A 9-cis-epoxycarotenoid dioxygenase inhibitor for use in the elucidation of abscisic acid action mechanisms

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Abstract

The plant hormone abscisic acid (ABA) accumulates in response to drought stress and confers stress tolerance to plants. 9-cis-Epoxycarotenoid dioxygenase (NCED), the key regulatory enzyme in the ABA biosynthesis pathway, plays an important role in ABA accumulation. Treatment of plants with abamine, the first NCED inhibitor identified, inhibits ABA accumulation. On the basis of structure–activity relationship studies of abamine, we identified an inhibitor of ABA accumulation more potent than abamine and named it abamineSG. An important structural feature of abamineSG is a three-carbon linker between the methyl ester and the nitrogen atom. Treatment of osmotically stressed plants with 100 μM abamineSG inhibited ABA accumulation by 77% as compared to the control, whereas abamine inhibited the accumulation by 35%. The expression of AB A-responsive genes and ABA catabolic genes was strongly inhibited in abamineSG-treated plants under osmotic stress. AbamineSG is a competitive inhibitor of the enzyme NCED, with a Ki of 18.5 μM. Although the growth of Arabidopsis seedlings was inhibited by abamine at high concentrations (>50 μM), an effect that was unrelated to the inhibition of ABA biosynthesis, seedling growth was not affected by 100 μM abamineSG. These results suggest that abamineSG is a more potent and specific inhibitor of ABA biosynthesis than abamine.

Introduction

Carotenoid cleavage dioxygenases (CCDs) produce various apocarotenoids that have important biological functions in animals and plants.1 CCDs catalyze the oxidative cleavage of double bonds at various positions in a variety of carotenoids. Several CCDs have been identified and characterized. An enzyme that cleaves β-carotene at the 15–15′ double bond produces vitamin A, which is essential for development and vision in animals.2 9-cis-Epoxycarotenoid dioxygenase (NCED) is the best-characterized CCD in plants. NCED from maize, the first carotenoid cleavage enzyme identified, catalyzes the cleavage of 9-cis-epoxycarotenoid at the 11–12 double bond to produce a precursor of the plant hormone abscisic acid (ABA).3, 4 CCD1 cleaves several carotenoids symmetrically at the 9–10 and 9′–10′ double bonds to yield C13-norisoprenoid compounds such as β-ionone,5 which plays a role in flower fragrance. Recently, it has been reported that CCD1 regulates the β-ionone content in petunia, tomato, and grape.6, 7, 8 CCD7 and CCD8 catalyze the sequential cleavage of β-carotene.9 As the max3/ccd7 and max4/ccd8 mutants of Arabidopsis show increased lateral branching, CCD7 and CCD8 appear to be involved in the biosynthesis of an unknown branch-inhibiting factor.10, 11, 12

ABA is involved in the regulation of many developmental processes in plants, accelerating abscission, inducing dormancy, and stimulating stomatal closure.13 ABA is also involved in responses to environmental stresses such as drought and high salinity.14 The levels of ABA rapidly increase more than 10-fold within a few hours of osmotic stress, conferring plants with stress tolerance. The accumulation of ABA in response to osmotic stress is thought to be regulated by NCED, the key regulatory enzyme in ABA biosynthesis (Fig. 1). NCED genes have been isolated from bean, cowpea, tomato, Arabidopsis, and avocado.15, 16, 17, 18, 19 These genes are upregulated by osmotic stress,15 but are not regulated by ABA.19, 20

In view of the importance of ABA in plants, it is worthwhile to synthesize and evaluate specific ABA biosynthesis inhibitors that would be useful tools for functional studies of ABA biosynthesis and the effects of ABA in higher plants. In such studies, one advantage of ABA biosynthesis inhibitors over ABA-deficient mutants is that an inhibitor can be applied to any type of plant. Moreover, ABA biosynthesis inhibitors provide a useful method to isolate mutants in which the genes involved in ABA signal transduction have been altered.

