HOME :: CHAPTER 20 :: Topic 20.3
Valerie Sponsel, Biology Department, University of Texas, San Antonio, TX, USA
Terpenoids are compounds made up of 5-carbon isoprenoid building blocks, joined head to tail. The GAs are diterpenoids that are formed from four such isoprenoid units. They are assembled to give a 20-carbon linear molecule, geranylgeranyl diphosphate (GGPP). The stepwise conversion of GGPP to the first-formed GA, GA12, is essentially similar in all plant species studied so far.
The subsequent formation of many different GAs from GA12 differs not only from species to species, but even in different organs within one species, or in response to different environmental conditions. The GA biosynthetic pathway can be divided into three stages, each residing in a different cellular compartment: the plastid, the endoplasmic reticulum, and the cytosol (Hedden and Phillips 2000).
Stage 1: Production of Terpenoid Precursors in Plastids
The basic biological isoprenoid unit is isopentenyl diphosphate (IPP) (Web Figure 20.3.A). For many years it was assumed that the early stages of the terpenoid biosynthetic pathway were the same for all types of terpenoids. This is now known to be untrue. In plants, IPP can be synthesized in either the cytosol or in the stroma of plastids, using different precursors at each site. The cytosolic pathway utilizes acetate and is named the mevalonate (MVA) pathway after the important intermediate, mevalonic acid. This is the only pathway that operates for terpenoid biosynthesis in fungi and animals. The mevalonate pathway is thus the source of IPP for GA biosynthesis in the fungus Gibberella zeae.
|Web Figure 20.3.A|
The plastidic pathway in the green parts of plants and in algae uses glyceraldehyde 3-phosphate and pyruvate, and again the pathway is named for an important intermediate, methyl erythritol phosphate (MEP) (Eisenreich et al. 2001). In Arabidopsis, it has been determined that IPP used in GA biosynthesis in green tissues comes predominantly from IPP synthesized in plastids by the MEP pathway (Kashara et al. 2002), and this is thought to be the source of IPP for GA biosynthesis in most plants. (One exception is known—in the endosperm of pumpkin seeds, IPP that is used for GA biosynthesis is formed from mevalonate.) An overview of the two pathways is shown in Web Figure 20.3.B.
|Web Figure 20.3.B Two biosynthetic pathways to terpenoids in plants. The MVA pathway is in the cytosol, the MEP pathway in the stroma of plastids. (Click image to enlarge.)|
Once synthesized, the 5-carbon units condense to produce intermediates of 10 carbons (geranyl diphosphate, GPP), 15 carbons (farnesyl diphosphate, FPP), and 20 carbons (geranylgeranyl diphosphate, GGPP) as shown in Web Figure 20.3.B. GGPP is a precursor of many diterpenoid compounds, including the phytol side chain of chlorophyll, and tetraterpenoids (40 carbons), including carotenoids. Therefore, any mutation or chemical that blocks the MEP pathway prior to GGPP will prevent the production of these pigments giving albino plants, and is most likely to be lethal/toxic. In fact, fosmidomycin, which blocks early in the MEP pathway, can be used as a herbicide. It is only after GGPP that, in most plants, the pathway is dedicated for GA biosynthesis.
There are two cyclization reactions that convert linear GGPP to ent-kaurene (Web Figure 20.3.C). The formation of a bicyclic intermediate ent-copalyl-diphosphate (CPP) is catalyzed by ent-copalyl diphosphate synthase (CPS), and the conversion of CPP to the tetracyclic ent-kaurene is catalysed by ent-kaurene synthase (KS). (The precursor of GAs is the enantiomeric form of kaurene, hence the prefix "ent." However, in this Web Topic, the prefix is dropped after its first use with each compound.)
Chemical inhibitors of CPS, such as AMO-1618 and chlormequat chloride (also known as cycocel) block GA biosynthesis at this point (see Rademacher 2000). This leads to severely depleted levels of bioactive GA; these chemicals are therefore powerful growth retardants (see Web Topic 20.1). Mutations that block either CPS or KS cause a severely dwarfed phenotype as long as there is no gene with overlapping function to provide additional isoforms of the enzyme. Thus, mutations in GA1 and GA2 that encode the first two enzymes in the committed part of GA biosynthesis in Arabidopsis, give extreme dwarfs.
Stage 2: Oxidation Reactions on the Plastid Envelope and ER
In the second stage of GA biosynthesis, the C-19 methyl (CH3) group of kaurene is oxidized in three steps to give ent-kaurenoic acid (KA) (see Web Figure 20.3.C). These oxidations are catalyzed by ent-kaurene oxidase (KO), which is said to be multifunctional because it can catalyze all three reactions. In Arabidopsis, KO is encoded by GA3, and mutations in this gene will again produce severely dwarfed plants.
Kaurenoic acid is then oxidized at C-7 and C-6 in two steps to give GA12-aldehyde. The conversion of KA to GA12-aldehyde involves the contraction of the B-ring from 6 carbons to 5 (Web Figure 20.3.C), which distinguishes the ent-gibberellane skeleton (that has a 6-membered B-ring) from that of ent-kaurene. GA12-aldehyde is then oxidized to GA12, which is the first-formed GA, and thus the precursor, of all the other GAs.
