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. 2020 Apr 30;15(4):e0232352.
doi: 10.1371/journal.pone.0232352. eCollection 2020.

Cytokinin biosynthesis genes expressed during nodule organogenesis are directly regulated by the KNOX3 protein in Medicago truncatula

Mahboobeh Azarakhsh  1   2 , Andrey M Rumyantsev  1 , Maria A Lebedeva  1 , Lyudmila A Lutova  1
Affiliations

Cytokinin biosynthesis genes expressed during nodule organogenesis are directly regulated by the KNOX3 protein in Medicago truncatula

Mahboobeh Azarakhsh et al. PLoS One. .

Erratum in

Abstract

Cytokinin is an important regulator of symbiotic nodule development. Recently, KNOTTED1-LIKE HOMEOBOX 3 transcription factor (TF) was shown to regulate symbiotic nodule development possibly via the activation of cytokinin biosynthesis genes. However, the direct interaction between the KNOX3 TF and its target genes has not been investigated up to date. Here, using EMSA analysis and SPR-based assay, we found that MtKNOX3 homeodomain directly binds to the regulatory sequences of the MtLOG1, MtLOG2, and MtIPT3 genes involved in nodulation in Medicago truncatula. Moreover, we showed that MtLOG2 and MtIPT3 expression patterns partially overlap with MtKNOX3 expression in developing nodules as it was shown by promoter:GUS analysis. Our data suggest that MtKNOX3 TF may directly activate the MtLOG1, MtLOG2, and MtIPT3 genes during nodulation thereby increasing cytokinin biosynthesis in developing nodules.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
Tissue specific expression of pMtKNOX3:GUS (A-E), pMtIPT3:GUS (F-J), and pMtLOG2:GUS (K-O) at different stages of nodule development (nodule primordia at 3 dpi (A, F, K), developing nodules at 7 dpi (B, G, L), developing nodules at 12 dpi (C, H, M) and mature nodules at 18–21 dpi (D-E, I-J, N-O)). Bars, 100 μm. Thickness of sections, 50 μm.
Fig 2
Fig 2. The localization of the putative MtKNOX3-binding sites in the regulatory sequences of the MtLOG1, MtLOG2, and MtIPT3 genes.
Exons are shown as blue blocks, while introns are represented by blue lines; 5’UTRs and 3’UTRs are shown as green blocks.
Fig 3
Fig 3. The results of EMSA experiment for the analysis of interaction between the homeodomain of MtKNOX3 and the regulatory sequences of the MtLOG2, MtIPT3, and MtLOG1 genes.
1- free ds-DNA, 2- ds-DNA with the protein, 3- mutated ds-DNA with the protein, 4- ds-DNA with the protein and 2000X of competitor DNA, 5- ds-DNA with the protein and 1000X of competitor DNA, 6- ds-DNA with the protein and 500X of competitor DNA. The protein amount is the same in all wells (1000 ng).
See this image and copyright information in PMC

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