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HYPER RECOMBINATION1 of the THO/TREX complex plays a role in controlling transcription of the REVERSION-TO-ETHYLENE SENSITIVITY1 gene in Arabidopsis.

Xu C, Zhou X, Wen CK - PLoS Genet. (2015)

Bottom Line: The hpr1-5 mutation reduced the total and nuclear RTE1 transcript levels to a similar extent, and RTE1 transcript reduction rate was not affected by hpr1-5 with cordycepin treatment, which prematurely terminates transcription.The defect in the THO-interacting TEX1 protein of TREX but not the mRNA export factor SAC3B also reduced the total and nuclear RTE1 levels.SERINE-ARGININE-RICH (SR) proteins are involved mRNA splicing, and we found that SR protein SR33 co-localized with HPR1 in nuclear speckles, which agreed with the association of human TREX with the splicing machinery.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) represses ethylene hormone responses by promoting ethylene receptor ETHYLENE RESPONSE1 (ETR1) signaling, which negatively regulates ethylene responses. To investigate the regulation of RTE1, we performed a genetic screening for mutations that suppress ethylene insensitivity conferred by RTE1 overexpression in Arabidopsis. We isolated HYPER RECOMBINATION1 (HPR1), which is required for RTE1 overexpressor (RTE1ox) ethylene insensitivity at the seedling but not adult stage. HPR1 is a component of the THO complex, which, with other proteins, forms the TRanscription EXport (TREX) complex. In yeast, Drosophila, and humans, the THO/TREX complex is involved in transcription elongation and nucleocytoplasmic RNA export, but its role in plants is to be fully determined. We investigated how HPR1 is involved in RTE1ox ethylene insensitivity in Arabidopsis. The hpr1-5 mutation may affect nucleocytoplasmic mRNA export, as revealed by in vivo hybridization of fluorescein-labeled oligo(dT)45 with unidentified mRNA in the nucleus. The hpr1-5 mutation reduced the total and nuclear RTE1 transcript levels to a similar extent, and RTE1 transcript reduction rate was not affected by hpr1-5 with cordycepin treatment, which prematurely terminates transcription. The defect in the THO-interacting TEX1 protein of TREX but not the mRNA export factor SAC3B also reduced the total and nuclear RTE1 levels. SERINE-ARGININE-RICH (SR) proteins are involved mRNA splicing, and we found that SR protein SR33 co-localized with HPR1 in nuclear speckles, which agreed with the association of human TREX with the splicing machinery. We reveal a role for HPR1 in RTE1 expression during transcription elongation and less likely during export. Gene expression involved in ethylene signaling suppression was not reduced by the hpr1-5 mutation, which indicates selectivity of HPR1 for RTE1 expression affecting the consequent ethylene response. Thus, components of the THO/TREX complex appear to have specific roles in the transcription or export of selected genes.

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Effect of sac3b-2 and tex1–4 alleles on RTE1 expression.(A) Phenotype of etiolated sac3b-2 and tex1–4 seedlings and wild-type (Col-0) and hpr1–5 seedlings. Ethylene dose–response assay of hypocotyl length (B) and normalized hypocotyl length (C) for wild-type (Col-0), sac3b-2, and tex1–4 seedlings. Mean difference and statistical significance were estimated by Scheffe test (α = 0.01). Ethylene concentration is presented as the logarithm of ethylene (in μL L-1). Phenotype of etiolated seedlings for the indicated genotypes (sac3b-2 and text1–4) in RTE1ox (D) and the etr1–2 allele (E), and hypocotyl length (F) and ERF1 level (G) for the corresponding seedlings. RTE1 level in RTE1ox seedlings with the sac3b-2 and text1–4 alleles (H) and in sac3b-2 and tex1–4 seedlings with and without the etr1–2 allele (I). (J) Immunofluorescence of RTE1 level in RTE1ox and the indicated genotypes with the sac3b-2 and tex1–4 alleles. (K) Nuclear RTE1 level in sac3b-2 and tex1–4. Blot: Commassie Blue staining to indicate the protein amount on the membrane. RTE1-Ab: a monoclonal antibody for RTE1. Data are mean±SD for seedling hypocotyls (n>30), and mean±SE for gene expression (at least 3 independent biological samples with 3 measurements for each).
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pgen.1004956.g008: Effect of sac3b-2 and tex1–4 alleles on RTE1 expression.(A) Phenotype of etiolated sac3b-2 and tex1–4 seedlings and wild-type (Col-0) and hpr1–5 seedlings. Ethylene dose–response assay of hypocotyl length (B) and normalized hypocotyl length (C) for wild-type (Col-0), sac3b-2, and tex1–4 seedlings. Mean difference and statistical significance were estimated by Scheffe test (α = 0.01). Ethylene concentration is presented as the logarithm of ethylene (in μL L-1). Phenotype of etiolated seedlings for the indicated genotypes (sac3b-2 and text1–4) in RTE1ox (D) and the etr1–2 allele (E), and hypocotyl length (F) and ERF1 level (G) for the corresponding seedlings. RTE1 level in RTE1ox seedlings with the sac3b-2 and text1–4 alleles (H) and in sac3b-2 and tex1–4 seedlings with and without the etr1–2 allele (I). (J) Immunofluorescence of RTE1 level in RTE1ox and the indicated genotypes with the sac3b-2 and tex1–4 alleles. (K) Nuclear RTE1 level in sac3b-2 and tex1–4. Blot: Commassie Blue staining to indicate the protein amount on the membrane. RTE1-Ab: a monoclonal antibody for RTE1. Data are mean±SD for seedling hypocotyls (n>30), and mean±SE for gene expression (at least 3 independent biological samples with 3 measurements for each).

