FISIOLOGIA VEGETAL COMPLEMENTAR

(2013/2014)






SECONDARY METABOLITES
production, accumulation and functions



Plant Physiology, Fifth Edition (2010), L. Taiz & E. Zeiger
Chapter 13 Secondary Metabolites and Plant Defense

Biochemistry & Molecular Biology of Plants (2000), B.B. Buchanan, W.
Gruissen, R. J. Jones. Chaper 24 Natural Products (Secondary Metabolites).
Historically, the compounds produced by
plants have been separated into primary
and secondary metabolites, or products.


Primary metabolites, by definition are molecules that are
found in all plant cells and are necessary for the life of the plant.
Examples of primary metabolites are simple sugars and
carbohydratea, amino acids, proteins, nucleic acids, fatty acids,
lipids as phospholipids, triglicerides.

Secondary metabolites, also called secondary products or
natural products are a large, diverse array of organic compounds
produced by plants that appear to have no direct function in their
growth and development.

GENERAL ECOLOGICAL FUNCTIONS OF SECONDARY METABOLITES

For many years the secondary metabolites were thought to be simply
functionless metabolites or metabolic wastes as their adaptative
significance was unknown.

Over the 20th and 21th centuries however, the study of the secondary
metabolites produced by plants, has shown their importance as
medicinal drugs, poisons, flavours and as a wide range of industrial
materials.

Today, we know that many secondary metabolites have important
ecological functions and roles in adapatation of the plants to the
environment and their protection against biotic and abiotic stress
factors. As examples, their functions include:

protection against intensive deleterious light radiation
protection against hervibores and microbial pathogens
as atractants for pollinators and seed-dispersing animals
as agents of plant-plant competition and plant-microbe symbioses

SECONDARY METABOLITES ARE DIVIDED INTO THREE MAJOR GROUPS:
Terpenoids; phenolics; and nitrogen-containing compounds (alkaloids) (Figure 13.1)

Figure 13.1 A simplified view of the major pathways of secondary-metabolite
biosynthesis and their interrelationships with primary metabolism.
TERPENOIDS
Terpenoids constitute the largest class of secondary metabolites,
with over 22.000 terpenoid compounds described. This group of
secondary metabolites is represented in all the plants studied up
to now.

Most of the diverse terpenoids are insoluble in water.

The simplest of the terpenoids is the hydrocarbon isoprene
(CH). All terpenoids can be classified according to their number of
isoprene units: 1 unit hemiterpenes; 2 units monoterpenes; 3 units
sesquiterpenes; 4 units diterpenes; 6 units triterpenes; 8 units
tetraterpenes; > eight units polyterpenes.

A single plant may sinthesize many different isoprenoids at different
locations within the plant for a variety of purposes at different times
during the course of its development.
GREAT SMOKY MONTAINS Tenessee and North Carolina

Blue haze, composed largely of isoprene, hovering the Great
Smoky Montains in North Carolina.

Isoprene itself is a gas emitted in significant quantities by the leaves
of many plant species and is largely responsible for the bluish haze
that hovers over wooded hills and montains in summer.
Isoprene is also a component of the smog.

This compound which is emitted only in the light, is made in
chloroplasts from carbon dioxide recently assimilated in organic
compounds by photosynthesis.

One may wonder, why plants produce and discharge such large
quantities of isoprene.

Studies have shown that isoprene emissions are highest on hot days,
and further that isoprene production may aid the plant to cope with
heat by stabilizing photosynthetic membranes within the plants cells.

Biosynthesis of terpenoids is compartmentalized, as is the production
of the terpenoid precursor IPP.

In particular, plants produce a much wide variety of terpenoids than do either animals
or microbes, a difference reflected in the complex organization of plant terpenoid
biosynthesis at the tissue, cellular, subcellular, and genetic levels.

Figure 24.3 (A). Scanning electron mocrograph of the leaf surface of Thymus sp. The round structures are
peltate glandular trichomes, in which monoterpenes and sesquiterpenes ares synthesized

Figure 24.3 (B). Light micrograph of a glandular trichome of spearmint, shown in longitudinal
section (C subcuticular space; S secretory cells; St stalk; B basal cell; E epidermal cell)

Figure 24.3 (C). Light micrograph of a secretory cavity in a lemon leaf, shown in cross section
(L Lumen; S secretory cell; Sh sheath cells; P parenchyma cell
The production of large quantities of terpenoid natural products as well as their
subsequent accumulation, emission, or secretion is almost always associated with
the presence of anatomically hight specialized structures.
The glandular trichomes (Figure 24 3A, B) and secretory cavities of leaves
(Figure 24 3C and the glandular epiderms of flower petals generate, store or emit
terpenoid essential oils that are important because they encourage pollination by
insects.
Figure 24.3 (D). Light micrograph of a resin duct in wood Jeffrey pine
shown in cross section.
(L Lumen; S secretory cell; Sh sheath cells; X secondary xylem.).

