Plant biochemistry

Plant biochemistry involves the study of plant life at molecular level

1. MICHAEL ADU-FRIMPONG (PhD, [email protected] ) BCH 309 PLANT BIOCHEMISTRY

2. Course Content Carbohydrates: carbohydrate stores in germination of seeds; storage carbohydrates (starch, sucrose and other reserve carbohydrates); structural carbohydrates (cellulose, hemicellulose, pectin); the biosynthesis of carbohydrates. Lipids: germination of oil seeds, the glyoxalate pathway and gluconeogenesis; chemistry of plant lipids: cutins, suberins and waxes; fatty acid biosynthesis. Nitrogen metabolism: nitrogen fixation (dinitrogenase); nitrogen uptake and reduction . Secondary metabolites: Terpenes (the mevalonic acid pathway); phenolic compounds (the shikimic acid pathway); saponins, cardiac glycosides, cyanogenic glycosides and glucosinades and alkaloids; functions Medicinally important secondary metabolites (e.g., glycosides, alkaloids, saponins, flavonoids). The biochemical basis of action, extraction and characterization of bioactive compounds from plants . Photosynthesis: Chloroplast structure; photoreceptors and transduction of light into chemical energy (the photosynthesis electron transport chain); carbon fixation; the C3, C2 and C4 cycles; CAM metabolism . Molecular and biochemical regulation of plant metabolic pathways activated in response to environmental cues: environmental stress, and interaction with pathogenic and symbiotic organisms. Cell wall formation (primary wall, wood), secondary metabolism (lignin, flavonoids, phenolics), wounding, plant defenses (phytoalexins, oxidative burst, hypersensitivity), responses to drought, flooding, salinity, pollutants (heavy metals, ozones).

3. INTRODUCTION Definition What is plant biochemistry? 1. Biochemistry is the study of molecular basis of life (Stryer, 1975) 2. Biochemistry is the study of the way in which chemical components are synthesised and utilised by organism in the life process (Godwin & Mercer, 1990) Plant Biochemistry is the study of molecular basis of plant life that includes the synthesis and utilization of compounds in the life process of plants (growth & development)

4. BIOMOLECULES What are the types of molecules studied in Biochemistry? The principal types of biological molecules or biomolecules are: Carbohydrates Lipids Proteins Nucleic acids Many of these molecules are complex molecules called polymers which are made up of monomeric subunits Biochemical molecules are principally based on carbon

5. glucose cellulose cell wall fatty acid phospholipid membrane amino acid p rotein subunit p rotein complex nucleotide DNA chromosome carbohydrates lipids proteins nucleic acids monomer polymer supramolecular structure Many important biomolecules are Polymers

6. Carbohydrates Carbohydrates are the most abundant biomolecules on Earth Carbohydrates are essentially hydrates of carbon ((i.e. they are composed of carbon and water with a composition of (CH 2 O)n)) Carbohydrates are also referred to as polyhydroxy aldehydes or ketones and their derivatives They are found mainly in foods from plant sources, such as fruits , vegetables , grain products , dry beans , etc. They are synthesised in green plants through photosynthesis Carbohydrates can be simple or complex

7. Carbohydrate stores in the germination of seeds Carbohydrate stores in seeds are mainly in the form of starch (long polysaccharide chain composing of glucose) The stored starch in the endosperm/ cotyledon tissues of seed provides energy and materials for germination ( beginning of the growth of a seed into seedling ) Besides, starch function to aid the young embryo to develop during early germination or growth

8. Storage Carbohydrates Carbohydrates serve as  energy sources and as essential structural components in organisms Carbohydrate reserves constitute the major part of edible portion of the plants 1. Sucrose Sucrose is a reserve carbohydrate in sugar beets and sugarcanes It is considered nonreducing sugar because the anomeric carbons of both monosaccharides in this compound are tied up in glycosidic linkages .

9. Storage carbohydrates 1. Sucrose

10. Storage carbohydrate cont’d 2. Starch Starch is a homopolysaccharide of D glucose residues It is found in the cytoplasm/chloroplast of plant cells Starch is extensively hydrated because it has many exposed hydroxyl (OH) groups available to hydrogen-bond with water Starch is a mixture of amylose (10-30%) and amylopectin (70-90%) Amylose is a linear polymer of D glucose residues that are connected via (  1  4) linkages

11. Storage carbohydrate cont’d 2. Starch cont’d The molecular weights of amylose chains vary from a few thousand to more than a million . Long chains of amylose tend to coil into helix Amylopectin is a branched polymer of D glucose residues that can weigh up to 200 million Da The glycosidic linkages between D glucose residues in amylopectin chains are also (  1  4) Additionally, branch point linkages between D glucose units are (  1 6 ) linkages, which occur about every 24 to 30 residues

12. Storage carbohydrate cont’d 2. Starch cont’d

13. Example of other carbohydrates reserve Fructans About 15% of all flowering plant species store fructans, which are linear and branched polymers of fructose Fructans are classified as prebiotics because they selectively promote the growth of beneficial bacteria such as Lactobacilli and Bifidobacteria Fructans are synthesised by the action of two or “more different fructosyltransferases that utilise sucrose as fructose donor ( Vijn e smeekens , 1999) As carbohydrate reserve, fructans seem to be involved in the protection of plants during stress conditions such as drought, high salinity or cold . Examples of fructans are inulin and phleins (found in grasses)

