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Plant J. 2019 Jul 17. doi: 10.1111/tpj.14443. [Epub ahead of print]

Affinity-based high-resolution analysis of DNA binding by VASCULAR-RELATED NAC-DOMAIN7 via fluorescence correlation spectroscopy.

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Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, 630-0192, Japan.
Institute of Transformative Bio-Molecules, Nagoya University, Nagoya, Aichi, 464-8602, Japan.
Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8562, Japan.
Graduate School of Science and Engineering, Saitama University, Saitama, Saitama, 338-8570, Japan.


VASCULAR-RELATED NAC-DOMAIN7 (VND7) is the master transcription factor for vessel element differentiation in Arabidopsis thaliana. To identify the cis-acting sequence(s) bound by VND7, we employed fluorescence correlation spectroscopy (FCS) to find VND7-DNA interactions quantitatively. This identified an 18-bp sequence from the promoter of XYLEM CYSTEINE PEPTIDASE1 (XCP1), a direct target of VND7. A quantitative assay for binding affinity between VND7 and the 18-bp sequence revealed the core nucleotides contributing to specific binding between VND7 and the 18-bp sequence. Moreover, by combining the systematic evolution of ligands by exponential enrichment (SELEX) technique with known consensus sequences, we defined a motif termed the Ideal Core Structure for binding by VND7 (ICSV). We also used FCS to search for VND7 binding sequences in the promoter regions of other direct targets. Taking these data together, we proposed that VND7 preferentially binds to the ICSV sequence. Additionally, we found that substitutions among the core nucleotides affected transcriptional regulation by VND7 in vivo, indicating that the core nucleotides contribute to vessel-element-specific gene expression. Furthermore, our results demonstrate that FCS is a powerful tool for unveiling the DNA-binding properties of transcription factors.


NAC transcription factor; VASCULAR-RELATED NAC-DOMAIN7; cis-acting sequence; competitor binding assay; fluorescence correlation spectroscopy; xylem vessel differentiation


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