The resin ducts of conifer species (Figure 24.3D), produce and accumulate a
defensive resin consisting of turpentine (monoterpene olefins) and rosin (diterpenoid
rosin acids).
Leaves of plants from some families often show several types of secretory structures, as uni- or
pluricellular glandular trichomes or secretory cavities, which can be found also at the other,
vegetative and reproductive organs.
Such glandular trichomes or secretory cavities have secretory cells with capacity to sinthesize
secondary metabolites which can appear as mixtures as essential oils, resins, mucilages,
alkaloids, and so one.
Such substances are used in pharmaceutic, food, and perfumery industries, or others. Trichomes,
particularly, besides production, secretion and storage of secondary metabolites, cover and coat
plant parts, protecting against the herbivores and some types of stresses like deleterious radiation
and drought.
Functions of secretory structures of plant leafs
There are two pathways for terpene biosynthesis

MEVALONIC ACID PATHWAY (Cytosolic pathway).
Three molecules of acetyl-CoA are joined together to form mevalonic acid
(Figure 13.2) This key six-carbon intermediate is then pyrophosphorylated,
decarboxylated, and dehydrated to yield isopentenyl diphosphate (IPP)* which
is the activated five-carbon building block of terpenes.


METHYLERYTHRITOL POSPHATE (MEP) PATHWAY (Plastid pathway).
This pathway operates in chloroplasts and other plastids.
Glyceraldehyde 3-phosphate and two carbon atoms derived from pyruvate
condense to form the five-carbon intermediate 1-deoxy-D-xylulose 5-
phosphate. After this intermediate is rearranged and reduced to 2-C-methyl-D-
erythritol 4-phosphate (MEP), it is eventualy converted into IPP (Figure 13.2).
TERPENOID BIOSYNTHESIS
(MONO- AND SESQUITERPENOIDS ARE THE MAJOR CONSTITUENTS OF THE MOST ESSENTIAL OILS)
Figure 13.2
Outline of terpene biosynthesis.

The basic 5-carbon units of
terpenes are synthesized by two
different pathways.

The phosphorylated intermediates,
IPP and DMPP, are combined to
make 10-carbon, 15-carbon, and
larger terpenes.
Biochemistry & Molecular Biology of Plants. Buchanan; Gruissen;Jones
Figure 24.4 - A via do acetato/mevalonato para
a formao do IPP, a unidade bsica, de cinco
tomos de carbono da biossntese dos terpenos.
A sntese de cada unidade de IPP requer trs
molculas de acetil-CoA.
HMG-CoA reductase


Is one of the most highly regulated enzymes in animals, being highly
responsible for the control of cholesterol biosynthesis.


In plants, HMG-CoA reductase is also highly regulated. It is located
in RE and, in many cases it is encoded by multiple members of a
small gene family which are expressed in complex patterns, with
individual genes exhibiting constitutive, tissue- or development-
specific, or hormone-inducible expression.


Specific HMG-CoA reductase genes are induced by wounding or
pathogen infection.


The activity of HMG-CoA reductase may be subject to
posttranslational regulation, for example, by a protein kinase
cascade that phosphorylates and thereby activates the enzyme.

Proteolytic degradation of HMG-CoA reductase protein and the rate
of turnover of the corresponding mRNA transcripts may also
influence enzyme activity.


The mechanisms of HMG-CoA reductase regulation and thereby
precise biochemical controls that facilitate the production of different
terpenoid families, are yet unknown. Such dificulties are due to the
fact of HMG-CoA reductase is associated with the ER membrane.


A model proposed to rationalize the selective participation of HMG-
CoA reductase in the biosynthesis of different mevalonate-derived
terpenoids is shown in Figure 24.5.

Biochemistry & Molecular Biology of Plants. Buchanan; Gruissen;Jones
Figure 24.5. Model for the membrane topology of HMG-CoA reductase (HMGR). The protein includes a
highly variable hydrophilic N-terminal sequence (blue), a conserved membrane anchor (orange), a highly
variable linker sequence (green and purple), and a highly conserved, cytosol-exposed, C-terminal ctalytic
domain (yellow). Isoforms of HMGR that are associated with elicitor-induced synthesis of sesquiterpenoid
phytoalexins contain N-linked glycosylation site exposed to the ER lumen. Differences in N-terminal
sequences and extent of glycosylation may affect targeting of HMGR to various ER domains to other
organelles of the endomembrane system
(ER endoplasmic reticulum; MVA mevalonic acid).
Prenyltransferase and terpene synthase reactions

Prenyltransferases enzymes generate the alylic diphosphate esters
geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and
geranylgeranyl diphosphate (GGPP).

Reactions that these compounds undergo (often cyclizations), which
are catalyzed by terpene synthases, yield a wide variety of
terpenoid compounds.



Repetitive addition of C5 units carried out by
prenyltransferases

IPP is utilized in a sequence of elongation reactions to produce a series of
prenyl diphosphate homologs, which serve as the immediate precursors of
the different families of terpenoids (Figures 24.7).