14. Inulin Inulin is a heterogeneous group of fructose-based polymers used as storage carbohydrates by numerous plant species Inulin is fermented by bacteria that normalize the colon and is considered a prebiotic (they improve GIT health) Plants that store inulin include wheat , onion , banana , dandelion root and garlic ( Niness , 1999) Chemically, inulin consists of a chain of 35–50 1,2-linked fructofuranose units terminated by one glucose unit Inulin is not metabolized by the body and is excreted unchanged

15. Inulin benefits and structure

16. Structural Carbohydrates Cellulose Cellulose contains glucose units bonded by β(1 4) This glycosidic bond conformation changes the three dimensional shape of cellulose compared with that of amylose The chain of glucose units is straight . This allows the chains to align next to each other to form a strong rigid structure The β -glucose molecules are joined together by condensation , i.e., the removal of water, forming β -(1, 4)-glycosidic linkages

17. Structural Carbohydrates cont’d Cellulose cont’d The glucose units are linked into straight chains each (about 100-1000 units) Weak hydrogen bonds form between parallel chains bind them into cellulose microfibrils Cellulose microfibrils arrange themselves into thicker bundles called microfibrils (usually known as fibres ) Other compounds such as hemicelluloses and calcium pectate can “glue” the cellulose fibres together to form complex structures like plant cell walls

18. Structural Carbohydrates cont’d Cellulose cont’d Cellulose is an insoluble fibre in our diet . Humans lack the cellulase enzyme to hydrolyse the β(1 4) glycosidic bond Whole grains are a good source of cellulose Cellulose assists with the digestive movement of food in the small and large intestines Some insects and animals can digest cellulose because of the presence of bacteria that produce cellulase

19. Structural Carbohydrates cont’d Cellulose cont’d

20. Structural Carbohydrates cont’d 2. Hemicellulose Hemicellulose (also known as polyose) is a polysaccharide found in plant cell walls that have β -(1 4)-linked backbones with an equatorial configuration ( Scheller and Uluvskov , 2010) Hemicellulose is defined as those polysaccharides that can be extracted by alkaline solutions Examples include xyloglucans , xylans , mannans and glucomannans and β -(1 3,1 4)-glucans Hemicellulose mostly contributes to strengthening the cell wall by interacting with cellulose and, in some walls, with lignin .

21. Structural Carbohydrates cont’d 2. Hemicellulose cont’d Monomeric units of hemicellulose are D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methylglucuronic, D-galacturonic and D-glucuronic acid

22. Structural Carbohydrates cont’d 3. Pectin (soluble fibre) Pectin is a mixture of polymers from sugar acids, D-galacturonic acid , which are connected by α -1, 4 glycosidic linkages Some of the carboxyl groups are esterified by methyl groups ( this is used to assess the purity of pectin ) Pectin is derived from various plant sources such as lemon peel, orange peel, apple pomace, carrots, guava, mangoes and papaya, etc. They are found in the primary cell walls of all seed bearing plants and aids to bind cells together Assignment: Write short notes on the extraction , purification, health benefits, side effects and applications of pectins (in food, cosmetics and pharmaceutical industries)

23. Structure of pectin 3. Pectin cont’d

24. Carbohydrate biosynthesis in plants The synthesis of carbohydrates in animal cells always employs precursors having at least three carbons , all of which are less oxidized than carbon in CO 2 In contrast, plants can synthesize carbohydrates from CO 2  and H 2 O by reducing CO 2  using energy and reducing power supplied respectively by ATP and NADPH (generated during the light-dependent reactions of photosynthesis ) Plants can use  CO 2   as the sole source of the carbon atoms  required for the biosynthesis of cellulose, starch , lipids and proteins , as well as many other organic components of plant cells

25. Carbohydrate biosynthesis in plants cont’d Green plants contain in their chloroplasts unique enzymatic machinery that catalyses the conversion of CO 2 to simple (reduced) organic compounds through a process called CO 2 / carbon assimilation Carbohydrates are formed in green plants by photosynthesis , which is the chemical combination, or "fixation", of carbon dioxide and water via utilization of energy from the absorption of visible light . The overall result is the reduction of carbon dioxide to carbohydrate and the formation of oxygen

26. G ermination of oilseeds Germinating oilseeds break down fatty acids (obtain from TAG hydrolysis) through β -oxidation to acetyl CoA in peroxisomes ( glyoxysomes ) Carbons in fatty acids are converted into soluble carbohydrates through the glyoxylate cycle and gluconeogenesis Also, the glycerol released by the hydrolysis of triacylglycerol is phosphorylated to glycerol 3-phosphate and subsequently oxidised to dihydroxyacetone phosphate , which is fed into the gluconeogenesis Thus, the utilisation of lipid reserves in the endosperm requires gluconeogenesis and transport of the resulting sugars to the germinating embryo

27. Glyoxylate cycle The glyoxylate cycle is the metabolic process through which plants synthesize hexoses from acetyl CoA Animals are unable to synthesize glucose from acetyl CoA , but plants are capable of gluconeogenesis from lipids , because they possess the enzymes of the glyoxylate cycle Glyoxylate cycle is located in glyoxysomes like β -oxidation , thus the name of the cycle Two starting reactions of the cycle are identical to those of the citric acid/tricarboxylic acid cycle

28. Glyoxylate cycle cont’d Acetyl CoA combines with oxaloacetate (catalysed by citrate synthase ) to produce citrate , which is then converted by aconitase to isocitrate Next, isocitrate is split by isocitrate lyase into succinate and glyoxylate Malate synthase catalyses condensation of glyoxylate with acetyl CoA to form malate Malate is oxidized by malate dehydrogenase to oxaloacetate , which completes the glyoxylate cycle

29. Glyoxylate cycle cont’d

30. Gluconeogenesis pathway Plants can direct acetyl CoA to gluconeogenesis via the glyoxylate cycle Gluconeogenesis is the synthesis of glucose from pyruvate or oxaloacetate In plants, the formation of oxaloacetate from pyruvate and TCA cycle intermediates is restricted to the mitochondrion/cytosol While the enzymes that convert Phosphoenolpyruvate (PEP) to glucose-6-phosphate are found in the cytosol Assignment: Explain why plants transport sugars in the form of sucrose but not glucose?