Mentions: We used ethylene dose–response assay to evaluate whether the loss-of-function sac3b-2 and tex1–4 mutations [15, 16, 24], resulting from T-DNA insertion, affect the ethylene response. Etiolated sac3b-2 and tex1–4 seedlings showed enhanced ethylene growth inhibition (Fig. 8A). Over a wide range of ethylene concentration, the hypocotyl was slightly shorter for etiolated mutant seedlings than the wild type (Col-0), with a shorter seedling for sac3b-2 than text1–4 (Fig. 8B). When normalized to the untreated seedling hypocotyl length, the relative hypocotyl length was slightly shorter for sac3b-2 than wild-type and tex1–4 seedlings. The sac3b-2 and tex1–4 mutations each conferred ethylene hypersensitivity to various degrees (Fig. 8C).


HYPER RECOMBINATION1 of the THO/TREX complex plays a role in controlling transcription of the REVERSION-TO-ETHYLENE SENSITIVITY1 gene in Arabidopsis.

Xu C, Zhou X, Wen CK - PLoS Genet. (2015)

Effect of sac3b-2 and tex1–4 alleles on RTE1 expression.(A) Phenotype of etiolated sac3b-2 and tex1–4 seedlings and wild-type (Col-0) and hpr1–5 seedlings. Ethylene dose–response assay of hypocotyl length (B) and normalized hypocotyl length (C) for wild-type (Col-0), sac3b-2, and tex1–4 seedlings. Mean difference and statistical significance were estimated by Scheffe test (α = 0.01). Ethylene concentration is presented as the logarithm of ethylene (in μL L-1). Phenotype of etiolated seedlings for the indicated genotypes (sac3b-2 and text1–4) in RTE1ox (D) and the etr1–2 allele (E), and hypocotyl length (F) and ERF1 level (G) for the corresponding seedlings. RTE1 level in RTE1ox seedlings with the sac3b-2 and text1–4 alleles (H) and in sac3b-2 and tex1–4 seedlings with and without the etr1–2 allele (I). (J) Immunofluorescence of RTE1 level in RTE1ox and the indicated genotypes with the sac3b-2 and tex1–4 alleles. (K) Nuclear RTE1 level in sac3b-2 and tex1–4. Blot: Commassie Blue staining to indicate the protein amount on the membrane. RTE1-Ab: a monoclonal antibody for RTE1. Data are mean±SD for seedling hypocotyls (n>30), and mean±SE for gene expression (at least 3 independent biological samples with 3 measurements for each).
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pgen.1004956.g008: Effect of sac3b-2 and tex1–4 alleles on RTE1 expression.(A) Phenotype of etiolated sac3b-2 and tex1–4 seedlings and wild-type (Col-0) and hpr1–5 seedlings. Ethylene dose–response assay of hypocotyl length (B) and normalized hypocotyl length (C) for wild-type (Col-0), sac3b-2, and tex1–4 seedlings. Mean difference and statistical significance were estimated by Scheffe test (α = 0.01). Ethylene concentration is presented as the logarithm of ethylene (in μL L-1). Phenotype of etiolated seedlings for the indicated genotypes (sac3b-2 and text1–4) in RTE1ox (D) and the etr1–2 allele (E), and hypocotyl length (F) and ERF1 level (G) for the corresponding seedlings. RTE1 level in RTE1ox seedlings with the sac3b-2 and text1–4 alleles (H) and in sac3b-2 and tex1–4 seedlings with and without the etr1–2 allele (I). (J) Immunofluorescence of RTE1 level in RTE1ox and the indicated genotypes with the sac3b-2 and tex1–4 alleles. (K) Nuclear RTE1 level in sac3b-2 and tex1–4. Blot: Commassie Blue staining to indicate the protein amount on the membrane. RTE1-Ab: a monoclonal antibody for RTE1. Data are mean±SD for seedling hypocotyls (n>30), and mean±SE for gene expression (at least 3 independent biological samples with 3 measurements for each).
Mentions: We used ethylene dose–response assay to evaluate whether the loss-of-function sac3b-2 and tex1–4 mutations [15, 16, 24], resulting from T-DNA insertion, affect the ethylene response. Etiolated sac3b-2 and tex1–4 seedlings showed enhanced ethylene growth inhibition (Fig. 8A). Over a wide range of ethylene concentration, the hypocotyl was slightly shorter for etiolated mutant seedlings than the wild type (Col-0), with a shorter seedling for sac3b-2 than text1–4 (Fig. 8B). When normalized to the untreated seedling hypocotyl length, the relative hypocotyl length was slightly shorter for sac3b-2 than wild-type and tex1–4 seedlings. The sac3b-2 and tex1–4 mutations each conferred ethylene hypersensitivity to various degrees (Fig. 8C).

