Carbohydrate Metabolism
Carbohydrates are the major source of energy for all living beings. Only green plants have the capability of carbon assimilation from the atmosphere through photosynthesis. The different types of carbohydrates present in the plants are: Monosaccharides Triose-D-glyceraldehyde, Dihydroxyacetone Tetrose- erythrose Pentose- ribose, deoxyribose, xylose, arabinose, ribulose, xylose Hexoses-glucose, fructose, mannose, galactose Heptoses-sedoheptulose Oligosaccharides Diasaccharides- maltose, cellobiose, sucrose Trisaccharides- Raffinose, gentianose Polysaccharides Structural polysaccharides- cellulose, hemicelluloses (xylans, mannans, glucomannans, galactans and arbinoglactans), pectins (galactans,arabinan, galacturonan) and chitin Storage polysaccharides-Starch (amylose and amylopectin), inulin Carbohydrates have several roles in living organisms beside energy transportation, structural, and storage. In plants more than 30% of carbohydrates are used in cell wall synthesis. Carbohydrates are actively involved in fertilization, immune system as well as protecting membranes of the cell. Metabolite Pool and exchange Metabolism is based on enzyme-catalyzed reactions or pathways which do not operate in isolation. Many metabolic pathways are interconnected and share intermediates. These intermediates are maintained at equilibrium with each other by reversible enzymatic reactions. In a cell there are ‘pools’ of different metabolites from where these can be withdrawn or added whenever required thus maintaining almost constant availability of these metabolite. “Metabolite pool” occurs in the subcellular compartments, such as plastid and cytosol. Interchange of the metabolites occurs through cell membrane.
Fig. Carbohydrate metabolism: An overview of the major pathways and organelles involved. TPT triose phosphate transporter, GPT glucose phosphate transporter
Fig: During daytime, occurrence of photosynthesis in chloroplasts adds to hexose monophosphate pool which is used for synthesis of transitory starch. During nighttime, carbon skeleton stored as starch is mobilized and translocated out of chloroplasts to other parts of plants, either to be used as the substrate for respiration or for storage in non-green plastids of storage organs or is translocated to be used as the source for respiration. Intercellular transport occurs in the form of sucrose
Three different pools of carbohydrates metabolites are
Hexose monophosphate pool-Hexose monophosphate pool has hexose monophosphate, glucose 1-phosphate, glucose 6-phosphate and fructose 6-phosphate. All these are interconvertible, and the enzymes required are phosphoglucomutase, glucose 6-phosphate isomerase Triose phosphate pool- this consist of interconvertible forms of triose, glyceraldehyde 3- phosphate and dihydroxyacetone phosphate, which are catalyzed by triose phosphate isomerase. Pentose phosphate pool- this include ribose 5-phosphate, ribulose 5-phosphate and xylulose-5-phosphate and the enzyme involved in interconversion is transketolase.
Fig. Hexose phosphate, triose phosphate, and pentose phosphate pools and exchange of intermediates in between these pools (both in cytosol and in plastid) Carbon compounds are added in these pools which are produced during photosynthesis or other metabolic pathways. Gluconeogenesis is also responsible for adding metabolite in these pools,
Fig: Metabolite pools involved in carbohydrate metabolism. Duplicate pathways are present in cytosol, chloroplast, and non-green plastid
Transport of metabolites Only some metabolite whose transporters are present on the membrane can be translocated. Two main transporters are Triose phosphate translocator (TPT): present on inner plastid membrane, antiporter type, responsible for exchange of triose phosphate (formed by calvin cycle) with inorganic phosphate between plastid and cytosol. If inorganic phosphate is absent then no exchange takes place. This can transport glucose and maltose but phosphorylated form of glucose and maltose cannot be translocated. Glucose 6-phosphate/phosphate translocator (GPT): is present and is localized in the inner envelop of the plastid. Transport of glucose 6-phosphate in exchange of inorganic phosphate takes place through GPT. It can also bring about exchange of triose phosphate and xylulose 5-phosphate with cytosolic inorganic phosphate. There is a direct correlation in between the metabolite pools of cytosol and plastids, which involves lot of exchange of sugars between these compartments. Since carbohydrates in plants are primarily transported in the form of sucrose and are stored as starch, study involving metabolism of sucrose and starch, both their synthesis and catabolism, is significant.