31. Gluconeogenesis pathway

32. Chemistry of plant lipids Lipids serves as structural components of cell membranes , which acts as permeable barriers to the external environment of cells In plants, lipids play important roles as signalling and energy storage compounds . Plant lipids include triacylglycerols , phospholipids , galactolipids and sphingolipids Extracellular lipids are specially significant in plants and are comprised of cutins , suberins and waxes (physical defenses)

33. Cutins, Waxes & Suberins Cutin, waxes and suberins are made of hydrophobic compounds ( have water-hating properties) These compounds are non-polar and serve as barrier protection against environmental stress Fatty acids are one type of hydrophobic compound

34. Cutins, Waxes & Suberins cont’d

35. Cutins They composed of long fatty acid chains (synthesized by epidermal cells) They are a major component of plant cuticle (cover epidermis of leaves , young shoots , etc.) P Plants’ cuticle often vary with the climate in which they live

36. Waxes Waxes are complex mixtures of long-chain lipids that are extremely hydrophobic They are synthesised by epidermal cells (coatings on leaves and stems) They exuded through pores in the epidermal cell wall by an unknown mechanism to outside layer of the cell

37. Suberins Suberins are also formed from fatty acids but has a different structure from cutin They are constituent of plant cell wall and are often within roots They can protect against pathogens and other damage The older parts of roots are usually more suberized

38. Suberins can form transport barriers between the soil and the roots

39. Fatty acid biosynthesis Fatty acids are present as constituents of membrane lipids in every cell , each cell must contain the enzymes for the synthesis fatty acids In plants the de novo synthesis of fatty acids always occurs in the plastids ( known as chloroplasts of green cells & leucoplast/chromoplast of non-green plants ) In plant cells, enzymes of fatty acid synthesis are also found in the membrane of the ER but they are involved only in the modification of fatty acids that have been synthesised earlier in the plastids These modifications include a chain elongation of fatty acids , as catalysed by elongases and the introduction of further double bonds by desaturases

40. Fatty acid is synthesised from acetyl CoA derived from photosynthesis Assignment 1. Indicate the role of acyl carrier protein (ACP) in fatty acid biosynthesis in plants 2. Explain why linoleic (omega 6) and linolenic acids (omega 3) are considered essential acids for humans

41. Nitrogen Metabolism Nitrogen metabolism is based on recycling of ammonia (NH 3) in its neutral or charged form, ammonium ion (NH 4 + ) Role of nitrogen in plants It is a major substance in plants next to water Constituent element of chlorophyll , alkaloids , nucleic acids , proteins , various vitamins , cytochromes , etc. Plays important roles in metabolism , growth , reproduction and heredity

42. Sources of Nitrogen Atmospheric dinitrogen (N 2 ) 78% of N 2 in the atmosphere Plants cannot utilize this form Some Bacteria , Blue Green Algae , leguminous plants Nitrates (NO 3 ) , Nitrites (NO 2 ), Ammonia (NH 3 ), Urea CO(NH 2 ) 2 Nitrate is chief form through which some plants obtain organic nitrogen Amino acids in the soil Many soil organisms use this form Some higher plants can use this form Organic Nitrogenous compounds in insects Insectivorous plants (Venus flytrap, brocchinia, waterwheel)

43. Nitrogen Fixation The process of reducing dinitrogen (N 2 ) in the atmosphere to ammonia so that plants can absorb nitrogen Types of nitrogen fixation

44. Biological fixation Fixation of atmospheric Nitrogen into nitrogenous compounds with the help of microorganisms (mostly bacteria) Two types Symbiotic This type of fixation of nitrogen is carried out by microorganisms ( Rhizobium spp ) in the soil which lives symbiotically ( via nodule formation ) inside the plants Non-symbiotic This fixation is carried out by free living microorganisms (like aerobic, anaerobic and blue green algae (cyanobacteria) as well as free living fungi and specialised nitrogen fixing bacteria )

45. Root system of Phaseolus vulgaris (bean) with a dense formation of nodules after infection with Rhizobium etli & Rhizobium gallicum

46. Non Biological Fixation It does not involve microorganisms This occurs during rainy season when lightning takes place to convert nitrogen and oxygen into nitrogen oxides (nitrates) 1. N 2 + O 2 lightning 2NO ( Nitric oxide ) 2. 2NO + O 2 oxidation 2NO 2 ( Nitrogen per oxide ) 3. 2NO 2 + H 2 O HNO 2 + HNO 3 4. 4NO 2 + 2H 2 O + O 2 4HNO 3 ( Nitric acid ) 5. CaO + 2HNO 3 Ca (NO 3 ) 2 + H 2 O ( Calcium nitrate ) 6. HNO 3 + NH 3 NH 4 NO 3 ( Ammonium nitrate ) 7. HNO 2 + NH 3 NH 4 NO 2 ( Ammonium nitrite )

47. Biochemistry of Nitrogen Fixation Basic requirements for Nitrogen fixation Nitrogenase and hydrogenase enzymes Protective mechanism against oxygen Ferredoxin (iron and sulphur containing proteins) Hydrogen releasing system or electron donor (pyruvic acid or glucose/sucrose) Constant supply of ATP Coenzymes and cofactors (TPP, CoA, inorganic phosphate and Mg 2+, Cobalt and Molybdenum) A carbon compound

