding sites. Five families show conservation of the motif along phylogenetic clades. Five families including the C2H2 zinc finger family show high diversity among motifs in plants, suggesting potential for neofunctionalization of duplicated transcription factors based on the motif recognized. For conserved motifs we tested if they remained conserved since at least 450 million years ago by determining the binding motifs of 17 orthologous transcription factors from 11 families in M. polymorpha using amplified DNA affinity purification sequencing. We detected nearly identical binding motifs as predicted from the angiosperm data. Taken together, the results show a large repertoire of overlapping binding sites within a TF family and species and a high degree of binding motif conservation for at least 450 million years. The results indicate more potential for evolution in cis- rather than trans-regulatory elements.

plant biology↗

bioRxiv2024

Metabolic fingerprinting reveals roles of Arabidopsis thaliana BGLU1, BGLU3 and BGLU4 in glycosylation of various flavonoids

Flavonoids are specialized metabolites that play important roles in plants, including interactions with the environment. The high structural diversity of this metabolite group is largely due to enzyme-mediated modifications of flavonoid core skeletons. In particular, glycosylation with different sugars is very common. In this study, the functions of the Arabidopsis thaliana glycoside hydrolase family 1-type glycosyltransferase proteins BGLU1, BGLU3 and BGLU4 were investigated, using a reverse genetics approach and untargeted metabolic fingerprinting. We screened for metabolic differences between A. thaliana wild type, loss-of-function mutants and overexpression lines and partially identified differentially accumulating metabolites, which are putative products and/or substrates of the BGLU enzymes. Our study revealed that the investigated BGLU proteins are glycosyltransferases involved in the glycosylation of already glycosylated flavonoids using different substrates. While BGLU1 appears to be involved in the rhamnosylation of a kaempferol diglycoside in leaves, BGLU3 and BGLU4 are likely involved in the glycosylation of quercetin glycosides in A. thaliana seeds. In addition, we present evidence that BGLU3 is a multifunctional enzyme that catalyzes other metabolic reactions with more complex substrates. This study deepens our understanding of the metabolic pathways and enzymes that contribute to the high structural diversity of flavonoids. HighlightThe proteins BGLU1, BGLU3 and BGLU4 are involved in glycosylations of different, already glycosylated flavonoids in Arabidopsis thaliana. BGLU3 appears to be multifunctional, acting on several complex substrates.

plant biology↗

bioRxiv2023

Generation and characterisation of an Arabidopsis thaliana f3h/fls1/ans triple mutant that accumulates eriodictyol derivatives

BackgroundFlavonoids are plant specialised metabolites, which derive from phenylalanine and acetate metabolism. They possess a variety of beneficial characteristics for plants and humans. Several modification steps in the synthesis of tricyclic flavonoids cause for the amazing diversity of flavonoids in plants. The 2-oxoglutarate-dependent dioxygenases (2-ODDs) flavanone 3-hydroxylase (F3H, synonym FHT), flavonol synthase (FLS) and anthocyanidin synthase (ANS, synonym leucoanthocyanidin dioxigenase (LDOX)), catalyse oxidative modifications to the central C ring. They are highly similar and have been shown to catalyse, at least in part, each others reactions. FLS and ANS have been identified as bifunctional enzymes in many species, including Arabidopsis thaliana, stressing the capability of plants to bypass missing or mutated reaction steps on the way to flavonoid production. However, little is known about such bypass reactions and the flavonoid composition of plants lacking all three central flavonoid 2-ODDs. ResultsTo address this issue, we generated a f3h/fls1/ans mutant, as well as the corresponding double mutants and investigated the flavonoid composition of this mutant collection. The f3h/fls1/ans mutant was further characterised at the genomic level by analysis of a nanopore DNA sequencing generated genome sequence assembly and at the transcriptomic level by RNA-Seq analysis. The mutant collection established, including the novel double mutants f3h/fls1 and f3h/ans, was used to validate and analyse the multifunctionalities of F3H, FLS1, and ANS in planta. Metabolite analyses revealed the accumulation of eriodictyol and additional glycosylated derivatives in mutants carrying the f3h mutant allele, resulting from the conversion of naringenin to eriodictyol by flavonoid 3-hydroxylase (F3H) activity. ConclusionsWe describe the in planta multifunctionality of the three central flavonoid 2-ODDs from A. thaliana and identify a bypass in the f3h/fls1/ans triple mutant that leads to the formation of eriodictyol derivatives. As (homo-)eriodictyols are known as bitter taste maskers, the annotated eriodictyol (derivatives) and in particular the observations made on their in planta production, could provide valuable insights for creating of novel food supplements.

