Abstract
The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.
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Claims 15
Claim · model-written summary
CaPUB1 was isolated from water-stressed hot pepper.
- Taxon
- hot pepper
Capsicum frutescense0 - Material
- Capsicum annuum L. cv Pukange0
- Gene
- C. annuum putative U-box protein 1 (CaPUB1)e0
- Outcome
- isolatede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1)”
Claim · model-written summary
CaPUB1 had E3 ubiquitin ligase activity.
- Taxon
- hot pepperCapsicum frutescenst0
- Gene
- CaPUB1e0
- Outcome
- E3 ubiquitin ligase activitye0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CaPUB1 possessed E3 ubiquitin ligase activity”
t0“Hot Pepper”
Claim · model-written summary
The CaPUB1 U-box motif was essential for enzyme activity.
- Taxon
- Hot PepperCapsicum frutescenst0
- Gene
- CaPUB1t0
- Outcome
- enzyme activitye0
- Qualifier
- U-box motife0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the U-box motif was indeed essential for its enzyme activity.”
t0“CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog”
Claim · model-written summary
Abiotic stresses induced CaPUB1 mRNA rapidly.
- Taxon
- Hot PepperCapsicum frutescenst0
- Treatment
- drought, high salinity, cold temperature, and mechanical woundinge0
- Gene
- CaPUB1e0
- Outcome
- CaPUB1 mRNAe0
- Direction
- induced rapidlye0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding”
t0“Hot Pepper”
Claim · model-written summary
Ethylene did not induce CaPUB1 mRNA.
- Taxon
- Hot PepperCapsicum frutescenst0
- Treatment
- ethylenee0
- Gene
- CaPUB1t0
- Outcome
- CaPUB1 mRNAe0
- Direction
- not in responsee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen”
t0“CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog”
Claim · model-written summary
Abscisic acid did not induce CaPUB1 mRNA.
- Taxon
- Hot PepperCapsicum frutescenst0
- Treatment
- abscisic acide0
- Gene
- CaPUB1t0
- Outcome
- CaPUB1 mRNAe0
- Direction
- not in responsee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen”
t0“CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog”
Claim · model-written summary
A bacterial pathogen did not induce CaPUB1 mRNA.
- Taxon
- Hot PepperCapsicum frutescenst0
- Treatment
- a bacterial pathogene0
- Gene
- CaPUB1t0
- Outcome
- CaPUB1 mRNAe0
- Direction
- not in responsee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen”
t0“CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog”
Claim · model-written summary
CaPUB1-expressing Arabidopsis had longer hypocotyls and roots.
- Taxon
- Arabidopsis
Arabidopsise0 - Treatment
- constitutively expressed the CaPUB1 genee0
- Gene
- CaPUB1e0
- Outcome
- hypocotyls and rootse0
- Direction
- markedly longere0
- Comparator
- wild typee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype.”
Claim · model-written summary
CaPUB1-expressing Arabidopsis grew more rapidly than wild type.
- Taxon
- ArabidopsisArabidopsise0
- Treatment
- constitutively expressed the CaPUB1 genee0
- Outcome
- grewe0
- Direction
- more rapidlye0
- Comparator
- wild typee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype.”
Claim · model-written summary
CaPUB1-expressing Arabidopsis bolted early.
- Taxon
- ArabidopsisArabidopsise0
- Treatment
- constitutively expressed the CaPUB1 genee0
- Outcome
- bolting phenotypee0
- Direction
- earlye0
- Comparator
- wild typee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype.”
Claim · model-written summary
CaPUB1 roots had more small cells and disordered cell layers.
- Taxon
- ArabidopsisArabidopsist0
- Organ / stage
- rootse0
- Treatment
- 35S::CaPUB1e0
- Outcome
- numbers of small-sized cellse0
- Direction
- increasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele.”
t0“Arabidopsis (Arabidopsis thaliana)”
Claim · model-written summary
CaPUB1 overexpression increased sensitivity to water stress.
- Taxon
- ArabidopsisArabidopsist0
- Treatment
- CaPUB1-overexpressinge0
- Outcome
- sensitivity to water stresse0
- Direction
- increasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity.”
t0“Arabidopsis (Arabidopsis thaliana)”
Claim · model-written summary
CaPUB1 overexpression increased sensitivity to mild salinity.
- Taxon
- ArabidopsisArabidopsist0
- Treatment
- CaPUB1-overexpressinge0
- Outcome
- sensitivitye0
- Direction
- increasede0
- Qualifier
- mild salinitye0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity.”
t0“Arabidopsis (Arabidopsis thaliana)”
Claim · model-written summary
RPN6 protein level was significantly reduced in CaPUB1 seedlings relative to wild type.
- Taxon
- ArabidopsisArabidopsist0
- Treatment
- 35SCaPUB1 seedlingse0
- Gene
- RPN6 proteine0
- Outcome
- level of RPN6 proteine0
- Direction
- reducede0
- Comparator
- wild typee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings as compared to the wild type.”
t0“Arabidopsis (Arabidopsis thaliana)”
Claim · model-written summary
RPN6 physically interacted with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro.
- Taxon
- ArabidopsisArabidopsist0
- Gene
- RPN6e0
- Outcome
- interacted physically with CaPUB1 and was ubiquitinatede0
- Qualifier
- in vitroe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro.”
t0“Arabidopsis (Arabidopsis thaliana)”