STARCH-SUCROSE METABOLISM Dr.Roshni Rajamohan Department of Botany Deshbandhu College Carbohydrates

• Plants are autotrophs and synthesize carbohydrates through photosynthesis, then use them to create biological macromolecules. • Plant‐derived carbohydrates (e.g., cellulose) dominate the biosphere and serve as a key sink for atmospheric carbon dioxide (CO2). • The primary use of the captured energy is the fixation and reduction of CO2via the Calvin–Benson cycle.

Dr. Roshni Rajamohan, Deshbandhu College Carbohydrates

• Plants store carbs as – vacuolar sucrose or fructans -but the most common storage form is Starch, a polymer of Glucose - bacteria, animals and fungi- store as glycogen, much branched polymer of glucose

Starch – chloroplast synthesized during day in chloroplasts stroma in photosynthetic tissues stored temporarily until night degraded at night In storage tissues, such as tubers or seeds, starch is synthesized in amyloplasts and stored for much longer periods

Dr. Roshni Rajamohan, Deshbandhu College Three interconvertible hexose phosphates make up the hexose phosphate pool

Dr. Roshni Rajamohan, Deshbandhu College Triose PO4 pool to Hexose PO4 pool

• DHAP & 3-PGAL–Triose PO4 pool • Aldolase condensation • Hexose pool

Fr-1, 6- PO4 Fr-1, 6- Phosphatase

Fr- 6- PO4 Hexose Isomerase

Gl- 6- PO4 Phospho gluco -Mutase

Gl- 1 - PO4

Dr. Roshni Rajamohan, Deshbandhu College Starch synthesis

• Starch synthesis involves chain elongation, branching, and debranching reactions • The first committed step in the starch biosynthetic pathway is the production of ADP‐glucose by ADP‐glucose pyrophosphorylase

• Glucose-1-phosphate + ATP ------> ADP-glucose + Ppi (imported from cytosol)

• Three enzymes are responsible for the synthesis of starch from ADP‐glucose: 1. starch synthases 2. starch branching enzymes- produces the α‐1,6 branches in starch 3. starch debranching enzymes

Dr. Roshni Rajamohan, Deshbandhu College Transporters located in the inner envelope membranes of plastids exchange metabolites between the cytosol and the plastid stroma. In the transport processes illustrated here, photosynthetic chloroplasts export triose phosphate in exchange for inorganic phosphate (Pi); this is a major flux in photosynthesis. Transporters also exchange pentose phosphates and phosphoenolpyruvate for Pi, and nonphotosynthetic plastids also have transporters mediating the movement of hexose phosphates . Adenylate cofactors can also be transported across the chloroplast envelope, as can maltose and glucose (the products of starch breakdown) and intermediates involved in nitrogen assimilation. All the transporters are reversible, and the direction of transport depends on the concentrations of metabolites on each side of the envelope STARCH

• Osmotically inert form of carbohydrate –polymer of GLUCOSE • Massive, compact, stable, insoluble semi-crystalline granules • Starch granules contain 2 distinct glucose polymers- Amylose and Amylopectin both of which are homopolymers of α‐1,4‐linked glucose • Why starch? • If a comparable number of hexose units accumulated in the plastid as sucrose, the stromal solution would contain too many solute particles, and water from the cytosol would flood in by osmosis, causing the plastid to swell and burst

Dr. Roshni Rajamohan, Deshbandhu College Dr. Roshni Rajamohan, Deshbandhu College Dr. Roshni Rajamohan, Deshbandhu College COMPONENTS OF STARCH

Amylose Amylopectin • Branched molecule • a linear polymer of glucose • α-(1,4)- and α-(1,6) linkages • An α-(1,4)-glucan (branch points) • smaller than amylopectin • branching pattern is not random- • has very few branch points one branching point per 24 to 30 glucose residues • 1,000-20,000 glucose residues • 1,00,000-10,00,000 glucose • Accounts residues • 30% or less of the starch • 70% or more of the starch • similar to glycogen

Starch Synthesis • EnzymesADP-glucose pyrophosphorylase and starch synthase— are found localized in the chloroplast stroma. • Starch synthesis in the chloroplast begins with the hexose phosphate pool generated by the PCR cycle

Fructose-6-P < ------hexose-phosphate isomerase → Glucose -6-P

phosphoglucomutase Glucose -6-P < ------→ Glucose -1-P

ADP-glucose phosphorylase ATP + Glucose-1-P ------→ ADP-Glucose + H2O + PPi

PPi + H2O ↔ 2Pi

Starch synthase ADP-Glucose + α-(1→4)-glucan ------→ α-(1→4)-glucosyl-glucan + ADP

Dr. Roshni Rajamohan, Deshbandhu College Branching enzyme(Q Enzyme)- Give rise to → α-(1→6) branching of amylopectin

Debranching Enzymes- trims the side chains and make amylopectin ready for package into starch granule and provides primers (α-(1→4)-glucan ) for ADP-Glucose addition to their non-reducing end

The aldehyde group can react with (reduce) organic and inorganic substrates and is, therefore, described as the reducing end of the molecule. Eg.Glucose, Fructose

Non-reducing end Reducing end

ADP-Glucose +

During the day, carbon dioxide (CO2) is assimilated through photosynthesis. Some is used for growth, and some is stored. At night, stored carbohydrate is used to support respiration and continued growth. Sucrose biosynthesis

• Sucrose is a major product of photosynthesis in green leaves and accounts for much of the CO2fixed during photosynthesis. • It also serves as the principal long‐distance transport compound in most plants and as a storage compound in some [including sugar beet (Beta vulgaris), sugar cane (Saccharum sp.), and carrot (Daucus carota)].

