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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

Interacting proteins of homeobox TFs identified by yeast-two hybrid analysis and their gene expression profiling.(A,B) The transformants in yeast strain were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction of proteins with OsHOX24 (A) and OsHOX22 (B). pGBKT7-p53 + pGADT7-T antigen represents positive control and pGBKT7-Lam + pGADT7-T antigen represents negative control. The graphical panels (right) represent quantitative β-galactosidase assay showing the lacZ reporter gene expression (β-galactosidase activity in Miller units) for interacting proteins of OsHOX24 (A) and OsHOX22 (B). Ortho-nitrophenyl-β-D-galactoside (ONPG) was used as substrate for β-galactosidase assay. The putative function and locus identifier of the interacting proteins are given on the left side. (C) Heat-map showing gene expression profiles of OsHOX24, OsHOX22, and genes encoding for their interacting proteins during various abiotic stress conditions. The heat-map has been generated by Genevestigator (v.3) using the publicly available abiotic stress related microarray data. The color scale representing fold change (log2 ratio) is shown below the heat-map.
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Figure 6: Interacting proteins of homeobox TFs identified by yeast-two hybrid analysis and their gene expression profiling.(A,B) The transformants in yeast strain were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction of proteins with OsHOX24 (A) and OsHOX22 (B). pGBKT7-p53 + pGADT7-T antigen represents positive control and pGBKT7-Lam + pGADT7-T antigen represents negative control. The graphical panels (right) represent quantitative β-galactosidase assay showing the lacZ reporter gene expression (β-galactosidase activity in Miller units) for interacting proteins of OsHOX24 (A) and OsHOX22 (B). Ortho-nitrophenyl-β-D-galactoside (ONPG) was used as substrate for β-galactosidase assay. The putative function and locus identifier of the interacting proteins are given on the left side. (C) Heat-map showing gene expression profiles of OsHOX24, OsHOX22, and genes encoding for their interacting proteins during various abiotic stress conditions. The heat-map has been generated by Genevestigator (v.3) using the publicly available abiotic stress related microarray data. The color scale representing fold change (log2 ratio) is shown below the heat-map.

Mentions: The deletion constructs of homeobox genes (OsHOX24ΔC and OsHOX22ΔC) were used as baits to identify their interacting proteins. Numerous transformants were obtained after large-scale transformation of OsHOX24ΔC and OsHOX22ΔC bait plasmid DNAs and screened on SD media lacking leucine, tryptophan and histidine. Selected transformants were screened by colony PCR and further grown on TDO (SD-Trp-Leu-His) and QDO (SD-Trp-Leu-His-Ade) media supplemented with or without X-α-Gal or X-β-Gal for reconfirmation. The growth of putative clones and blue color development in colonies was observed on TDO and QDO selection media, which also indicated activation of reported genes (Mel1 and lacZ). The sequencing of plasmid DNAs of selected confirmed clones resulted in the identification of interacting proteins of candidate homeobox TFs. At least nine and five proteins were identified as interacting proteins of OsHOX24 and OsHOX22, respectively. OsHOX24 was found to interact with protein fragments belonging to GRAM domain TF, expressed protein, high mobility group protein (HMG1/2), eukaryotic translation initiation factor I, DUF domain protein, endoplasmic reticulum (ER) lumen protein retaining receptor and enzymes like sucrose synthase and phenylalanine ammonia lyase (Figure 6A). OsHOX22 was found to interact with protein fragments belonging to an expressed protein, pentatricopeptide repeat protein, hypoxia-responsive family protein, universal stress protein domain containing protein and UDP-glucuronosyl and UDP-glucosyl transferase domain containing protein (Figure 6B).


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)

Interacting proteins of homeobox TFs identified by yeast-two hybrid analysis and their gene expression profiling.(A,B) The transformants in yeast strain were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction of proteins with OsHOX24 (A) and OsHOX22 (B). pGBKT7-p53 + pGADT7-T antigen represents positive control and pGBKT7-Lam + pGADT7-T antigen represents negative control. The graphical panels (right) represent quantitative β-galactosidase assay showing the lacZ reporter gene expression (β-galactosidase activity in Miller units) for interacting proteins of OsHOX24 (A) and OsHOX22 (B). Ortho-nitrophenyl-β-D-galactoside (ONPG) was used as substrate for β-galactosidase assay. The putative function and locus identifier of the interacting proteins are given on the left side. (C) Heat-map showing gene expression profiles of OsHOX24, OsHOX22, and genes encoding for their interacting proteins during various abiotic stress conditions. The heat-map has been generated by Genevestigator (v.3) using the publicly available abiotic stress related microarray data. The color scale representing fold change (log2 ratio) is shown below the heat-map.
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Related In: Results  -  Collection

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Figure 6: Interacting proteins of homeobox TFs identified by yeast-two hybrid analysis and their gene expression profiling.(A,B) The transformants in yeast strain were grown on SD-Trp-Leu (DDO medium) and SD-Trp-Leu-His-Ade medium (QDO medium) for confirmation of interaction of proteins with OsHOX24 (A) and OsHOX22 (B). pGBKT7-p53 + pGADT7-T antigen represents positive control and pGBKT7-Lam + pGADT7-T antigen represents negative control. The graphical panels (right) represent quantitative β-galactosidase assay showing the lacZ reporter gene expression (β-galactosidase activity in Miller units) for interacting proteins of OsHOX24 (A) and OsHOX22 (B). Ortho-nitrophenyl-β-D-galactoside (ONPG) was used as substrate for β-galactosidase assay. The putative function and locus identifier of the interacting proteins are given on the left side. (C) Heat-map showing gene expression profiles of OsHOX24, OsHOX22, and genes encoding for their interacting proteins during various abiotic stress conditions. The heat-map has been generated by Genevestigator (v.3) using the publicly available abiotic stress related microarray data. The color scale representing fold change (log2 ratio) is shown below the heat-map.
Mentions: The deletion constructs of homeobox genes (OsHOX24ΔC and OsHOX22ΔC) were used as baits to identify their interacting proteins. Numerous transformants were obtained after large-scale transformation of OsHOX24ΔC and OsHOX22ΔC bait plasmid DNAs and screened on SD media lacking leucine, tryptophan and histidine. Selected transformants were screened by colony PCR and further grown on TDO (SD-Trp-Leu-His) and QDO (SD-Trp-Leu-His-Ade) media supplemented with or without X-α-Gal or X-β-Gal for reconfirmation. The growth of putative clones and blue color development in colonies was observed on TDO and QDO selection media, which also indicated activation of reported genes (Mel1 and lacZ). The sequencing of plasmid DNAs of selected confirmed clones resulted in the identification of interacting proteins of candidate homeobox TFs. At least nine and five proteins were identified as interacting proteins of OsHOX24 and OsHOX22, respectively. OsHOX24 was found to interact with protein fragments belonging to GRAM domain TF, expressed protein, high mobility group protein (HMG1/2), eukaryotic translation initiation factor I, DUF domain protein, endoplasmic reticulum (ER) lumen protein retaining receptor and enzymes like sucrose synthase and phenylalanine ammonia lyase (Figure 6A). OsHOX22 was found to interact with protein fragments belonging to an expressed protein, pentatricopeptide repeat protein, hypoxia-responsive family protein, universal stress protein domain containing protein and UDP-glucuronosyl and UDP-glucosyl transferase domain containing protein (Figure 6B).

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