Inhibitors of GA biosynthesis

Figure 11. Inhibitors of GA biosynthesis AMO-1618 ( 2 -isopropyl-4 -(trimethyl-ammonium chloride)-5 -methylphenylpiperidine-l-carboxylate) and N,N,N-tri-methyl-l-methyl-(2f, , 6 -trimethylcyclohex-2 -en-l -yl)prop-2-enylammonium...

Inhibitors of GA biosynthesis have long held an interest for the agrochemical industry as growth retardants. They are also important experimental tools that serve as alternatives to the use of mutants in studying GA function [125]. In this regard, they suffer in some instances from lack of specificity, but have an advantage over mutants in that the timing and sites of inhibition within the plant can be more controlled. 

Fig. 3. Impaired arrangements of CMT arrays in cells of gibberellin (GA)-deficient mutant (d5) maize roots (a,b) and in cells of cv. LG-11 maize roots treated with inhibitor of GA biosynthesis 2S, 3S paclobutrazol (e,f). For more details on these treatments see [119]. Note that CMT arrays show abundant holes in the GA-deficient root cells (arrows). Addition of exogenous gibberellic acid rescues these lesions and well ordered transverse CMT arrays appear in cells of both d5 mutants (c,d) as well as of paclobutrazol-treated LG-11 root cells (g, h). Bar represents 10 pm.

A complementary approach is the use of chemical inhibitors of GA-biosynthesis. Surprisingly this method using synthetic plant growth retardants has only recently been used (20, 21, 23) to study GA-biosynthesis in the absence of the normal fungal metabolites. 

Some plant growth retardants can inhibit ABA biosynthesis in the fungus Cerco-spora rosicola [22]. The most effective were paclobutrazol, ancymidol and decylimidazole [21]. CCC, Alar and paclobutrazol also act as inhibitors of GA biosynthesis and both ABA and GA are synthesized via the isoprenoid pathway. However, different growth inhibitors act at different points on the pathway and thus may inhibit either or both ABA and GA biosynthesis. 

A second problem encountered in studies using inhibitors of GA biosynthesis or GA-deficient mutants — and one that is often not fully appreciated — is that flower primordia may have formed although further development to macroscop-ically visible flower buds is blocked. We have observed this in our studies on the CRP Thlaspi arvense. Application of CCC to thermoinduced plants blocks both stem elongation and the appearance of flower buds. However, microscopic examination of apices showed that flower primordia were formed about the same time after the end of the cold treatment as the non-CCC treated plants (K. Dusbabek and J. Metzger, unpublished data). We have also observed a similar phenomenon in a GA-deficient dwarf mutant of this species. 

As pointed out earlier there are a number of examples in which inhibitors of GA biosynthesis block flower initiation in inductive conditions, indicating that under certain circumstances GAs are limiting for flower initiation. There are at least three... 

Since GAs as diterpenes share many intermediates in the biosynthetic steps leading to other terpenoids, eg, cytokinins, ABA, sterols, and carotenoids, inhibitors of the mevalonate (MVA) pathway of terpene synthesis also inhibit GA synthesis (57). Biosynthesis of GAs progresses in three stages, ie, formation of / Akaurene from MVA, oxidation of /-kaurene to GA 2" hyde, and further oxidation of the GA22-aldehyde to form the different GAs more than 70 different GAs have been identified. 

Most of the GA-synthesis inhibitors characterized so far affect two segments of the compHcated pathway from MVA to the many different GAs identified. The cycHzation reactions that produce / Akaurene are inhibited by the onium growth retardants, and the oxidations of /-kaurene to /-kaurenoic acid are sensitive to heterocycHc triazoles such as paclobutrazol and similar compounds. Other enzymes in the pathway are points for pathway dismption by as yet undeveloped GA biosynthesis inhibitors (236). 

Reddy et al29 studied the molecule N6,N6-dimethyl-2,6-diaminobenz[cd]-indole, which is shown in Figure 6. The aim was to assess its suitability as an inhibitor of thymidylate synthase, an enzyme which is important in DNA biosynthesis. Three structures were investigated N1H, which is the amine form depicted, and N2H, the imine form, in anti- and syn-conformations, i.e. with the proton on Ni away from or towards the N2 proton, respectively. In the gas phase it was found that the order of stability is N1H > anti-N2H > syn-N2H, in a ratio of 73.1 20.3 6.6. Solvation then further favours the N1H form, and the ratios in solution are calculated to be 98.5 0.5 1.0. Unfortunately, the syn-N2H form is likely to be the conformation that binds most strongly to the enzyme, and so the calculations indicate that this molecule is unlikely to be a suitable inhibitor. 

Plants possessing genes Ih and Is contain very low levels of GA-like activity [ 17] and respond as well as wild-type plants (which were dwarfed with the GA-synthesis inhibitor, AMO 1618) to all GA-precursors examined, including e r-kaurene [6], suggesting that these genes block GA-biosynthesis prior to e kaurene. Neither Is nor Ih plants appear to possess impaired carotenoid production, suggesting that the... 

The simple concept that AMO-1618 and related retardants were growth inhibitors solely by virtue of their effects on GA biosynthesis has been questioned by Douglas... 

Although growth retardants acting on GA biosynthesis beyond e /-kaurenoic acid are not available, there is interest in developing such compounds since they may offer more specificity than the current inhibitors. The enzymes catalyzing the steps between ezz/-kaurenoic acid and GAi2-aldehyde are microsomal oxygenases of... 

These PGRs suppress growth only. The developmental sequence of the plant continues, however, new plant organs develop in miniature size. Examples of this type include paclobutrazol, fluprimidol, uniconazole, and tetcyclasis. These compounds have primary action as GA biosynthesis inhibitors and are far more effective in suppressing internode elongation than leaf size. 

Other Bioregulators. It should be mentioned that the physiological ly differently acting phytohormones abscisic acid cuid indolylacetic acid, which have different physiological activity, generate response patterns which exhibit little similarity either to each other or to those of the GA biosynthesis inhibitors. 

McAdam, BE, Catella-Lawson, F, Mardini, lA, Kapoor, S, Lawson, JA and FitzGerald, GA (1999) Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2 the human pharmacology of a selective inhibitor of COX-2. Proc Natl Acad Sci USA, 96, 272-277. 

Other plant hormones, such as auxins and GAs. Finally, selected compounds were assayed using a cress hypocotyl elongation test. It has been demonstrated that cress is very sensitive to an internal deficiency of BRs and is therefore a useful species for evaluating BR biosynthesis inhibitors [7-8],... 

Isoenzymes of enzymes involved in the first step of a branched biosynthetic pathway may differ in their sensitivity to inhibitors. Thus the enzyme aspartate kinase catalyses in Escherichia coli the first step in the synthesis of lysine, methionine and threonine. Three isoenzymes occur, the synthesis and activity of one is suppressed by L-lysine, the activity of the second is depressed by L-threonine and the activity of the third by homoserine (an intermediate in methionine biosynthesis). Thus accumulation of L-lysine or L-threonine suppresses their own further S3mthesis but does not prevent the activity of the aspartokinase isoenzyme involved in methionine synthesis. Again peroxidase isoenzymes differ in their activity in destroying indol-3yl-acetic acid (lAA) and the pattern of peroxidase isoenzymes can be altered by feeding lAA or gibberellic acid (GA) to plant tissues. 

---