Figure 24.7
The major subclasses of terpenoids are
biosynthesized from the basic five-carbon
unit, IPP, and from the initial prenyl (allylic)
diphosphate, dimethylallyl diphosphate,
which is formed by isomerization of IPP. In
reactions catalyzed by prenyltransferases,
monoterpenes (C
10
), sesquiterpenes (C
15
),
and diterpenes (C
20
) are derived from the
corresponding intermediates by sequential
head-to-tail addition of C
5
units, Triterpenes
(C
30
), are formed from two (C
15
), (farnesyl)
units joined head-to-head, and tetraterpenes
(C
40
), are formed from two (C
20
),
(geranylgeranyl) units joined head-to-head*.

* See Figure 24.2.
Biochemistry & Molecular Biology of Plants.
Buchanan; Gruissen;Jones
The enzyme limonene
synthase is a model for
monoterpene synthase
action (Figure 24.9)
Other monoterpene synthase reactions

Synthases that produce acyclic olefin products (e.g., myrcene)
and bicyclic products (-pinene and -pinene) from GPP are also
known, as are the enzymes that transform GPP to oxygenated
derivatives such as 1,8-cineole and bornyl diphosphate (Figure
24.10), the precursor of camphor.

Monoterpene synthases have the ability to produce more than
one product. For example, pinene synthase has the ability to
produce both -pinene and -pinene.

Figure 24.10

Structures of monoterpenes,
including insecticidal compounds
(- e -pinene, pyrethrin),
pollinator attractants (linalool e 1,8-
cineole) and antiherbivory agents
(1,8-cineole).
Monoterpene functions


Monoterpenes have quite different functions. Thus
linalool and 1,8-cineole (Figure 24.10) emited by
flowers serve as attractants for pollinators, including
bees, moths and bats.


1,8-Cineole and camphor act as foliar feeding
deterrents to large herbivores and also may provide
a competitive advantage to several angiosperm
species as allelopatic agents that inhibit germination
of seeds of other species.
Biochemistry & Molecular Biology of Plants. Buchanan; Gruissen;Jones
-Pinene and -pinene are among the most common monoterpenes produced by
plants and are principal components of turpentine of the pines and spruces, being
toxics to bark beetles and their pathogenic fungal symbionts, which cause serious
damage to conifer species worldwide.

Many conifers respond to bark beetle infestation by up-regulating synthesis of
monoterpenes, a process analogue to the production of antimicrobial phytoalexins,
when under pathogen attack (Figure 24.11).
Mentha spicata
( Hortel )
Mentha x piperita
( Hortel pimenta )
Secondary transformations of the basic parent skeletons produced by the terpenoid synthases
most commonly involve oxidation, reduction, isomerization, and conjugation reactions which impart
functional properties to the terpenoid molecules and are responsible for generating the myriad
different terpenoids produced by plants.
The conversion of (-)-limonene to (-)-menthol in peppermint and carvone in spearmint illustrates the
biochemistry of terpenoid modification (Figure 24.16).
Figure 24.16(A). Essential oil synthesis in spearmint and peppermint.
In spearmint (-)-limonene is converted to (-)-carvone by a two-step pathway.
In peppermint (-)-limonene is converted to (-)-isopiperitenone which is modified to form an intermediate
(+)-Pulegone which then, is converted in (-)-menthol and related compounds.
Biochemistry & Molecular
Biology of Plants. Buchanan;
Gruissen;Jones
Biossntese de alguns dos principais constituintes do leo essencial de Mentha piperita
In peppermint (-)-
limonene is converted
to (-)-isopiperitenone
which is modified to
form an intermediate
(+)-Pulegone which
then, is converted in
(-)-menthol and
related compounds.

Figure 24.16(B)
MONOTERPENOS
alguns dos constituintes
mais volteis dos leos
essenciais de PAM
Methods in Plant
Biochemistry: Vol. 7
Terpenoids (1991). Dey,
PM & Harborn, JB;
Academic Press Limited
MONOTERPENOS alguns
dos constituintes mais
volteis dos leos essenciais
de PAM
Methods in Plant Biochemistry:
Vol. 7 Terpenoids (1991). Dey,
PM & Harborn, JB; Academic
Press Limited
Some terpenoid skeletons are extensively
decorated (Figure 24.17)
Figure 24.17(A). Terpenoids formed by secondary transformations of
parent cyclic compounds. The yellow highlighting delineates the
terpenoid portion of the molecule taxol.
Figure 24.17(B). Terpenoids formed by secondary transformations of
parent cyclic compounds
END OF THE TERPENOIDS SECTION

ALKALOIDS
(alkaloids; cyanogenic glycosides; glucosinolates, and non-protein amino acids)
The alkaloids are a large family of more than 15,000 nitrogen-containing
secondary metabolites. They are found in approximately 20% of vascular
plant species. The nitrogen atom in these compounds is usually part of a
heterocyclic ring , a ring that contains both nitrogen and carbon atoms.

