Plant Physiology, Sixth Edition

Plant Physiology, Sixth Edition
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APPENDIX 3
APPENDICES Hormone Biosynthetic Pathways
HOME Despite their diverse chemical structures, most of the known plant
SITE SEARCH hormones are derived from three main types of metabolic precursors:
amino acids, isoprenoid compounds, and lipids (Figure A3.1). The amino
About the Book acids tryptophan and methionine serve as precursors for IAA (indole-3-
Errata & Updates acetic acid) and ethylene, respectively. The isoprenoid pathway gives rise
to five classes of plant hormones: cytokinins, brassinosteroids, gibberellins,
abscisic acid, and strigolactones. And finally, jasmonic acid is synthesized
from a lipid precursor.
Figure A3.1 Three categories of hormones based on their

biosynthetic precursors.
The major intermediates and subcellular locations of most of the

biosynthetic pathways have now been elucidated, although new details
continue to emerge. Of particular interest to researchers is how the
individual hormone biosynthetic pathways share common intermediates and
where synthesis of one hormone is regulated by another hormone; such
regulation and interactions have profound effects on plant development.
Here we provide some additional details about the biosynthetic pathways

beyond the treatments in the textbook, for those students who wish to
further their understanding of plant hormone biochemistry. For some of the
hormones, such as auxin and ethylene, we discuss the biosynthetic
pathways and their regulation in greater depth than in the textbook, while
for others, such as gibberellin, ABA, and brassinolide, we present diagrams
of more complete versions of the pathways.
I. Hormones Derived from Amino Acids
http://6e.plantphys.net/app03.html[12-May-18 9:32:52 PM]

Auxins
Indole-3-acetic acid (IAA) is structurally related to the amino acid
tryptophan, and plants convert tryptophan to IAA by several pathways. The
tryptophan biosynthetic pathway in plants is shown in Figure A3.2. Much
of what we know about the biosynthesis of IAA has been learned from
studies of Arabidopsis mutants.
Figure A3.2 The tryptophan biosynthetic pathway provides

precursors for IAA biosynthesis. In most plants, tryptophan
synthesis takes place in the chloroplast. The branchpoint
precursor for tryptophan-independent IAA biosynthesis is indole.
Indole pyruvic acid is thought to be an intermediate in the
pathway.
The indole-3-pyruvic acid (IPyA) pathway (Figure A3.3) is the principal

IAA biosynthetic pathway in plants (Ljung 2013). In Arabidopsis, IPyA is
formed from tryptophan by tryptophan aminotransferase (TAA1, TAR). IPyA
is then converted to IAA by the YUCCA flavin monooxygenases (Dai et al.
2013).

Figure A3.3 Tryptophan-dependent pathways of IAA

biosynthesis in Arabidopsis. Dashed arrows indicate that neither
a gene nor an enzyme activity has been identified in
Arabidopsis. TRP, tryptophan; IAM, indole-3-acetamide; IPyA,
indole-3-pyruvic acid; IAOx, indole-3-acetaldoxime; IG, indole-
3-methylglucosinolate; TRM, tryptamine; IAN, indole-3-
acetonitrile. (After Normanly 2010.)
In Arabidopsis and other members of the Brassicaceae that produce indole

glucosinolate defense compounds (see textbook Chapter 23), indole-3-
acetaldoxime (IAOx) is synthesized from tryptophan by cytochrome P450
enzymes (see Figure A3.3). IAOx is then converted to indole glucosinolates.
When the indole glucosinolate pathway is genetically interrupted, as in
superroot mutants, IAOx is converted by an unknown, low affinity process
to indole-3-acetonitrile (IAN). In response to biotic stresses, myrosinases
hydrolyze sugar conjugates from indole glucosinolates to release IAN,
which can then be converted to IAA by nitrilases (see Figure A3.3). Some
evidence of similar activity has been observed in species outside of the
Brassicaceae, but this activity is no longer thought to represent a major
IAA biosynthetic pathway.
In some bacteria that generate IAA during their interactions with plant
roots, indole-3-acetamide (IAM) is generated as an intermediate. Low
levels of IAM have been shown to be present in Arabidopsis, maize, rice,
and tobacco (Sugawara et al. 2009; Novák et al. 2012), and plant IAM
hydrolases have been shown to convert exogenously applied IAM to IAA.
However, there is no evidence that this is a major IAA biosynthetic
pathway.
A tryptophan-independent pathway of IAA synthesis, starting with indole,

has been partially characterized in several plant species using precursors
labeled with stable isotopes. IAN and IPyA are possible intermediates.
However, the enzymatic processes involved are unknown and the relevance

of this proposed pathway to normal growth processes has not been

established.
As described in textbook Chapter 15. IAA can be temporarily inactivated by

conjugation to amino acids or catabolized via conjugation to sugars and
oxidation by dioxygenase for auxin (DAO) and other oxygenases.
Ethylene
In vivo experiments have shown that plant tissues convert l-
[ 14 C]methionine to [ 14 C]ethylene, and that the ethylene is derived from
carbons 3 and 4 of methionine (Figure A3.4). The CH 3—S group of
methionine is recycled via the Yang cycle in the cytosol. Without this
recycling, the amount of reduced sulfur present would limit the available
methionine and the synthesis of ethylene. S-adenosylmethionine (AdoMet),
which is synthesized from methionine and adenosine triphosphate (ATP), is
an intermediate in the ethylene biosynthetic pathway, and the immediate
precursor of ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC)
(Figure A3.4).
Figure A3.4 Ethylene biosynthetic pathway and the Yang cycle.

