Abstract
Nitrate reductases (NRs) catalyze the first step in the reduction of nitrate to ammonium. NR activity is regulated by sumoylation through the E3 ligase activity of AtSIZ1. However, it is not clear how NRs interact with AtSIZ1 in the cell, or how nitrogen sources affect NR levels and their cellular localization. Here, we show that the subcellular localization of NRs is modulated by the E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 and that NR protein levels are regulated by nitrogen sources. Transient expression analysis of GFP fusion proteins in onion epidermal cells showed that the NRs NIA1 and NIA2 localize to the cytoplasmic membrane, and that AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately, whereas NRs and AtSIZ1 localize to the nucleus when co-expressed. Nitrate did not affect the subcellular localization of the NRs, but it caused AtSIZ1 to move from the nucleus to the cytoplasm. NRs were not detected in ammonium-treated cells, whereas the localization of AtSIZ1 was not altered by ammonium treatment. NR protein levels increased in response to nitrate but decreased in response to ammonium. In addition, NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants. NR protein degradation occurred later in cop1-4 than in the wild-type, although the NR proteins did not interact with COP1. Therefore, AtSIZ1 controls nuclear localization of NR proteins, and ammonium negatively regulates their levels. The function and stability of NR proteins might be post-translationally modulated by ubiquitination.
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Claims 14
Claim · model-written summary
NIA1 and NIA2 localized to the cytoplasmic membrane when expressed separately.
- Taxon
- onion
Allium cepae1 - Gene
- NIA1 and NIA2e0
- Outcome
- localize to the cytoplasmic membranee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the NRs NIA1 and NIA2 localize to the cytoplasmic membrane”
e1“in onion epidermal cells”
Claim · model-written summary
AtSIZ1 localized to nucleoplasm including nuclear bodies when expressed separately.
- Taxon
- onionAllium cepae1
- Gene
- AtSIZ1e0
- Outcome
- localizes to the nucleoplasm, including nuclear bodiese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately”
e1“in onion epidermal cells”
Claim · model-written summary
Co-expressed NRs and AtSIZ1 localized to the nucleus.
- Taxon
- onionAllium cepae1
- Treatment
- co-expressede0
- Gene
- NRs and AtSIZ1e0
- Outcome
- localize to the nucleuse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“NRs and AtSIZ1 localize to the nucleus when co-expressed”
e1“in onion epidermal cells”
Claim · model-written summary
Nitrate did not affect NR subcellular localization.
- Taxon
- onionAllium cepae1
- Treatment
- Nitratee0
- Gene
- NRse0
- Outcome
- subcellular localization of the NRse0
- Direction
- did not affecte0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Nitrate did not affect the subcellular localization of the NRs”
e1“in onion epidermal cells”
Claim · model-written summary
Nitrate moved AtSIZ1 from nucleus to cytoplasm.
- Taxon
- onionAllium cepae2
- Treatment
- Nitratee1
- Gene
- AtSIZ1e0
- Outcome
- move from the nucleus to the cytoplasme0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“it caused AtSIZ1 to move from the nucleus to the cytoplasm”
e1“Nitrate did not affect the subcellular localization of the NRs”
e2“in onion epidermal cells”
Claim · model-written summary
NRs were not detected in ammonium-treated cells.
- Taxon
- onionAllium cepae1
- Treatment
- ammonium-treatede0
- Gene
- NRse0
- Outcome
- NRs were not detectede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“NRs were not detected in ammonium-treated cells”
e1“in onion epidermal cells”
Claim · model-written summary
Ammonium did not alter AtSIZ1 localization.
- Taxon
- onionAllium cepae1
- Treatment
- ammonium treatmente0
- Gene
- AtSIZ1e0
- Outcome
- localization of AtSIZ1e0
- Direction
- not alterede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“localization of AtSIZ1 was not altered by ammonium treatment”
e1“in onion epidermal cells”
Claim · model-written summary
Nitrate increased NR protein levels.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“NR protein levels increased in response to nitrate”
e1“in onion epidermal cells”
Claim · model-written summary
Ammonium decreased NR protein levels.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“NR protein levels increased in response to nitrate but decreased in response to ammonium”
e1“in onion epidermal cells”
Claim · model-written summary
A 26S proteasome inhibitor increased NR protein levels.
- Taxon
- onionAllium cepae1
- Treatment
- 26S proteasome inhibitore0
- Gene
- NRe0
- Outcome
- NR protein levelse0
- Direction
- increasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“NR protein levels increased in response to a 26S proteasome inhibitor”
e1“in onion epidermal cells”
Claim · model-written summary
NR protein levels increased in cop1-4 plants.
Verbatim
e0“NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants”
Claim · model-written summary
NR protein levels increased in DN-COP1-overexpressing transgenic plants.
Verbatim
e0“NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants”
Claim · model-written summary
NR protein degradation was delayed in cop1-4 relative to wild type.
Verbatim
e0“NR protein degradation occurred later in cop1-4 than in the wild-type”
e1“transgenic plants”
Claim · model-written summary
NR proteins did not interact with COP1.
- Taxon
- plantsViridiplantaee1
- Gene
- NR proteinse0
- Outcome
- did not interact with COP1e0
Verbatim
e0“the NR proteins did not interact with COP1”
e1“transgenic plants”
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