As their name would suggest, most alkaloids are alkaline. At pH values
commonly found in the cytosol (pH 7.2) or the vacuole (pH 5-6), the nitrogen
atom is protonated; hence alkaloids are positively charged and generaly
water soluble
Alkaloids Have Marked Physiological Effects on Animals

As a group, alkaloids are best known for their striking pharmacological effects on vertebrate animals.

Usually alkaloids are synthesized from one of a few common amino acids in particular lysine, tyrosine,
tryptophan, phenylalanine or arginine. However the carbon skeleton of some alkaloids contains a component derived
from the terpene pathway. Table 13.2 lists the major alkaloid types, their amino acid precursors and main human uses.
Several different alkaloid types, including nicotine and its relatives (Figure 13.14) are derived
from ornitine, an intermediate in arginine biosynthesis.

The B vitamin, nicotinic acid (niacin) is a precursor of pyridine (six-membered) ring of this
alkaloid; the pyrrolidine (five-membered) ring of nicotine arises from ornitine (Figure 13.15).

Nicotinic acid is also a constituent of NAD and NADP, which serve as electron carriers in
metabolism.
Structure of the anticholinergic tropane
alkaloid atropine from Hyoscyamus niger

Atropine dilate pupils, then extracts of
this plant were used by Ladies for beauty
and seduction. It was used by Cleopatra.
Structure of the tropane alkaloid
cocaine, a central nervous system
stimulant derived from Erythroxylon
coca.
History of the human use of alkaloids
Alkaloids have a 3000-year history of humans use and
a lot of them are important therapeutic compounds of
the modern medicine. For example, the use of latex of
the opium (Papaver somniferum) either in potions or
poisons can be traced back at least 1400 to 1200 B.C.
Figure 24.21 (A) - Structures of the alkaloids codeine and
morfine from opium poppy Papaver somniferum. Asymetric
(chiral) carbons are highlighted with red dots.

Codeine: antitussive and analgesic in modern medicines
Figure 24.21 (B)
The frog bufo
marinus accumulates
a considerable
amount of morphine
in its skin.
Figure 24.23 Structure of diacetyl
morphine, commonly known as heroin.
Figure 24.19. (A) The piperidine alkaloid
coniine, the first alkaloid to be synthesized, is
extremely toxic, causing paralysis of motor nerve
endings.

(B) in 399 B.C., the philosopher Socrates was
executed by consuming na extract of coniine-
containing poisonous hemlock. This depiction of
the event The Death of Socrates, was painted
by Jacques-Louis David in 1787.
Figure 24.27 Structure of the steroid alkaloid
glycoside -solanine from Solanum tuberosum
(potato). The aglycone solanidine is derived from
cholesterol.

The steroid alkaloid -solasodine, a
cholinesterase inhibitor found in potato tuber, is
the trace toxic constituent thought to be
responsible for the teratogenicity of sprouting
potatoes (Figure 24.27).
Various alkaloids are toxic to insects or function as feeding deterrents. For example, nicotine found in
tobacco, was one of the first insecticides used by humans and remains one of the most effective (Figure
24.22). Another effective insect toxin is caffeine, found in seeds and leaves of cocoa, coffe, cola, mat,
and tea (Figure 24.26). Herbivory has been found to stimulate nicotine biosynthesis in wild tobacco plants.
Figure 24.22. Structure of nicotine from
Nicotiana tabacum. The asymmetric chiral
carbon is highlighted with a red dot.
Figure 24.26. Structure of the purine alkaloid
caffeine from Coffea arabica
Physiologically active alkaloids participate in plant chemical defences

The role of chemical defence for alkaloids in plants is supported by their wide range of physiological
effects in animals and by the antibiotic activities many alkaloids possess.

Taking into consideration the following examples, alkaloids can be viewed as a part of the chemical defense
system of the plant that evolved under the selection pressure of predation.
Production of selected alkaloids in plant cell culture
Berberine Coptis japonica 7.0 g/l 12% dry weight
Jatrorrhizine Berberis wilsoniae 3.0 g/l 12% dry weight
Raucaffricine Rauwolfia serpentina 1.6 g/l 03% dry weight
Alkaloid biosynthesis research has been greatly aided by development of
techniques for culturing plant cells.
Figure 24.31 Structure of the monoterpenoid
indole alkaloid vinblastine from Catharantus roseus
Figure 24.32 The imidazole alkaloid pilocarpine from
Pilocarpus jaborandi
Plant cell suspension cultures
were considered an alternative
source of industrially significant
secondary metabolites, particularly
alkaloids of pharmaceutical
importance.

However, many important
compounds such vincristine,
vinblastine (Figure 24.31),
pilocarpine (Figure 24.32)
morphine, and codeine, among
many others, are not synthesized
to any appreciable extent in cell
culture.