Although carotenoid biosynthesis inhibitors such as fluridone and norflurazon have been used as ABA biosynthesis inhibitors,21, 22 these compounds cause lethal damage during plant growth because carotenoids play an important role in protecting photosynthetic organisms against damage by photooxidation.23 Therefore, the use of these inhibitors in the investigation of ABA functions is limited to narrow physiological aspects. Abamine is a novel inhibitor of ABA biosynthesis that targets NCED and does not cause lethal damage.24, 25 Thus, abamine could be used to examine a broad range of physiological aspects involved in the functions of ABA. Abamine has already helped reveal that ABA plays a role in the control of the number of nodules on roots of leguminous plants.26

However, abamine has some points to be improved. Treatment with abamine suppresses the levels of ABA accumulation in Arabidopsis plants exposed to osmotic stress by 40% at the maximum. Moreover, the growth of Arabidopsis seedlings is inhibited by abamine at high concentrations, an effect that is unrelated to the inhibition of ABA biosynthesis. To overcome these problems, we designed and synthesized derivatives of abamine and carried out structure–activity relationship studies on these molecules. This approach led to the identification of an ABA biosynthesis inhibitor that is more potent and specific than abamine–abamineSG.

Section snippets

A screen for potent ABA biosynthesis inhibitors

Table 1 shows the structures of abamine and the abamine derivatives that were tested in this study. The abamine derivatives have a modified phenyl ring of the N-benzyl group (compounds 14), a modified linker between the ester and the nitrogen atom (compounds 58), or a modified alkyl group at the ester moiety (compounds 914).

To screen the above chemicals for ABA biosynthesis inhibitory activity more potent than that of abamine, we determined the ABA content of Arabidopsis plants that were

Discussion

To develop more potent ABA biosynthesis inhibitors, abamine derivatives were synthesized (Table 1) and evaluated for their effects on osmotic-stress-induced ABA accumulation (Fig. 2). On the basis of a study of structure–activity relationships, we developed abamineSG, an ABA biosynthesis inhibitor that is more potent and specific than abamine (Fig. 3). The ABA content in mannitol-stressed plants treated with 100 μM abamine was 10 times that in unstressed plants, but the ABA content in

Chemicals

9′-cis-Neoxanthin and all-trans-violaxanthin for the NCED assay were purified from spinach (Spinacea oleracea) leaves. β-Carotene for the CCD assay was purchased from Sigma (USA). Carotenoid standards were purchased from Wako Pure Chemical (Japan).

Synthesis of abamine derivatives

Abamine derivatives were synthesized essentially as reported by Han et al.24 The chemical data for compound 6 (abamineSG) and compound 8 are as follows:

4-[[3-(3,4-Dimethoxyphenyl)allyl](4-fluorobenzyl)amino]butyric acid methyl ester (6): (69%), pale

Acknowledgments

We thank Ms. Yoko Miura for synthesis of chemicals, Dr. Takemichi Nakamura for technical advice on LC–MS/MS analysis, Drs. Michele C. Loewen and Steven H. Schwartz for the CCD7 plasmid, and Drs. Peter Beyer and Salim Al-Babili for the CCD1 plasmid. This work was supported in part by the Bioarchitect Research Program at RIKEN.

References and notes (35)

  • F. Bouvier et al.

    Trends Plant Sci.

    (2005)
  • S.H. Schwartz et al.

    J. Biol. Chem.

    (2001)
  • S.H. Schwartz et al.

    J. Biol. Chem.

    (2004)
  • J. Booker et al.

    Curr. Biol.

    (2004)
  • S.Y. Han et al.

    Bioorg. Med. Chem. Lett.

    (2004)
  • N. Kitahata et al.

    Bioorg. Med. Chem.

    (2005)
  • J. von Ligtig et al.

    Biochim. Biophys. Acta

    (2005)
  • S.H. Schwartz et al.

    Science

    (1997)
  • B.C. Tan et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1997)
  • A.J. Simkin et al.

    Plant Physiol.

    (2004)
  • A.J. Simkin et al.

    Plant J.

    (2005)
  • S. Mathieu et al.

    J. Exp. Bot.

    (2005)
  • K. Sorefan et al.

    Gene Dev.

    (2003)
  • K. Baibridge et al.

    Plant J.

    (2005)
  • J.A.D. Zeevaart et al.

    Annu. Rev. Plant Physiol. Plant Mol. Biol.

    (1988)
  • K. Shinozaki et al.

    Plant Physiol.

    (1997)
  • X. Qin et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1999)
  • Cited by (0)

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