48. Nitrogenase enzyme This enzyme plays key role in reducing dinitrogen to ammonia (found in cyanobacteria ) Active in anaerobic condition Made up of two protein subunits Non haem iron protein (Fe-protein or dinitrogen reductase ) Iron molybdenum protein ( Mo/Fe-protein or nitrogenase ) Fe protein reacts with ATP to reduce second subunit which ultimately reduces N 2 into ammonia Hydrogen produced is catalysed into protons and electrons by hydrogenase ( H 2 2H+ + 2e)

49. Nitrogenase enzyme cont’d

50. Pathway of nitrogen fixation in root nodules Both the metalloproteins , nitrogenase (Mo-Fe-protein) and dinitrogenase reductase (Fe-protein) are essential for nitrogenase activity. Fe-protein interacts with ATP and Mg ++ , and Mo-Fe-protein catalyses the reduction of N 2  to NH 3 , H +  to H 2  and acetylene to ethylene NB. What is the function of leghaemoglobin?

51. Uptake and reduction of nitrogen by plants (Nitrogen cycle) Legend: The nitrogen cycle; (1) uptake of nitrogen by plants from the atmosphere , (2) uptake of ammonium and nitrate by plants from soil and water , (3) ammonification, (4) nitrification, (5) denitrification, (6) nitrate immobilization by soil sorption, (7) nitrate leaching from the soil, (8) release of ammonia (NH 3 ), gaseous nitrogen and nitrous oxide to the atmosphere

52. Metabolites Primary metabolites are m olecules that are essential for growth and development of plants Examples are carbohydrates , proteins , lipids , nucleic acids , cytochromes and chlorophylls Secondary metabolites are a large variety of compounds with no apparent function for growth and development of an organism Possibly over 250,000 secondary metabolites occur in plants Classified based on common biosynthetic pathways where a chemical is derived Four major classes are alkaloids , glycosides , phenolics and terpenoids

53. Ecological functions of secondary metabolites They are more limited in distribution and are usually found in specific families Chemical warfare to protect plants from the attacks by predators , pathogens or competitors Attract pollinators or seed dispersal agents Important for abiotic stresses ( drought , salinity , low or high temperatures ) Medicine ( terpenes , alkaloids , tannins , cardiac glycosides , etc.)

54. Secondary metabolites are derived from primary metabolites

55. Terpenoids Isoprene, Farnesol, Chlorophyll , β- Carotene 1. Terpenes The chemist Leopold S. Ruzicka (born 1887) showed that many compounds found in nature were formed from multiples of five carbons arranged in the same pattern as an isoprene molecule (obtained by pyrolysis of natural rubber) He called these compounds ‘terpenes’

56. Biological isoprene unit The isoprene units in terpenes do not come from isoprene They come from isopentenyl pyrophosphate (isoprenoid precursor) Isopentenyl pyrophosphate (5 carbons) is obtained from acetate (2 carbons) via mevalonate (6 carbons) Isopentenyl pyrophosphate (basis for all terpenoids)

57. Terpenes Terpenes are natural products that are structurally related to isoprene Isoprene (2-methyl-1,3-butadiene)

58. Classification of Terpenes Type of Terpene Number of Carbon Atoms Isoprene units hemiterpene C 5 one terpene C 10 two sesquiterpene C 15 three diterpene C 20 four triterpene C 30 six tetraterpene C 40 eight

59. Classification of Terpenes Hemiterpenes consist of a single isoprene unit . Isoprene itself is considered the only hemiterpene, but oxygen-containing derivatives such as prenol and isovaleric acid are hemiterpenoids. Monoterpenes consist of two isoprene units and have the molecular formula C 10 H 16 . Examples of monoterpenes are geraniol , limonene and terpineol . Sesquiterpenes consist of three isoprene units and have the molecular formula C 15 H 24 . Examples of sesquiterpenes are humulene , farnesenes and farnesol . Diterpenes are composed of four isoprene units and have the molecular formula C 20 H 32 . They are derived from geranylgeranyl pyrophosphate . Examples of diterpenes are cafestol , kahweol , cembrene and taxadiene (precursor of taxol).

60. Classification of Terpenes cont’d Sesterterpenes , terpenes having 25 carbons and five isoprene units are rare relative to the other sizes, example: geranylfarnesol Triterpenes consist of six isoprene units and have the molecular formula C 30 H 48 . The linear triterpene squalene , the major constituent of shark liver oil , is derived from the reductive coupling of two molecules of farnesyl pyrophosphate . Sesquarterpenes are composed of seven isoprene units and have the molecular formula C 35 H 56 . Sesquarterpenes are typically microbial in their origin. Examples of sesquarterpenes are ferrugicadiol and tetraprenylcurcumene .

