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Isolation and functional characterization of an Ethylene Response Factor (RhERF092) from rose (Rosa hybrida)

DOI:10.1007/s11240-019-01719-y
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Muhammad Ali Khan at Abdul Wali Khan University Mardan
Muhammad Ali Khan
Muhammad Imtiaz at Abdul Wali Khan University Mardan
Muhammad Imtiaz
Adil Hussain at Abdul Wali Khan University Mardan
Adil Hussain
Fazal Jalal at Abdul Wali Khan University Mardan
Fazal Jalal
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Abstract and Figures

Improper post-harvest handling results in significant losses to cut rose flowers. The phytohormone ethylene plays a key role in the growth of rose and flower opening. In this study we isolated and characterized an Ethylene Response Factor (RhERF092) from rose (Rosa hybrida) cv. “Samantha” and investigated its role in flower opening and development. RhERF092 was originally detected in a microarray experiment with a significant increase in its expression in rose petals after 1 h of ethylene treatment. Sequence analysis showed the presence of a canonical AP2/EREBP domain and a C-terminus trans-activation domain. Phylogenetic analysis showed that RhERF092 is an orthologue of the Arabidopsis ERF1 (AT3G23240) belonging to subgroup IX of the ERF gene family. Confocal laser scanning microscopy showed RhERF092: GFP expression in the nucleus. Basal expression of RhERF092 is highest expression at stage 0 of flower opening which gradually decreases and remains constant till senescence. Ectopic expression of RhERF092 in Arabidopsis caused various ethylene-related aberrations in plant development including stunted growth, abortion of apical dominance, production of lateral tillers from rosette nodes, branches from the aerial nodes, and sterile inflorescence. Arabidopsis 35SRhERF092 plants were found to be sensitive to the ethylene precursor ACC with significantly reduced hypocotyl and root system with significant reduction in the expression of genes involved in cell proliferation, cell expansion and cell cycle such as ARGOS, ARGOS-LIKE, SIM, JAGGED, AN3, CYCD3-1, and CYCD3-2as compared to WT plants. Taken together these results indicate that RhERF092 regulates ethylene-specific responses in rose and Arabidopsis. The rose Ethylene Response Factor RhERF092 is a transcription factor that regulates essential ethylene-related development of rose flowers and mediates abiotic stress responses in Arabidopsis.
Amino acid sequence alignment of RhERF092 with ERF orthologues of other plant species. Amino acid sequence alignment of RhERF092 showed high similarity with ERF of other plant species. The ORF region contained the trademark AP2/EREBP domain starting from amino acid 87 to amino acid 159 (red square). (Color figure online)
Amino acid sequence alignment of RhERF092 with ERF orthologues of other plant species. Amino acid sequence alignment of RhERF092 showed high similarity with ERF of other plant species. The ORF region contained the trademark AP2/EREBP domain starting from amino acid 87 to amino acid 159 (red square). (Color figure online)
… 
Phylogenetic analysis of 12 rose ERF genes. The phylogenetic analysis of 12 rose ERF genes was conducted with 122 ERF genes from the model plant Arabidopsis thaliana. RhERF genes were divided into seven groups (black arrows) including GVI-L, GV, GX, GI (responsible for disease resistance and wax accumulation in plants) GVIII, and GIX (responsible for signaling of phytohormones such as ethylene, JA, ABA and drought/salt responses) and GIII (responsible for freezing, drought and salt stress responses). Two transcriptomic elements RU05029 and RU15319 were unique to Rosa hybrida only. RhERF092 (Ru27155) was classified into group IX along with 17 Arabidopsis ERF genes (red arrow). (Color figure online)
Phylogenetic analysis of 12 rose ERF genes. The phylogenetic analysis of 12 rose ERF genes was conducted with 122 ERF genes from the model plant Arabidopsis thaliana. RhERF genes were divided into seven groups (black arrows) including GVI-L, GV, GX, GI (responsible for disease resistance and wax accumulation in plants) GVIII, and GIX (responsible for signaling of phytohormones such as ethylene, JA, ABA and drought/salt responses) and GIII (responsible for freezing, drought and salt stress responses). Two transcriptomic elements RU05029 and RU15319 were unique to Rosa hybrida only. RhERF092 (Ru27155) was classified into group IX along with 17 Arabidopsis ERF genes (red arrow). (Color figure online)
