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  • Nerolidol production in agroinfiltrated tobacco: Impact of protein stability and membrane targeting of strawberry (Fragraria ananassa) NEROLIDOL SYNTHASE1.

Nerolidol production in agroinfiltrated tobacco: Impact of protein stability and membrane targeting of strawberry (Fragraria ananassa) NEROLIDOL SYNTHASE1.

Plant science : an international journal of experimental plant biology (2018-01-25)
Paola Andrade, David Manzano, Karla Ramirez-Estrada, Daniel Caudepon, Montserrat Arro, Albert Ferrer, Michael A Phillips
ABSTRACT

The sesquiterpene alcohol nerolidol, synthesized from farnesyl diphosphate (FDP), mediates plant-insect interactions across multiple trophic levels with major implications for pest management in agriculture. We compared nerolidol engineering strategies in tobacco using agroinfiltration to transiently express strawberry (Fragraria ananassa) linalool/nerolidol synthase (FaNES1) either at the endoplasmic reticulum (ER) or in the cytosol as a soluble protein. Using solid phase microextraction and gas chromatography-mass spectrometry (SPME-GCMS), we have determined that FaNES1 directed to the ER via fusion to the transmembrane domain of squalene synthase or hydroxymethylglutaryl - CoA reductase displayed significant improvements in terms of transcript levels, protein accumulation, and volatile production when compared to its cytosolic form. However, the highest levels of nerolidol production were observed when FaNES1 was fused to GFP and expressed in the cytosol. This SPME-GCMS method afforded a limit of detection and quantification of 1.54 and 5.13 pg, respectively. Nerolidol production levels, which ranged from 0.5 to 3.0 μg/g F.W., correlated more strongly to the accumulation of recombinant protein than transcript level, the former being highest in FaNES-GFP transfected plants. These results indicate that while the ER may represent an enriched source of FDP that can be exploited in metabolic engineering, protein accumulation is a better predictor of sesquiterpene production.

MATERIALS
Product Number
Brand
Product Description

Supelco
SPME-OC Fiber Assembly, Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), df 75 μm (Coating thickness includes 65 μm coating + 10 μm OC (overcoating)), needle size 23 ga, pkg of 3 ea, fiber L 1 cm
Supelco
SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), needle size 23 ga, StableFlex, for use with autosampler
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 7 μm (PDMS), needle size 23 ga, for use with autosampler
Supelco
SPME fiber assembly Carboxen/Polydimethylsiloxane (CAR/PDMS), df 85 μm (CAR/PDMS), needle size 23 ga, StableFlex, for use with autosampler
Supelco
SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), needle size 23 ga, StableFlex, for use with manual holder/autosampler, fiber L 2 cm
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (PDMS), for use with autosampler, needle size 24 ga
Supelco
SPME fiber assembly Carboxen/Polydimethylsiloxane (CAR/PDMS), df 75 μm (CAR/PDMS), for use with manual holder, needle size 24 ga
Supelco
SPME Portable Field Sampler, coating PDMS/DVB
Supelco
SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), needle size 24 ga, StableFlex, for use with manual holder/autosampler
Supelco
SPME fiber assembly Carboxen/Polydimethylsiloxane (CAR/PDMS), df 75 μm (CAR/PDMS), needle size 24 ga, for use with autosampler
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 30 μm (PDMS), needle size 24 ga, for use with autosampler
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 7 μm (PDMS), needle size 24 ga, for use with manual holder
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 7 μm (PDMS), needle size 24 ga, for use with autosampler
Supelco
SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), needle size 24 ga, StableFlex, for use with autosampler
Supelco
SPME fiber assembly polyacrylate (PA), df 85 μm (PA), for use with manual holder, needle size 24 ga
Supelco
SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), needle size 24 ga, for use with manual holder
Supelco
SPME Sampling Stand, for use with 15 mL vials
Supelco
SPME Portable Field Sampler, coating PDMS
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (PDMS), needle size 24 ga, for use with manual holder
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (PDMS), for use with autosampler, needle size 23 ga
Supelco
SPME fiber assembly Carboxen/Polydimethylsiloxane (CAR/PDMS), df 75 μm (CAR/PDMS), needle size 23 ga, for use with autosampler
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 30 μm (PDMS), needle size 24 ga, for use with manual holder
Supelco
SPME fiber assembly Carboxen/Polydimethylsiloxane (CAR/PDMS), df 75 μm (CAR/PDMS), needle size 23 ga, for use with manual holder
Supelco
SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), for use with autosampler, needle size 23 ga, metal alloy fiber, fiber L 1 cm
Supelco
SPME fiber assembly Carboxen/Polydimethylsiloxane (CAR/PDMS), df 85 μm (CAR/PDMS), for use with autosampler, needle size 23 ga, metal alloy fiber
Supelco
SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), for use with autosampler, needle size 23 ga, metal alloy fiber, fiber L 2 cm
Supelco
SPME fiber assembly polyacrylate (PA), df 85 μm (PA), for use with autosampler, needle size 24 ga
Supelco
SPME Sampling Stand, for use with 4 mL vials
Supelco
SPME fiber assembly Carboxen/Polydimethylsiloxane (CAR/PDMS), df 85 μm (CAR/PDMS), needle size 24 ga, StableFlex, for use with autosampler
Supelco
SPME Portable Field Sampler, coating CAR/PDMS