Sucrose Metabolism
Sucrose is the major form of carbohydrates which is translocated from source to sink in sieve elements of plants. Characteristic features of sucrose It is the most ubiquitous and abundant disaccharide (α-D-glucopyranosyl-β-D- fructofuranoside) in plant tissues. Two monosaccharides (α-D-glucopyranose and β-D-fructofuranose) by joining C-1 of α- D-glucose to C-2 of β-D-fructose by 1α→2β glycosidic bond forms sucrose. It is a non reducing sugar since glycosidic bond joins the carbonyl carbons of glucose and fructose. Fructose protects potentially reactive groups from oxidation and from non-specific enzyme attack making it structurally stable. As it stable, highly soluble, and relatively inert molecule, it becomes quite suitable for transport (other forms of carbohydrates such as raffinose, verbascose, stachyose (in members of family Cucurbitaceae) and sorbitol (in many plants of family Rosaceae) are also translocated). In some plants, e.g., beetroot (Beta vulgaris), sugarcane (Saccharum sp.), and carrot (Daucus carota), sucrose can also be stored. It only has osmotic effect in a cell and does not affect other biological processes. Biosynthesis
Sucrose synthesis occurs only in plants and bacteria. In plants, sucrose is synthesized mainly in cytosol of photosynthetic tissues or in germinating seeds. In mesophyll cells, source for triose phosphates is the Calvin cycle which occurs in chloroplasts. Triose phosphate is precursor of sucrose synthesis
Fig. Synthesis of sucrose in a mesophyll cell. Glucose is activated by UDP to form UDP- glucose. Activated glucose is transferred to fructose 6-phosphate resulting in synthesis of sucrose phosphate. The two reactions catalyzed by fructose 1,6-biphosphatase and sucrose- phosphate phosphatase are irreversible. These two reactions make sucrose synthesis irreversible
Fig. Synthesis of sucrose: Activation of glucose occurs by transferring glucose molecules from glucose 1-phosphate to UTP resulting in synthesis of UDP-glucose. Reaction is catalyzed by UDP-glucose pyrophosphorylase. The reaction is coupled with release of PPi (pyrophosphate) and becomes irreversible due to hydrolysis of sucrose-phosphate to sucrose. Activated glucose is transferred from UDP-glucose to fructose 6-phosphate resulting in synthesis of sucrose 6-phosphate (phosphate is linked to 6-C of fructose) in a reversible reaction catalyzed by the enzyme sucrose-phosphate synthase. Sucrose-phosphate phosphatase catalyzes hydrolysis of sucrose-phosphate resulting in formation of sucrose and release in Pi. This is an exothermic reaction coupled with °′ −1 release of energy (ΔG = −18.4 kJ.mol ). Release of Pi is necessary for sucrose biosynthesis because it makes the reactions irreversible. Additionally, recycling of Pi to chloroplast is essential.
Both allosteric modulation and covalent modulation of the enzyme protein due to phosphorylation regulates SPS activity. SPS is allosterically modulated by glucose 6-phosphate and Pi. Glucose 6- phosphate activates the enzyme, while its activity is inhibited by Pi. An increase in cytosolic hexose monophosphates is coupled with reduction in Pi, thus activating SPS.