… 
Expression profile of RhERF092. Expression profile of RhERF092 in rose flower using quantitative real time PCR analysis after 1, 6, 12, 18 and 24 h of ethylene treatment (a). Relative expression of RhERF092 in different growth stages of flower development (b). Maximum transcript accumulation occurred at stage 0 (flower bud is tightly closed). RhACT5 was used as internal control. Each experiment was repeated three times with three technical replicate of each treatment. Significant differences were calculated using student’s t test at P ≤ 0.05 and error bars show standard deviation
Expression profile of RhERF092. Expression profile of RhERF092 in rose flower using quantitative real time PCR analysis after 1, 6, 12, 18 and 24 h of ethylene treatment (a). Relative expression of RhERF092 in different growth stages of flower development (b). Maximum transcript accumulation occurred at stage 0 (flower bud is tightly closed). RhACT5 was used as internal control. Each experiment was repeated three times with three technical replicate of each treatment. Significant differences were calculated using student’s t test at P ≤ 0.05 and error bars show standard deviation
… 
Sub-cellular localization of RhERF092. The cellular localization of RhERF092 was determined using 35SCaMV-RhERF092-GFP recombinant protein cloned in onion epidermal cells. Green fluorescence signals were detected scattered throughout the cells transformed with the control plasmid containing GFP only whereas the 35SCaMV-RhERF092-GFP fusion protein was detected in nucleus only
Sub-cellular localization of RhERF092. The cellular localization of RhERF092 was determined using 35SCaMV-RhERF092-GFP recombinant protein cloned in onion epidermal cells. Green fluorescence signals were detected scattered throughout the cells transformed with the control plasmid containing GFP only whereas the 35SCaMV-RhERF092-GFP fusion protein was detected in nucleus only
… 
Ectopic expression of RhERF092 in Arabidopsis. Real time PCR analysis showing significantly higher expression of the transgene in all three transgenic lines as compared to WT plants (a). Transgenic Arabidopsis plants showed a variety of ethylene-related phenotypic aberrations with an overall reduced phenotype (b). Phenotypic aberrations such as branches at different aerial nodes, inflorescence at lateral aerial nodes, stems with significantly smaller internode length, bunch or tuft of tillers and leaves at the crown were common features (c)
+3
Ectopic expression of RhERF092 in Arabidopsis. Real time PCR analysis showing significantly higher expression of the transgene in all three transgenic lines as compared to WT plants (a). Transgenic Arabidopsis plants showed a variety of ethylene-related phenotypic aberrations with an overall reduced phenotype (b). Phenotypic aberrations such as branches at different aerial nodes, inflorescence at lateral aerial nodes, stems with significantly smaller internode length, bunch or tuft of tillers and leaves at the crown were common features (c)
… 
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Plant Cell, Tissue and Organ Culture (PCTOC) (2020) 140:157–172
https://doi.org/10.1007/s11240-019-01719-y
ORIGINAL ARTICLE
Isolation andfunctional characterization ofanEthylene Response
Factor (RhERF092) fromrose (Rosa hybrida)
Muhammad AliKhan1,2· MuhammadImtiaz1,2· AdilHussain1· FazalJalal1· SikandarHayat3· SayedHussain1·
FazalSaid1· MehboobAlam4· RabiaAmir5
Received: 6 January 2019 / Accepted: 14 October 2019 / Published online: 29 October 2019
© Springer Nature B.V. 2019
Abstract
Improper post-harvest handling results in significant losses to cut rose flowers. The phytohormone ethylene plays a key role in
the growth of rose and flower opening. In this study we isolated and characterized an Ethylene Response Factor (RhERF092)
from rose (Rosa hybrida) cv. “Samantha” and investigated its role in flower opening and development. RhERF092 was
originally detected in a microarray experiment with a significant increase in its expression in rose petals after 1h of ethylene
