Abstract
Heterologous expression systems based on tobacco BY-2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway((R))-compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher (3)H-IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease-deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration-dependent reversal of (3)H-IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.
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Claims 6
Claim · model-written summary
A Schizosaccharomyces pombe system with knockout mutants and expression vectors was developed for plant transporter analysis.
- Taxon
- Arabidopsis
Arabidopsist0 - Outcome
- comparative studies of plant transport proteinse0
- Value
- all ABC and putative auxin transport genese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway((R))-compatible expression vectors for functional analysis and subcellular localization of recombinant proteins.”
t0“Arabidopsis”
Claim · model-written summary
ABCB19 showed higher IAA export than ABCB1.
- Taxon
- ArabidopsisArabidopsist0
- Gene
- ABCB19e0
- Outcome
- (3)H-IAA export activitye0
- Direction
- highere0
- Comparator
- ABCB1e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“ABCB19 showed a higher (3)H-IAA export activity than ABCB1.”
t0“Arabidopsis”
Claim · model-written summary
PIN1 activity exceeded PIN2 despite similar protein expression.
- Taxon
- ArabidopsisArabidopsist0
- Gene
- PIN1e0
- Outcome
- activitye0
- Direction
- highere0
- Qualifier
- similar protein expression levelse0
- Comparator
- PIN2e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“PIN1 showed higher activity than PIN2 with similar protein expression levels.”
t0“Arabidopsis”
Claim · model-written summary
AUX1 expression increased net auxin uptake.
- Taxon
- ArabidopsisArabidopsist0
- Treatment
- Expression of AUX1e0
- Gene
- AUX1e0
- Outcome
- net auxin uptake activitye0
- Direction
- increasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Expression of AUX1 in a permease-deficient vat3 mutant resulted in increased net auxin uptake activity.”
t0“Arabidopsis”
Claim · model-written summary
ABCB4 showed a concentration-dependent reversal of IAA transport.
- Taxon
- ArabidopsisArabidopsist0
- Gene
- ABCB4e0
- Outcome
- concentration-dependent reversal of (3)H-IAA transporte0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“ABCB4 expressed in mam1pdr1 displayed a concentration-dependent reversal of (3)H-IAA transport”
t0“Arabidopsis”
Claim · model-written summary
Structural modeling suggested three substrate interaction sites in ABCB4 versus two in ABCB19.
- Taxon
- ArabidopsisArabidopsist0
- Gene
- ABCB4e0
- Outcome
- substrate interaction sitese0
- Value
- threee0
- Qualifier
- twoe0
- Comparator
- ABCB19e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19”
t0“Arabidopsis”
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