Cell culture continues, however, to
provide biochemists with a rich
source of certain alkaloid
biosynthesis enzymes and
convenient system with which to
study enzyme regulation.
Alkaloids are in most cases formed from L-amino
acids (e.g., tryptophan, tyrosine, phenylalanine, lysine
and arginine) either alone or in combination with a
steroidal, secoiridoid (e.g., secologanin), or other
terpenoid-type moiety.

The L-tryptophane-derived monoterpenoid indole alkaloid
ajmalicine was the first alkaloid for which the biosynthesis
was clarified at the enzyme level (Figure 24.33). In that
study plant cells suspension of Cataranthus roseus,
cultures were used.

In plants, the biosynthesis of ajmalicine and more that
1800 other monoterpenoid indole alkaloids begins with
the decarboxylation of the amino acid L-trypthophan by
tryptophan decarboxylase, to form tryptamine.
Tryptamine is derived from L-tryptophan by decarboxylatin
throug the action of tryptophan decarboxylase,
The secoridoid secologanin is derived in multi steps from
the monoterpene geraniol.
BIOSYNTHESIS OF ALKALOIDS
Figure 24.33 Biosynthesis of
the monoterpenoid indole
alkaloid related compounds
in Catharanthus roseus.
Tryptamine, by action of
strictosidine synthase,
is sterereospecifically
condensed with the
secoiridoid secaloganin
to form 3-strictosidine.
Figure 24.34 Strictosidine,
the product of tryptamine and
secologanin, is the precursor
for many species-specific
alkaloids.
Strictosidine can then be
enzymatically permutated in
a species-specific manner
to form a multitude of
diverse structures (Figure
24.34).
Figure 24.36. The Berberis wilsoniae plant (left) and cell suspension culture (right).
The cell suspension culture derives its color from optimized production of the highly
oxidized benzylisoquinoline alkaloid berberine. Plant cell cultures (like this one)
that produce large quantities of alkaloids have led to complete elucidation of
several alkaloid biosynthetic pathways.
The berberine synthesis pathway has been defined completely

Berberine is a tetrahydrobenzylisoquinoline (antimicrobial), the first alkaloid for which each
biosynthetic enzyme has been identified, isolated, and characterized from the primary
metabolite precursor to the end product alkaloid, in Berberis (barberry) cell suspension
cultures (Figure 24.36).
Uses of berberine in traditional
and contemporaneous medicine:

Antifungi (Candida albicans; Estafilococus etc.)
Antidiabetic (Diabetes mellitus)
Antilipidemic
Hepatoprotective
Hearth anticongestive
Anticancer
Antidepressive
Neuroprotector
Anti-HIV
Biosynthesis of
berberine from two
molecules of L-tyrosine.
SAM, S-adenosylmethionine
SAH, S-adenosylhomocystine
Berberis wilsoniae
Berberis vulgaris
Berberis sp.
Figure 24-38. (S)-Reticuline has been called the chemical chamaleon. Depending on how the molecule is
twisted and turned before undergoing enzymatic oxidation, a vast array of tetrahydrobenzylisoquinoline-
derived alkaloids of remarkably different structures can be formed
END OF ALKALOIDS
PHENOLIC COMPOUNDS
PHENOLIC COMPOUNDS
Phenolic compounds (or simply phenolics)
are metabolites that contain a phenol group: a
hydroxyl functional group on an aromatic ring.
Plant phenolics are a chemically heterogenous group of nearly 10,000
individual compounds.

Some phenolics are only soluble in organic solvents, some are water-soluble
carboxylic acids and glycosides, and others are large, insoluble polymers.

Phenolics play a variety of roles in the plant. Many serve as defenses
against hervibores and pathogens. Others function in mechanical support; in
attracting pollinators and fruit dispersers; in absorving harmful UV radiation,
or in reducing the growth of nearby competing plants.
Figure 24.45

Phenylpropanoid and
phenylpropanoid-acetate
skeletons and representative
plant compounds based on
those structures
Figure 24.46 The aromatic amino acids phanylalanine and
tyrosine are derivatives of the shikimic-chorismic acid pathway
Figure 24.47
The shikimate-derived skeleton
(A) forms the core of gallic acid
(B), a component of hydrolyzable
tannins, includind castalagin (C)
from chestnut (D)
Phenylalanine is an intermediate in the biosynthesis
of most plant phenolics.
From a metabolic point of view, plant phenolics constitute a heterogeneous group
of metabolites as they are synthesized by different routes.

Two basic pathways are involved (Figure 13.6):
(1) the shikimic acid pathway; and (2) the malonic acid pathway:

(1) participates in the biosynthesis of most plant phenolics using shikimic acid as
an intermediate ;
(2) although an important source of phenolic secondary metabolites in fungi and
bacteria, is of less importance in higher plants.

The shikimic acid pathway, converts simple carbohydrate precursors derived from
glycolysis and the pentose phosphate pathway into the three aromatic amino acids:
phenylalanine, tyrosine, and tryptophane.

