Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun 25;39(2):147-153.
doi: 10.5511/plantbiotechnology.22.0122a.

VND-INTERACTING2 effectively inhibits transcriptional activities of VASCULAR-RELATED NAC-DOMAIN7 through a conserved sequence

Aili Ailizati  1 , Isura Sumeda Priyadarshana Nagahage  2 , Atsuko Miyagi  1 , Toshiki Ishikawa  1 , Maki Kawai-Yamada  1 , Taku Demura  3 , Masatoshi Yamaguchi  1
Affiliations

VND-INTERACTING2 effectively inhibits transcriptional activities of VASCULAR-RELATED NAC-DOMAIN7 through a conserved sequence

Aili Ailizati et al. Plant Biotechnol (Tokyo). .

Abstract

An Arabidopsis NAC domain transcription factor VND-INTERACTING2 (VNI2) was originally isolated as an interacting protein with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel element differentiation. VNI2 inhibits transcriptional activation activity of VND7 by forming a protein complex. Here, to obtain insights into how VNI2 regulates VND7, we tried to identify the amino acid region of VNI2 required for inhibition of VND7. VNI2 has an amino acid sequence similar to the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR (ERF)-associated amphiphilic repression (EAR) motif, conserved in transcriptional repressors, at the C-terminus. A transient expression assay showed that the EAR-like motif of VNI2 was not required for inhibition of VND7. The C-terminal deletion series of VNI2 revealed that 10 amino acid residues, highly conserved in the VNI2 orthologs contributed to effective repression of the transcriptional activation activity of VND7. Observation of transgenic plants ectopically expressing VNI2 showed that the identified 10 amino acid sequence strongly affected xylem vessel formation and plant growth. These data indicated that the 10 amino acid sequence of VNI2 has an important role in its transcriptional repression activity and negative regulation of xylem vessel formation.

Keywords: Arabidopsis thaliana; NAC domain transcription factor; protein-protein interaction; transcriptional repressor; xylem vessel formation.

PubMed Disclaimer

Figures

None
Figure 1. The EAR-like motif of VNI2 is not required for inhibition. (A) A schematic representation of VNI2 and the alignment of sequences of the EAR motif of transcription factors. White letters indicate the EAR-like motif in VNI2 or EAR motifs. (B) Amino acid sequences of the EAR-like motif of VNI2. Residues 216 (aspartic acid) and 218 (asparagine) were replaced by alanine residues in VNI2m1. The leucine residues at position 217 and 219 were replaced by alanine in VNI2m2. White letters in VNI2 indicate the EAR-like motif. White letters in VNI2m1 and VNI2m2 indicate the replaced amino acids. (C) A schematic representation of the effector and reporter constructs used for the transient reporter assays. The reporter construct contained the promoter of XCP1 and a firefly LUC reporter gene. The effector constructs contained either the MCS fragment or NAC domain transcription factors driven by CaMV35S promoter. (D) Results of the transient expression assays. Firefly LUC activity was normalized using the activity of Renilla LUC. Values and error bars indicate means±SD (n=4). Different letters indicate significant differences at p<0.05, as determined by one-way ANOVA with Tukey’s HSD test.
None
Figure 2. The 201–210 amino acid region of VNI2 contributes to effective inhibition of VND7. (A) Schematic diagram of the C-terminal deletion series of the VNI2. (B) Results of the transient expression assay. Firefly LUC activity was normalized using the activity of Renilla LUC. Values and error bars indicate means±SD (n=4). Different letters indicate significant differences at p<0.05, as determined by one-way ANOVA with Tukey’s HSD test.
None
Figure 3. The 201–210 amino acid region of VNI2 affects xylem vessel formation and plant growth. (A) Thirty-five-day-old T1 transgenic plants. Bar=5 cm. (B) Ratio of heights of the 35-day-old transgenic plants. (C) Dark-field images of the first true leaves of 14-day-old plants. Yellow boxes indicate the positions of main vein. Bars=1 mm.
None
Figure 4. Conservation of the amino acid sequences among VNI2 orthologs. (A) The 10 amino acid sequences of VNI2 and its orthologs. Carubv: Capsella rubella, Brara: Brassica rapa, Potri: Populus trichocarpa, Ciclev: Citrus clementina, Eucgr: Eucalyptus grandis. White letters indicate the amino acids identical to those of VNI2. (B) The positions of amino acid residues substituted in VNI21–210. Residues 204 (isoleucine) and 206 (phenylalanine) were replaced by alanine residues in VNI21–210 m. White letters indicate the replaced amino acid residues. Asterisks indicate the stop codon. (C) Results of the transient expression assay. Firefly LUC activity was normalized using the activity of Renilla LUC. Values and error bars indicate means±SD (n=4). Different letters indicate significant differences at p<0.05, as determined by one-way ANOVA with Tukey’s HSD test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ailizati A, Nagahage ISP, Miyagi A, Ishikawa T, Kawai-Yamada M, Demura T, Yamaguchi M (2021) An Arabidopsis NAC domain transcription activator VND7 negatively regulates VNI2 expression. Plant Biotechnol (Tokyo) 38: 415–420 - PMC - PubMed
    1. Asahina M, Azuma K, Pitaksaringkarn W, Yamazaki T, Mitsuda N, Ohme-Takagi M, Yamaguchi S, Kamiya Y, Okada K, Nishimura T, et al. (2011) Spatially selective hormonal control of RAP2.6L and ANAC071 transcription factors involved in tissue reunion in Arabidopsis. Proc Natl Acad Sci USA 108: 16128–16132 - PMC - PubMed
    1. Duval M, Hsieh TF, Kim SY, Thomas TL (2002) Molecular characterization of AtNAM: A member of the Arabidopsis NAC domain superfamily. Plant Mol Biol 50: 237–248 - PubMed
    1. Endo H, Yamaguchi M, Tamura T, Nakano Y, Nishikubo N, Yoneda A, Kato K, Kubo M, Kajita S, Katayama Y, et al. (2015) Multiple classes of transcription factors regulate the expression of VASCULAR-RELATED NAC-DOMAIN7, a master switch of xylem vessel differentiation. Plant Cell Physiol 56: 242–254 - PubMed
    1. Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J 34: 733–739 - PubMed

LinkOut - more resources