The homeodomain of the Raphanus sativus WOX4 binds to the promoter of the LOG3 cytokinin biosynthesis gene

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Abstract

BACKGROUND: The WOX4 transcription factor plays a crucial role in maintaining the organisation of cambium meristem during secondary growth, but its direct targets are unknown.

AIM: The aim of our work were to study the effect of WOX4 overexpression on the root development and gene expression in radish (Raphanus sativus L.), a root crop related to Arabidopsis thaliana, and to search for direct targets of the WOX4 in radish.

MATERIALS AND METHODS: Radish line 19 of the St. Petersburg State University radish genetic collection was used. Plants were grown on Murashige–Skoog medium and then in soil at 23оС and 16 h of daylight. Total DNA was extracted from radish seedlings using the CTAB method. The PCR-amplified full-length RsWOX4-2 gene, gene fragments or homeobox sequence were cloned into the vectors for overexpression (pB7WG2D), RNA interference (pH7GWIWG2) and yeast one-hybrid assay (pDEST22), respectively, using the Gateway system. The vectors for overexpression and RNA interference of RsWOX4-2 were transformed into Escherichia coli DH10B and then into Agrobacteium rhizogenes Arqua chemically competent cells. Radish seedlings were transformed with A. rhizogenes containing vectors for overexpression and RNA interference of RsWOX4-2, and GUS-overexpressing A. rhizogenes was used as a control. Total RNA from transgenic radish roots was extracted with Trizol reagent. RNA reverse transcription was performed using dT-18 primers and RevertAid reverse transcriptase. qPCR was performed using the Eva Green reagent kit on a CFX96 thermocycler with fluorescence detection system. Results were processed using the 2–ΔΔCT method. Yeast transformation was performed using the competent Saccharomyces cerevisiae cells of Y2H Gold strain. For the yeast one-hybrid assay, the obtained yeast colonies transformed with plasmids containing TF homeodomain sequence and promoter regions of genes were grown on DDO and TDO selective media with different concentrations of 3-amino-1,2,4-triazole. Statistical processing based on Student’s t-test and graphing were performed using the ggplot2 package for the R programming language (v.4.0.2).

RESULTS: Overexpression of the RsWOX4-2 gene affects the structure of the radish root stele and alters the number of vessels and cambium cells. Overexpression and RNA interference of the RsWOX4-2 causes changes in the expression levels of putative target genes with the WOX family transcription factor conserved binding sites in their promoters. Using the yeast one-hybrid assay, we have shown that the DNA-binding homeodomain of RsWOX4-2 interacts with the TAATCC site in the promoter of the RsLOG3 gene, which encodes the enzyme for cytokinin biosynthesis.

CONCLUSIONS: We have demonstrated the effect of RsWOX4-2 overexpression on radish root stele and gene expression and identified the RsLOG3 as the putative direct target of the WOX4 transcription factor in radish.

About the authors

Xenia A. Kuznetsova

Saint Petersburg State University

Email: kskuz95@mail.ru
ORCID iD: 0000-0003-3180-5306
SPIN-code: 3864-3062
Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034

Irina E. Dodueva

Saint Petersburg State University

Email: wildtype@yandex.ru
ORCID iD: 0000-0001-5282-718X
SPIN-code: 8061-2388

Cand. Sci. (Biology), Associate Professor

Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034

Lyudmila A. Lutova

Saint Petersburg State University

Author for correspondence.
Email: la.lutova@gmail.com
ORCID iD: 0000-0001-6125-0757
SPIN-code: 3685-7136

Dr. Sci. (Bioligy), Professor

Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034

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Supplementary files

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2. Fig. 1. Effect of RsWOX4-2 (a) and RsWOX14 (c) overexpression on radish root development compared to GUS overexpression (control, b). c, cambium; px, primary xylem; sx, secondary xylem

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3. Fig. 2. Effect of RsWOX4 and RsWOX14 overexpression (oe) on the number of xylem (a) and cambium (b) cells in the root stele compared to GUS overexpression. Cell counting was performed in a 200 × 200 μm part of a root sector in 10 slices. The significance level was assessed using Student’s t-test. c, sectors of root with RsWOX14 (left) and GUS (right) overexpression. c, cambium

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4. Fig. 3. Predicted interactions between WOX4 (a) and WOX14 (b) and their putative targets, plotted using the STRING program. Lines of different colours indicate different types of interactions predicted for protein products of these genes: sea-wave — known interactions based on information from databases; crimson — known experimentally confirmed interactions; green — predicted gene neighbour interactions; red — predicted interactions based on gene fusion studies; blue — predicted interactions based on gene co-occurrence studies; light green — other types of interactions based on textmining; dark — other types of interactions based on co-expression studies; light purple — other types of interaction based on protein homology. Circles of different colours indicate different protein products of genes; the structure of the corresponding proteins is depicted in the centre of each circle

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5. Fig. 4. Relative expression levels of candidate genes (genes that were upregulated upon overexpression (oe) of RsWOX4-2 and downregulated upon RsWOX4-2 RNAi) upon overexpression (crimson) and RNAi (blue) of the RsWOX4 gene compared to plants with the GUS overexpression (grey). As for the other 33 genes studied in this work, their expression or have insignificant levels, or were not changed complementary in the roots with overexpression and RNAi of RsWOX4-2. Error bars indicate the error of the mean. The significance level was assessed by Student’s t-test

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6. Fig. 5. Yeast one-hybrid assay results for the assessment of interactions between the promoter regions of the RsLOG3 and the RsARR18 genes and the RsWOX4 TF homeodomain. Confirmed interaction is highlighted in green. Colonies were grown on media with different concentrations of 3-AT, the series of photos shows the growth of colonies on media with 10 mM 3-AT. *The plasmids pHisLeu-pAtLHY and pDEST22-NAM, with promoter and TF sites, respectively, which have previously been shown to interact with each other , were used as control 3 (positive control)

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7. Kuznetsova_suppl1
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8. Kuznetsova_suppl2
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