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Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.

Klose H, Günl M, Usadel B, Fischer R, Commandeur U - Biotechnol Biofuels (2013)

Bottom Line: However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development.These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany. commandeur@molbiotech.rwth-aachen.de.

ABSTRACT

Background: Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development.

Results: Here, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.

Conclusion: We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

No MeSH data available.


Related in: MedlinePlus

Effects of Ethanol induction on transgenic plants. Activity on 4MUC measured over time after ethanol induction in six-week-old soil-grown plants (A). Plants were induced at t0 by applying 2% ethanol in 100 ml irrigation water, and cellulase activity was monitored over a 96 h time course. Ethanol dose response in alcR::TrCel5A lines (B). Transgenic lines were monitored for cellulase activity 24 h after watering. Values represent the mean of three plants per independent transgenic line. Effect of sequential ethanol induction (C). Six-week-old soil-grown plants from the homozygous line F6.5 were induced using 2% ethanol at t0 and again after 48 h (asterisks). Cellulase activity was monitored over 144 h. Wild-type plants were monitored in parallel, and no cellulase activity was observed throughout the time course. Comparison of cellulose content and cellulase activity after repeated induction with ethanol (D). A relative value of 100% cellulase activity represents a conversion of 27 nmol 4MU min-1 mg-1, whereas a value of 100% for cellulose content represents 140 μg glucose per mg alcohol insoluble residue (AIR). For all panels, values represent the mean of three plants per independent transgenic line. Error bars the show standard deviation of the mean after subtraction of wild-type control data.
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Figure 4: Effects of Ethanol induction on transgenic plants. Activity on 4MUC measured over time after ethanol induction in six-week-old soil-grown plants (A). Plants were induced at t0 by applying 2% ethanol in 100 ml irrigation water, and cellulase activity was monitored over a 96 h time course. Ethanol dose response in alcR::TrCel5A lines (B). Transgenic lines were monitored for cellulase activity 24 h after watering. Values represent the mean of three plants per independent transgenic line. Effect of sequential ethanol induction (C). Six-week-old soil-grown plants from the homozygous line F6.5 were induced using 2% ethanol at t0 and again after 48 h (asterisks). Cellulase activity was monitored over 144 h. Wild-type plants were monitored in parallel, and no cellulase activity was observed throughout the time course. Comparison of cellulose content and cellulase activity after repeated induction with ethanol (D). A relative value of 100% cellulase activity represents a conversion of 27 nmol 4MU min-1 mg-1, whereas a value of 100% for cellulose content represents 140 μg glucose per mg alcohol insoluble residue (AIR). For all panels, values represent the mean of three plants per independent transgenic line. Error bars the show standard deviation of the mean after subtraction of wild-type control data.

Mentions: We examined the effects of alcohol induction on plant growth and TrCel5A expression. In all assays T2 TrCel5A plants were compared to wild-type plants. Initial induction of TrCel5A activity using 2% ethanol led to a maximum expression level of 0.4 U mg-1 TSP on azoCMC and 5 nmol 4MU min--1 mg--1 after 24 h (Figure 4A). Further analysis showed that significant induction could be achieved with 0.1% ethanol (without basal expression) but that optimal expression levels required 2% ethanol (Figure 4B). There was no improvement in cellulase expression in transgenic plants when 5% ethanol was used, and concentrations greater than 5% were detrimental to both wild-type and transgenic plants, resulting in physiological stress symptoms such as leaf yellowing and curling (data not shown).


Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.

Klose H, Günl M, Usadel B, Fischer R, Commandeur U - Biotechnol Biofuels (2013)

Effects of Ethanol induction on transgenic plants. Activity on 4MUC measured over time after ethanol induction in six-week-old soil-grown plants (A). Plants were induced at t0 by applying 2% ethanol in 100 ml irrigation water, and cellulase activity was monitored over a 96 h time course. Ethanol dose response in alcR::TrCel5A lines (B). Transgenic lines were monitored for cellulase activity 24 h after watering. Values represent the mean of three plants per independent transgenic line. Effect of sequential ethanol induction (C). Six-week-old soil-grown plants from the homozygous line F6.5 were induced using 2% ethanol at t0 and again after 48 h (asterisks). Cellulase activity was monitored over 144 h. Wild-type plants were monitored in parallel, and no cellulase activity was observed throughout the time course. Comparison of cellulose content and cellulase activity after repeated induction with ethanol (D). A relative value of 100% cellulase activity represents a conversion of 27 nmol 4MU min-1 mg-1, whereas a value of 100% for cellulose content represents 140 μg glucose per mg alcohol insoluble residue (AIR). For all panels, values represent the mean of three plants per independent transgenic line. Error bars the show standard deviation of the mean after subtraction of wild-type control data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643885&req=5

Figure 4: Effects of Ethanol induction on transgenic plants. Activity on 4MUC measured over time after ethanol induction in six-week-old soil-grown plants (A). Plants were induced at t0 by applying 2% ethanol in 100 ml irrigation water, and cellulase activity was monitored over a 96 h time course. Ethanol dose response in alcR::TrCel5A lines (B). Transgenic lines were monitored for cellulase activity 24 h after watering. Values represent the mean of three plants per independent transgenic line. Effect of sequential ethanol induction (C). Six-week-old soil-grown plants from the homozygous line F6.5 were induced using 2% ethanol at t0 and again after 48 h (asterisks). Cellulase activity was monitored over 144 h. Wild-type plants were monitored in parallel, and no cellulase activity was observed throughout the time course. Comparison of cellulose content and cellulase activity after repeated induction with ethanol (D). A relative value of 100% cellulase activity represents a conversion of 27 nmol 4MU min-1 mg-1, whereas a value of 100% for cellulose content represents 140 μg glucose per mg alcohol insoluble residue (AIR). For all panels, values represent the mean of three plants per independent transgenic line. Error bars the show standard deviation of the mean after subtraction of wild-type control data.
Mentions: We examined the effects of alcohol induction on plant growth and TrCel5A expression. In all assays T2 TrCel5A plants were compared to wild-type plants. Initial induction of TrCel5A activity using 2% ethanol led to a maximum expression level of 0.4 U mg-1 TSP on azoCMC and 5 nmol 4MU min--1 mg--1 after 24 h (Figure 4A). Further analysis showed that significant induction could be achieved with 0.1% ethanol (without basal expression) but that optimal expression levels required 2% ethanol (Figure 4B). There was no improvement in cellulase expression in transgenic plants when 5% ethanol was used, and concentrations greater than 5% were detrimental to both wild-type and transgenic plants, resulting in physiological stress symptoms such as leaf yellowing and curling (data not shown).

Bottom Line: However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development.These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany. commandeur@molbiotech.rwth-aachen.de.

ABSTRACT

Background: Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development.

Results: Here, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.

Conclusion: We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

No MeSH data available.


Related in: MedlinePlus