treatment. Sequence analysis showed the presence of a canonical AP2/EREBP domain and a C-terminus trans-activation
domain. Phylogenetic analysis showed that RhERF092 is an orthologue of the Arabidopsis ERF1 (AT3G23240) belonging
to subgroup IX of the ERF gene family. Confocal laser scanning microscopy showed RhERF092: GFP expression in the
nucleus. Basal expression of RhERF092 is highest expression at stage 0 of flower opening which gradually decreases and
remains constant till senescence. Ectopic expression of RhERF092 in Arabidopsis caused various ethylene-related aberrations
in plant development including stunted growth, abortion of apical dominance, production of lateral tillers from rosette nodes,
branches from the aerial nodes, and sterile inflorescence. Arabidopsis 35SRhERF092 plants were found to be sensitive to
the ethylene precursor ACC with significantly reduced hypocotyl and root system with significant reduction in the expres-
sion of genes involved in cell proliferation, cell expansion and cell cycle such as ARGOS, ARGOS-LIKE, SIM, JAGGED,
AN3, CYCD3-1, and CYCD3-2as compared to WT plants. Taken together these results indicate that RhERF092 regulates
ethylene-specific responses in rose and Arabidopsis.
Key message
The rose Ethylene Response Factor RhERF092 is a transcription factor that regulates essential ethylene-related development
of rose flowers and mediates abiotic stress responses in Arabidopsis.
Keywords Rose· Flower opening· Ethylene· RhERF092· Transcriptional factor
Introduction
Rose (Rosa hybrida) is a woody perennial plant of the genus
Rosa with over 100 species, within the family Rosaceae
forming a large group of shrubs, and climbing or trailing
plants with stems often armed with sharp prickles. Flowers
vary in color and size. Generally, they are harvested and cut
when in bud, and held in refrigerated conditions until ready
for display at their point of sale.
Ethylene, unlike the rest of the plant hormones; is a gas-
eous hormone. It is the only member of its class and has
the simplest structure of all the plant growth substances.
It plays a key role in seed germination, leaf senescence,
fruit ripening and abscission, response to biotic and abi-
otic stresses, and in the triple response (Abeles etal. 2012;
Arteca and Arteca 1999). Flower senescence in many plant
species has been associated with ethylene biosynthesis and
homeostasis. The climacteric rise of endogenous ethylene
in ornamental flowers has been shown to play a regulatory
Communicated by Sergio Rosales-Mendoza.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1124 0-019-01719 -y) contains
supplementary material, which is available to authorized users.
* Muhammad Ali Khan
malikhan@awkum.edu.pk
Extended author information available on the last page of the article
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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... This distinction was less evident in respect to dry biomass but a reliable difference still remained in all the lines. It is known that ARGOS genes are related to auxin hormones [25] that, in turn, activate expansion growth owing to influence on expansin proteins [5,26]. It is possible that such an influence of the gene's products was not apparent under standard culturing conditions when the cells actively divide and grow but it could produce a beneficial effect on growth parameters upon exposure to stress agents. ...
Growth and stress resistance of tobacco hairy roots with constitutive expression of ARGOS-LIKE gene / Рост и стрессоустойчивость волосовидных корней табака с конститутивной экспрессией гена ARGOS-LIKE
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... In most recent reports, the gene isolation and characterization was carried out in various plants such as Gossypium arboretum (Mushtaq et al. 2020), Rosa hybrida (Khan et al. 2020) and Arabidopsis thaliana (Ziyuan et al. 2020). Our laboratory has a comprehensive research programme on molecular interventions in A. paniculata Srinath et al. 2020Srinath et al. , 2021. ...
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Isolation of 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of methyl erythritol diphosphate (MEP) pathway, in silico analysis and differential tissue specific ApHDR expression in Andrographis paniculata (Burm. f) Nees
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