1BCMB 8020 April 11,2006StarchMost significant carbon reserve in plants (i.e. amount made& distribution) Major component of crop plant yield in the worldMajor source of calories in the human dietImportant raw material for industrial processes (e.g. potato & maize)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_place/biocoach/images/plants/plant.gifPlant Cell 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.ohio-state.edu/hcs300/jpeg/THYLAK.JPG2Summary of the Calvin cycle (the reductive pentose phosphate cycle) of photosynthesisFig. 15.16 from Horton et al., 2006, Prentice HallPhotosynthesis, storage starch, sucroseAccumulation and breakdown of starch in Brassicaripening seeds 16 dpp24 dppStarch exists as starch granules (<1 to >100 µM diameter)Glucose polymer arranged in 3D semicrystalline structureStarch granules in leaves and storage organs differ in their macro structure. Structural studies of starch are made on storage organstarch (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).starch granulehttp://mse.iastate.edu/images/microscopy/bot7.jpg3Starch is made up of two types of polymers: amylose and amylopectinAmylose is a linear homopolymer of α1,4-linked glucose with a DP of ~1000Amylose may have a low level of branching (~one branch per 1000 residues) with an α1,6-linkage. Amylose makes up ~35% of starch (range of 11-36% depending on plant and organ). In solution amylose forms hydrogen bound with other amylosemolecules to yield rigid gels.Amylopectin is highly branched form of “amylose”. The linear α1,4-linked glucose backbone is branched at every ~20 residues by an α1,6-linkage which is extended by α1,4-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).AmylopectinAmylose helixAmylose forms complexes with iodine, butanol & nitro compounds: designated V amyloseV amylose in the crystalline state is a left-handed helix of 13 Å diameter, pitch of 8 Å, and 6 residues per turn (i.e. n = 6; h (rise) = 1.33 Å). Thus, it is a 6(-1.33) helix.V amylose helicesAmylopectin 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 crystals of Starch A and B are left-handed helices of 6(-3.55) and 6(-3.47) respectively.4Amylopectin Starch AAmylopectin Starch BBiosynthesis of starch in the chloroplastFig. 15.16 from Horton et al., 2006, Prentice HallThree 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 + PPiADPglucose pyrophosphorylase (ADPGPPase)ADP-Glc + amylose(n) → ADP + amylose(n+1)starch synthase (SS)Amylose + Amylose → amylopectin + amylosestarch branching enzyme (SBE)Starch debranching enzyme (isoamylase; glycogen 6-glucanohydrolase)All species studied have multiple isoforms for each of the starch biosynthetic enzymes. Multiple genes also exist.5Glucose-1-P + ATP → ADP-Glc + PPiADPglucose 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 3-phosphoglycerate and (-) regulated by PO4-. ADPGPPase is a heterotetramer of 2 large (54-60KD) and 2small (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)ADP-Glc + amylose(n) → ADP + amylose(n+1)starch synthase (SS)Starch Synthase catalyzes α1,4-linkage between non-reducing 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.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 amylopectinSoluble SS mutant: Chlamydomonas STA3: mutant has more short chains (DP 2-7) and fewer long chainsSSIII also present in some plants/tissuesSS isoforms have conserved C-terminal region of ~60kD similar to glycogen synthase from bacteria6Amylose + Amylose → amylopectin + amylosestarch branching enzyme (SBE)SBE hydrolyzes α1,4-linkage in glucan chain in stable double helical conformation & catalyzes formation of α1,6-linkage between reducing end of “cut” chain and glc in another chain.At least 2 SBE gene families have been identified in maize, rice and pea. They share sequence similarity with bacterial glycogen-branching enzymes. Family A has lower