BCMB 8020 April 11 2006 Major source of calories in the human diet Starch Most significant carbon reserve in plants i e amount made distribution Major component of crop plant yield in the world Important raw material for industrial processes e g potato maize Plant Cell Starch is synthesized in plastids in leaves from photosynthate during the day assimilation starch a transitory reserve carbohydrate mobilized at night largely to storage organs seeds and fruits http www phschool com science biology p lace biocoach images plants plant gif Fig 1 15 b Principles of Biochemistry Fourth Edition Horton H R Moran L A Scrimgeour K G Perry M D Rawn J D 2006 Starch in storage organs seeds fruits tubers storage roots is synthesized in plastids amyloplasts from sucrose that is imported from the rest of the plant Plastid from young strawberry that is intermediate between a chloroplast presence of thylakoids and an amyoplast large starch grains http www hcs ohiostate edu hcs300 jpeg THYLAK JPG 1 Summary of the Calvin cycle the reductive pentose phosphate cycle of photosynthesis Photosynthesis storage starch sucrose Fig 15 16 from Horton et al 2006 Prentice Hall 16 dpp Accumulation and breakdown of starch in Brassica ripening seeds 24 dpp Starch exists as starch granules 1 to 100 M diameter starch granule Glucose polymer arranged in 3D semicrystalline structure Starch granules in leaves and storage organs differ in their macro structure Structural studies of starch are made on storage organ starch i e not transient starch Once extracted H bonds form between linear regions of starch amylose to yield rigid gels concentration DP and temperature dependent http mse iastate edu images microscopy bot7 jpg 2 Starch is made up of two types of polymers amylose and amylopectin Amylose is a linear homopolymer of 1 4 linked glucose with a DP of 1000 Amylose may have a low level of branching one branch per 1000 residues with an 1 6 linkage Amylose makes up 35 of starch range of 11 36 depending on plant and organ In solution amylose forms hydrogen bound with other amylose molecules to yield rigid gels Amylopectin is highly branched form of amylose The linear 1 4 linked glucose backbone is branched at every 20 residues by an 1 6 linkage which is extended by 1 4 linked linkages A single amylopectin molecule has one reducing end contains branch clusters at every 7 10 nm is 15 nm wide and 200 400 nm long i e has 20 40 clusters Amylopectin Amylose forms complexes with iodine butanol nitro compounds designated V amylose Amylose helix V amylose in the crystalline state is a left handed helix of 13 diameter pitch of 8 and 6 residues per turn i e n 6 h rise 1 33 Thus it is a 6 1 33 helix Amylopectin has a regular clustering of double helices formed by twisting of short chains 11 13 glucans in Starch A cereals 16 18 glucans in Starch B tubers Structures based on electron diffraction data from V amylose helices crystals of Starch A and B are left handed helices of 6 3 55 and 6 3 47 respectively 3 Amylopectin Starch B Amylopectin Starch A Biosynthesis of starch in the chloroplast Fig 15 16 from Horton et al 2006 Prentice Hall Three enzymes are directly required for Starch Biosynthesis A fourth enzyme has been proposed to play a major role Biosynthesis occurs in the plastid Glucose 1 P ATP ADP Glc PPi ADPglucose pyrophosphorylase ADPGPPase ADP Glc amylose n ADP amylose n 1 starch synthase SS Amylose Amylose amylopectin amylose starch branching enzyme SBE Starch debranching enzyme isoamylase glycogen 6glucanohydrolase All species studied have multiple isoforms for each of the starch biosynthetic enzymes Multiple genes also exist 4 Glucose 1 P ATP ADP Glc PPi ADPglucose pyrophosphorylase ADPGPPase ADP Glc synthesized in the plastid although some ADPGPPase may also act cytosolically in some tissues plants e g maize barely endosperm ADPGPPase is allosterically regulated by 3phosphoglycerate and regulated by PO4 ADPGPPase is a heterotetramer of 2 large 54 60KD and 2 small 51 55 kd subunits cDNA s isolated for both subunits show sequence homologies suggest they arose from a common ancestor gene Multiple genes encode the large subunit differ in tissue specific expression and multiple genes can encode the small subunit Both subunits required for activity Small subunit thought to be main catalytic activity large subunit is regulatory although it also has activity Large subunit mutants maize shrunken2 sh2 endosperm pea rugosusb rb Small subunit mutants brittle2 bt2 endosperm GBSSI mutant waxy wx mutants in maize rice sorghum Amaranthus potato amylose free amf pea LAM mutant make little of no amylose Thus GBSSI probably important for amylose synthesis and in synthesis of long amylose like stretches of amylopectin SSII mutant pea RUG5 mutant has few chains of 15 45 residues but rather has many very short chains DP 15 and many very long chains Concluded that SSII elongates very short chains to create the chains that form the basis of the clusters in amylopectin ADP Glc amylose n ADP amylose n 1 starch synthase SS Starch Synthase catalyzes 1 4 linkage between nonreducing end of glucan chain Glc from ADP Glc SS can use both amylose and amylopectin as acceptors Priming event not known some evidence for protein primer some evidence for de novo synthesis Both granule bound SS GBSSI and soluble SS are found in amyloplasts GBSSI makes amylose in planta and has low activity in vitro GBSSII which may be granule bound or soluble has high activity in vivo Any plant species probably has several active soluble SS Soluble SS mutant Chlamydomonas STA3 mutant has more short chains DP 2 7 and fewer long chains SSIII also present in some plants tissues SS isoforms have conserved C terminal region of 60kD similar to glycogen synthase from bacteria 5 Amylose Amylose amylopectin amylose starch branching enzyme SBE SBE hydrolyzes 1 4 linkage in glucan chain in stable double helical conformation catalyzes formation of 1 6linkage between reducing end of cut chain and glc in another chain Structure of SBE have been modeled based on structure of amylase which has a central 8 barrel structure involved in hydrolysis Since the loop between strand 8 and helix 8 is similar in all Family A isoforms distinct from Family B isoforms in length sequence it is thought to be involved in determining branch length SBE Family A mutants pea rugosus r maize amylose extender ae At least 2 SBE gene families have been identified in maize rice and pea They share sequence similarity