Abstract
The leaves of the carnivorous plant Byblis guehoi (B. guehoi) are densely covered with structurally simple sessile trichomes, each composed of eight flattened cells arranged in a ring. These trichomes exhibit robust protein exocytosis and endocytosis, making them a suitable system for studying these processes. To track protein secretion, we established an Agrobacterium-mediated transformation protocol using root explants. Calli were induced on Byblis Callus Induction Medium (BCIM), and transgenic shoots were regenerated within two months on hygromycin-containing Byblis Selection and Regeneration Medium (BSRM). In addition, a temporary immersion system (TIS) was implemented to rapidly propagate transgenic lines, enhancing growth and facilitating acclimation. Four 35S promoter-driven constructs expressing GUS, RUBY, mGFP5, or CLV3sp-mGFP5 validated the protocol, yielding transformation efficiencies of 24.9-59.4% and regeneration efficiencies of 22.3-73.7%. Confocal microscopy revealed predominantly cytoplasmic signals for mGFP5 and plasma membrane-localized signals for CLV3sp-mGFP5 in epidermal cells. Unexpectedly, both constructs exhibited GFP signals at the plasma membrane and in the extracellular space of sessile trichome cells, regardless of the presence of a signal peptide. Western blot analysis further confirmed that mGFP5 was secreted into the mucilage independently of signal peptide presence. These findings reveal signal peptide-independent extracellular protein accumulation in glandular trichomes, deviating from the classical ER-Golgi pathway. Given that sessile trichomes of B. guehoi possess a specialized cuticular pore lacking a rigid cell wall, this structural feature may provide a plausible interface for non-classical protein export. This study establishes a rapid transformation-propagation pipeline for B. guehoi, highlighting its potential as an experimental platform for investigating protein secretion in plant glandular cells.
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Claims 9
Claim · model-written summary
A transformation protocol regenerated transgenic Byblis shoots within two months.
- Taxon
- Byblis guehoiByblis guehoit0
- Treatment
- hygromycin-containing Byblis Selection and Regeneration Medium (BSRM)e0
- Outcome
- transgenic shoots were regeneratede0
- Value
- within two monthse0
Verbatim
e0“transgenic shoots were regenerated within two months on hygromycin-containing Byblis Selection and Regeneration Medium (BSRM)”
t0“Byblis guehoi”
Claim · model-written summary
The temporary immersion system enhanced growth and facilitated acclimation of transgenic lines.
- Taxon
- Byblis guehoiByblis guehoit0
- Treatment
- temporary immersion system (TIS)e0
- Outcome
- enhancing growth and facilitating acclimatione0
Not reportedComparator
Verbatim
e0“a temporary immersion system (TIS) was implemented to rapidly propagate transgenic lines, enhancing growth and facilitating acclimation”
t0“Byblis guehoi”
Claim · model-written summary
The four constructs yielded transformation efficiencies of 24.9–59.4%.
- Taxon
- Byblis guehoiByblis guehoit0
- Outcome
- transformation efficienciese0
- Value
- 24.9-59.4%e0
Verbatim
e0“Four 35S promoter-driven constructs expressing GUS, RUBY, mGFP5, or CLV3sp-mGFP5 validated the protocol, yielding transformation efficiencies of 24.9-59.4%”
t0“Byblis guehoi”
Claim · model-written summary
The constructs yielded regeneration efficiencies of 22.3–73.7%.
- Taxon
- Byblis guehoiByblis guehoit0
- Outcome
- regeneration efficienciese0
- Value
- 22.3-73.7%e0
Verbatim
e0“regeneration efficiencies of 22.3-73.7%”
t0“Byblis guehoi”
Claim · model-written summary
mGFP5 signals were predominantly cytoplasmic in epidermal cells.
- Taxon
- Byblis guehoiByblis guehoit0
- Organ / stage
- epidermal cellse0
- Gene
- mGFP5e0
- Outcome
- predominantly cytoplasmic signalse0
Not reportedDirection
Verbatim
e0“predominantly cytoplasmic signals for mGFP5 and plasma membrane-localized signals for CLV3sp-mGFP5 in epidermal cells”
t0“Byblis guehoi”
Claim · model-written summary
CLV3sp-mGFP5 signals were localized to the plasma membrane in epidermal cells.
- Taxon
- Byblis guehoiByblis guehoit0
- Organ / stage
- epidermal cellse0
- Gene
- CLV3sp-mGFP5e0
- Outcome
- plasma membrane-localized signalse0
Not reportedDirection
Verbatim
e0“plasma membrane-localized signals for CLV3sp-mGFP5 in epidermal cells”
t0“Byblis guehoi”
Claim · model-written summary
Both constructs showed GFP at the plasma membrane and extracellular space of sessile trichome cells regardless of signal peptide.
- Taxon
- Byblis guehoiByblis guehoit0
- Organ / stage
- sessile trichome cellse0
- Gene
- GFPe0
- Outcome
- GFP signals at the plasma membrane and in the extracellular spacee0
- Qualifier
- regardless of the presence of a signal peptidee0
Not reportedDirection
Verbatim
e0“both constructs exhibited GFP signals at the plasma membrane and in the extracellular space of sessile trichome cells, regardless of the presence of a signal peptide”
t0“Byblis guehoi”
Claim · model-written summary
mGFP5 was secreted into mucilage independently of signal peptide presence.
- Taxon
- Byblis guehoiByblis guehoit0
- Gene
- mGFP5e0
- Outcome
- secreted into the mucilagee0
- Qualifier
- independently of signal peptide presencee0
Not reportedDirection
Verbatim
e0“mGFP5 was secreted into the mucilage independently of signal peptide presence”
t0“Byblis guehoi”
Claim · model-written summary
The authors propose the specialized cuticular pore as a possible interface for non-classical protein export.
- Taxon
- Byblis guehoiByblis guehoit0
- Organ / stage
- sessile trichomese0
- Outcome
- non-classical protein exporte0
- Qualifier
- specialized cuticular pore lacking a rigid cell walle0
Verbatim
e0“sessile trichomes of B. guehoi possess a specialized cuticular pore lacking a rigid cell wall, this structural feature may provide a plausible interface for non-classical protein export”
t0“Byblis guehoi”