This document summarizes research on polyphenol oxidase pathways and their role in plant defense. It discusses how phenolic compounds accumulate at infection sites and are activated by polyphenol oxidases to form quinones, which act as antibiotics or make plant tissues unpalatable. The document outlines the biochemical properties and occurrence of phenolic compounds and polyphenol oxidases in plants. It describes several ways phenolic compounds contribute to plant defense, including acting as feeding deterrents or interfering with nutrient uptake in herbivores. The document provides examples of research showing induced defense enzyme activities, including polyphenol oxidase, in resistant plant genotypes compared to susceptible ones when infected.

1. Biochemistry of induced resistance-
Polyphenol oxidase pathways
Submitted by: Simran Bhatia
(H-2021-06-D)
Ph.D.- Entomology II Year
ENT 607
Submitted to:
Dr. SC Verma
Dr. VGS Chandel

2. Introduction
◦ Phenolic compounds are the most widely distributed secondary metabolites present in the plant
kingdom. Their functions in plants are related to structure, protection, adaptation to environment, and
interaction with various biotic and abiotic factors.
◦ The first step of the defense mechanism in plants involves a rapid accumulation of phenols at the
infection site (Matern and Kneusal,1988). The role of phenolic compounds in defense is related to
their antibiotic, antinutritional or unpalatable properties.
◦ In plants, PPOs are nearly ubiquitous and located in chloroplasts. The loss of sub-cellular
compartmentalization, due to senescence, wounding, interactions with pests and pathogens and
handling during postharvest processing and storage, results in contact between PPOs and vacuolar
phenolic substrates
◦ This ultimately leads to a reaction known as enzymatic browning, negatively affecting colour, flavour,
nutritional properties and shelf life of food products. However, in a few cases, enzymatic browning
might be beneficial, as it leads to the formation of compounds conferring characteristic flavours
◦ Due to the economic importance of browning, PPOs have been extensively studied in relation to their
physico-chemical properties.
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4. What are phenolic compounds?
◦ The terms ‘phenol’ and ‘polyphenol’ can be defined chemically as substances that possess an
aromatic ring bearing one (phenol) or more (polyphenol) hydroxyl substituents.
◦ Synthesized via two different routes:
◦ The shikimate pathway (in plants)
◦ The acetate-mevalonate pathway (in fungi and bacteria)
◦ They are heterogeneous group.
◦ Some are water soluble only in organic solvents
◦ Some are water soluble carboxylic acids and glycosides
◦ Some are insoluble polymer
◦ Many serves as defense compounds against herbivores and pathogens
◦ Other function in attracting pollinators and fruit dispensers
◦ Polyphenol oxidase can form quinones which act as anti-nutritional proteins interfering with
digestibility and nutrient uptake of insects or produce proteinase-inhibitors leading to cross-
linking and polymerization of cells leading to herbivore defense.
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5. Occurrence of phenolics?
◦ They are usually found as esters or glycosides rather than as free compounds.
◦ Polyphenols (relatively hydrophilic) usually accumulate in the central vacuoles of guard cells,
epidermal cells and the subepidermal cells of leaves and shoots.
◦ Some are found covalently linked to the plant cell wall (lignin) and some found in waxes (related to
lipidic structures) or on the external surfaces (cuticle) of plant organs (Lattanzio et al., 2006).
◦ Biosynthesis of phenolic compounds occurs at various sites in plant cells, such as the chloroplasts,
the cytoplasm and the endoplasmic reticulum membrane.
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7. PPO biochemical properties
Conversion of phenolic substrates to o-quinones by ppos occurs by means of two oxidation
steps.
1. The first is the hydroxylation of the ortho-position adjacent to an existing hydroxyl group
(“monophenol oxidase” or “monophenolase” activity, also referred to as hydroxylase activity).
2, The second is the oxidation of o-dihydroxybenzenes to o-benzoquinones (“diphenol
oxidase” or “diphenolase activity”, also referred to as catecholase or oxidase activity)
Figure : Simplified schematization of browning process. PPO, polyphenol oxidases.