plant biology↗

bioRxiv2023

Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets

BackgroundSugar beet (Beta vulgaris subsp. vulgaris) and its crop wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNA sequences represent the fastest evolving parts of the genome, they likely impact genomic variability and contribute to the separation of beet gene pools. Hence, we investigated if innovations and losses in the repeatome can be linked to chromosomal differentiation and speciation. ResultsWe traced genome- and chromosome-wide evolution across sugar beet and twelve wild beets comprising all sections of the beet genera Beta and Patellifolia. For this, we combined data from short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive data framework for our beet panel that spans the complete scale from DNA sequence to chromosome up to the genome. Genome sizes and repeat profiles reflect the separation of the beet species into three gene pools. These gene pools harbor repeats with contrasting evolutionary patterns: We identified section- and species-specific repeat emergences and losses, e.g. of the retrotransposons causal for genome expansions in the section Corollinae/Nanae. Since most genomic variability was found in the satellite DNAs, we focused on tracing the 19 beetSat families across the three beet sections/genera. These taxa harbor evidence for contrasting strategies in repeat evolution, leading to contrasting satellite DNA profiles and fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia species to the formation of patchwork chromosomes in Corollinae/Nanae species. ConclusionsWe show that repetitive DNA sequences are causal for genome size expansion and contraction across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably among beet taxa, leading to the evolution of distinct chromosomal setups. These differences likely contribute to the barriers in beet breeding between the three gene pools. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genome variability, and chromosomal differentiation/evolution and provide a theoretical basis for understanding barriers in crop breeding.

genomics↗

bioRxiv2023

Transcription factors operate on a limited vocabulary of binding motifs in Arabidopsis thaliana

Predicting gene expression from promoter sequence requires understanding of the different signal integration points within a promoter. Sequence-specific transcription factors (TFs) binding to their cognate TF binding motifs control gene expression in eukaryotes by activating and repressing transcription. Their interplay generates complex expression patterns in reaction to environmental conditions and developmental cues. We hypothesized that signals are not only integrated by different TFs binding various positions in a promoter, but also by single TF binding motifs onto which multiple TFs can bind. Analyzing 2,190 binding motifs, we identified only 76 core TF binding motifs in plants. Twenty-one TF protein families act highly specific and bind a single conserved motif. Four TF families are classified as semi-conserved as they bind up to four motifs within a family, with divisions along phylogenetic groups. Five TF families bind diverse motifs. Expression analyses revealed high competition within TF families for the same binding motif. The results show that singular binding motifs act as signal integrators in plants where a combination of binding affinity and TF abundance likely determine the output.

systems biology↗

bioRxiv2023

Pangenome of cultivated beet and crop wild relatives reveals parental relationships of a tetraploid wild beet

Most crop plants, including sugar beet (Beta vulgaris subsp. vulgaris), suffer from domestication bottlenecks and low genetic diversity caused by extensive selection for few traits. However, crop wild relatives (CWRs) harbour useful traits relevant for crop improvement, including enhanced adaptation to biotic and abiotic stresses. Especially polyploids are interesting from an evolutionary perspective as genes undergo reorganisation after the polyploidisation event. Through neo-and subfunctionalisation, novel functions emerge, which enable plants to cope with changing environments and extreme/harsh conditions. Particularly in the face of climate change, specific stress and pathogen resistances or tolerances gain importance. To introduce such traits into breeding material, CWRs have already been identified as an important source for sustainable breeding. The identification of genes underlying traits of interest is crucial for crop improvement. For beets, the section Corollinae contains the tetraploid species Beta corolliflora (2n=4x=36) that harbours salt and frost tolerances as well as a wealth of pathogen resistances. The number of beneficial traits of B. corolliflora is increased compared to those of the known diploids in this section (all 2n=2x=18). Nevertheless, neither the parental relationships of B. corolliflora have been resolved, nor are genomic resources available to steer sustainable, genomics-informed breeding. To benefit from the resources offered by polyploid beet wild relatives, we generated a comprehensive pangenome dataset including B. corolliflora, Beta lomatogona, and Beta macrorhiza, as well as a more distant wild beet Patellifolia procumbens (2n=2x=18). Joined analyses with publicly available genome sequences of two additional wild beets allowed the identification of genomic regions absent from cultivated beet, providing a sequence database harbouring traits relevant for future breeding endeavours. In addition, we present strong evidence for the parental relationship of the B. corolliflora wild beet as an autotetraploid emerging from B. macrorhiza.

