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. 2020 Feb 21:11:128.
doi: 10.3389/fpls.2020.00128. eCollection 2020.

Genome-Wide Identification of the MdKNOX Gene Family and Characterization of Its Transcriptional Regulation in Malus domestica

Peng Jia  1 , Chenguang Zhang  1 , Libo Xing  1 , Youmei Li  1 , Kamran Shah  1 , Xiya Zuo  1 , Dong Zhang  1 , Na An  1   2 , Mingyu Han  1 , Xiaolin Ren  1
Affiliations

Genome-Wide Identification of the MdKNOX Gene Family and Characterization of Its Transcriptional Regulation in Malus domestica

Peng Jia et al. Front Plant Sci. .

Abstract

Knotted1-like Homeobox (KNOX) proteins play important roles in regulating plant growth, development, and other biological processes. However, little information is available on the KNOX gene family in apple (Malus domestica Borkh.). In this study, 22 KNOX genes were identified in the apple genome. The gene structure, protein characteristics, and promoter region were characterized. The MdKNOX family members were divided into three classes based on their phylogenetic relationships. Quantitative real-time PCR analysis revealed that the majority of MdKNOX genes exhibited strongly preferential expression in buds and were significantly up-regulated during the flower induction period. The transcript levels of MdKNOX genes were responsive to treatments with flowering- and stress-related hormones. The putative upstream regulation factor MdGRF could directly bind to the promoter of MdKNOX15 and MdKNOX19, and inhibit their transcriptional activities, which were confirmed by yeast one-hybrid and dual-luciferase assays. The results provide an important foundation for future analysis of the regulation and functions of the MdKNOX gene family.

Keywords: Knotted1-like Homeobox; Malus domestica; flower induction; growth-regulating factor; yeast one-hybrid.

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Figures

Figure 1
Figure 1
Multiple sequences alignment of MdKNOX proteins.
Figure 2
Figure 2
Analysis of MdKNOX gene structure. An unrooted neighbor-joining tree was constructed derived from MdKNOX protein sequences (left) and exon–intron composition analysis (right).
Figure 3
Figure 3
Neighbor-joining tree representing phylogenetic relationships among KNOX genes from apple, Arabidopsis, and rice.
Figure 4
Figure 4
Analysis of evolutionary relationships among KNOX gene family members. Relative positive positions are depicted according to apple chromosomes, colored lines indicate syntenic regions of the apple genome (A). Synteny analysis of KNOX genes between Arabidopsis and apple; relative positive positions are depicted according to apple and Arabidopsis chromosomes, colored lines indicate syntenic regions of the apple and Arabidopsis genomes (B).
Figure 5
Figure 5
MdKNOX gene expression profiles in different tissues. The heat map was generated using MEV software. Relative expression profiles are based on quantitative real-time PCR data.
Figure 6
Figure 6
MdKNOX gene expression profiles during the flower induction period. Samples were collected at 30, 50, and 70, days after full bloom (DAFB). Each value represents the mean ± standard error of three replicates. Asterisks (*) means significant difference at the 0.05 level.
Figure 7
Figure 7
MdKNOX transcript levels in response to 6-benzylaminopurine (6-BA) and gibberellic acid (GA3) treatments. Samples were collected at 30, 50, 70 days after full bloom (DAFB) after 6-BA or GA3 treatment, with water used as a control. Each value represents the mean± standard error of three replicates. Different letters means significant difference at the 0.05 level.
Figure 8
Figure 8
MdKNOX expression levels in response to SA and ABA treatments. Samples were collected at 30, 50, 70 days after full bloom (DAFB) after SA or ABA treatment, with water used as a control. Each value represents the mean ± standard error of three replicates. Different letters means significant difference at the 0.05 level.
Figure 9
Figure 9
Predicted cis-elements in the MdKNOX promoters. The 1.5 kb sequence upstream from the start codon of MdKNOX genes was analyzed using the PlantCARE database.
Figure 10
Figure 10
MdGRF binds to MdKNOX15 and MdKNOX19 promoters. (A) Promoter sequences of MdKNOX15 and MdKNOX19 genes used for yeast one-hybrid (Y1H) assay. The putative GRF binding sites, core CAG repeats or its reverse complementary sequence CTG were marked with gray shadow. (B) The yeast strains were grown on SD/-Leu and SD/-Leu/+ AbA medium for 3 d.
Figure 11
Figure 11
MdGRF inhibited the promoter activities of MdKNOX15 and MdKNOX19. (A) Schematic diagram of the reporter vector and effector vector. (B) Dual-luciferase assays showing that MdGRF inhibits the transcription MdKNOX15 and MdKNOX19 in tobacco leaves. The MdGRF effector vector (MdGRF-GFP) or the control effector vector (GFP) with the reporter vector containing the MdKNOX promoter (proKNOX15 and proKNOX19) were infiltrated into tobacco leaves for analysis of Rluc/Fluc activity. Each experiment was performed in three replicates. Asterisks (*) indicates a significant difference (p <0.05) compared with the control.
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References

    1. Azarakhsh M., Kirienko A. N., Zhukov V. A., Lebedeva M. A., Dolgikh E. A., Lutova L. A. (2015). KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule development. J. Exp. Bot. 66, 7181–7195. 10.1093/jxb/erv414 - DOI - PMC - PubMed
    1. Bürglin T. R., Affolter M. (2016). Homeodomain proteins: an update. Chromosoma 125, 497–521. 10.1007/s00412-015-0543-8 - DOI - PMC - PubMed
    1. Bailey T. L., Williams N., Misleh C., Li W. W. (2006). MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res. 34, W369–W373. 10.1093/nar/gkl198 - DOI - PMC - PubMed
    1. Barton M. K., Poethig R. S. (1993). Formation of the shoot apical meristem in Arabidopsis thaliana: an analysis of development in the wild type and in the shoot meristemless mutant. Development 119, 823–831.
    1. BellesBoix E., Hamant O., Witiak S. M., Morin H., Traas J., Pautot V. (2006). KNAT6: An arabidopsis homeobox gene involved in meristem activity and organ separation. Plant Cell. 18, 1900. 10.1105/tpc.106.041988 - DOI - PMC - PubMed