The reactions from kaurenoic acid to GA12 are catalyzed by another multifunctional enzyme, ent-kaurenoic acid oxidase (KAO). In Arabidopsis, there is no known phenotype associated with a mutated KAO, probably because there are two stem-expressed genes with overlapping functions. As discussed in chapter 20, mutations in the stem-expressed NA gene that encodes KAO in pea yields a so-called nana phenotype that is very small.
All the enzymes involved in the second phase of GA biosynthesis are monooxygenases that utilize cytochrome P450 in their reactions, and are typically associated with membranes. As mentioned earlier, enzymes that catalyze the first part of the pathway are in the chloroplast stroma. Kaurene must therefore exit the chloroplast, because in Arabidopsis, KO is located on the plastid envelope while KAO is located in the endoplasmic reticulum (Helliwell et al. 2001). Actually, kaurene can be volatile, and not only does it exit the chloroplast, it can also diffuse from the plant and affect the growth of neighboring plants (Otsuka et al. 2004).
Paclobutrazol and other inhibitors of P450 monooxygenases specifically inhibit this second stage of GA biosynthesis (see Web Topic 20.1). These compounds are growth retardants and are used for ornamental plants—especially potted ones—to produce shorter plants that are easier to manage and more attractive.
Stage 3: Gibberellins from GA12 or GA53 Are Produced in the Cytosol
All subsequent steps from GA12 are carried out by a group of soluble dioxygenases in the cytosol. These enzymes require 2-oxoglutarate and molecular oxygen as co-substrates, and they use Fe2+ and ascorbate as cofactors.
The specific steps in the modification of GA12 vary from species to species, and between organs of the same species. Typically, the reactions in vegetative tissue (young seedlings and stems) are more conserved between species and follow a similar basic plan of C-20 oxidation along with hydroxylation at C-3, and eventually, at C-2. Hydroxylation at C-13 may or may not occur.
In contrast, in maturing seeds there are many additional modifications of GAs—for example, hydroxylations at carbons 1, 11, 12, or 15 that consequently lead to the production of the large number of polyhydroxylated GAs. Many of the 136 known GAs have been identified only in seeds, and closely related species often have seed GAs with similar patterns of hydroxylation. In vegetative tissues, we will discuss only the major GA pathway in each of three examples: pea, maize and Arabidopsis. Often minor pathways are also present, giving a metabolic network rather than a single pathway, but only the major pathway in specific plants is described here. The basic pathway from GA12 in vegetative tissues is as follows:
1. Hydroxylation at C-13. This is a major metabolic event in pea and maize pathways (but not in the main pathway in Arabidopsis). 13-Hydroxylation converts GA12 to GA53.
2. Successive oxidation at C-20. This occurs in four steps, and in the final step C-20 is lost, resulting in the first-formed, C19-GA. As mentioned in Web Topic 20.1, this loss of C-20 results in the formation of a lactone between C-4 and C-10. In Arabidopsis, this sequence is GA12 (CH3 at C-20) to GA15 (CH2OH at C-20 in the open-lactone form) to GA 24 (CHO at C-20) to GA9 (C-20 has been lost) (see Web Figure 20.3.C). In pea and maize, the sequence is identical, giving the 13-hydroxylated counterparts of the Arabidopsis GAs, namely GA53 (CH3 at C-20) to GA44 (CH2OH at C-20 in the open-lactone form) to GA19 (CHO at C-20) to GA20 (C-20 has been lost). The four sequential oxidations are all catalyzed by the same multifunctional GA 20-oxidase (GA20ox).
3. 3β-Hydroxylation. In Arabidopsis, this converts GA9 to the bioactive GA, GA4. In pea and maize, the 13-OH counterpart of GA9, namely GA20, is converted to bioactive GA1. Both GA4 and GA1 are C19-GAs with a 4,10-lactone, a carboxylic acid group at C-6, and a 3β-OH group. These are the structural requirements for bioactivity that were detailed in Web Topic 20.1 and in Chapter 20.
4. 2β-Hydroxylation. The bioactive GAs can be deactivated by hydroxylation at C-2, converting GA4 to GA34 in Arabidopsis, and GA1 to GA8 in pea/maize.
As we can see from Web Figure 20.3.C, in Arabidopsis there are 12 reactions in the linear pathway from the earliest committed precursor (GGPP) to the bioactive GA, GA4. In pea and maize, there are 13 steps to the bioactive GA, GA1, because of the additional hydroxylation step at C-13.
Inhibitors of the third stage of the GA biosynthetic pathway interfere with enzymes that utilize 2-oxoglutarate as a co-substrate. Among these, prohexadione calcium (BX-112) is especially useful because it specifically inhibits GA 3-oxidase, and it is a growth retardant, marketed as Apogee. It is used to control the canopy size of apple and orange trees and reduce the amount of pruning required. (see Web Topic 20.1).
Gibberellins can be converted into conjugates, most commonly with glucose. The linkage of the GA moiety to glucose occurs either via a hydroxyl group to produce a GA-O-glucosyl ether, or via the COOH at C-6, yielding a GA-glucosyl ether. Gibberellin conjugates are frequently found in Phaseolus vulgaris (bean) seeds during the later stages of seed maturation.
When applied to bioassay plants, the GA-O-glucosyl ethers show little-to-no activity, indicating that they are deactivation products. In contrast, the GA-glucosyl esters may be hydrolyzed within a bioassay plant, yielding the free GA. Hence, the formation of glucosyl ester conjugates may be a mechanism for sequestration of bioactive GA that can be released later, as required by the plant.
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