Abstract
KEY MESSAGE: Modification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callus Metabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.
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Claims 8
Claim · model-written summary
Six near-miss motifs were identified within a 120-bp upstream region of rice PSY1.
- Taxon
- rice
Oryza sativat0 - Gene
- PHYTOENE SYNTHASE 1t0
- Outcome
- six motifse0
- Value
- six motifse0
- Qualifier
- within a 120-bp regione0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides”
t0“PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus”
Claim · model-written summary
Editing four motifs to match BCH2 elements allowed the promoter to drive GFP expression in callus.
- Taxon
- riceOryza sativat0
- Organ / stage
- calluse0
- Treatment
- mutated four motifse0
- Gene
- GFPe0
- Outcome
- GFP expression in calluse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants”
t0“rice callus”
Claim · model-written summary
The four-motif promoter also drove GFP expression in seeds of regenerated plants.
- Taxon
- riceOryza sativat0
- Organ / stage
- seedse0
- Treatment
- mutated four motifse0
- Gene
- GFPe0
- Outcome
- GFP expression in callus and in seeds of regenerated plantse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants”
t0“rice callus”
Claim · model-written summary
The 4 M promoter failed to drive PSY1 expression.
- Taxon
- riceOryza sativat0
- Treatment
- 4 M promotere0
- Gene
- PSY1e0
- Outcome
- PSY1 expressione0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The 4 M promoter was not sufficient to drive PSY1 expression”
t0“rice callus”
Claim · model-written summary
The six-motif promoter plus PDS transgene drove PSY1 expression.
- Taxon
- riceOryza sativat0
- Treatment
- 6 M promotere0
- Gene
- PSY1e0
- Outcome
- PSY1 expressione0
- Qualifier
- in combination with a PDS transgenee0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene”
t0“rice callus”
Claim · model-written summary
The six-motif promoter with PDS produced orange callus with confirmed carotenoid accumulation.
- Taxon
- riceOryza sativat0
- Organ / stage
- callus tissuee0
- Treatment
- 6 M promotere1
- Outcome
- accumulation of carotenoidse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis”
t0“rice callus”
e1“resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene”
Claim · model-written summary
Uncorrected promoter plus PDS produced callus lacking carotenoids.
- Taxon
- riceOryza sativat0
- Treatment
- uncorrectede0
- Outcome
- callus that lacked carotenoidse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids”
t0“rice callus”
Claim · model-written summary
The 4 M promoter plus PDS produced callus lacking carotenoids.
- Taxon
- riceOryza sativat0
- Treatment
- 4 M variants of the promoter plus a PDS transgenee0
- Outcome
- callus that lacked carotenoidse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids”
t0“rice callus”
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