61. Classification of Terpenes cont’d Tetraterpenes contain eight isoprene units and have the molecular formula C 40 H 64 . Biologically important tetraterpenes include the acyclic lycopene , the monocyclic gamma-carotene , and the bicyclic alpha- and beta-carotenes Polyterpenes consist of long chains of many isoprene units , example is natural rubber Norisoprenoids , examples C 13 -norisoprenoids 3-oxo- α- ionol present in Muscat of Alexandria leaves and 7,8-dihydroionone derivatives , such as megastigmane-3,9-diol and 3-oxo-7,8-dihydro- α- ionol found in Shiraz leaves (both grapes in the species Vitis vinifera )

62. Some examples of different types of terpenoids/terpenes

63. Things to remember about terpenes The number of C atoms is a multiple of 5, C 5 , C 10 , C 15 , C 20 , C 25 , C 30 , C 35 , C 40 2. Each group of 5 C is an isoprene subunit 3. They can be saturated or unsaturated 4. Many contain O atoms as well 5. What they all have in common is 1 & 2 above

64. Mevalonic acid pathway for synthesis of terpenes Biosynthetic precursors of terpenoid natural compounds

65. Some medicinal importance of terpenes

66. Phenolic compounds Phenols are organic compounds which contain a hydroxyl group (OH) directly bonded to a phenyl (aromatic or benzene) ring Phenolic compounds are the substances containing benzoic molecules with one or more hydroxylic groups and their derivatives If molecule contains two or more hydroxylic groups it is known as polyphenols Phenolics can be mainly classified into 2 groups: 1. The flavonoids 2. The non-flavonoids Typical phenolic compounds are lignin and suberin

67. Phenolic compounds Thousands of phenolic structures known They are found in vascular plants , Bryophytes , Fungi , Algae , Bacteria Account for 40% of organic carbon circulating in the biosphere Evolution of vascular plants: in cell wall structures, plant defense, features of woods and barks , flower colour and flavours Phenol Polyphenol

68. General biosynthetic pathways of phenolic compounds

69. Simplified pathways of phenolic compounds

70. Phenolic compounds are mainly derived from phenylpropanoid and phenylpropanoid-acetate pathways

71. Significance of phenolic compounds to plants

72. Classification of phenolic compounds Bark, leaves & fruit of oak, rhubarb, walnut, grapes, cocoa, etc

73. Flavonoids (various compounds found in many fruits and vegetables)

74. Some medicinal importance of phenolic compounds

75. Saponins They are plant constituents which bring about frothing in an aqueous solution . Historically, they are used for their detergent properties As plant glycosides, saponins share in varying degrees, two common characteristics: (a) They foam in aqueous solution (b) They cause haemolysis of red blood cells when injected into blood stream (highly toxic) but harmless when taken orally The aglycones (non- sugar unit) of the saponins are collectively referred to as Sapogenins . The more poisonous saponins are often called Sapotoxins Assignment: Explain mechanism underlying haemolysis of red blood cells by saponins

76. Properties of saponins Saponins form colloidal solution in water (hydrophilic colloids) which froths upon shaking These substances modify and lower the surface tension and therefore foam when shaken This has led to their use to increase the foaming of beer Practical industrial applications of saponins include their use in cleaning industrial equipment and fine fabrics and as powerful emulsifiers of certain resins , fats and fixed oils

77. Structure of saponins The general formula of saponins is (С 5 Н 8 ) 6 Based on the structure of the aglycone ( an organic compound, such as a phenol or alcohol combined with the sugar portion of a glycoside) or sapogenin , two kinds of saponin are recognized: 1.The steroidal type 2.The triterpenoid type Both of these have a glycosidal linkage at C-3 and have a common biosynthetic origin via mevalonic acid and isoprene units

78. Chemical composition of saponins

79. Some medicinal importance of saponins

80. Cardiac glycosides These are an important class of naturally occurring drugs whose actions include both beneficial and toxic effects on the heart (t hey have specific action on heart) Generally, glycosides are non-reducing organic compounds that on hydrolysis with acids , alkalis or enzymes yield A sugar part (or glycone, formed from one or more sugar units ) A non-sugar part (or aglycone , also called genin ) Historically, they were used to assassinate people ( via arrow poisons ) Currently, they are used; T o treat congestive heart failure (dropsy) F or treatment of atrial fibrillation and flutter The aglycone structure is important for their activity

81. Classifications of glycosides Based on their therapeutic effects 1. Congestive heart failure (CHF) and cardiac muscle stimulators such as: Digitalis glycosides: digoxin , digitoxin , gitoxin (Fox glove leaves) Ouabain : Strophanthus gratus seeds K-strophanthin : Strophanthus kombe seeds Scillaren A,B which isolated from red and white Squill bulbs Convolloside : Convallaria majalis ( Lily of the Valley)

82. Classifications of glycosides cont’d Based on the type of lactone ring 1. Cardenolide (one double bond, lactone ring): Has five member lactone ring (unsaturated) attached at C-17 β position of steroidal nucleus Examples: Digitalis glycosides: Digoxin , Digitoxin and Gitoxin Strophanthus gratus glycoside: Ouabain Strophanthus kombe glycoside: K- strophanthin 2. Bufadienolide : (contain two double bonds, lactone ring): Has six member (unsaturated) lactone ring attached at C-17 alpha –position Example: Squill bulb glycoside and Scillaren

83. Cardiac glycosides cont’d Group of steroidal glycosides act as cardiotonic agent Major sources are Digitalis purpurea (foxglove) , Digitalis lanata (foxglove) and Strophanthus kombe (vine used as arrow poison by Africans) They increase tone, excitability and contractility of cardiac muscles General properties of cardiac glycosides Amorphous (formless) powder Bitter taste Solubility in H 2 O Insolubility in organic solvents Very toxic compounds Odourless except saponin (glycyrrhizin)

84. Cardioactive glycosides A small group of plant glycosides act directly on the heart muscle These include (but are not limited to cardiac glycosides or cardenolides ) Cardenolides are steroidal glycosides which exert a slowing and strengthening effect on the failing cardiac muscle