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↗

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

Phased grapevine genome sequence assembly of an Rpv12 carrier for exploration of Rpv12 associated positional and functional Plasmopara viticola resistance genes

The downy mildew disease caused by the oomycete Plasmopara viticola is a serious threat for grapevine and can cause enormous yield losses in viticulture. The quantitative trait locus Rpv12, mediating resistance against P. viticola, was originally found in Asian Vitis amurensis. This locus and its genes were analyzed here in detail. A haplotype-separated genome sequence of the diploid Rpv12-carrier Gf.99-03 was created and annotated. The defense response against P. viticola was investigated in an infection time-course RNA-Seq experiment, revealing approximately 600 up-regulated Vitis genes during host-pathogen interaction. The Rpv12 regions of the resistance conferring and the sensitivity encoding Gf.99-03 haplotypes were structurally and functionally compared to each other. Two different clusters of resistance-related genes were identified within the Rpv12 locus. One cluster carries a set of four differentially expressed genes with three ACCELERATED CELL DEATH 6-like genes. The other cluster carries a set of six resistance gene analogues related to qualitative pathogen resistance. The Rpv12 locus and its candidate genes for P. viticola resistance provide a precious genetic resource for P. viticola resistance breeding. Newly developed co-segregating simple sequence repeat markers in close proximity to the R-genes enable its improved applicability in marker-assisted grapevine breeding.

plant biology↗

bioRxiv2022

A fully phased interspecific grapevine rootstock genome sequence representing V. riparia and V. cinerea and allele-aware annotation of the phylloxera resistance locus Rdv1

The phylloxera resistant rootstock cultivar Borner is an interspecific hybrid derived from Vitis riparia and V. cinerea and a valuable resource for Vitis disease resistances. We created a fully phased, high-quality Borner genome sequence named BoeRC using long PacBio reads. Comprehensive gene annotation of both Borner haplotypes, designated BoeRip and BoeCin, was applied to describe the phylloxera resistance locus Rdv1. Using a mapping population derived from a susceptible V. vinifera breeding line and Borner, the Rdv1 locus was further delimited. Rdv1, which is derived from V. cinerea and included in the haplotype BoeCin, was compared with sequences of phylloxera-susceptible and phylloxera-tolerant cultivars. Between flanking regions that display high synteny, we detected and precisely characterized a diverse sequence region that covers between 202 to 403 kbp in different haplotypes. In BoeCin, five putative disease resistance genes were identified that represent likely candidates for conferring resistance to phylloxera.

genomics↗

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↗

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

Characterization of the Brassica napus flavonol synthase gene family reveals bifunctional flavonol synthases

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyses the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

plant biology↗

bioRxiv2021

Functional characterisation of banana (Musa spp.) 2-oxoglutarate-dependent dioxygenases involved in flavonoid biosynthesis

Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well-known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

molecular biology↗

bioRxiv2021

Transcriptomic analysis of temporal shifts in berry development between two grapevine cultivars of the Pinot family reveals potential ripening-regulative genes

BackgroundGrapevine cultivars of the Pinot family represent clonally propagated mutants with major phenotypic and physiological differences, such as different colour or shifted ripening time, as well as changes in important viticultural traits. Specifically, the cultivars Pinot Noir (PN) and Pinot Noir Precoce (PNP, early ripening) flower at the same time, but vary in the beginning of berry ripening (veraison) and, consequently, harvest time. In addition to genotype, seasonal climatic conditions (i.e. high temperatures) also affect ripening times. To reveal possible regulatory genes that affect the timing of veraison onset, we investigated differences in gene expression profiles between PN and PNP throughout berry development with a closely meshed time series and over two separate years. ResultsThe difference in the duration of berry formation between PN and PNP was quantified to be approximately two weeks under the growth conditions applied, using plant material with a proven PN and PNP clonal relationship. Clusters of co-expressed genes and differentially expressed genes (DEGs) were detected which reflect the shift in the timing of veraison onset. Functional annotation of these DEGs fit to observed phenotypic and physiological changes during berry development. In total, we observed 3,342 DEGs in 2014 and 2,745 DEGs in 2017 between PN and PNP, with 1,923 DEGs across both years. Among these, 388 DEGs were identified as veraison-specific and 12 were considered as berry ripening time regulatory candidates. The expression profiles revealed two candidate genes for ripening time control which we designated VviRTIC1 and VviRTIC2 (VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP. ConclusionsMany of the 1,923 DEGs show highly similar expression profiles in both cultivars if the patterns are aligned according to developmental stage. In our work, putative genes differentially expressed between PNP and PN which could control ripening time as well as veraison-specific genes were identified. We point out connections of these genes to molecular events during berry development and discuss potential candidate genes which may control ripening time. Two of these candidates were observed to be differentially expressed in the early berry development phase. Several down-regulated genes during berry ripening are annotated as auxin response factors / ARFs. Conceivably, general changes in auxin signaling may cause the earlier ripening phenotype of PNP.

developmental biology↗

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↗