The amino acid methionine is the precursor of ethylene. The
rate-limiting step in the pathway is usually the conversion of
AdoMet to ACC, which is catalyzed by the enzyme ACC
synthase. The last step in the pathway, the conversion of ACC
to ethylene, requires oxygen and is catalyzed by the enzyme
ACC oxidase. The CH3—S group of methionine is recycled via
the Yang cycle and thus conserved for continued synthesis.
Besides being converted to ethylene, ACC can be conjugated to
N-malonyl ACC. AOA, aminooxyacetic acid; AVG, aminoethoxy-

vinylglycine. (After McKeon et al. 1995.)
The role of ACC became evident in experiments in which plants were

treated with [ 14 C]methionine. Under anaerobic conditions, ethylene was
not produced from the [ 14 C]methionine, and 14 C-labeled ACC accumulated
in the tissue. On exposure to oxygen, however, ethylene production
surged. The labeled ACC was rapidly converted to ethylene in the presence
of oxygen by various plant tissues, suggesting that ACC is the immediate
precursor of ethylene in higher plants and that oxygen is required for the
conversion.
In general, when ACC is supplied exogenously to plant tissues, ethylene

production increases substantially. This observation indicates that the
synthesis of ACC is usually the biosynthetic step that limits ethylene
production in plant tissues. Exceptions include tissues with high rates of
ethylene synthesis, such as ripening fruits (see below).
ACC synthase (ACS), the enzyme that catalyzes the conversion of AdoMet
to ACC (see Figure A3.4), has been characterized in many types of tissues
of various plants. ACC synthase is an unstable, cytosolic enzyme. Its level
is regulated by environmental and internal factors, such as wounding,
drought stress, flooding, and auxin. Because ACC synthase is present in
such low amounts in plant tissues (0.0001% of the total protein of ripe
tomato) and is very unstable, it is difficult to purify the enzyme for
biochemical analysis.
ACC synthase is a member of a subfamily of carbon-sulfur lyases encoded

by a multigene family that is differentially regulated by various inducers of
ethylene biosynthesis. In tomato, for example, there are at least ten ACC
synthase genes, different subsets of which are induced by auxin, wounding,
and/or fruit ripening. The Arabidopsis genome contains nine ACC synthase
genes. An analysis of purified proteins encoded by eight of these genes
revealed a diversity of kinetic properties (for example, various affinities for
the substrate AdoMet), suggesting that these isoforms might be optimized
for different roles in various tissues and cell types (Yamagami et al. 2003).
The deduced crystal structure of ACC synthase from both apple and tomato
reveals a dimeric protein with shared active sites, similar to
aminotransferases (Capitani et al. 1999; Huai et al. 2001).
ACC oxidase catalyzes the last step in ethylene biosynthesis: the

conversion of ACC to ethylene in the cytosol (see Figure A3.4). In tissues
that show high rates of ethylene production, such as ripening fruit, ACC
oxidase activity can be the rate-limiting step in ethylene biosynthesis. Like
ACC synthase, ACC oxidase is encoded by a family of differentially
regulated genes. For example, in ripening tomato fruits and senescing
petunia flowers, the mRNA levels of a subset of ACC oxidase genes are
highly elevated. Both soluble and membrane-associated isoforms have been
identified. Differential subcellular compartmentation of ACC oxidase
isoforms suggests that the soluble and membrane-associated isoforms may
have different substrates. One hypothesis is that the membrane-associated
ACC oxidases, which are localized at the ER, may also regulate auxin
homeostasis in the ER, because ACC oxidase can oxidize auxin in vitro.
The deduced amino acid sequences of ACC oxidases revealed that these
enzymes belong to the Fe 2+ /ascorbate oxidase superfamily. This sequence
similarity suggested that ACC oxidase might require Fe 2+ and ascorbate for

activity—a requirement that has been confirmed by biochemical analysis of

the protein. The requirement of ACC oxidase for cofactors presumably
explains why purification of this enzyme eluded researchers for so many
years. A soluble ACC oxidase was crystalized as a tetramer, but it is still
unknown if the soluble isoform functions as a monomer or oligomer.
Experimental evidence indicates that the membrane-associated ACC
oxidase functions as a monomer.
Researchers have studied the catabolism of ethylene by supplying 14 C2H 4

