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Characterization of Rice Homeobox Genes, OsHOX22 and OsHOX24, and Over-expression of OsHOX24 in Transgenic Arabidopsis Suggest Their Role in Abiotic Stress Response.

Bhattacharjee A, Khurana JP, Jain M - Front Plant Sci (2016)

Bottom Line: The recombinant full-length and truncated homeobox proteins were found to be localized in the nucleus.Many of these genes were found to be involved in transcriptional regulation and various metabolic pathways.Altogether, our results suggest the possible role of OsHOX22/OsHOX24 homeobox proteins as negative regulators in abiotic stress responses.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
Homeobox transcription factors are well known regulators of plant growth and development. In this study, we carried out functional analysis of two candidate stress-responsive HD-ZIP I class homeobox genes from rice, OsHOX22, and OsHOX24. These genes were highly up-regulated under various abiotic stress conditions at different stages of rice development, including seedling, mature and reproductive stages. The transcript levels of these genes were enhanced significantly in the presence of plant hormones, including abscisic acid (ABA), auxin, salicylic acid, and gibberellic acid. The recombinant full-length and truncated homeobox proteins were found to be localized in the nucleus. Electrophoretic mobility shift assay established the binding of these homeobox proteins with specific DNA sequences, AH1 (CAAT(A/T)ATTG) and AH2 (CAAT(C/G)ATTG). Transactivation assays in yeast revealed the transcriptional activation potential of full-length OsHOX22 and OsHOX24 proteins. Homo- and hetero-dimerization capabilities of these proteins have also been demonstrated. Further, we identified putative novel interacting proteins of OsHOX22 and OsHOX24 via yeast-two hybrid analysis. Over-expression of OsHOX24 imparted higher sensitivity to stress hormone, ABA, and abiotic stresses in the transgenic Arabidopsis plants as revealed by various physiological and phenotypic assays. Microarray analysis revealed differential expression of several stress-responsive genes in transgenic lines as compared to wild-type. Many of these genes were found to be involved in transcriptional regulation and various metabolic pathways. Altogether, our results suggest the possible role of OsHOX22/OsHOX24 homeobox proteins as negative regulators in abiotic stress responses.

No MeSH data available.


Related in: MedlinePlus

Transactivation and dimerization properties of homeobox proteins.(A) Schematic representation of full-length homeobox proteins (BD::OsHOX24 and BD::OsHOX22) and truncated (C-terminal acidic region removed) fusion constructs (BD::OsHOX24ΔC and BD::OsHOX22ΔC) used. (B) Transactivation assay of full-length and truncated (ΔC) homeobox proteins in yeast. The transformants grown on SD-Trp (SD-T, left) medium; SD-Trp-His medium (SD-T-H, middle) and SD-Trp-His-Ade (SD-T-H-A, right) medium are shown. (C) Dimerization assay of full-length (fl) and truncated (ΔC) homeobox proteins. The deletion constructs, BD::OsHOX24ΔC and BD::OsHOX22ΔC, were co-transformed with different combinations of full-length (AD::OsHOX24fl, AD::OsHOX22fl) and deletion constructs (AD::OsHOX24ΔC, AD::OsHOX22ΔC) of homeobox proteins in yeast, as indicated on left side panel. The transformants were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction. pGBKT7-p53 + pGADT7-T antigen represents positive control. Empty pGBKT7 vector (BD) represents negative control for transactivation assay and pGBKT7 + pGADT7 represents negative control for dimerization assay.
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Figure 5: Transactivation and dimerization properties of homeobox proteins.(A) Schematic representation of full-length homeobox proteins (BD::OsHOX24 and BD::OsHOX22) and truncated (C-terminal acidic region removed) fusion constructs (BD::OsHOX24ΔC and BD::OsHOX22ΔC) used. (B) Transactivation assay of full-length and truncated (ΔC) homeobox proteins in yeast. The transformants grown on SD-Trp (SD-T, left) medium; SD-Trp-His medium (SD-T-H, middle) and SD-Trp-His-Ade (SD-T-H-A, right) medium are shown. (C) Dimerization assay of full-length (fl) and truncated (ΔC) homeobox proteins. The deletion constructs, BD::OsHOX24ΔC and BD::OsHOX22ΔC, were co-transformed with different combinations of full-length (AD::OsHOX24fl, AD::OsHOX22fl) and deletion constructs (AD::OsHOX24ΔC, AD::OsHOX22ΔC) of homeobox proteins in yeast, as indicated on left side panel. The transformants were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction. pGBKT7-p53 + pGADT7-T antigen represents positive control. Empty pGBKT7 vector (BD) represents negative control for transactivation assay and pGBKT7 + pGADT7 represents negative control for dimerization assay.

Mentions: OsHOX24 and OsHOX22 proteins were found to be rich in acidic amino acids at the C-terminal region, which could possibly contribute to their transactivation property. Thus, we investigated the transcriptional activation property of these HD-ZIP I TFs in yeast. The complete ORFs and C-terminal deletion constructs (ΔC) of OsHOX24 and OsHOX22 were cloned in yeast expression vector containing DNA binding domain (Figure 5A). The colonies of transformed yeast cells grew uniformly on SD-Trp selection medium. The growth of yeast transformants on SD-Trp-His and SD-Trp-His-Ade selection media, even with increasing serial dilution, confirmed the transactivating nature of full-length homeobox proteins (Figure 5B). In contrast, yeast transformants harboring OsHOX24ΔC and OsHOX22ΔC, and empty bait vector control, did not grow in either of the selection media. This suggested that C-terminal region of full-length homeobox proteins was responsible for their transcriptional activation property, because these proteins could drive the expression of HIS3 and ADE2 reporter genes even in the absence of any interacting protein in yeast.