85. Structures of cardiac glycosides

86. Some medicinal importance of cardiac glycosides

87. Cyanogenic glycosides (Cyanide glycosides) Cyanogenesis is the ability of certain living organisms, plants in particular , to produce hydrocyanic acid (HCN, prussic acid ) Cyanogenesis in plants is a chemical defense mechanism against organism (that damage or feed on plant tissues) via release of HCN gas, which is toxic They are distributed in over 2000 plant species belonging to 110 families ( eg . , bitter almonds , wild cherry ) These compounds, in presence of enzymes such as β -glucosidase , lose their sugar portion to form a cyanohydrin which, in the presence of water and hydroxynitrile lyase , can undergo hydrolysis to give benzaldehyde and the highly toxic hydrogen cyanide (HCN)

88. Cyanogenic glycosides cont’d The sugar portion of the molecule may be a monosaccharide or a disaccharide such as gentiobiose or vicianose . If a disaccharide, enzymes present in the plant may bring about hydrolysis in two stages, as in the case of amygdalin They are derivatives of α -hydroxynitrile or 2-hydroxynitrile (cyanohydrins) In all cases, the first sugar attached to the aglycone is β -D-glucose Most cyanogenic glycosides are biosynthetically derived from the amino acids such as valine , leucine , isoleucine , tyrosine or phenylalanine

89. Overview of biosynthetic and catabolic pathway of cyanogenic pathway

90. Glucosinolates They constitute a natural class of organic compounds that contain sulfur and nitrogen and are derived from glucose and an amino acid . They are water-soluble anions and belong to the glucosides (found in cabbage , horseradish , cauliflower , mustard seed, broccoli ) They are considered as the main secondary metabolites in cruciferous crops Every glucosinolate contains a central carbon atom which is bonded via a sulfur atom to the glycone group , and via a nitrogen atom to a sulfonated oxime group In addition, the central carbon is bonded to a side group, i.e. different glucosinolates have different side groups

91. Glucosinolates cont’d Central carbon Glucosinolates are destroyed by enzymatic hydrolysis due to enzyme called myrosinase In normal conditions, thiocyanates and isothiocyanates are formed Under pH 4, nitriles and elementary sulphur are generated Glucosinolates are heat- unstable , so they are partially destroyed by cooking

92. Biosynthesis and degradation of Glucosinolates

93. Some medicinal importance of Glucosinolates

94. Alkaloids Are naturally occurring chemical compounds containing nitrogen atoms Are physiologically active, insoluble or sparingly soluble in water Appear crystalline solids , while a few of them are amorphous (uncrystallized) Usually classified according to the nature of the basic chemical structures from which they are derived Form double-salts with compounds of Hg , Au , Pt and other heavy metals

95. Alkaloids cont’d Possible functions 1. Poisonous agents protecting the plant against insects and herbivores 2. End products of detoxification of alkaloids can metabolically lock up of compounds that are harmful to the plant 3. Regulatory growth factors 4. Reserve substances capable of supplying nitrogen or other elements necessary for plants economy

96. Chemical classification of alkaloids ALKALOIDS TRUE ALKALOIDS PROTO/AMINO ALKALOIDS PSEUDO ALKALOIDS Include heterocyclic nitrogen (derived from amino acids) Simple amines (derived from amino acids) Not derived from amino acids, but from Acetyl CoA

97. Main classes of alkaloid precursors and derivatives

98. Some medicinal importance of alkaloids

99. Biochemical basis of action of bioactive compounds from plants

100. Extraction and characterisation of bioactive compounds from plants

101. P hotosynthesis In 1780 Joseph Priestly discovered photosynthesis: “…plants can “restore air which has been injured by the burning of candles.” “…the air would neither extinguish a candle, nor was it all inconvenient to a mouse which I put into it.”

102. Photosynthesis cont’d Photosynthesis provides essentially all free energy in biological systems by converting solar energy into chemical energy Carbohydrates are formed from light-driven reactions that collectively appear deceptively simple: CO 2 + H 2 O + light  C n H 2n O n + O 2

103. Chloroplast structure Photosynthesis occurs in specialised organelles called chloroplasts Photosynthetic machinery in eukaryotic cells 10–500/cell Contain their own DNA Carry out protein synthesis in the organelle Contain three membranes Outer Inner Thylakoid

104. Chloroplast absorption Absorbs maximum amounts of light at 400 and 700 nm

105. Photosynthesis electron transport chain Photosynthesis consists of two sets of reactions: the LIGHT and DARK reactions The LIGHT -driven reactions are the primary events of photosynthesis; these occur in the thylakoid membrane. The DARK reactions occur in the stroma The light-reactions of photosynthesis generate high-energy electrons that are used to form NADPH On their way to NADPH , these high-energy electrons flow through a membrane-bound “ electron transport ” pathway, generating a proton motive potential ( Δ p) from which ATP is made

106. Photosynthesis electron transport chain cont’d

107. Photosynthetic electron transport system Convert solar energy into chemical energy and carbohydrate sugar precursors Occurs in five steps: 1. Four photons absorbed by chlorophyll molecule Water is oxidised to O 2 while 4H + are released and contribute to the proton motive force 2. Electron transport via carrier molecules 8H + are translocated for every 4e – donated

108. Photosynthetic electron transport system cont’d Occurs in five steps (continued): 3. Photon absorption by photosystem I (PSI, pigment complex) NADPH is generated 4. Chloroplast ATP synthase produces ATP Although 12H + are used to produce 3 ATP, only 8H + are translocated and 3 ATP are generated 5. ATP and NADPH are used by enzymes in the Calvin cycle to drive carbon fixation 3CO 2  + 3RuBp + 6NADPH + 5H 2 O + 9  ATP  → glyceraldehyde-3-phosphate (G3P) + 2H +  + 6NADP +  + 9ADP + 8Pi (Pi = inorganic phosphate)