to plant tissues and tracing the radioactive compounds produced. Carbon
dioxide, ethylene oxide, ethylene glycol, and the glucose conjugate of
ethylene glycol have been identified as metabolic breakdown products.
However, because certain cyclic olefin compounds, such as 1,4-
cyclohexadiene, have been shown to block ethylene breakdown without
inhibiting ethylene action, ethylene catabolism does not appear to play a
significant role in regulating the level of the hormone (Raskin and Beyer
1989).
Not all the ACC found in a tissue is converted to ethylene. ACC can also be
converted to a conjugated form, N-malonyl ACC (see Figure A3.4), which
does not appear to break down and accumulates in the tissue, primarily in
the vacuole. A second, minor conjugated form of ACC, 1-(γ-L-
glutamylamino)cyclopropane-1-carboxylic acid (GACC), has also been
identified. The conjugation of ACC may play an important role in the control
of ethylene biosynthesis, in a manner analogous to the conjugation of
auxin and cytokinin.
Several species of soil bacteria express an enzyme called ACC deaminase

that hydrolyzes ACC to ammonia and α-ketobutyrate (Glick 2005). These
bacteria can promote plant growth by sequestering and cleaving ACC made
and excreted by plants, thereby lowering the level of ethylene to which the
plants are exposed. ACC deaminase has also been expressed in transgenic
plants to lower the level of ethylene produced.
Salicylic acid
As described in Chapter 24, Salicylic acid (SA) is a major stress response
signaling molecule that is increasingly characterized as a hormone (see
textbook Chapter 15). SA is synthesized from phenylalanine in a pathway
that starts with conversion to trans-cinnamic acid by phenylalanine
ammonia lyase followed by conversion to salicylic acid via a benzoate
intermediate. SA can be conjugated to glucose or aspartate, and is active
in volatile form when methylated to form methyl salicylate.
II. Hormones Synthesized via Isoprenoid

Pathways
Isoprenoid compounds (also referred to as terpenoids) are synthesized
from isoprene subunits via the mevonolate and methylerythritol phosphate
pathways in the chloroplasts and cytosol of plants. Isoprenoid precursors
are used to synthesize four major plant hormones.
Cytokinins
As described in textbook Chapter 15, the first committed step in cytokinin

biosynthesis is the transfer of the isopentenyl (iP) group of dimethylallyl

diphosphate (DMAPP) to an adenosine moiety by an isopentenyl transferase
(IPT) (Figure A3.5). There are nine different IPT genes in Arabidopsis,
seven of which form a unique group or clade not found in animals. This
group of IPTs function in cytokinin biosynthesis. The other two IPT genes
encode encoding enzymes used in tRNA modification (tRNA-IPTs) that are
similar to those found in animals that produce tRNA cytokinins (Kakimoto
2001; Takei et al. 2001). The possibility that free cytokinins derived from
tRNA are functionally important in plants has been explored extensively,
and has now been largely discounted.
Figure A3.5 Simplified biosynthetic pathway for cytokinin

biosynthesis. The first committed step in cytokinin biosynthesis
is the addition of the isopentenyl (iP) side chain from DMAPP
(dimethylallyl diphosphate) to an adenosine moiety. The
products of this reaction (iPRDP or iPRTP) are converted to
zeatin by a cytochrome P450 monooxygenase (CPY735A).
Dihydrozeatin (DHZ) cytokinins are made from the various
forms of trans-zeatin by an unknown enzyme (not shown). The

ribotide and riboside forms of trans-zeatin can be

interconverted, and free trans-zeatin can be formed from the
riboside by enzymes of the general purine metabolism. In
addition, the LONELY GUY (LOG) enzyme can convert zeatin
ribotide (but only the monophosphate) directly to the free base
form. Note that iP and DHZ ribotides can also be converted to
the corresponding ribosides and free base forms in a similar
manner (not shown). Inset: The pathway for cytokinin
biosynthesis via Agrobacterium Ipt. The plant and bacterial Ipt
enzymes differ in the adenosine substrate used and the side
chain donor; the plant enzyme appears to utilize both ADP and
ATP and couples this to DMAPP, and the bacterial enzyme
utilizes AMP and couples this to HMBDP (1-hydroxy-2-methyl-2-
(E)-butenyl 4-diphosphate). Note that the product of the
Agrobacterium Ipt reaction is a zeatin ribotide.
The proteins encoded by the Arabidopsis IPT genes were expressed in E.

coli and analyzed. It was found that with the exception of the two genes
most closely related to the animal and bacterial tRNA-IPT genes, these
genes encoded proteins capable of synthesizing free cytokinins. Unlike
Agrobacterium Ipt (note that the names for bacterial proteins are
capitalized without italics), however, the Arabidopsis enzymes utilize
adenosine triphosphate (ATP) and adenosine diphosphate (ADP)
preferentially over AMP, and use DMAPP as the source of the side chain
rather than HMBDP (see Figure A3.5).
The chloroplast methylerythritol phosphate pathway is the primary source

