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

Abstract

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.

Nicotiana benthamiana XYLEM CYSTEINE PROTEASEgenes facilitate tracheary element formation in interfamily grafting

“…Only VND7 could bind to the NbXCP1 promoter (Supplemental Figure S4, Figure 4C). The target sequence of VND7 (Tamura et al, 2019) was identified in the NbXCP1 promoter (Figure 4C). An equivalent sequence was present in the NbXCP2 promoter, although it contained a mismatch of one terminal nucleotide.…”

“…XYN1 was verified expression on the predominantly in xylem tissues and encodes xylanase. The xylem transport patterns were affected in AtXYN1 modified lines (Zeilinger et al, 1996;Suzuki et al, 2002;Endo et al, 2019). In the module of xylem formation, genes associated with the generation and scavenging of reactive oxygen species (ROS) were found (Figure 3, Supplemental Table S7).…”

Genome-Wide Identification of Direct Targets of ZjVND7 Reveals the Putative Roles of Whole-Genome Duplication in Sour Jujube in Regulating Xylem Vessel Differentiation and Drought Tolerance

“…The results showed that the motifs in the diploids and autotetraploids were different. However, the binding motifs in both diploid and autotetraploid sour jujube have a continuous core sequence, CTTNAAG, which is conserved with core sequence published by Tamura et al (2019).…”

“…Putative target genes of VND have been reported in A. thaliana (Tamura et al, 2019), maize (Dong et al, 2020), and poplar (Ohtani et al, 2011). These target genes were predicted to be related to the regulation of xylem development, programmed cell death, cell wall synthesis, metabolic enzymes, and signal transduction.…”

Single-Molecule Nanotechnologies: An Evolution in Biological Dynamics Detection

“…Dynamic parameters of the fluorescently labeled molecules can be discerned by fitting the autocorrelation curve with a suitable model function . In contrast to smFRET measurements, smFCS is not limited to a detection size of 1–10 nm and is sensitive to distances shorter than 1 nm, , making it applicable to a wide range of investigations in the biological sciences such as obtaining reaction rates, diffusion coefficients, and affinity constants. − …”