The shikimic acid pathway is present in plants, fungi and bacteria but is not found
in animals. Animals have no way to synthesize phenylalanine, tyrosine, and
tryptophane, which are therefore essential nutrients in animal diets.
The most abundant classes of phenolic secondary compounds
in plants are derived from phenylalanine via phenylalanine
ammonia lyase (PAL) which eliminates an ammonia molecule
to form cinnamic acid (Figure 13.7).

PAL is situated at a branch point between primary and
secondary metabolites, being an important regulatory enzyme
whose activity is increased by environmental factors as low
nutrient levels, light (through its effect on phytochromes), and
fungal infection.

PAL is encoded by multiple genes, some of which are
expressed only in specific tissues and other ones only under
certain environmental conditions.

The point of control of PAL appears to be the initiation of
transcription which is triggered by environmental factors that,
thereby stimulates the synthesis of phenolic compounds.
Figure 13.7 Outline of phenolic biosynthesis from phenylalanine onward. The formation
of many plant phenolics, including simple phenylpropanoids, coumarins, benzoic acid
derivatives, lignin, anthocyanins, isoflavones, condensed tannins, and other flavonoids,
begin with phenylalanine.
Reactions subsequent to that catalyzed by PAL lead to the
addition of more hydroxyl groups and other substituents. The
metabolites trans-cinnamic acid, p-coumaric acid, and their
derivatives are simple phenolic compounds called
phenylpropanoids because they contain a benzene ring:
Phenylpropanoids are widespread in vascular
plants and appear to function in various capacities.
Their structures include:

(A) simple phenylpropanoids

(B) phenylpropanoid lactones (cyclic esters) called
coumarins

(C) Benzoic acid derivatives
(A) - simple phenylpropanoids, such as trans-
cynnamic acid, p-coumaric acid, and their
derivatives, such as caffeic acid which
have a basic phenylpropanoid carbon
skeleton (Figure 13.8A)


(B) - phenylpropanoid lactones (cyclic esters)
called coumarins, which also have a
phenylpropanoid carbon skeleton (Figure
13.8B)


(C) - Benzoic acid derivatives, which have a
carbon skeleton formed from
phenylpropanoids by the cleavage of two-
carbon fragment from the side chain
(Figure 13.8C)
Figure 13.8 Simple phenolic compounds (phenylpropanoids) play a variety of roles in plants.
(A) Caffeic acid and ferulic acid may be released into the soil and inhibit the growth of neighboring
plants. (B) Psoralen is a furanocoumarin that exhibits phototoxicity to insect herbivores.
(C) Salicylic acid is a plant hormone that is involved in systemic resistence to plant pathogens.
UVs activates some simple phenolics
Certain coumarins, called furanocoumarins, have an
attached furan ring (Figure 13.8B). These compounds are
not toxic until they are activated by light. They become toxic
when they are activated to a high-energy electron state by
sunlight in the UV-A region of the spectrum (320-400 nm).

Activated furanocoumarins can insert themselves into the
double helix of DNA and bind to pyrimidine bases cytosine
and thymine, thus blocking transcription and repair and
leading eventually to cell dead.

Phototoxic furanocoumarins are especially abundant in
members of Apiaceae (Umbeliferae) family, includind celery
(aipo), parsnip (cenoura branca), and parsley (salsa).
Phototoxic furanocoumarins are especially abundant in
members of Apiaceae (Umbeliferae) family, includind celery
(aipo), parsnip (cenoura branca), and parsley (salsa).
Figure 24.73 A linear furanocoumarin, 8-methoxypsoralen, sensitizes human skin
to UV-A light. This compound, present in external tissues of Heracleum species,
causes severe blistering on skin contact followed by exposiure to UV-irradiation
Figure 24.72
Structures of (A)
coumarin (from clover),
and (B) a synthetic
coumarin, the
rodenticide Warfarin
Figure 24.74
Structures of the linear
furanocoumarin psoralen,
the angular furano-
coumarin angelicin, the
pyranocoumarin seselicin,
and the pyrone
substituted coumarin 4-
hydroxycoumarin.
The release of phenolics in the soil
may limit the growth of other plants
Simple phenylpropanoids and benzoic acid
derivatives are frequently cited as having allelopatic
activity.

Compounds such as caffeic acid and ferulic acid
are found in soil in appreciable amounts and have
been shown in laboratory experiments to inhibit the
germination and growth of many plants.

Such compounds can be released from leaves,
roots and decaing litter.
Lignin is a highly complex phenolic macromolecule
Lignin is a highly branched polymer of
phenylpropanoid groups. It plays both primary and
secondary roles in plants. Its precise structure is
not known as this polymer is covalently linked to
celulose and other polysaccarides of the cell wall.

Lignin is generally formed from three phenylpropanoid
alcohols: coniferyl, coumaryl, and sinapyl alcohols, all of which
are synthesized from phenylalanine via various cinnamic acid
derivatives.

The proportions of the three phenylpropanoid alcohols in lignin
vary among the species, plant organs, and even layers in a
single cell wall.