Sucrose Catabolism Sucrose is the major form of carbohydrate that is transported in plants from source (e.g., photosynthetic tissues) to sinks (non-photosynthetic tissues). In a cell, sucrose is generally stored in vacuoles. Larger size of vacuoles is advantageous for storage. Whenever cell requires more energy it withdraws sucrose from vacuole. Sucrose may be transported in apoplastic or symplastic way, depending upon the type of the plant or the tissues involved. Enzymes involved in sugar catabolism are invertase and sucrose synthase (SS). Sucrose synthase catalyzes reversible reaction and thus is responsible for synthesis of sucrose also. SS is more significant during catabolism of sucrose, either when it is used as an energy source or is metabolized for starch biosynthesis. Invertase is hydrolytic enzyme, which hydrolyzes sucrose to glucose and fructose. Different isoforms of invertases
Apoplastic Vacuolar Cytosolic Acidic pH-5 Acidic pH-5 Alkaline pH-7.5
Present in apoplast Conc. is high where sucrose is either consumed as an hexoses are energy source after being hydrolyzed accumulated e.g.fruits, by cytosolic invertase or is meristems transported across tonoplast to be stored in the vacuole. It has significant cell expansion, sugar Cytosolic invertases are important role when sucrose storage, and in for plant growth, especially roots. transport is regulation of cold- apoplastic. induced sweetening.
Sucrose synthase Invertase It utilizes UDP for catalyzing sucrose Hydrolysis of sucrose by invertase yields glucose breakdown, releasing UDP-glucose and and fructose and then phosphorylated by ATP fructose. UDP-glucose is converted to glucose 1-phosphate by the enzyme UDP- glucose pyrophosphorylase. During the reaction enzyme use PPi and UTP is synthesized. i.e. ATP independent During cellulose and callose synthesis SS Invertase activity is zero where starch is to activity is higher e.g. potato tuber synthesized. E.g. potato tuber It is bound to membrane Different isoforms are present in cytosol, vacuole and apoplast
Fig: Sucrose catabolism in sink cell by sucrose synthase and invertase
Starch Metabolism
Starch is the dominant form of carbohydrates which is used by almost half of world’s population as the energy source. Starch is a polysaccharide, consisting of α-D-glucose molecules, which are linked through α-1,4-glycosidic linkages. These linkages protect aldehyde groups of glucose molecules against oxidation, except that of terminal glucose molecule, which is unprotected and is called reducing end of the molecule. Additionally, α-1,6-glycosidic linkages are also present, which add to branching of the molecule.
Amylose is a homopolysaccharide consisting of α-D-glucose which are joined by α-1-4- glycosidic linkages to make it a linear polymer. Occasional branching after almost 1000 glucosyl residues occurs due to the presence of α-1-6-glycosidic linkages. Due to the presence of extensive –OH groups, there are hydrogen bonds which makes the molecule to form helical ring. Ring of the molecule is planar; however, binding of C-O linkages of glycosidic bonds results in single helical formation with six glycosyl residues in each helix In amylopectin, α-D glucosyl residues are joined by α-(1-4)-glycosidic bonds similar to those in amylose. However, unlike amylase, amylopectin is a highly branched polymer because of the presence of α-(1-6)-glycosidic linkages after every 20–26 glucosyl residues. Hydrogen bonds are formed in between hydroxyl groups of adjacent chains as a result of which double helical structure is formed in amylopectin either in between adjacent branches of the molecule itself or with amylose polymer
Starch Biosynthesis Plants synthesize starch in plastids. In addition to chloroplasts (synthesis of transient starch), starch is also synthesized and stored in amyloplasts of storage organs of the plant, including in grains and tubers. Basic mechanism of starch synthesis, in chloroplasts and amyloplasts, is similar. There is a hexose monophosphate pool also in plastids. Fructose 6-phosphate is converted to glucose 6-phosphate which is further converted to glucose 1-phosphate catalyzed by phosphohexoisomerase and phosphoglucomutase, respectively. Mutant for any of these enzymes will result in impaired starch synthesis. The first committed step-in starch biosynthesis is catalyzed by ADP-glucose pyrophosphorylase (AGPase), which includes activation of glucose to ADP-glucose. Pyrophosphate released during the reaction is immediately hydrolyzed to produce two molecules of Pi, which ensures irreversibility of the reaction. Glucose 1−phosphate+ATP→ADP−glucose+PPi