of the DMAPP used in cytokinin biosynthesis by plant IPT enzymes. This
pathway occurs in plastids, which is where the majority of IPT enzymes are
localized in plants. Thus, the primary site of cytokinin biosynthesis in plants
is in the plastids. However, in Arabidopsis, at least one IPT protein, IPT3,
can be modified by the addition of a farnesyl moiety (Galichet et al. 2008).
Farnesylation is the addition of a hydrophobic farnesyl group (a long-chain
lipid molecule made from isoprene subunits) to C-terminal cysteine(s) of
the target protein, which can alter its subcellular localization, often
targeting the protein to a membrane. The farnesylation of IPT3 directs it to
the nucleus, rather than to the plastids, where the non-farnesylated IPT3
protein is localized. Furthermore, the IPT4 protein is found in the
cytoplasm, and the IPT7 protein is found in mitochondria. Thus, the plastids
are the primary sites of cytokinin biosynthesis, but not the only sites.
The expression patterns of the IPT genes indicate that cytokinin is produced
at multiple locations throughout the plant (Miyawaki et al. 2004). The
expression of a subset of IPT genes is down-regulated by cytokinin,
indicating that cytokinin exerts a negative feedback control on its own
biosynthesis. IPT gene expression is also affected by other regulatory
inputs, including auxin, nitrate, and the meristem identity gene
SHOOTMERISTEMLESS (STM).
The immediate products of the IPT reaction are iP-ribotides. The isoprene
side chain of iP-ribotides is subsequently trans-hydroxylated by the P450
monooxygenases CYP735A1 and CYP735A2 to yield zeatin ribotides (Takei
et al. 2004). Cytokinin nucleotides can be converted to their most active
free base forms via dephosphorylation and deribosylation. Such
interconversions may involve enzymes common to purine metabolism. In
addition to these enzymes, the monophosphate forms of cytokinin ribotides

can be directly converted to the free base forms by the

phosphoribohydrolase LONELY GUY (LOG) (see Figure A3.5), which was
identified in a rice mutant that displayed shoot meristem defects (Kurakawa
et al. 2007). LOG expression is localized to the tip of shoot meristems and
the enzyme likely fine-tunes the spatial distribution of bioactive cytokinins
to regulate meristem activity. Zeatin can be oxidized; it can also be
conjugated to form O-glucosides or N-glucosides. These reactions are
shown in Figures A3.6 and A3.7.
Figure A3.6 Cytokinin oxidase irreversibly degrades some

cytokinins.

Figure A3.7 Cytokinins can be conjugated to various molecules

at the positions shown. The conjugates shown in red are
irreversible and result in an inactive cytokinin. The conjugates
shown in blue cause inactivation of the corresponding cytokinin,
but are reversible. The conjugates shown in green are active
cytokinins, albeit less so than the corresponding free bases. The
ribose moieties are reversible, but the methylthiol modification
appears to be irreversible. (B) Example of the conjugation of
trans-zeatin to glucose at the side chain to form an O-linked
conjugate (top) and on the adenine ring to form an N-linked
conjugate (bottom).
Brassinosteroids
Our knowledge of the brassinosteroid (BR) biosynthetic pathway is the
result of a combination of genetic and biochemical analyses (Fujioka and
Yokota 2003). For the biochemical studies, periwinkle (Catharanthus
roseus) cell cultures were used, as they produce BRs in relatively high
amounts. Radiolabeled BR intermediates were used in feeding experiments,
and their metabolic derivatives were identified by gas chromatography–
mass spectroscopy. Coupling this type of analysis to genetic studies of BR-
deficient mutants in Arabidopsis, tomato, and other species has allowed the

identification of the complete biosynthetic pathways.
A simplified version of the BR biosynthetic pathway, starting with the sterol

progenitor campesterol, is shown in Figure A3.8. Campesterol is first
converted to campestanol in several steps. Campestanol is then converted
to castasterone through one of two pathways called the early- and late C-6
oxidation pathways, after which castasterone is converted to brassinolide
The early and the late C-6 oxidation pathways coexist and can be linked at
different points in Arabidopsis, pea, and rice, although the early C-6
oxidation branch is not detected in some plant species (Fujioka and Yokota
2003). An alternative, campestanol-independent route has also been
described. The presence of several linked pathways increases the
complexity of BR biosynthesis and may provide an advantage under
different physiological conditions, such as various types of stress.
Figure A3.8 Simplified pathways for BL biosynthesis and

catabolism. The precursor for BL biosynthesis is campesterol.

The sequence of biosynthetic events is represented by black
arrowheads. Solid arrows indicate single reactions; dashed
arrows represent multiple reactions. As shown, castasterone, the
immediate precursor of BL, can be synthesized from two parallel
pathways: the early and the late C-6 oxidation pathways. In the
early C-6 oxidation pathway, oxidation at C-6 of the B ring
occurs before the addition of vicinal hydroxyls at C-22 and C-23
of the side chain (refer to BL structure in textbook Figure 15.24.
In the late C-6 oxidation pathway, C-6 is oxidized after the
introduction of hydroxyls at the side chain and C-2 of the A
ring. Both the early and the late pathways may be linked at
various points, creating a biosynthetic network rather than a
linear pathway. The Arabidopsis enzymes that catalyze the
different steps are indicated.
The main BR biosynthesis genes have been isolated and characterized.

