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In planta expression of A. cellulolyticus Cel5A endocellulase reduces cell wall recalcitrance in tobacco and maize.

Brunecky R, Selig MJ, Vinzant TB, Himmel ME, Lee D, Blaylock MJ, Decker SR - Biotechnol Biofuels (2011)

Bottom Line: Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion.Tobacco and maize plants were healthy and developed normally compared with the wild type (WT).Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Center, National Renewable Energy Laboratory, 1617 Cole Boulevard, MS 3323, Golden, CO 80401, USA. roman.brunecky@nrel.gov.

ABSTRACT
The glycoside hydrolase family 5 endocellulase, E1 (Cel5A), from Acidothermus cellulolyticus was transformed into both Nicotiana tabacum and Zea mays with expression targeted to the cell wall under a constitutive promoter. Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion. Tobacco and maize plants were healthy and developed normally compared with the wild type (WT). After thermochemical pretreatment and enzyme digestion, transformed plants were clearly more digestible than WT, requiring lower pretreatment severity to achieve comparable conversion levels. Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

No MeSH data available.


Related in: MedlinePlus

Localization of E1 in transgenic corn stover. Confocal microscopic images of sectioned WT and E1-containing corn stover. All images were obtained by using immunofluorescence confocal laser microscopy. An E1cd primary antibody and an Alexa Fluor 488 antimouse secondary antibody were used and spectrally deconvoluted to show antibody in red and nonspecific autofluoresence in green. (a) Original magnification ×200 image of E1-transgenic maize leaf tissue indicating that the transgenic tissue exhibits a broad expression of E1 throughout the plant. Scale bar, 50 μm. (b) Original magnification ×200 confocal microscopic image of leaf tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 50 μm. (c) Original magnification ×600 image of E1-transgenic maize stem tissue. This image indicates that E1-transgenic tissue exhibits specific expression of E1 in the lignified sclerenchyma cells in the bottom portion of the image. Scale bar, 25 μm. (d) Original magnification ×600 confocal microscopic image of stem tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 25 μm.
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Figure 8: Localization of E1 in transgenic corn stover. Confocal microscopic images of sectioned WT and E1-containing corn stover. All images were obtained by using immunofluorescence confocal laser microscopy. An E1cd primary antibody and an Alexa Fluor 488 antimouse secondary antibody were used and spectrally deconvoluted to show antibody in red and nonspecific autofluoresence in green. (a) Original magnification ×200 image of E1-transgenic maize leaf tissue indicating that the transgenic tissue exhibits a broad expression of E1 throughout the plant. Scale bar, 50 μm. (b) Original magnification ×200 confocal microscopic image of leaf tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 50 μm. (c) Original magnification ×600 image of E1-transgenic maize stem tissue. This image indicates that E1-transgenic tissue exhibits specific expression of E1 in the lignified sclerenchyma cells in the bottom portion of the image. Scale bar, 25 μm. (d) Original magnification ×600 confocal microscopic image of stem tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 25 μm.

Mentions: Imaging cross-sections of both tobacco and maize plant cell walls obtained by anti-E1 immunomicroscopy clearly showed differences in the localization of E1 between these two plant types. The transgenic E1 is broadly distributed throughout the sclerenchyma-type cell walls of maize (Figure 8), whereas the E1 tobacco shows specific localization to the inner surface of the cell wall (Figure 9). In Figure 9, we can see localization of the E1 signal to interior regions of the plant cell wall. This result was expected, as E1 was expressed under the control of the CaMV 35S constitutive promoter and was targeted to the apoplast using the soybean variant-specific protein VSPβ secretion signal peptide.


In planta expression of A. cellulolyticus Cel5A endocellulase reduces cell wall recalcitrance in tobacco and maize.

Brunecky R, Selig MJ, Vinzant TB, Himmel ME, Lee D, Blaylock MJ, Decker SR - Biotechnol Biofuels (2011)

Localization of E1 in transgenic corn stover. Confocal microscopic images of sectioned WT and E1-containing corn stover. All images were obtained by using immunofluorescence confocal laser microscopy. An E1cd primary antibody and an Alexa Fluor 488 antimouse secondary antibody were used and spectrally deconvoluted to show antibody in red and nonspecific autofluoresence in green. (a) Original magnification ×200 image of E1-transgenic maize leaf tissue indicating that the transgenic tissue exhibits a broad expression of E1 throughout the plant. Scale bar, 50 μm. (b) Original magnification ×200 confocal microscopic image of leaf tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 50 μm. (c) Original magnification ×600 image of E1-transgenic maize stem tissue. This image indicates that E1-transgenic tissue exhibits specific expression of E1 in the lignified sclerenchyma cells in the bottom portion of the image. Scale bar, 25 μm. (d) Original magnification ×600 confocal microscopic image of stem tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037329&req=5

Figure 8: Localization of E1 in transgenic corn stover. Confocal microscopic images of sectioned WT and E1-containing corn stover. All images were obtained by using immunofluorescence confocal laser microscopy. An E1cd primary antibody and an Alexa Fluor 488 antimouse secondary antibody were used and spectrally deconvoluted to show antibody in red and nonspecific autofluoresence in green. (a) Original magnification ×200 image of E1-transgenic maize leaf tissue indicating that the transgenic tissue exhibits a broad expression of E1 throughout the plant. Scale bar, 50 μm. (b) Original magnification ×200 confocal microscopic image of leaf tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 50 μm. (c) Original magnification ×600 image of E1-transgenic maize stem tissue. This image indicates that E1-transgenic tissue exhibits specific expression of E1 in the lignified sclerenchyma cells in the bottom portion of the image. Scale bar, 25 μm. (d) Original magnification ×600 confocal microscopic image of stem tissue from a WT control corn stover stalk showing no expression of E1 enzyme. Scale bar, 25 μm.
Mentions: Imaging cross-sections of both tobacco and maize plant cell walls obtained by anti-E1 immunomicroscopy clearly showed differences in the localization of E1 between these two plant types. The transgenic E1 is broadly distributed throughout the sclerenchyma-type cell walls of maize (Figure 8), whereas the E1 tobacco shows specific localization to the inner surface of the cell wall (Figure 9). In Figure 9, we can see localization of the E1 signal to interior regions of the plant cell wall. This result was expected, as E1 was expressed under the control of the CaMV 35S constitutive promoter and was targeted to the apoplast using the soybean variant-specific protein VSPβ secretion signal peptide.

Bottom Line: Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion.Tobacco and maize plants were healthy and developed normally compared with the wild type (WT).Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Center, National Renewable Energy Laboratory, 1617 Cole Boulevard, MS 3323, Golden, CO 80401, USA. roman.brunecky@nrel.gov.

ABSTRACT
The glycoside hydrolase family 5 endocellulase, E1 (Cel5A), from Acidothermus cellulolyticus was transformed into both Nicotiana tabacum and Zea mays with expression targeted to the cell wall under a constitutive promoter. Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion. Tobacco and maize plants were healthy and developed normally compared with the wild type (WT). After thermochemical pretreatment and enzyme digestion, transformed plants were clearly more digestible than WT, requiring lower pretreatment severity to achieve comparable conversion levels. Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

No MeSH data available.


Related in: MedlinePlus