PPi+H2O→2Pi
Fig. Starch synthesis catalyzed by starch synthase. Glucose from ADP-glucose is added to C-4 (nonreducing end of starch primer) catalyzed by starch synthase
Fig. Starch synthesis in amyloplasts of heterotrophic tissues. In cereal grain endosperm, ADP- glucose produced in cytosol is transported to plastids (1). In non-cereal storage organs, glucose 1- phosphate (G1P) and glucose 6-phosphate (G6P) are transported across plastid membrane. Transporters are present in the inner envelope of plastids Two enzymes for starch biosynthesis are ADP-glucose pyrophosphorylase is a heterotetrameric (α2β2) protein, consisting of small and large subunits. These are encoded by two different nuclear genes. Catalytic activity is present in small subunits, while large subunits are believed to have regulatory role. Activity of AGPase is allosterically regulated by the key metabolites of carbon assimilation. PGA is an activator of the enzyme, while Pi is the inhibitor. starch synthase (SS), which catalyzes transfer of glucose from ADP-glucose to preexisting starch primer as amylose or amylopectin. Glucose is added to the C-4 of the glucosyl residue at the nonreducing end of starch. ADP−glucose+α−glucann→α−glucann+1+ADP
Starch synthase remains bound with the growing starch granule. Five isoforms of starch synthase encoded by five genes have been identified, i.e., GBSS (granule-bound starch synthase), SSI, SSII, SSIII, and SSIV. GBSS is tightly associated with starch granule and is responsible for synthesis for amylose SS isoforms are the soluble forms, which are partly bound with starch granules and are located in the plastid stroma.
Fig. Pathway for starch biosynthesis. AGPase ADP-glucose pyrophosphorylase, GBSS granule- bound starch synthase, SSS soluble starch synthase, SBE starch branching enzyme Starch branching enzyme (SBE) is responsible for introducing the branches in the molecule.
Transitory Starch Transitory starch refers to temporary storage of reduced carbon in the form of starch. It is produced in chloroplasts and generally does not last for more than 24 h. Fate of intermediates of Calvin cycle is determined by ratio of PGA and Pi in stroma of chloroplasts. Glucose cannot be stored since it adds to osmotic concentration, and too much glucose accumulation will lead to lowering of water potential of plastids resulting in water uptake leading to their bursting. Starch is osmotically inactive and can be accumulated. Transient starch biosynthesis acts as buffer.
Fig. Synthesis of transitory starch in chloroplast. Two irreversible reactions are responsible for synthesis of starch, which are catalyzed by fructose 1,6-biphosphatase and by ADP-glucose pyrophosphorylase. Pyrophosphate (PPi) hydrolysis by pyrophosphatase makes the reaction irreversible. ADP-glucose pyrophosphorylase (AGPase) catalyzes the synthesis of ADP- glucose, which is a precursor for starch synthesis. AGPase is sensitive to the ratio of PGA/Pi with PGP promoting the activity of enzyme and Pi inhibiting the activity. High rate of ATP synthesis during light reaction results in shortage of Pi in plastids, thereby increasing PGA/Pi ratio. During daytime starch synthesis is favored in plastids which is stored as transient starch. This results in decreased export of triose-P. Decrease in synthesis of sucrose in cytosol also results in decreased release of Pi, and it is not imported into plastid which results in decreasing the ratio of PGA/Pi. This promotes synthesis of starch in the plastids catalyzed by fructose 1,6-biphosphatase (1), phosphohexoisomerase (2), phosphoglucomutase (3), ADP- glucose pyrophosphorylase (4), pyrophosphatase (5), starch synthase (6), and starch branching enzyme (7)
Starch Catabolism
Starch, whether stored temporarily in chloroplasts (transient starch) or stored in amyloplasts of cereal grain or tubers (storages starch), needs to be utilized either as a source of energy or as a source for carbon skeleton. Degradation pathways are different in different organs, and there are distinct pathways which operate within the same organ. Range of enzymes with many isoforms are involved in degradation of starch along with the related glucans which vary from tissue to tissue. The large polymeric molecule is cleaved into small monomeric molecules, glucose by either of the two classes of enzymes, phosphorolytic enzymes or hydrolytic enzymes.