DET2 encodes a protein with high amino acid sequence identity to
mammalian steroid 5α-reductases (Li et al. 1996). Mammalian steroid 5α-
reductases catalyze an NADPH-dependent conversion of testosterone to
dihydrotestosterone, a key step in steroid metabolism that is essential for
normal embryonic development of male external genitalia and the prostate.
All other known genes involved in the conversion of campestanol to BL
encode cytochrome P450 monooxygenases (CYPs).
The amount of active BRs is also regulated by metabolic processes that

inactivate BL. Several types of reactions result in BL inactivation, including
epimerization, oxidation, hydroxylation, sulfonation, and conjugation to
glucose or lipids (Fujioka and Yokota 2003). Our limited knowledge in this
area is based on experiments in which plants are fed radiolabeled BRs, and
the resulting labeled products are identified and endogenous metabolites
analyzed; however, the relevance of these compounds to the BR pathway
in the plant is still not clear. Two types of BR catabolic enzymes have been
described in planta, along with their catalyzed reactions (see Figure A3.8).
One is the CYP protein BAS1, which has a steroid 26-hydroxylase activity
and leads to the accumulation of an inactive 26-hydroxy-BL (Neff et al.
1999). The second type of enzyme belongs to the family of UDP-
glycosyltransferases (UGTs) that also regulate glucosylation of multiple
plant hormones (Husar et al. 2011; Poppenberger et al. 2005).
Overexpression of the aforementioned catabolic genes in plants causes a
BR-deficient phenotype.
Gibberellins
Gibberellins (GAs) are terpenoid compounds produced in many parts of a
plant, often in cells that are undergoing division and/or elongation. For
example, studies using a reporter version of a gene encoding an enzyme in
the GA pathway showed it to be expressed in immature seeds, shoot
apices, root tips, and anthers of Arabidopsis plants (Silverstone et al.
1997), providing evidence that GAs are synthesized in these locations.
GAs, like all terpenoid compounds, are made from five-carbon isoprenoid
building blocks. GAs are diterpenoids that are formed from four
isoprenoid units. The GA biosynthetic pathway can be divided into three
stages, each residing in a different cellular compartment: plastid, ER, or
cytosol (Figure A3.9).

Figure A3.9 The three stages of GA biosynthesis. In stage 1,

geranylgeranyl diphosphate (GGPP) is converted to ent-kaurene.
In stage 2, ent-kaurene is converted to GA12. In many plants,
GA12 is converted to GA53 by hydroxylation at C-13. In stage 3
in the cytosol, GA12 or GA53 is converted, via parallel pathways,
to other GAs. This conversion proceeds with a series of
oxidations at C-20, resulting in the eventual loss of C-20 and
the formation of C 19-GAs. A 3β-hydroxylation reaction then
produces GA4 and GA1 as the bioactive GAs in each pathway. In
most plants the 13-hydroxylation pathway predominates,
although in Arabidopsis and some other plants, the non-13-OH-
pathway is the main pathway. OL, open lactone. See the table
for full names and subcellular locations of the enzymes.
In stage 1, which occurs in plastids, four isoprenoid units are assembled to

give a 20-carbon linear molecule, geranylgeranyl diphosphate (GGPP). In
addition to being a precursor of GAs, GGPP is an intermediate in the
synthesis of compounds that are important for photosynthesis, so chemicals
or mutations that block stage 1 kill plants. For the synthesis of GAs, GGPP

is converted into a tetracyclic compound, ent-kaurene, in two steps, which

are catalyzed by ent-copalyl-diphosphate synthase (CPS) and ent-kaurene
synthase (KS).
In stage 2, which occurs on the plastid envelope and in the endoplasmic

reticulum, ent-kaurene is converted, in a stepwise manner, to the first-
formed GA, GA 12 . Two important enzymes in this part of the pathway are
ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO). The
pathway to GA 12 is essentially the same in all plant species studied so far.
In stage 3, which occurs in the cytosol, GA 12 is converted, through a

series of oxidative reactions, first into other C20 -GAs, and then into C19 -
GAs, including the bioactive GA(s). There are two major stage 3 pathways.
They both have the same series of oxidative reactions, but all
intermediates in one pathway have a hydroxyl (-OH) group at C-13 (and so
it is called the 13-hydroxylation pathway), whereas intermediates in the
other pathway do not (and so it is referred to as the non-13-hydroxylation
pathway). The series of oxidative reactions occur in the A-ring, and are the
same in both stage 3 pathways.
In the following discussion the reactions leading to bioactive GA (GA4 in the

non-13-hydroxylation pathway and GA 1 in the 13-hydroxylation pathway)
are referred to as “biosynthesis.” Further metabolism of the bioactive GA is
referred to as “deactivation.”
Some enzymes in the GA pathway are highly regulated
Three enzymes in stage 3 of the pathway are discussed in detail because

they catalyze steps that are closely regulated. These are GA 20-oxidase
(GA20ox) and GA 3-oxidase (GA3ox), which catalyze the steps prior to
bioactive GA, and GA 2-oxidase (GA2ox), which is involved in GA
deactivation. All three of these enzymes are classified as dioxygenases.
GA 20-oxidase. This multifunctional enzyme catalyzes the three-step

