Abstract
High-molecular-weight glutenin subunit (HMW GS) proteins are major components of the gluten matrix, which is the physical basis of bread-making in wheat. Epigenetic and transcriptional regulations of HMW GS genes were studied both in silico and in wet lab to understand their tissue (endosperm) specific expression. Our co-expressional network analysis identified key transcription factor (TF) genes that regulate HMW GS genes. We also show here that HMW GS genes are inhibited in vegetative tissues by histone deacetylation as revealed by strong GUS expression in vascular tissues of transgenic barley seedlings harbouring HMW GS gene promoter::uidA-reporter gene fusions upon treatment with a histone deacetylase inhibitor. A novel method termed in vivo DNA affinity purification (IP) has been developed here for the isolation of histones and transcription factors binding to target DNA regions. The technique is based on the biolistic introduction of biotinylated PCR probes amplified from HMW GS gene promoters into wheat leaves. Twenty-four hours later, the probe is cross-linked with interacting factors and subsequently re-purified from plant nuclear extracts. Many proteins, ribosomal proteins and histones have so far been isolated. No lysine-acetylated histone protein fragments were found which further highlight the inhibiting effect of histone deacetylation on HMW GS gene expression.
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Acknowledgements
The excellent on-line nano-LC-MS/MS analysis performed by Éva Gulyás (Biological Research Centre, Szeged Hungary) is highly appreciated.
Funding
This research has been supported by grants OTKA K100881 and NKFI PD_16 no. 121322.
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Online Resource 1
TF composition of identified genetic programmes that represent distinct sub-networks within an improved focused transcriptional co-expression network modelling the HMW GS gene circuit. (XLSX 11 kb)
Online Resource 2
RT-PCR detection of barley alpha tubulin 2 gene in cDNA of TSA-treated and untreated transgenic barley seedling stems. GP stands for non-transgenic ‘Golden Promise’ barley. The positive control (+) was non-transgenic barley genomic DNA, while the negative control (−) was water. The original total RNA samples were also tested for genomic DNA contamination. (PNG 166 kb)
Online Resource 3
RT-PCR detection of Glu-1Bx7 or Glu-1By9 HMW GS gene promoter-driven uidA gene in cDNA of TSA-treated and untreated transgenic barley seedling stems. GP stands for non-transgenic ‘Golden Promise’ barley. The positive control (+) was pCambia1391z plasmid, while the negative control (−) was water. The original total RNA samples were also tested for genomic DNA contamination. (PNG 154 kb)
Online Resource 4
Complete list and characterisation of peptide fragments obtained by in vivo DNA affinity purification from ‘Bánkúti 1201’ wheat using LMW1, HMW1 and HMW2 PCR-products as probes. (XLS 558 kb)
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Éva, C., Szőke-Pázsi, K., Makai, S. et al. In Vivo DNA Affinity Purification and Histone Deacetylase Inhibitor Treatment Proves the Role of Histone Acetylation in the Expression Regulation of High-Molecular-Weight Glutenin Genes. Plant Mol Biol Rep 36, 750–763 (2018). https://doi.org/10.1007/s11105-018-1117-8
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DOI: https://doi.org/10.1007/s11105-018-1117-8