109. Photosynthetic electron transport system cont’d

110. Phototranslocation and NADPH production

111. Energy Conversion by Photosystems I and II Four electrons are required to oxidise two H 2 O molecules to form O 2 and NADPH Light energy is converted to redox energy Photons are absorbed by chlorophyll and excite electrons from ground state to a higher orbital

112. Photosystem II (PSII) P680 reaction center Absorbs light energy at 680 nm Oxidises H 2 O  O 2 Can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea ( DCMU)

113. Photosystem I (PSI) P700 reaction center Absorbs light energy at 700 nm Generates NADPH for carbohydrate synthesis Large protein complex embedded in the chloroplast membranes Fe-S cluster is the final electron acceptor to ferroredoxin

114. Paraquat: A potent herbicide Accepts electrons from PSI and donates them to O 2 Blocks NADPH production Generates superoxide anion (O 2 – ) and hydrogen peroxide (H 2 O 2 ) Assignment Identify other herbicides that can inhibit photophosphorylation in plants

115. Light-Harvesting Complexes (LHCs) Act as “solar panels ” to capture light energy for photooxidation in reaction center (central part of photosystem) complexes Array of proteins and chlorophyll molecules (chromophores-part of a molecule responsible for its colour ) embedded in the thylakoid membrane and participate in energy transfer reactions Two types: LHCI LHCII Outnumbers LHCI Major light-gathering antenna in photosynthetic membrane

116. LHCII structure

117. Z-Scheme of photosynthetic electron transport system A series of photosystems, each requiring an input of energy from light absorption at PSII and PSI reaction center complexes Z-Scheme describes the oxidation/reduction changes that occur during the  light reactions  of photosynthesis Photon absorption by PSII results in electron flow from water to plastoquinone (PQ) This process translocates H + Photon is absorbed by PSI and provides energy to reduce NADP + to NADPH PC transports electrons from cytochrome b6f to PSI reaction center

118. Z-Scheme of photosynthetic electron transport cont’d OEC-Oxygen evolving complex

119. Protein Components of the photosynthetic electron transport system Pheophytin Plastoquinone PQA PQB Plastoquinol

120. Phosphorylation generates ATP Chloroplast ATP synthase complex consists of two components: CF o (membrane bound F o complex) CF 1 (catalytic F 1 complex) Has the same binding change mechanism as mitochondrial ATP synthase Proton gradient is needed

121. Cyclic photophosphorylation Occurs between PSI and cytochrome b 6 f It is the  movement  of the electrons in a cyclic manner for synthesising ATP molecules Proton gradient is independent of water oxidation Active when electron flow to NADP + is rate-limiting Ferroredoxin mediated reduction of PQ B acts as an alternative electron acceptor Controls ATP:NADPH

122. Cyclic photophosphorylation cont’d

123. Difference between cyclic and non-cyclic photophosphorylation

124. Rate of photooxidation can be regulated

125. Carbohydrate biosynthesis in plants (The Calvin cycle)

126. The Calvin Cycle: Overview Is controlled by light Generates: 3-phosphoglycerate glyceraldehyde-3-phosphate dihydroxyacetone phosphate These metabolites can be used to make fructose-1,6-bisphosphate fructose-6-phosphate

127. The Calvin (C3) cycle part 1 Three stages: 1. Synthesis of a C 6 molecule from Rubisco ( ribulose-1,5-bisphosphate carboxylase/oxygenase ) which is cleaved to 2 molecules of 3-phosphoglycerate - Carbon fixation 2. 3-phosphoglycerate is reduced to glyceraldehyde-3-phosphate- reduction ATP and NADPH are reactants 3. Ribulose-1, 5-bisphosphate is resynthesised- regeneration “Carbon shuffle” reactions

128. Stage 1: Fixation

129. Stage 2: Reduction

130. Production of hexose sugars

131. Stage 3: Regeneration of Ribulose-1,5,-Bisphosphate

132. Stage 3: Regeneration of Ribulose-1,5,-Bisphosphate cont’d

133. The Calvin Cycle part 2

134. The Calvin cycle regulation via light

135. Thioredoxin-mediated reduction of Calvin cycle enzymes PSI photon absorption leads to reduced ferroredoxin Assignment : Indicate factors that can slow down Calvin cycle

136. C 2 pathway (Glycolate pathway/cycle) Occurs in plants Converts 2- phosphoglycolate to glycolate in the stroma Glycolate is exported to plant cell organelles peroxisomes , where it is oxidized to form glyoxylate Transamination of glyoxylate in peroxisomes produces glycine , which is then translocated to mitochondria

137. C 4 versus Crassulacean acid metabolism ( CAM) pathway C 4 Found in tropical plants One cell type is required for CO 2 uptake and another for rubisco-mediated carboxylation Captures CO 2 in the form of oxaloacetate in mesophyll cells and then delivers it to bundle sheath cells as malate , another C 4 intermediate CAM In desert succulent plants Captures CO 2 at night then fixes CO 2 in the day

138. C 4 pathway/cycle

139. Molecular and biochemical regulation of plant metabolic pathways activated in response to environmental cues In complex multicellular organisms such as higher plants and animals, metabolism , growth and development of the various organs are coordinated by the emission of signal compounds Plant signal compounds are termed phytohormones Some of the phytohormones (e.g., brassinosteroids ) resemble animal hormones in their structure, but others are completely different in structure Like animal hormones, phytohormones also have many different signal functions They control the adjustment of plant metabolism to environmental conditions, such as water supply , temperature , and day length , as well as regulate plant development

