Winkled pea mutant deficient in a starch branching enzyme Waxy mutant in maize: amylopectin replaces amylose due to deficient in granule bound starch synthase
Wild type potato Transgenic potato with a bacterial ADPG pyrophosphorylase not subject to feedback inhibition Iodine stained potato slices
Shrunken maize mutant: deficient in sucrose synthase Sucrose Glucose + Fructose Invertase Sucrose synthase Fructose + UDPG UTP Synthesis of starch or cellulose Sucrose-P synthase
Starch degradation during cereal grain germination
Cell walls determine the shape and size of a cell. Protoplasts are always spherical, but walled cells are different in shape and size.
Involvement of cell walls in cell differentiation Shape and size of cells Aging and senescence Vascular system formation Fiber formation Abscission formation Fruit ripening Defense against pathogens and predators Sensing changes in the environment
Layers of walls: middle lamella, primary walls and secondary walls A new wall begins in the “phragomosome”/”phragmoplast” in a dividing cell.
From sugar to polysaccharides: Glucose (sugar) -------> glucan (such as (1-->4)glucan, cellulose) Mannose -------> mannan Glactose -------> galactan Xylose -------> xylan Xylose and glucose -------> xyloglucan (hemicellulose) Glactose ----> oxidized to glacturonic acid ------> galacturonan Techniques used to determine the structure of complexpolysaccharides: 1. Gas-liquid chromatography (GLC)--composition 2. Mass spectrometry--structure 3. Nuclear magnetic resonance---interacting groups 4. Electrone microscopy---”seeing is believing”
Cellulose fibrils in the primary cell wall are oriented perpendicular to the main axis of the cell. H-bondings between the cellulose fibrils provide the strength of the wall.
Cellulose microfibrils are assembled at the surface of plasms memberane. Cellulose synthase “rosettes” in action!
Cellulose synthase First isolated from certain bacteria (Acetobactor xylinum and Agrobacterium tumefaciens) mutants--CesA genes Apparent homologs of CesA have been isolated from cotton, Arabidopsis, etc.
Thickness of walls remains the same when a cell expands
Biological sources of cell wall degrading enzymes Rice straw composts Guts of herbivorous animals Guts of wood/grass consuming insects Guts of grass consuming fishes and aquatic organisms
Integrative Processes Gene Cloning Microbes Isolation Composts Microbes from ATCC etc. Data Bases Recombinant Protein Characterization Transformation into energy crops Gene Selection Protein Production Environments Metagenomic Libraries Proteomics Protein Engineering Protein isolation, purification Genomic libraries Enzymatic treatments by adding to feedstock In planta deconstruction & bioconversion
SDS-PAGE zymogram of endoglucanse activities from Geobaccillus spp. Substrate: 0.1% CMC Cellulases: endoglucanases, exoglucanases (such as CBH), -glucosidases
2-D gel zymogram detection for endoglucanse activities Substrate: 0.4% CMC
Cloning of endoglucanase genes via activity staining Geobaccillus spp. Geobaccillus thermodentrificans
Endoglucanase activities in gut tissues of Taiwanese grasshopper
SDS-PAGE zymogram of xylanase from Geobaccillus spp. Substrate: Birch wood xylan
Xalanase activities in gut tissues of Taiwanese grasshopper
Laccase activities in Basdiomyctous fungi 1,4 benzenediol + O 2 ------> quinone + H 2 O (Laccase is a ligninase)
Detection of laccase activity on SDS-PAGE Source: Pycnoporus cinnabarinus
Take home messages Lignocellulosic materials are virtually the cell walls of plants Plant cell walls are complicated interlocking polymers of cellulose, hemicellulose, lignins, pectins and proteins A combination of enzymes are needed to “deconstruct” the cell wall complex Much effort is needed to search for novel cell wall degrading enzymes/microbes
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