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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 tobacco shown in confocal microscopic images of sectioned WT and E1-containing tobacco. 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 blue. (a) Negative control WT tobacco confocal image stack at ×600 magnification. Note the absence of E1 antibody staining in this image (red). (b) E1cd-transformed tobacco image stack at ×1,200 magnification. (c and d) Individual sections of the confocal image stack presented in (b) illustrate that E1cd antibody stained E1cd-transformed tobacco. Arrows indicate (1) deconvoluted E1 antibody signal present within a tobacco cell wall section (red) and (2) E1 antibody staining present on the inside of a cell wall (red). Blue coloring denotes the residual plant autofluorescence signal. Scale bar in (a), 25 μm; original magnification ×600. Scale bars in (b-d), 50 μm; original magnification, ×1,200.
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Figure 9: Localization of E1 in transgenic tobacco shown in confocal microscopic images of sectioned WT and E1-containing tobacco. 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 blue. (a) Negative control WT tobacco confocal image stack at ×600 magnification. Note the absence of E1 antibody staining in this image (red). (b) E1cd-transformed tobacco image stack at ×1,200 magnification. (c and d) Individual sections of the confocal image stack presented in (b) illustrate that E1cd antibody stained E1cd-transformed tobacco. Arrows indicate (1) deconvoluted E1 antibody signal present within a tobacco cell wall section (red) and (2) E1 antibody staining present on the inside of a cell wall (red). Blue coloring denotes the residual plant autofluorescence signal. Scale bar in (a), 25 μm; original magnification ×600. Scale bars in (b-d), 50 μm; original magnification, ×1,200.

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 tobacco shown in confocal microscopic images of sectioned WT and E1-containing tobacco. 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 blue. (a) Negative control WT tobacco confocal image stack at ×600 magnification. Note the absence of E1 antibody staining in this image (red). (b) E1cd-transformed tobacco image stack at ×1,200 magnification. (c and d) Individual sections of the confocal image stack presented in (b) illustrate that E1cd antibody stained E1cd-transformed tobacco. Arrows indicate (1) deconvoluted E1 antibody signal present within a tobacco cell wall section (red) and (2) E1 antibody staining present on the inside of a cell wall (red). Blue coloring denotes the residual plant autofluorescence signal. Scale bar in (a), 25 μm; original magnification ×600. Scale bars in (b-d), 50 μm; original magnification, ×1,200.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Localization of E1 in transgenic tobacco shown in confocal microscopic images of sectioned WT and E1-containing tobacco. 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 blue. (a) Negative control WT tobacco confocal image stack at ×600 magnification. Note the absence of E1 antibody staining in this image (red). (b) E1cd-transformed tobacco image stack at ×1,200 magnification. (c and d) Individual sections of the confocal image stack presented in (b) illustrate that E1cd antibody stained E1cd-transformed tobacco. Arrows indicate (1) deconvoluted E1 antibody signal present within a tobacco cell wall section (red) and (2) E1 antibody staining present on the inside of a cell wall (red). Blue coloring denotes the residual plant autofluorescence signal. Scale bar in (a), 25 μm; original magnification ×600. Scale bars in (b-d), 50 μm; original magnification, ×1,200.
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