oxidation of carbon atom-20 in GA 12 , producing GA 9 in the non-13-
hydroxylation pathway, and in GA 53 , producing GA 20 in the 13-
hydroxylation pathway. The sequential oxidation of C-20 proceeds from
CH 3 in GA 12 to CH 2OH, and then to CHO, before C-20 is eliminated,
yielding a C19 -GA (either GA 9 or GA 20 ). In most plants the GA 20-oxidase
is encoded by a small gene family, so there are several isoforms all
catalyzing the same sequence of reactions. The individual isoforms are
expressed in different parts of the plant and/or may be regulated differently
by external signals, such as day length. Because there is gene redundancy,
a mutation in any one of the GA20ox genes gives only partially dwarf
(semi-dwarf) plants.
GA 3-oxidase. This enzyme catalyzes the 3β-hydroxylation of GA 9, giving

GA 4, or of GA 20 , giving GA 1. This reaction is necessary for bioactivity, as
the 3β-OH group facilitates binding of the GA molecule into the pocket of
the GA receptor. In many plants, the GA 3-oxidase is encoded by a small
gene family. The dwarf character trait identified by Gregor Mendel that
distinguished tall and dwarf pea plants is a caused by a mutation in a gene
(LE) that encodes a GA 3-oxidase expressed in stems. A second GA 3-
oxidase, expressed in reproductive tissue, is needed for normal pod and
seed development. Nowadays, most cultivated pea plants are le mutants;
they have short stems but normal pod and seed size, allowing for
convenient harvesting and high yield.

GA 2-oxidase. Addition of a 2-OH group in β-orientation prevents GA

binding in the GA receptor pocket, so 2β-hydroxylation deactivates a GA. In
pea, a genotype identified because it grew taller than wild-type plants
contains a mutation in a gene termed SLENDER (SLN), which encodes a GA
2-oxidase. Seedlings of the sln mutant contain more GA 1 than wild-type
plants, because deactivation of the hormone is blocked.
Gibberellin regulates its own metabolism
Part of a plant’s response to bioactive GA is to depress GA biosynthesis and

stimulate deactivation, thereby preventing excessive stem elongation.
Depression of biosynthesis is achieved through down-regulation (inhibition
of expression) of some of the GA20ox and GA3ox genes. This effect of GA
on its own biosynthesis is termed negative feedback regulation.
Enhanced GA deactivation is also important for maintaining GA
homeostasis. This enhancement is achieved by up-regulating the
expression of some of the GA2ox genes encoding the enzyme that
deactivates GA. The ability of GA to promote the expression of genes
involved in its own deactivation is termed positive feed-forward
regulation.
GA1 and GA4 have intrinsic bioactivity for stem growth

Seminal studies with GA biosynthetic mutants (also referred to as GA-
deficient mutants) in the 1980s achieved two important goals. Not only did
they provide a way for the pathways of GA metabolism to be definitively
established, but these studies also determined that GA 1 is the major
bioactive GA for stem growth in pea and maize, and that its precursors
have no intrinsic biological activity.
LE and le are two alleles of the gene that encodes a GA 3-oxidase in pea. If
GA 20 is applied to the le mutant, it is not bioactive (the plants remain
dwarf), whereas GA 1 is bioactive, and rescues the mutant phenotype (the
plants grow tall). Gibberellin A 8 is also inactive. We can infer from this
information, and from knowledge of the GA metabolic pathway in pea
(GA20 → GA 1 → GA 8), that GA 20 is inactive unless it can be converted to
GA 1 within the plant, and that GA 1 has intrinsic bioactivity (Ingram et al.
1984).
A study of other pea mutants has confirmed that the height of pea plants is
directly correlated with the amount of endogenous GA 1. The na mutant of
pea, in which stage 2 is blocked, is almost completely devoid of GA 1. As a
consequence it achieves a stature of only about 1 cm at maturity. In
contrast, the seedlings of the sln mutant contain elevated levels of GA 1
because of impaired GA deactivation, and these mutant plants are actually
taller than the wild type.
In Arabidopsis and several members of the Cucurbitaceae (e.g., pumpkin

and cucumber), applied GA 1 has less biological activity than GA 4.
Therefore, in Arabidopsis and probably also in these cucurbits, GA 4 is the
main biologically active GA, and stem length is correlated with GA 4 levels.
Plant height can be manipulated by overexpressing or blocking the
expression of certain genes in the GA biosynthetic pathway, to achieve
optimal plant size. This approach was pioneered using Arabidopsis and is
now feasible in crop plants too.
Abscisic Acid

Abscisic acid (ABA) biosynthesis begins in chloroplasts and other plastids.

