Expression analysis of flavonoid biosynthesis genes during Arabidopsis thaliana silique and seed development with a primary focus on the proanthocyanidin biosynthetic pathway

Kleindt CK, Stracke R, Mehrtens F, Weisshaar B. Expression analysis of flavonoid biosynthesis genes during Arabidopsis thaliana silique and seed development with a primary focus on the proanthocyanidin biosynthetic pathway. BMC Research Notes. 2010;3(1): 255.Background: The coordinated activity of different flavonoid biosynthesis genes in Arabidopsis thaliana results in tissue-specific accumulation of flavonols, anthocyanins and proanthocyanidins (PAs). These compounds possess diverse functions in plants including light-attenuation and oxidative stress protection. Flavonoids accumulate in a stimulus- and/or development-dependent manner in specific parts of the plant. PAs accumulate in the seed coat (testa). Findings: We describe the biological material and the preparation of total RNA for the AtGenExpress developmental silique and seed series. AtGenExpress ATH1 GeneChip expression data from the different stages were reanalyzed and verified using quantitative real time PCR (qPCR). We observed organ-specific transcript accumulation of specific flavonoid biosynthetic genes consistent with previously published data and our PA compound accumulation data. In addition, we investigated the regulation of PA accumulation in developing A. thaliana seeds by correlating gene expression patterns of specific flavonoid biosynthesis genes with different seed embryonic developmental stages and organs and present two useful marker genes for isolated valve and replum organs, as well as one seed-specific marker. Conclusions: Potential caveats of array-based expression data are discussed based on comparisons with qPCR data. Results from ATH1 microarray and qPCR experiments revealed a shift in gene activity from general flavonoid biosynthesis at early stages of seed development to PA synthesis at late (mature) stages of embryogenesis. The examined PA accumulation-associated genes, including biosynthetic and regulatory genes, were found to be exclusively expressed in immature seeds. Accumulation of PAs initiates at the early heart stage of silique and seed development. Our findings provide new insights for further studies targeting the PA pathway in seeds

Natural variation in flavonol accumulation in Arabidopsis is determined by the flavonol glucosyltransferase BGLU6.

Ishihara H, Tohge T, Viehöver P, Fernie AR, Weisshaar B, Stracke R. Natural variation in flavonol accumulation in Arabidopsis is determined by the flavonol glucosyltransferase BGLU6. Journal of Experimental Botany. 2016;67(5):1505-1517

Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling

The genes MYB11, MYB12 and MYB111 share significant structural similarity and form subgroup 7 of the Arabidopsis thaliana R2R3-MYB gene family. To determine the regulatory potential of these three transcription factors, we used a combination of genetic, functional genomics and metabolite analysis approaches. MYB11, MYB12 and MYB111 show a high degree of functional similarity and display very similar target gene specificity for several genes of flavonoid biosynthesis, including CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE and FLAVONOL SYNTHASE1. Seedlings of the triple mutant myb11 myb12 myb111, which genetically lack a complete subgroup of R2R3-MYB genes, do not form flavonols while the accumulation of anthocyanins is not affected. In developing seedlings, MYB11, MYB12 and MYB111 act in an additive manner due to their differential spatial activity; MYB12 controls flavonol biosynthesis mainly in the root, while MYB111 controls flavonol biosynthesis primarily in cotyledons. We identified and confirmed additional target genes of the R2R3-MYB subgroup 7 factors, including the UDP-glycosyltransferases UGT91A1 and UGT84A1, and we demonstrate that the accumulation of distinct and structurally identified flavonol glycosides in seedlings correlates with the expression domains of the different R2R3-MYB factors. Therefore, we refer to these genes as PFG1–3 for ‘PRODUCTION OF FLAVONOL GLYCOSIDES’

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

5openInternationalInternational coauthor/editorBananas (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.openBusche, M.; Acatay, C.; Martens, S.; Weisshaar, B.; Stracke, R.Busche, M.; Acatay, C.; Martens, S.; Weisshaar, B.; Stracke, R

A systematic survey in Arabidopsis thaliana of transcription factors that modulate circadian parameters

<p>Abstract</p> <p>Background</p> <p>Plant circadian systems regulate various biological processes in harmony with daily environmental changes. In <it>Arabidopsis thaliana</it>, the underlying clock mechanism is comprised of multiple integrated transcriptional feedbacks, which collectively lead to global patterns of rhythmic gene expression. The transcriptional networks are essential within the clock itself and in its output pathway.</p> <p>Results</p> <p>Here, to expand understanding of transcriptional networks within and associated to the clock, we performed both an <it>in silico </it>analysis of transcript rhythmicity of transcription factor genes, and a pilot assessment of functional phenomics on the <it>MYB</it>, <it>bHLH</it>, and <it>bZIP </it>families. In our <it>in silico </it>analysis, we defined which members of these families express a circadian waveform of transcript abundance. Up to 20% of these families were over-represented as clock-controlled genes. To detect members that contribute to proper oscillator function, we systematically measured rhythmic growth <it>via </it>an imaging system in hundreds of misexpression lines targeting members of the transcription-factor families. Three transcription factors were found that conferred aberrant circadian rhythms when misexpressed: <it>MYB3R2</it>, <it>bHLH69</it>, and <it>bHLH92</it>.</p> <p>Conclusion</p> <p>Transcript abundance of many transcription factors in Arabidopsis oscillates in a circadian manner. Further, a developed pipeline assessed phenotypic contribution of a panel of transcriptional regulators in the circadian system.</p