Characterization of Rice Homeobox Genes, OsHOX22 and OsHOX24, and Over-expression of OsHOX24 in Transgenic Arabidopsis Suggest Their Role in Abiotic Stress Response.

Bhattacharjee A, Khurana JP, Jain M - Front Plant Sci (2016)

Transactivation and dimerization properties of homeobox proteins.(A) Schematic representation of full-length homeobox proteins (BD::OsHOX24 and BD::OsHOX22) and truncated (C-terminal acidic region removed) fusion constructs (BD::OsHOX24ΔC and BD::OsHOX22ΔC) used. (B) Transactivation assay of full-length and truncated (ΔC) homeobox proteins in yeast. The transformants grown on SD-Trp (SD-T, left) medium; SD-Trp-His medium (SD-T-H, middle) and SD-Trp-His-Ade (SD-T-H-A, right) medium are shown. (C) Dimerization assay of full-length (fl) and truncated (ΔC) homeobox proteins. The deletion constructs, BD::OsHOX24ΔC and BD::OsHOX22ΔC, were co-transformed with different combinations of full-length (AD::OsHOX24fl, AD::OsHOX22fl) and deletion constructs (AD::OsHOX24ΔC, AD::OsHOX22ΔC) of homeobox proteins in yeast, as indicated on left side panel. The transformants were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction. pGBKT7-p53 + pGADT7-T antigen represents positive control. Empty pGBKT7 vector (BD) represents negative control for transactivation assay and pGBKT7 + pGADT7 represents negative control for dimerization assay.
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Figure 5: Transactivation and dimerization properties of homeobox proteins.(A) Schematic representation of full-length homeobox proteins (BD::OsHOX24 and BD::OsHOX22) and truncated (C-terminal acidic region removed) fusion constructs (BD::OsHOX24ΔC and BD::OsHOX22ΔC) used. (B) Transactivation assay of full-length and truncated (ΔC) homeobox proteins in yeast. The transformants grown on SD-Trp (SD-T, left) medium; SD-Trp-His medium (SD-T-H, middle) and SD-Trp-His-Ade (SD-T-H-A, right) medium are shown. (C) Dimerization assay of full-length (fl) and truncated (ΔC) homeobox proteins. The deletion constructs, BD::OsHOX24ΔC and BD::OsHOX22ΔC, were co-transformed with different combinations of full-length (AD::OsHOX24fl, AD::OsHOX22fl) and deletion constructs (AD::OsHOX24ΔC, AD::OsHOX22ΔC) of homeobox proteins in yeast, as indicated on left side panel. The transformants were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction. pGBKT7-p53 + pGADT7-T antigen represents positive control. Empty pGBKT7 vector (BD) represents negative control for transactivation assay and pGBKT7 + pGADT7 represents negative control for dimerization assay.
Mentions: OsHOX24 and OsHOX22 proteins were found to be rich in acidic amino acids at the C-terminal region, which could possibly contribute to their transactivation property. Thus, we investigated the transcriptional activation property of these HD-ZIP I TFs in yeast. The complete ORFs and C-terminal deletion constructs (ΔC) of OsHOX24 and OsHOX22 were cloned in yeast expression vector containing DNA binding domain (Figure 5A). The colonies of transformed yeast cells grew uniformly on SD-Trp selection medium. The growth of yeast transformants on SD-Trp-His and SD-Trp-His-Ade selection media, even with increasing serial dilution, confirmed the transactivating nature of full-length homeobox proteins (Figure 5B). In contrast, yeast transformants harboring OsHOX24ΔC and OsHOX22ΔC, and empty bait vector control, did not grow in either of the selection media. This suggested that C-terminal region of full-length homeobox proteins was responsible for their transcriptional activation property, because these proteins could drive the expression of HIS3 and ADE2 reporter genes even in the absence of any interacting protein in yeast.

Bottom Line: The recombinant full-length and truncated homeobox proteins were found to be localized in the nucleus.Many of these genes were found to be involved in transcriptional regulation and various metabolic pathways.Altogether, our results suggest the possible role of OsHOX22/OsHOX24 homeobox proteins as negative regulators in abiotic stress responses.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
Homeobox transcription factors are well known regulators of plant growth and development. In this study, we carried out functional analysis of two candidate stress-responsive HD-ZIP I class homeobox genes from rice, OsHOX22, and OsHOX24. These genes were highly up-regulated under various abiotic stress conditions at different stages of rice development, including seedling, mature and reproductive stages. The transcript levels of these genes were enhanced significantly in the presence of plant hormones, including abscisic acid (ABA), auxin, salicylic acid, and gibberellic acid. The recombinant full-length and truncated homeobox proteins were found to be localized in the nucleus. Electrophoretic mobility shift assay established the binding of these homeobox proteins with specific DNA sequences, AH1 (CAAT(A/T)ATTG) and AH2 (CAAT(C/G)ATTG). Transactivation assays in yeast revealed the transcriptional activation potential of full-length OsHOX22 and OsHOX24 proteins. Homo- and hetero-dimerization capabilities of these proteins have also been demonstrated. Further, we identified putative novel interacting proteins of OsHOX22 and OsHOX24 via yeast-two hybrid analysis. Over-expression of OsHOX24 imparted higher sensitivity to stress hormone, ABA, and abiotic stresses in the transgenic Arabidopsis plants as revealed by various physiological and phenotypic assays. Microarray analysis revealed differential expression of several stress-responsive genes in transgenic lines as compared to wild-type. Many of these genes were found to be involved in transcriptional regulation and various metabolic pathways. Altogether, our results suggest the possible role of OsHOX22/OsHOX24 homeobox proteins as negative regulators in abiotic stress responses.

No MeSH data available.


Related in: MedlinePlus