The complete pathway is depicted in Figure A3.10. Several ABA-deficient
mutants have been identified that have lesions at specific steps of the
pathway. These mutants exhibit abnormal phenotypes that can be
corrected by the application of exogenous ABA. For example, flacca (flc)
and sitiens (sit) are “wilty” mutants of tomato, in which the tendency of the
leaves to wilt (due to an inability to close their stomata) can be prevented
by the application of exogenous ABA. The aba mutants of Arabidopsis also
exhibit a “wilty” phenotype. These and other mutants have been useful in
elucidating the details of the pathway and in cloning the genes encoding
ABA biosynthetic enzymes (Wasilewska et al. 2008).
Figure A3.10 ABA biosynthesis and metabolism. In higher

plants, ABA is synthesized via the terpenoid pathway (see
textbook Chapter 13), as are cytokinins, brassinosteroids, and
gibberellins (see textbook Chapter 15. Some ABA-deficient
mutants that have been helpful in elucidating the pathway are
shown at the steps at which they are blocked. The pathways for
ABA catabolism include conjugation to form ABA-β-D-glucosyl
ester or oxidation to form phaseic acid and then dihydrophaseic
acid. NCED, 9-cis-epoxycarotenoid dioxygenase; ZEP, zeaxanthin
epoxidase. The pathway occurs in two compartments, (A)

plastid; (B) cytosol.
The pathway begins with isopentenyl diphosphate (IPP)—the biological

isoprene unit that is also a precursor of cytokinins, gibberellins, and
brassinosteroids—and leads to the synthesis of the C40 xanthophyll (i.e.,
oxygenated carotenoid) violaxanthin (see Figure A3.10). Synthesis of
violaxanthin is catalyzed by zeaxanthin epoxidase (ZEP), the enzyme
encoded by the ABA1 locus in Arabidopsis. This discovery provided
conclusive evidence that ABA synthesis occurs via the “indirect” or
carotenoid pathway, rather than by modification of a C15 isoprenoid, as in
the “direct pathway” of some phytopathogenic fungi. Maize (corn; Zea
mays) mutants (termed viviparous, vp) that are blocked at other steps in
the carotenoid pathway also have reduced levels of ABA and exhibit
vivipary—the precocious germination of seeds in the fruit while still
attached to the plant. Vivipary is a feature of many ABA-deficient seeds.
Violaxanthin is converted to another C40 compound, trans-neoxanthin,

under stress conditions, by a reaction dependent on the product of the
Arabidopsis ABA4 locus. Isomerization by an as-yet unidentified enzyme or
set of enzymes, is followed by the cleavage of both 9-cis-violaxanthin and
9-cis-neoxanthin by 9-cis-epoxycarotenoid dioxygenase (NCED) to
form the C15 compound xanthoxin, a growth inhibitor that has
physiological properties similar to those of ABA. This is the first committed
step for ABA biosynthesis, and it is a rate-limiting regulatory step.
A major cause of the inactivation of free ABA is oxidation to phaseic acid

(PA) and 4′-dihydrophaseic acid (DPA) (see Figure A3.10B). ABA
increases the expression of the oxidative enzymes in some tissues,
resulting in negative feedback regulation of ABA levels. This constitutes a
major part of the regulation of ABA levels, such that mutants lacking these
oxidases accumulate far more ABA than lines overexpressing any of the
biosynthetic enzymes.
Catabolism of GAs
The ABA degradation product phaseic acid is usually inactive, or it exhibits
greatly reduced activity, in bioassays. However, phaseic acid can induce
stomatal closure in some species, and it is as active as ABA in inhibiting
gibberellic acid–induced α-amylase production in barley aleurone layers.
These effects suggest that phaseic acid may be able to bind to some ABA
receptors. In contrast to phaseic acid, the other product of ABA
degradation, DPA, has no detectable activity in any of the bioassays tested.
Free ABA can also be inactivated by covalent conjugation to another

molecule, such as a monosaccharide. A common example of an ABA
conjugate is ABA-β-D-glucosyl ester (ABA-GE). Conjugation not only
renders ABA inactive as a hormone, it also alters its polarity and cellular
distribution. Whereas free ABA is localized in the cytosol, ABA-GE
accumulates in vacuoles and the apoplastic space, and might serve as a
storage form of the hormone.
Dehydration stress results in relocalization of ABA-GE from vacuoles to the

endoplasmic reticulum, where it may be cleaved by β-glucosidases.
Dehydration also rapidly activates the β-glucosidases, apparently by
inducing polymerization of these enzymes into larger complexes. Consistent
with the importance of ABA-GE as a source of ABA, mutants lacking the β-

glucosidases have lower ABA levels and impaired stomatal regulation,

germination, and stress responses.
Strigolactones
Like abscisic acid, strigolactones, the hormones that enhance root growth
and inhibit shoot branching, are synthesized via a carotenoid breakdown
product (see textbook Chapter 15, Figure 15.25). In the plastid, trans-β-
carotene is converted in three steps to carlactone (Figure A3.11A).
Carlactone is then exported to the cytosol where it is converted by the
MAX1 cytochrome P450 to active strigolactone. Several active
strigolactones have been discovered (Figure A3.11B). Also shown is G24,
a commonly used synthetic analog. Although several of the genes
regulating the pathway have been identified, the details of the reactions
have yet to be elucidated.
Figure A3.11 (A) Strigolactone is synthesized via a carotenoid

breakdown product. (B) Some strigolactone structures.