Twenty-Five Years of Propagation in Suspension Cell Culture Results in Substantial Alterations of the Arabidopsis Thaliana Genome

Pucker B, Rückert C, Stracke R, Viehöver P, Kalinowski J, Weisshaar B. Twenty-Five Years of Propagation in Suspension Cell Culture Results in Substantial Alterations of the Arabidopsis Thaliana Genome. Genes. 2019;10(9): 671.Arabidopsis thaliana is one of the best studied plant model organisms. Besides cultivation in greenhouses, cells of this plant can also be propagated in suspension cell culture. At7 is one such cell line that was established about 25 years ago. Here, we report the sequencing and the analysis of the At7 genome. Large scale duplications and deletions compared to the Columbia-0 (Col-0) reference sequence were detected. The number of deletions exceeds the number of insertions, thus indicating that a haploid genome size reduction is ongoing. Patterns of small sequence variants differ from the ones observed between A. thaliana accessions, e.g., the number of single nucleotide variants matches the number of insertions/deletions. RNA-Seq analysis reveals that disrupted alleles are less frequent in the transcriptome than the native ones

A de novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny

Pucker B, Holtgräwe D, Rosleff Sörensen T, Stracke R, Viehöver P, Weisshaar B. A de novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny. PLoS One. 2016;11(10): e0164321.Arabidopsis thaliana is the most important model organism for fundamental plant biology. The genome diversity of different accessions of this species has been intensively studied, for example in the 1001 genome project which led to the identification of many small nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). In addition, presence/absence variation (PAV), copy number variation (CNV) and mobile genetic elements contribute to genomic differences between A. thaliana accessions. To address larger genome rearrangements between the A. thaliana reference accession Columbia-0 (Col-0) and another accession of about average distance to Col-0, we created a de novo next generation sequencing (NGS)-based assembly from the accession Niederzenz-1 (Nd-1). The result was evaluated with respect to assembly strategy and synteny to Col-0. We provide a high quality genome sequence of the A. thaliana accession (Nd-1, LXSY01000000). The assembly displays an N50 of 0.590 Mbp and covers 99% of the Col-0 reference sequence. Scaffolds from the de novo assembly were positioned on the basis of sequence similarity to the reference. Errors in this automatic scaffold anchoring were manually corrected based on analyzing reciprocal best BLAST hits (RBHs) of genes. Comparison of the final Nd-1 assembly to the reference revealed duplications and deletions (PAV). We identified 826 insertions and 746 deletions in Nd-1. Randomly selected candidates of PAV were experimentally validated. Our Nd-1 de novo assembly allowed reliable identification of larger genic and intergenic variants, which was difficult or error-prone by short read mapping approaches alone. While overall sequence similarity as well as synteny is very high, we detected short and larger (affecting more than 100 bp) differences between Col-0 and Nd-1 based on bi-directional comparisons. The de novo assembly provided here and additional assemblies that will certainly be published in the future will allow to describe the pan-genome of A. thaliana

Singlet oxygen, flavonols and photoinhibition in green and senescing silver birch leaves

During autumn senescence, deciduous trees degrade chlorophyll and may synthesize flavonols. We measured photosynthetic parameters, epidermal flavonols, singlet oxygen production in vivo and photoinhibition of the photosystems (PSII and PSI) from green and senescing silver birch (Betula pendula) leaves. Chlorophyll a fluorescence and P700 absorbance measurements showed that the amounts of both photosystems decreased throughout autumn senescence, but the remaining PSII units stayed functional until ~ 90% of leaf chlorophyll was degraded. An increase in the chlorophyll a to b ratio, a decrease in > 700 nm absorbance and a blue shift of the PSI fluorescence peak at 77 K suggest that light-harvesting complex I was first degraded during senescence, followed by light-harvesting complex II and finally the photosystems. Senescing leaves produced more singlet oxygen than green leaves, possibly because low light absorption by senescing leaves allows high flux of incident light per photosystem. Senescing leaves also induced less non-photochemical quenching, which may contribute to increased singlet oxygen production. Faster photoinhibition of both photosystems in senescing than in green leaves, under high light, was most probably caused by low absorption of light and rapid singlet oxygen production. However, senescing leaves maintained the capacity to recover from photoinhibition of PSII. Amounts of epidermal flavonols and singlet oxygen correlated neither in green nor in senescing leaves of silver birch. Moreover, Arabidopsis thaliana mutants, incapable of synthesizing flavonols, were not more susceptible to photoinhibition of PSII or PSI than wild type plants; screening of chlorophyll absorption by flavonols was, however, small in A. thaliana. These results suggest that flavonols do not protect against photoinhibition or singlet oxygen production in chloroplasts

Nonsense Mutation Inside Anthocyanidin Synthase Gene Controls Pigmentation in Yellow Raspberry (Rubus idaeus L.)

Rafique MZ, Carvalho E, Stracke R, et al. Nonsense Mutation Inside Anthocyanidin Synthase Gene Controls Pigmentation in Yellow Raspberry (Rubus idaeus L.). Frontiers in Plant Science. 2016;7: 1892

Sugar Beet BeetMap-3, and Steps to Improve the Genome Assembly and Genome Sequence Annotation (W875)

Weisshaar B, Himmelbauer H, Schmidt T, et al. Sugar Beet BeetMap-3, and Steps to Improve the Genome Assembly and Genome Sequence Annotation (W875). Presented at the Plant and Animal Genome XXIV Conference, San Diego, USA