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.
Highlights
Lignocellulosic biomass is a valuable bioethanol production source for substitution of established fuel supply
Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis
MGDG on its own cannot make lamellar membranes whereas along with DGDG, proteins and xanthophylls, lamellar membranes are formed with intrinsic curvature stress [7]
Summary
Lignocellulosic biomass is a valuable bioethanol production source for substitution of established fuel supply. Photosynthetic energy transduction and the generation of plant biomass rely on the thylakoid membranes inside chloroplasts. Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) constitute more than 80% of total galactolipids in thylakoid membranes. MGDG and DGDG are important for all oxygenic photosynthetic organisms. These two galactolipids of thylakoid membrane are most abundant lipids in the biosphere [5,6]. Hydrogen bond formation between the galactolipid head groups of MGDG and DGDG is important for these lipids to associate with photosystem I (PSI) and photosystem II (PSII). An optimal lipid-protein ratio is maintained which is crucial for development, maturation process, fluidity and integrity of thylakoid membrane [8,9,10,11]
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