Bottom Line: The hpr1-5 mutation reduced the total and nuclear RTE1 transcript levels to a similar extent, and RTE1 transcript reduction rate was not affected by hpr1-5 with cordycepin treatment, which prematurely terminates transcription.The defect in the THO-interacting TEX1 protein of TREX but not the mRNA export factor SAC3B also reduced the total and nuclear RTE1 levels.SERINE-ARGININE-RICH (SR) proteins are involved mRNA splicing, and we found that SR protein SR33 co-localized with HPR1 in nuclear speckles, which agreed with the association of human TREX with the splicing machinery.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) represses ethylene hormone responses by promoting ethylene receptor ETHYLENE RESPONSE1 (ETR1) signaling, which negatively regulates ethylene responses. To investigate the regulation of RTE1, we performed a genetic screening for mutations that suppress ethylene insensitivity conferred by RTE1 overexpression in Arabidopsis. We isolated HYPER RECOMBINATION1 (HPR1), which is required for RTE1 overexpressor (RTE1ox) ethylene insensitivity at the seedling but not adult stage. HPR1 is a component of the THO complex, which, with other proteins, forms the TRanscription EXport (TREX) complex. In yeast, Drosophila, and humans, the THO/TREX complex is involved in transcription elongation and nucleocytoplasmic RNA export, but its role in plants is to be fully determined. We investigated how HPR1 is involved in RTE1ox ethylene insensitivity in Arabidopsis. The hpr1-5 mutation may affect nucleocytoplasmic mRNA export, as revealed by in vivo hybridization of fluorescein-labeled oligo(dT)45 with unidentified mRNA in the nucleus. The hpr1-5 mutation reduced the total and nuclear RTE1 transcript levels to a similar extent, and RTE1 transcript reduction rate was not affected by hpr1-5 with cordycepin treatment, which prematurely terminates transcription. The defect in the THO-interacting TEX1 protein of TREX but not the mRNA export factor SAC3B also reduced the total and nuclear RTE1 levels. SERINE-ARGININE-RICH (SR) proteins are involved mRNA splicing, and we found that SR protein SR33 co-localized with HPR1 in nuclear speckles, which agreed with the association of human TREX with the splicing machinery. We reveal a role for HPR1 in RTE1 expression during transcription elongation and less likely during export. Gene expression involved in ethylene signaling suppression was not reduced by the hpr1-5 mutation, which indicates selectivity of HPR1 for RTE1 expression affecting the consequent ethylene response. Thus, components of the THO/TREX complex appear to have specific roles in the transcription or export of selected genes.

Show MeSH
Related in: MedlinePlus