Dr. Roshni Rajamohan, Deshbandhu College Fate of sucrose in sink tissues

Sucrose needs to be metabolized or stored Why ?

Fate of sucrose 1. It can be respired. 2. It can be stored as sucrose, starch or another carbohydrate, or converted to a lipid. 3. It can be used for growth and the synthesis of cellcomponents.

During sucrose synthesis, the anomeric carbon of fructose (C‐2) is joined to the anomeric carbon of glucose (C-1) by a glycosidic bond. This bond protects the reducing ends of both monomers

By joining the carbonyl carbons of glucose and fructose in a stable glycosidic bond, sucrose formation prevents these groups Sucrose is a nonreducing sugar from becoming oxidized through non enzymatic reactions with other cellular Glucose + Fructose = Sucrose components and best suitable as a transport molecule. • Uridine diphosphate‐glucose(UDP‐glucose is one of the two substrates required for sucrose synthesis (the other is fructose‐6‐phosphate)

UDP‐glucose pyrophosphorylase • Glucose-1-phosphate + UTP ------→ UDP-glucose + Ppi

PPi + H2O ↔ 2 Pi

Sucrose‐phosphate synthase • UDP-glucose +Fructose-6-phosphate<------> Sucrose-6-phosphate + UDP

Sucrose-phosphate phosphatase

• Sucrose-6-phosphate+ H2O ------> Sucrose + Pi

Dr. Roshni Rajamohan, Deshbandhu College Sucrose‐phosphate synthase UDP-glucose +Fructose-6-phosphate <------> Sucrose-6-phosphate + UDP

Sucrose‐phosphate synthase is subject to allosteric modulation by glucose 6‐phosphate--- which activates the

enzyme by Pi , which inhibits it.

Dr. Roshni Rajamohan, Deshbandhu College • The formation of sucrose by this route has a large negative free energy change (ΔGo′= –25 kJ mol–1), which renders this sequence of reactions is not spontaneous and is essentially irreversible in vivo. Sucrose synthase UDP-glucose + fructose ------→ Sucrose + UDP

• Under normal conditions SS operates in the reverse direction to

break down sucrose Sucrose synthase Sucrose + UDP ------→ Fructose + UDP−glucose

UDP−glucose pyrophosphorylase UDP-glucose + PPi ------→ UTP + glucose-1-P

ADP−glucose phosphorylase Glucose -1-P + ATP ------→ ADP-glucose + H2O+ PPi

Starch synthase ADP-Glucose + α-(1→4)-glucan ------→ α-(1→4)-glucosyl-glucan (STARCH) + ADP Dr. Roshni Rajamohan, Deshbandhu College SUCROSE DEGRADATION • Sucrose is transported through symplastic or apoplastic pathways to the sink tissues where it is broken down. • Sucrose can be degraded by either Sucrose synthase or Invertase

Sucrose synthase catalyzes a reversible reaction that can synthesize or degrade sucrose. In plant cells, this enzyme is associated primarily with sucrose degradation.

The reaction catalyzed by invertase is irreversible and so leads only to sucrose degradation.

Dr. Roshni Rajamohan, Deshbandhu College Hexokinase uses ATP to phosphorylate glucose and fructose, the products of invertase. Both enzymes often appear in the same tissues. In addition, Sucrose synthase and UDP‐glucose pyrophosphorylase both sucrose synthase and invertase occur as multiple act together to generate hexose phosphate by an isoenzymes, each of which probably functions in a specific ATP‐independent pathway tissue or cellular compartment. It is considered that sucrose synthase activity tends to be associated with the utilization of sucrose for the biosynthesis of non-structural carbohydrate polymers like starch, and of structural carbohydrate polymers like cellulose and callose Dr. Roshni Rajamohan, Deshbandhu College INVERTASE TYPES

• Invertases - acid invertases (acidic pH optima) or alkaline invertases (neutral or alkaline pH optima). • Operate in different cellular compartments: Acid invertases are present in both the vacuole and the apoplst Vacuolar invertase- hydrolyzes sucrose stored in the vacuole, with the subsequent transport of the free hexoses to the cytosol for metabolism. Eg., developing fruits, and in rapidly growing tissues, such as the elongation zones of roots and hypocotyls Apoplastic invertase - is bound firmly to the cell wall matrix and hydrolyzes apoplastic sucrose delivered to sink tissues. plant reproductive tissues. Repression of cell wall invertases interferes with pollen development, causing male sterility and decreased seed size in important crops like maize (Zea mays) and rice (Oryza sativa) Alkaline invertases are present in the cytosol and plastid. Cytosolic invertases are also important for plant growth, particularly roots

Plants also possess proteinaceous invertase inhibitors that interact with the acid invertases of the apoplast and the vacuole. These inhibitors likely repress invertase activity Dr. Roshni Rajamohan, Deshbandhu College