Flavonoids
There are four major groups of flavonoids
The flavonoids are one of the largest classes of plant
phenolics. The basic carbon skeleton of a flavonoid contais
15 carbon atoms arranged in two aromatic rings conected by
a three-carbon bridge:
The flavonoid structure results from
two separate biosynthetic pathways:
the shikimic acid pathway, and the
malonic acid pathway (Figure 13.9).

Flavonoids are classified primarily on
the basis of the degree of oxidation
of the three-carbon bridge. The four
major groups of flavonoids are:
the anthocyanins, the flavones, the
flavonols, and the isoflavones.
Anthocyanins are colored flavonoids that attract animals.
Anthocyanins are the most widespread group of pigmented
flavonoids, being responsible for most of the red, pink, purple,
and blue colors observed in flowers and fruits.
Anthocyanins are glycosides that can have various sugars at
position 3 (Figure 13.10A) and sometimes elsewhere. Without
their sugars anthocyanins are known as anthocyanidins
(Figure 13.10B).
Figure 13.10 The structures of anthocyanins (A) and
anthocyanidins (B). The colors of anthocyanidins
depend in part on the substituents attached to the ring B.
An increase in the number of hydroxyl groups shifts
absorption to a longer wavelength and gives a bluer
color. Replacement of a hydroxyl group with a methoxyl
group (-OCH) shifts absorption to a slightly shorter
wavelength, resulting in redder color.
Anthocyanin color is influenced by many factors, including the number of
hydroxyl and methoxyl groups in ring B of the anthocyanidin, the presence
of aromatic acids esterified in the main skeleton, and the pH of the cell
vacuole in which the anthocyanins are stored.

Anthocyanins may also exist in supramolecular complexes along with
chelated metal ions and flavone copigments.

The most common anthocyanidins and their colors are shown in Table 13.1.
Figure 24.66
Selected anrhocyanin pigments: pelargonidin, cyanidine, and delphinidin,
from geranium, rose and larkspur, respectively
Flavones and flavonols may protect against damage by UV light
Flavones and flavonols are other groups of flavonoids found in flowers.

These flavonoids absorb light at shorter wavelengths than do anthocyanins
what make them not visible to the human eye.
However, insects such as bees, which see farther into the ultraviolet range of
the spectrum than humans do, may respond to flavones and flavonols as
visual attractant cues (Figure 13.11).
Figure 13.11 Black-eyed Susan (Rudbeckia sp.) as seen by
humans (A) and as it might appear to honeybees (B).
(A) To humans, the flowers have yellow rays and a brown
central disc.
(B) To bees, the tips of the rays appear light yellow, the inner
portion of the rays dark yellow and the central disk black.

UV-absorving flavonols are found in the inner parts of the rays,
but not in the tips. The distribution of flavonols in the rays
creates a bulls-eye pattern visible to honeybees, which
presumably helps them locate pollen and nectar.

Special lighting was used to simulate the spectral sensitivity of the honeybee visual system.
Flavonols in a flower often form symmetric patterns of strips,
spots or concentric circles called nectar guides, which are
thought to help indicate the location of pollen and nectar.

Flavones and flavonols are not restricted to flowers; they are also present
in the leaves of all green plants. These two classes of flavonoids protect
cells from excessive UV-B radiation (280-320 nm) because they accumulate
in the epidermal layers of leaves and stems and absorb light strongly in the
UV-B region while allowing the visible wavelengths (PAR) to pass through
uninterrupted.

Exposure of plants to increased UV-B light has been demonstrated to
increase the synthesis of flavones and flavonols. UV-B radiation is known
to induce mutations in the DNA as well as oxidative stress, which has
potential to damage cellular macromolecules.

When secreted into the soil by legume root, these flavonoids, mediate the
interaction of legumes and rhizobacteria, their nitrogen-fixing symbionts.

Flavonoids also play a regulatory role in plant development as modulators
of polar auxin transport.

Specific flavonoids can
functin to protect plants
against UV-B irradiation, a
role sometimes ascribed
to kaempferol.






Figure 24.67
Kaempferol, a UV-B protectant,
is present in many plants such
as soybean (Glycine max).
Isoflavonoids have widespread pharmacological activity
The isoflavones (isoflavonoids) are a group of flavonoids
in which the position of one aromatic ring (ring B) is shifted
(figure 13.7).

Some Isoflavones have anti-estrogenic effects, as their
ring system has a three-dimensional structure similar to
that of steroids, allowing these substances to bind to
estrogen receptors.

Isoflavones may also be responsible for the anticancer
benefits of foods prepared from soybeans.

Isoflavones have also role as phytoalexins antimicrobial
compounds synthesized in response to bacterial or fungal
infection.

