Abstract
Soybean is an excellent candidate for sustainable agriculture due to its production of nutritious, versatile beans and the ability to form symbiotic organs called root nodules that perform nitrogen fixation. As demand for both yield and sustainable agriculture continue to increase, root nodules offer an attractive alternative to expensive and environmentally harmful nitrogen fertilizers. Understanding root nodule formation may open genetic engineering avenues for optimizing nitrogen fixation performance and transferring the nodule-formation ability to other plants. A major determinant of nodule numbers and quality in soybean is microRNA 160 (miR160), which dictates developmental stage-specific auxin sensitivity by targeting repressor auxin response factor (ARF) transcription factors during nodule development. However, the specific downstream targets of the miR160-ARF signaling module remain unknown. This project seeks to resolve these targets by evaluating the cellular, spatiotemporal, and DNAbinding activity of ARF16-2 (a key target of miR160 in soybean nodules), by confocal microscopy, DamID-Seq, and protein-DNA interaction studies. ARF16-2promoter:GUS or tdT transcriptional fusions revealed that ARF16-2 is transcriptionally active in dividing and differentiating tissues such as primary root tips, lateral root primordium, and emerging nodules. In addition, ARF16-2 promoter:GUS or tdT fusions show that this gene is active in the parenchyma and root stele of mature nodules. Interestingly, ARF16- 2 promoter:16-2 CDS:tdT translational fusions show a reduction of tdT signal in mature nodule tissues, underscoring post-transcriptional regulation of ARF16-2, likely by miR160 during nodule maturation. Compared to known activator ARFs, ARF16-2 does not appear to bind the conserved AuxRE TGTCTC motif with the same affinity as activator ARFs, suggesting alternative binding sites or functionality. In addition, ARF16- 2 appears to function as a monomer compared to the activator ARFs that appear to act as dimers. We hypothesize that ARF16-2 might form heterodimers with activator ARFs to reduce transactivation of target genes. When the genomic binding profile of ARF16-2 is identified and evaluated, we expect to identify key downstream genes affected by its activity during nodule development. A protein-DNA binding assay that can be performed using a simple UV trans-illuminator developed as part of this research could be accessible for teaching labs to demonstrate specificity of bio-molecular interactions.
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Claims 9
Claim · model-written summary
ARF16-2 is transcriptionally active in primary root tips, lateral root primordium and emerging nodules.
- Taxon
- Soybean
Glycine maxt0 - Organ / stage
- primary root tips, lateral root primordium, and emerging nodulese0
- Gene
- ARF16-2e0
- Outcome
- transcriptionally activee0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“ARF16-2promoter:GUS or tdT transcriptional fusions revealed that ARF16-2 is transcriptionally active in dividing and differentiating tissues such as primary root tips, lateral root primordium, and emerging nodules.”
t0“Soybean”
Claim · model-written summary
ARF16-2 is active in parenchyma and root stele of mature nodules.
- Taxon
- SoybeanGlycine maxt0
- Organ / stage
- parenchyma and root stele of mature nodulese0
- Gene
- ARF16-2e0
- Outcome
- activee0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“ARF16-2 promoter:GUS or tdT fusions show that this gene is active in the parenchyma and root stele of mature nodules.”
t0“Soybean”
Claim · model-written summary
ARF16-2 translational fusion signal was reduced in mature nodule tissues.
- Taxon
- SoybeanGlycine maxt0
- Organ / stage
- mature nodule tissuese0
- Gene
- ARF16- 2e0
- Outcome
- tdT signale0
- Direction
- reductione0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“ARF16- 2 promoter:16-2 CDS:tdT translational fusions show a reduction of tdT signal in mature nodule tissues”
t0“Soybean”
Claim · model-written summary
The reduced translational signal suggests post-transcriptional ARF16-2 regulation, likely by miR160 during nodule maturation.
- Taxon
- SoybeanGlycine maxt0
- Gene
- ARF16-2e0
- Outcome
- post-transcriptional regulation of ARF16-2e0
- Qualifier
- likely by miR160 during nodule maturatione0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“underscoring post-transcriptional regulation of ARF16-2, likely by miR160 during nodule maturation.”
t0“Soybean”
Claim · model-written summary
ARF16-2 appears to bind the conserved AuxRE TGTCTC motif with lower affinity than activator ARFs.
- Taxon
- SoybeanGlycine maxt0
- Gene
- ARF16-2e0
- Outcome
- bind the conserved AuxRE TGTCTC motife0
- Direction
- does not appear to binde0
- Comparator
- activator ARFse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Compared to known activator ARFs, ARF16-2 does not appear to bind the conserved AuxRE TGTCTC motif with the same affinity as activator ARFs”
t0“Soybean”
Claim · model-written summary
The binding result suggests alternative binding sites or functionality for ARF16-2.
- Taxon
- SoybeanGlycine maxt0
- Gene
- ARF16-2t0
- Outcome
- alternative binding sites or functionalitye0
- Qualifier
- suggestinge0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“suggesting alternative binding sites or functionality.”
t0“ARF16-2 by microRNA160 During Soybean”
Claim · model-written summary
ARF16-2 appears to function as a monomer whereas activator ARFs appear to act as dimers.
- Taxon
- SoybeanGlycine maxt0
- Gene
- ARF16- 2e0
- Outcome
- function as a monomere0
- Comparator
- activator ARFse0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“ARF16- 2 appears to function as a monomer compared to the activator ARFs that appear to act as dimers.”
t0“Soybean”
Claim · model-written summary
The authors hypothesize that ARF16-2 heterodimerization with activator ARFs reduces target-gene transactivation.
- Taxon
- SoybeanGlycine maxt0
- Gene
- ARF16-2e0
- Outcome
- transactivation of target genese0
- Direction
- reducee0
- Qualifier
- might form heterodimers with activator ARFse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“We hypothesize that ARF16-2 might form heterodimers with activator ARFs to reduce transactivation of target genes.”
t0“Soybean”
Claim · model-written summary
A protein-DNA binding assay using a simple UV trans-illuminator was developed as part of the research.
- Taxon
- SoybeanGlycine maxt0
- Outcome
- protein-DNA binding assaye0
- Value
- simple UV trans-illuminatore0
- Qualifier
- could be accessible for teaching labse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“A protein-DNA binding assay that can be performed using a simple UV trans-illuminator developed as part of this research could be accessible for teaching labs to demonstrate specificity of bio-molecular interactions.”
t0“Soybean”