Despite their diverse chemical structures, most of the known plant hormones are derived from three main types of metabolic precursors: amino acids, isoprenoid compounds, and lipids (Figure A3.1). The amino acids tryptophan and methionine serve as precursors for IAA (indole-3-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

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-[¹⁴C]methionine to [¹⁴C]ethylene, and that the ethylene is derived from carbons 3 and 4 of methionine (Figure A3.4). The CH₃—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 [¹⁴C]methionine. Under anaerobic conditions, ethylene was not produced from the [¹⁴C]methionine, and ¹⁴C-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 Fe²⁺/ascorbate oxidase superfamily. This sequence similarity suggested that ACC oxidase might require Fe²⁺ 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 ¹⁴C₂H₄ 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 C₁₉-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, GA₁₂. Two important enzymes in this part of the pathway are ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO). The pathway to GA₁₂ is essentially the same in all plant species studied so far.

In stage 3, which occurs in the cytosol, GA₁₂ 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 GA₁ 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 GA₁₂, producing GA₉ in the non-13-hydroxylation pathway, and in GA₅₃, producing GA₂₀ in the 13-hydroxylation pathway. The sequential oxidation of C-20 proceeds from CH₃ in GA₁₂ to CH₂OH, and then to CHO, before C-20 is eliminated, yielding a C₁₉-GA (either GA₉ or GA₂₀). 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 GA₉, giving GA₄, or of GA₂₀, giving GA₁. 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 GA₁ 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 GA₁ 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 GA₂₀ is applied to the le mutant, it is not bioactive (the plants remain dwarf), whereas GA₁ is bioactive, and rescues the mutant phenotype (the plants grow tall). Gibberellin A₈ is also inactive. We can infer from this information, and from knowledge of the GA metabolic pathway in pea (GA₂₀ → GA₁ → GA₈), that GA₂₀ is inactive unless it can be converted to GA₁ within the plant, and that GA₁ 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 GA₁. The na mutant of pea, in which stage 2 is blocked, is almost completely devoid of GA₁. 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 GA₁ 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 GA₁ has less biological activity than GA₄. Therefore, in Arabidopsis and probably also in these cucurbits, GA₄ is the main biologically active GA, and stem length is correlated with GA₄ 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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