Phosphorolytic Starch Degradation
The enzymes, which use Pi to break the glycosidic bonds, are called phosphorolytic enzymes. At least three categories of enzymes are involved in phosphorolytic degradation of starch. This includes starch phosphorylase, debranching enzyme, and glucosyltransferase. Starch phosphorylase cleaves one glucose molecule from the nonreducing end of the polysaccharide as glucose 1-phosphate. Unlike the hydrolytic enzymes, amylases, during phosphorolytic degradation of starch, energy of glycosidic bond is conserved as the phosphate esters. In further mobilization, one ATP is consumed less, thus saving one ATP per molecule of glucose. Reaction catalyzed by starch phosphorylase is as follows: α−(Glucan)n+Pi→α(Glucan)n−1+Glucose 1−phosphate
Fig. Phosphorolytic starch breakdown Hydrolytic Starch Degradation Enzymes which use water to break glycosidic bonds are called hydrolytic enzymes. Amylases carry out hydrolytic degradation of glycosidic bonds. There are different amylases which act at different sites of starch. Amylases, which hydrolyze starch at the end of the starch molecule, are called exoamylases. The other category of amylases includes endoamylases, which hydrolyze starch at the interior of starch molecule. β-amylases belong to exoamylase category, which split off two glucose molecules from the nonreducing end of starch polymer as maltose.
Fig. Starch polymers amylose/amylopectin is hydrolyzed by α-amylase and β-amylase enzymes to straight-chain and branched-chain oligosaccharides. Limit dextrins (pullulanase) act on the chains (each with two glucosyl residues) linked by α-1,6-glycosidic linkages, while isoamylase also acts on α-1,6-glycosidic linkages of branched oligosaccharides with large molecular weight
.
Summary Carbohydrates are the primary source of energy for most of the living beings including plants. In plants these are stored primarily in the form of starch, since it is the inert form, and it does not influence the osmotic status of the organelle in storage tissue. Starch is stored in plastids. In chloroplast, starch is temporarily stored during daytime (transitory starch) which is mobilized during night. Starch is also stored in amyloplasts of grains, as well as of tubers and roots of some plants. Both plastidial and cytosolic hexose monophosphate pools, consisting of glucose 1- phosphate, glucose 6-phosphate, and fructose 6-phosphate, contribute to carbohydrate metabolism. Sucrose is synthesized in cytosol of the cell, using triose phosphates. Export of triose phosphates from plastids is facilitated by the antiporters, localized in the inner envelope of the organelle. Triose phosphates from plastids are exchanged with cytosolic inorganic phosphate (P i). Sucrose synthesis requires activation of glucose as UDP-glucose, which involves UDP-glucose pyrophosphorylase. Sucrose-phosphate synthase results in synthesis of sucrose phosphate, which is hydrolyzed by sucrose phosphatase to sucrose, with simultaneous release of Pi. This step-in sucrose synthesis makes the reaction irreversible. Sucrose is mainly hydrolyzed by sucrose synthase and invertase. Three isoforms of invertase are significant, apoplastic and vacuolar invertase, which function at acidic pH, while pH optima of cytosolic invertase are neutral or alkaline. Starch is synthesized in the plastids of storage organs, as well in chloroplasts (during day). Starch, which includes both amylose (linear polysaccharide) and amylopectin (branched polysaccharide), generally is present in semicrystalline granule form. In most cases ratio of amylose and amylopectin is 1:3; however, the ratio may vary in starches from different sources. Starch synthesis requires activated form of glucose as ADP-glucose. Synthesis of ADP-glucose is catalyzed by ADP-glucose pyrophosphorylase. This step is the dedicated step-in starch biosynthesis. Glucose as ADP-glucose is added to the nonreducing end of a starch primer. Reaction is catalyzed by starch synthase. Addition of branching to the polymer is catalyzed by branching enzyme. Excess branching of the molecule is removed by disproportion enzyme also known as D-enzyme. Starch is degraded both by phosphorolytic and hydrolytic enzymes. Phosphorolytic cleavage requires starch phosphorylases and debranching enzyme. α-amylase, β-amylases, debranching enzyme, and glucosidase are hydrolytic enzymes.
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