140. Molecular and biochemical regulation of plant metabolic pathways activated in response to environmental cues cont’d Light sensors such as phytochromes , which recognize red and far-red light , and cryptochromes and phototropin monitoring blue light , control the growth and differentiation of plants depending on the intensity and quality of light Signal transduction chains have not been fully resolved for any of the phytohormones or light sensors in plants Partial results indicate that certain components of the signal transduction chain in plants may be similar to those in animals The phytohormone receptors and light sensors apparently act as a multicomponent system , wherein, the signal transduction chains are interwoven to a network

141. Environmental stress Biological stress is not easily defined but it implies adverse effects on an organism Like all other living organisms, the plants are subjected to various environmental stresses such as water deficit and drought , cold , heat , salinity and air pollution , etc. The concept of stress is associated with stress tolerance , thus d egree of tolerance differs with different plant species According to Levitt (1972) , `` Stress is any change in environmental conditions that might reduce or adversely change plant’s growth and development `` According to Jones (1989) `` Adverse force or influence that tends to inhibit normal systems from functioning ”

142. Types of stress

143. Plant stress Plants can respond to stress in several ways Plants may escape the effects of stress by completing their growth during less stressful periods or they may suffer injury if the stress is present and they cannot cope Stress resistant plants can tolerate a particular stress Many p lants have the capacity to resist stress through either stress avoidance or stress tolerance

144. Stress resistant or stress t olerance Plants may become stress tolerant through- Adaptation Heritable modifications to increase fitness CAM plants’ physiological adaptations to low H 2 O environment Acclimation Non heritable physiological and biochemical gene expression Cold-hardy plants’ adaptation to high H 2 O environment

145. Stages of response to stress 1 . Reception 2. Transduction 3. Response

146. Environmental stresses to which plants may be subjected to High Temperature (Heat) Low Temperature (Chilling, Freezing) Water Deficits (Drought, Low water potential) Salinity Excess water (Flooding, Anoxia) Chemical (Heavy metals, Air Pollutants) Radiation (Visible, Ultraviolet) Pathogens Competition

147. Response of plant at different parts

148. Interaction with pathogenic and symbiotic organisms

149. Interaction of plants with pathogenic and symbiotic organisms

150. Defenses against pathogens A plant ’ s first line of defense against infection is the barrier presented by the epidermis and periderm If a pathogen penetrates the dermal tissue, the second line of defense is a chemical attack that kills the pathogen and prevents its spread This second defense system is enhanced by the inherited ability to recognize certain pathogen

151. Cell wall formation A plant cell wall was first observed and named simply as a “wall” by Robert Hooke in 1665 In 1804, Karl Rudolphi and J.H.F. Link proved that cells have independent cell walls A cell wall is a structural layer that surrounds some types of cells, situated outside the cell membrane It can be tough , flexible and rigid which provides cell with both structural support and protection

152. Properties of plant cell wall R igidity Tensile strength Hydraulic turgor pressure (exerted by fluid in the cell that presses the cell membrane against the cell wall) Inflation P ermeability Primary cell wall-30 to 60 kDa CO 2 , H 2 O– apoplastic flow pH

153. Cell wall layers Cell wall thickness -0.1μm to several μm L ayers 1.Primary cell wall T hin, flexible, extensible Contains cellulose, hemicellulose and pectins Permeable Size 30 to 60 kDa 2. S econdary cell wall Thick Present inside primary cell wall Contains lignin for strength and density of wood Cellulose fibres

154. Cell wall layers cont’d 3. M iddle lamella Outermost layer between adjacent plant cells Contains pectins Gives stability (form plasmodesmata) Made up of calcium and magnesium pectates

155. Composition Primary cell wall Cellulose Hemicellulose (xyloglycan) and pectin Plant epidermis cutin and wax Secondary cell wall cellulose: 35-50% Xylan: 20-35% Lignin : 10-25%

156. Cell wall f ormation Middle lamella – first formed from cell plate during cytokinesis (i s the physical process of cell division, which divides the cytoplasm of a parental cell into two daughter cells) Primary cell wall- composed of cellulose fibrils , produced at plasma membrane by cellulose synthase complex Microfibrils– held by hydrogen bonds (tensile strength) Secondary cell wall– constructed between plasma membrane and primary wall Plasmodesmata (microscopic channels that traverse the cell walls of plant cells) – interconnecting channels of cytoplasm that connect protoplasts (cells in plant whose cell wall as been removed)

157. Wood f ormation Wood formation is a complex  biological process , involving five major developmental steps Cell division from a secondary  meristem  called the  vascular cambium C ell expansion (cell elongation and radial enlargement) S econdary cell wall   deposition Programmed cell death Heartwood   formation

158. F unctions of plant cell wall To give cell rigidity and strength To act as a physical barrier to the plant To prevent cell swelling and bursting as a result of osmotic pressure To promote cell to cell signalling through their cell walls Assignment Indicate the roles of phytohormones such as gibberellins , cytokinin , abscisic acid, ethylene and jasmonic acid, methyl jasmonate, Brassinosteroids, peptide hormones and salicylic acid in plants

159. Lignin and flavonoids in cell wall formation

160. Wounding in plants Plants damaged by insects can release volatile chemicals to warn other plants of the same species Arabidopsis can be genetically engineered to produce volatile components that attract predatory mites

161. Plant defenses (phytoalexins, oxidative burst, hypersensitivity)

162. Plant responses to drought

163. Plant responses to flooding, salinity, pollutants (heavy metals, ozones )