10. Significance of phenolic compounds
UV sunscreen
Signal compounds
Pigmentation
Plant growth
Plant defense
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12. Phenolics and plant defense
◦ Phytoanticipins
◦ Phytoalexins
◦ Cell wall defense
◦ Feeding deterrents
◦ Defense hormones
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13. Phytoanticipins and
Phytoalexins
◦ Phytoanticipins are preformed antimicrobial compounds in
plants that are unique in action for their property of being
synthesized even before the attack of pathogen or infection, i.e.,
they exist in healthy plants in their biologically active forms
(constitutive).
◦ Phytoalexins are low molecular weight antimicrobial
compounds that are produced by plants as a response to
biotic and abiotic stresses. As such they take part in an
intricate defense system which enables plants to control invading
microorganisms.
◦ Gossypol from cotton, casbene from castor bean, and
rishitin from plants in the Solanaceae are examples of
phytoalexins biosynthesized by the malonic acid pathway

14. Feeding deterrents
◦ Toxic to many herbivores by reducing the growth and survivorship.
◦ Tannins can bind digestion enzymes in the gut of herbivores and also form complex polymers when bound to proteins
which are difficult to digest, thus decreasing the nutritional value of the plant material.
◦ Antifeedant proanthocyanidin (condensed tannins) in red sorghum deter birds from feeding on the seed
◦ White sorghum deficient in these compounds is eaten by birds.
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15. Impairs the nutrient uptake
Affect digestibility of plant
tissues
Disturbances in gut redox
state, proliferation of
oxyradicals that damage
proteins, lipids
ROLE OF DEFENSE ENZYMES
(Zhu et al 2008)

16. Mechanism of action of Silicon in
releasing of PPO enzymes in plants
◦ Oxidation of phenols catalyzed by polyphenol oxidase (PPO)
and peroxidase (POD) is a potential defense mechanism in
plants against herbivorous insects.
◦ Quinones formed by oxidation of phenols bind covalently
to leaf proteins, and inhibit the protein digestion in
herbivores, in addition, quinones also exhibit direct toxicity
to insects.
◦ Alkylation of amino acids reduces the nutritional value of
plant proteins for insects, which in turn negatively affects the
insect growth and development.
◦ Phenols also play an important role in cyclic reduction of
reactive oxygen species (ROS) such as superoxide anion and
hydroxide radicals, H2O2, and singlet oxygen, which in turn
activate a cascade of reactions leading to the activation of
defensive enzymes.
◦ Simple phenolics (salicylates) act as antifeedant to insect
herbivores such as Operophtera brumata (L.)

20. Fluctuations in POD activities of resistant and susceptible
black gram genotypes in response to Bemisia tabaci
 Nine black gram genotypes were used
 KU 99-20 and NDU 5-7: Moderately resistant
 IPU 02-043, KU 7-602, KU 7-605, KU7-618 and Mash 1-1: Susceptible
 KU 7-504 and KU 7-505:Highly susceptible
(Taggar et al 2012)
Activity of peroxidase at 30 and 50 DAS

21.  Five groundnut genotypes: ICGV
86699, ICGV 86031, ICG 2271 and
ICG 1697: Moderately resistant and
JL 24: susceptible genotype
 The activity of the defensive
enzymes (peroxidase, polyphenol
oxidase, phenylalanine ammonia
lyase, superoxide dismutase,
peroxidase, and catalase) and the
amounts of total phenols, hydrogen
peroxide, malondialdehyde and
proteins were recorded at 6 days
after infestation
(War et al 2013)
Defensive Responses in Groundnut Against Chewing and Sap-
Sucking Insects
• Induction of enzyme activities and the
amounts of secondary metabolites
were greater in all the insect- resistant
genotypes infested with H. armigera
and A. craccivora than in the
susceptible check JL 24
• The resistant genotypes suffered lower
insect damage and resulted in lower
Helicoverpa larval survival and weights
than those larvae fed on the susceptible
check JL 24
• The number of aphids was significantly
lower on insect- resistant genotypes
than on the susceptible check JL 24
(War et al 2013)

22. Upregulation of defense enzymes in chickpea
genotypes against Helicoverpa armigera
 Ten chickpea genotypes were used
 Resistant genotypes showed Increased activity of various defense enzymes such as:
 PPO
 PAL
 POD
 SOD
(Kaur et al 2015)