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Active fungal GH115 α-glucuronidase produced in Arabidopsis thaliana affects only the UX1-reactive glucuronate decorations on native glucuronoxylans.

Chong SL, Derba-Maceluch M, Koutaniemi S, Gómez LD, McQueen-Mason SJ, Tenkanen M, Mellerowicz EJ - BMC Biotechnol. (2015)

Bottom Line: The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2.Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions.Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, University of Helsinki, P.O. Box 27, Helsinki, 00014, Finland. sun-li.chong@helsinki.fi.

ABSTRACT

Background: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2.

Results: The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta.

Conclusions: Active GH115 α-glucuronidase has been produced for the first time in plants. The cell wall-targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.

No MeSH data available.


Analysis of ScAGU115 enzyme activity in Arabidopsis transgenic and WT plants. (A) The soluble and wall-bound proteins were analyzed for α-glucuronidase activity using a commercial assay. The error bar represents the standard deviation of three biological replicates. (B) and (C) TLC analysis of XUXX incubated with soluble (B) or wall-bound (C) protein. XUXX, a xylotetraose carrying MeGlcA at penultimate xylose from the nonreducing end; C, water blank; M1, marker for XUXX and MeGlcA; M2, marker for xylose (Xyl), xylobiose (Xyl2), xylotriose (Xyl3), and xylotetraose (Xyl4).
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Fig4: Analysis of ScAGU115 enzyme activity in Arabidopsis transgenic and WT plants. (A) The soluble and wall-bound proteins were analyzed for α-glucuronidase activity using a commercial assay. The error bar represents the standard deviation of three biological replicates. (B) and (C) TLC analysis of XUXX incubated with soluble (B) or wall-bound (C) protein. XUXX, a xylotetraose carrying MeGlcA at penultimate xylose from the nonreducing end; C, water blank; M1, marker for XUXX and MeGlcA; M2, marker for xylose (Xyl), xylobiose (Xyl2), xylotriose (Xyl3), and xylotetraose (Xyl4).

Mentions: To test if the expressed protein was active, α-glucuronidase activity was determined from the soluble proteins extracted from the inflorescence stems. The specific α-glucuronidase activity in the soluble proteins extracted from lines 4, 5, and 10 was about five to six fold higher than the background WT level, whereas no activity above the WT level was detected in line 6 (Figure 4A). Activity was also detected in wall-bound proteins, at an even higher level than in the soluble fractions (Figure 4A), confirming that there were significant amounts of wall-bound ScAGU115 enzyme in the transgenic lines.Figure 4


Active fungal GH115 α-glucuronidase produced in Arabidopsis thaliana affects only the UX1-reactive glucuronate decorations on native glucuronoxylans.

Chong SL, Derba-Maceluch M, Koutaniemi S, Gómez LD, McQueen-Mason SJ, Tenkanen M, Mellerowicz EJ - BMC Biotechnol. (2015)

Analysis of ScAGU115 enzyme activity in Arabidopsis transgenic and WT plants. (A) The soluble and wall-bound proteins were analyzed for α-glucuronidase activity using a commercial assay. The error bar represents the standard deviation of three biological replicates. (B) and (C) TLC analysis of XUXX incubated with soluble (B) or wall-bound (C) protein. XUXX, a xylotetraose carrying MeGlcA at penultimate xylose from the nonreducing end; C, water blank; M1, marker for XUXX and MeGlcA; M2, marker for xylose (Xyl), xylobiose (Xyl2), xylotriose (Xyl3), and xylotetraose (Xyl4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4472178&req=5

Fig4: Analysis of ScAGU115 enzyme activity in Arabidopsis transgenic and WT plants. (A) The soluble and wall-bound proteins were analyzed for α-glucuronidase activity using a commercial assay. The error bar represents the standard deviation of three biological replicates. (B) and (C) TLC analysis of XUXX incubated with soluble (B) or wall-bound (C) protein. XUXX, a xylotetraose carrying MeGlcA at penultimate xylose from the nonreducing end; C, water blank; M1, marker for XUXX and MeGlcA; M2, marker for xylose (Xyl), xylobiose (Xyl2), xylotriose (Xyl3), and xylotetraose (Xyl4).
Mentions: To test if the expressed protein was active, α-glucuronidase activity was determined from the soluble proteins extracted from the inflorescence stems. The specific α-glucuronidase activity in the soluble proteins extracted from lines 4, 5, and 10 was about five to six fold higher than the background WT level, whereas no activity above the WT level was detected in line 6 (Figure 4A). Activity was also detected in wall-bound proteins, at an even higher level than in the soluble fractions (Figure 4A), confirming that there were significant amounts of wall-bound ScAGU115 enzyme in the transgenic lines.Figure 4

Bottom Line: The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2.Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions.Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, University of Helsinki, P.O. Box 27, Helsinki, 00014, Finland. sun-li.chong@helsinki.fi.

ABSTRACT

Background: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2.

Results: The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta.

Conclusions: Active GH115 α-glucuronidase has been produced for the first time in plants. The cell wall-targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.

No MeSH data available.