Title

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

Authors

Authors

D. Verma; S. X. Jin; A. Kanagaraj; N. D. Singh; J. Daniel; P. E. Kolattukudy; M. Miller;H. Daniell

Comments

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Abbreviated Journal Title

PLoS One

Keywords

PLANT-CELL WALL; ECTOPIC EXPRESSION; TRANSGENIC PLANTS; THYLAKOID; LIPIDS; PHOTOSYSTEM-II; ARABIDOPSIS; EXPANSINS; MEMBRANES; PROTEIN; BIOMASS; Multidisciplinary Sciences

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.

Journal Title

Plos One

Volume

8

Issue/Number

2

Publication Date

1-1-2013

Document Type

Article

Language

English

First Page

13

WOS Identifier

WOS:000316849500077

ISSN

1932-6203

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