III. Lipids
Jasmonic Acid (JA)
Several biologically active fatty acid derivatives are formed during fatty acid
oxidation in plants and animals. In animals, the arachidonic acid cascade
generates numerous important metabolic mediators known as eicosanoids,
including prostaglandins and leukotrienes. Higher plants have a similar
linolenate cascade (also called the “octadecanoid pathway”), which leads to
jasmonate (JA) biosynthesis (see textbook Chapter 23). The first step in
jasmonate biosynthesis is the peroxidation of α-linolenic acid (18:3) by 13-
lipoxygenase to form (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT)
(Figure A3.12). 13-HPOT is converted to cis-(+)-12-oxophytodienoic acid
(OPDA) by the action of allene oxide synthase—yielding (13S)-12,13-
epoxy-octadecatrienoic acid (12,13-EOT)—and allene oxide cyclase. These
steps in JA biosynthesis occur in plastids.

Figure A3.12 Jasmonic acid is synthesized in two different

organelles, the plastid and the peroxisome, via the octadecanoid
pathway.
The subsequent reactions all occur in the peroxisomes. First, the

cyclopentenone ring of OPDA is reduced to 12-oxophytoenoic acid (OPC-8)
by OPDA reductase. Next, three β-oxidation cycles, involving oxidation,
hydration, oxidation, and thiolysis are thought to shorten the carboxylic
side chain of OPC-8 to produce the 12-carbon JA (Acosta et al. 2009).
More recently, the isoleucine (Ile) conjugate of jasmonic acid (JA-Ile) has
been shown to be a primary active form of JA. Conversion of JA to JA-Ile
occurs in the cytosol via the GH3 family member jasmonic acid-amido
transferase 1 (JAR1). In Arabidopsis, the conjugated form of JA is thought
to be the most active (Staswick et al. 2004). The carboxylic acid of

jasmonic acid can also be methylated by a cytosolic methyl transferase to

form the volatile defense signaling molecule methyl jasmonate (MeJA; see
Figure A3.12).
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Plant Physiology, Sixth Edition

Figure A3.1 Three categories of hormones based on their

The major intermediates and subcellular locations of most of the

Here we provide some additional details about the biosynthetic pathways

I. Hormones Derived from Amino Acids

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Figure A3.2 The tryptophan biosynthetic pathway provides

The indole-3-pyruvic acid (IPyA) pathway (Figure A3.3) is the principal

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Figure A3.3 Tryptophan-dependent pathways of IAA

In Arabidopsis and other members of the Brassicaceae that produce indole

A tryptophan-independent pathway of IAA synthesis, starting with indole,

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of this proposed pathway to normal growth processes has not been

As described in textbook Chapter 15. IAA can be temporarily inactivated by

Figure A3.4 Ethylene biosynthetic pathway and the Yang cycle.

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vinylglycine. (After McKeon et al. 1995.)

The role of ACC became evident in experiments in which plants were

In general, when ACC is supplied exogenously to plant tissues, ethylene

ACC synthase is a member of a subfamily of carbon-sulfur lyases encoded

ACC oxidase catalyzes the last step in ethylene biosynthesis: the

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activity—a requirement that has been confirmed by biochemical analysis of

Researchers have studied the catabolism of ethylene by supplying 14 C2H 4

Several species of soil bacteria express an enzyme called ACC deaminase

II. Hormones Synthesized via Isoprenoid

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biosynthesis is the transfer of the isopentenyl (iP) group of dimethylallyl

Figure A3.5 Simplified biosynthetic pathway for cytokinin

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ribotide and riboside forms of trans-zeatin can be

The proteins encoded by the Arabidopsis IPT genes were expressed in E.

The chloroplast methylerythritol phosphate pathway is the primary source

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can be directly converted to the free base forms by the

Figure A3.6 Cytokinin oxidase irreversibly degrades some

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Figure A3.7 Cytokinins can be conjugated to various molecules

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identification of the complete biosynthetic pathways.

A simplified version of the BR biosynthetic pathway, starting with the sterol

Figure A3.8 Simplified pathways for BL biosynthesis and

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catabolism. The precursor for BL biosynthesis is campesterol.

The main BR biosynthesis genes have been isolated and characterized.

The amount of active BRs is also regulated by metabolic processes that

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Figure A3.9 The three stages of GA biosynthesis. In stage 1,

In stage 1, which occurs in plastids, four isoprenoid units are assembled to

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is converted into a tetracyclic compound, ent-kaurene, in two steps, which

In stage 2, which occurs on the plastid envelope and in the endoplasmic

In stage 3, which occurs in the cytosol, GA 12 is converted, through a

In the following discussion the reactions leading to bioactive GA (GA4 in the

Some enzymes in the GA pathway are highly regulated

Three enzymes in stage 3 of the pathway are discussed in detail because

GA 20-oxidase. This multifunctional enzyme catalyzes the three-step

GA 3-oxidase. This enzyme catalyzes the 3β-hydroxylation of GA 9, giving

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GA 2-oxidase. Addition of a 2-OH group in β-orientation prevents GA

Gibberellin regulates its own metabolism

Part of a plant’s response to bioactive GA is to depress GA biosynthesis and

GA1 and GA4 have intrinsic bioactivity for stem growth