Isoflavonoids, which are found mostly in legumes, have
several different biological activities, namely insecticide,
pesticide and piscicide.
Figure 24.69
Flavonoids perform diverse functions in alfalfa (Medicago sativa).
The flavonoids apigenin and luteolin function as signaling molecules that induce
Nod gene expression in compatible Thizobium bacteria, facilitating the development
of nitrogen-fixing root nodules. The phytoalexin isoflavonoid medicarpin participates
in inducible plant defence.
The flavonoids apigenin and luteolin serve as signal molecules in legume
rhizobium bacteria interactions, facititing nitrogen fixation (Figure 24.69).
In a related function,
isoflavonoids are
involved in inducible
defense against fungal
attack in alfalfa (e.g.,
medicarpine; Fig. 24.69)
and other species.
Various flavonoids have been also studied extensively from the perspectives of
health protection and pharmacological utility, for which mammalian enzyme
systems have been used to assess flavonoid activity.

Flavonoids have been analyzed as modulators of immune and
inflammatory responses, for their impact as smooth muscle function,
and as anticancer, antiviral, antitoxic, and hepatoprotective agents.
There is considerable current interest in the use of isoflavonoids in
cancer prevention.
Dietary consumption of the
isoflavonoids daidzein and
genistein, which are present in
soybeans, is thought to reduce
substantially the incidence of
breast and prostate cancer in
humans.
Tannins deter feeding by herbivores
Tannins are a second category of plant phenolic polymers
(besides lignin) with defensive properties.

Tannins bind the collagen proteins of animal hides, thereby
increasing their resistence to heat, water and microbes,
converting the raw hide into leather in a process known as
tanning.

There are two categories of tannins:
(A) Condensed tannins
(B) - Hydrolizable tannins
(A) Condensed tannins
Are compounds formed by polymerization of flavonoid units (Figure 12.12A)
They are common constituents of woody plants.

Because condensed tannins can often be hydrolyzed into anthocyanidins by
treatment of strong acids, they are sometimes called pro-anthocyanidins.
(B) - Hydrolizable tannins
Are heterogenous polymers containing phenolic acids, especially gallic
acid, and simple sugars (Figure 13.12B).

Hydrolizable tannins are smaller than condensed tannins and may be
hydrolyzed more easily; only diluted acid is needed. Most tannins have
molecular masses between 600 and 3000 Da.
Figure 13.12 Structure of two types of tannins.

(A) The general structure of a condensed tannin, where n represents
usually 1 to 10. There may also be a third hydroxyl group on ring B.

(B) The hydrolyzable tannin from Rhus semialata consists of glucose and
eight molecules of gallic acid.
Condensed tanins such as the proanthocyanidins add a
distinct bitterness or astringency to the taste of certain plant
tissues and function as antifeedants (Figure 24.68).
Figure 24.68. Red sorghum produces proanthocyanidin antifeedant compounds-
condensed tannins, which deter birds from feeding on the seed. White sorgum, which
is deficient in these compounds, is rapidly consumed by birds. Similar compounds are
present in the heartwood of Douglas fir (not shown).
Figure 24.47
The shikimate-derived skeleton
(A) forms the core of gallic acid
(B), a component of hydrolyzable
tannins, includind castalagin (C)
from chestnut (D)
Tannins are general toxins that can reduce the growth and
survivor of many herbivores, when included in the animal diets.

Tannins are general antifeedants to a great variety of animals.
Unripe fruits, for instance, frequently have very high tannin
levels, which deter feeding on the fruits until their seeds are
mature enough for dispersal.

Humans often prefer a certain level of astringency in tannin-
containing foods, such as apples, blackberries, tea, and grapes.

The tannins in red wine have been shown to block the formation
of endothelin-1, a signaling molecule that makes boold vessels
constrict.

This effect of wine tannins may account for the often-touted health benefits of red
wine, especially the reduction in the risk of hearth disease associated with moderate
red wine consumption.
Other phenolic compound with such effect is the stilbene phenylpropanoid resveratrol

Moderate amounts of specific tannins may have health effects
for humans, but the defensive properties of most tanins are
due to their toxicity, which is generally attributed to their ability
to bind proteins nonspecifically.


Plant tannins bind proteins in the guts of hebivores by forming
hydrogen bonds between their hydroxyl groups and
electronegative sites on the proteins (Figure 13.13A).


Tannins and other phenolics can also bind to dietary proteins
in a covalent fashion (Figure 13.13B).

Figure 13.13 Proposed
mechanisms for the interaction
of tannins with proteins.


(A) Hydrogen bonds may form
between the phenolic
hydroxyl groups of the
tannin and electronegative
sites on the protein.

(B) Phenolic hydroxyl groups
may bind covalently to
proteins following activation
by oxidative enzymes, such
as polyphenol oxidase.
The foliage of many plants contains enzymes that oxidize
phenolics to their quinone forms in the guts of herbivores.
Quinones are highly reactive electrophilic molecules that
readily react with the nucleophilic NH and SH group of
proteins.

Therefore, tannins can have a negative effect on herbivore
protein nutrition, besides inactivation o digestive enzymes and
creation of complex aggregates tannins-plant protein difficult to
digest.

Proline has a high afffinity to tannins. Some animals are
tolerant to tanins due to their capacity in secreting salivary
proteins rich in proline content (25 to 45%).
END