Expression of Fungal Cutinase and Swollenin in Tobacco Chloroplasts Reveals Novel Enzyme Functions and/or Substrates

Loading...
Thumbnail Image
Penn collection
Departmental Papers (Dental)
Degree type
Discipline
Subject
Dentistry
Funder
Grant number
License
Copyright date
Distributor
Related resources
Author
Verma, Dheeraj
Jin, Shuangxia
Kanagaraj, Anderson
Singh, Nameirakpam D.
Daniel, Jaiyanth
Kolattukudy, Pappachan E.
Miller, Michael
Daniell, Henry
Contributor
Abstract

In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.

Advisor
Date Range for Data Collection (Start Date)
Date Range for Data Collection (End Date)
Digital Object Identifier
Series name and number
Publication date
2013-02-25
Journal title
PLoS One
Volume number
Issue number
Publisher
Publisher DOI
Journal Issue
Comments
At the time of publication, author Henry Daniel was affiliated with the University of Central Florida. Currently, he is a faculty member at the School of Dental Medicine at the University of Pennsylvania.
Recommended citation
Collection