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Specific Oxidative Cleavage of Carotenoids by VP14 of Maize

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Science  20 Jun 1997:
Vol. 276, Issue 5320, pp. 1872-1874
DOI: 10.1126/science.276.5320.1872

Abstract

The plant growth regulator abscisic acid (ABA) is formed by the oxidative cleavage of an epoxy-carotenoid. The synthesis of other apocarotenoids, such as vitamin A in animals, may occur by a similar mechanism. In ABA biosynthesis, oxidative cleavage is the first committed reaction and is believed to be the key regulatory step. A new ABA-deficient mutant of maize has been identified and the corresponding gene, Vp14, has been cloned. The recombinant VP14 protein catalyzes the cleavage of 9-cis-epoxy-carotenoids to form C25 apo-aldehydes and xanthoxin, a precursor of ABA in higher plants.

Apocarotenoids, which are compounds derived from the oxidative cleavage of carotenoids, are widely distributed in nature and have important metabolic and hormonal functions in diverse organisms. In Mucoraceous fungi, trisporic acid is a mediator of sexual processes (1). Retinal serves as a photosensory pigment in animals (2), green algae (3), andHalobacterium (4). Retinoids, which are vitamin A derivatives, are morphogens in animals (5) and have important clinical applications.

Apocarotenoids may be formed by random cleavage caused by photooxidation or lipoxygenase co-oxidation. However, regulation of the synthesis of biologically active apocarotenoids requires a more precise mechanism for their synthesis. Enzymatic cleavage of carotenoids at a specific position of the polyene chain has been proposed as a method for the synthesis of several apocarotenoids. The most definitive illustration of enzymatic cleavage is the production of β-cyclocitral (C10) from β-carotene by the cyanobacteriumMicrocystis (6). In other organisms, however, enzymatic cleavage of carotenoids has been difficult to demonstrate in vitro. For this reason, the cleavage reaction in vitamin A biosynthesis remains controversial. Cleavage of β-carotene by a 15,15′-dioxygenase to produce two molecules of retinal has been reported (7,8). However, there is also evidence for asymmetric cleavage of β-carotene. After asymmetric cleavage, additional carbons are removed from the larger product, by a mechanism similar to β-oxidation, to form one molecule of retinoic acid (9).

Abscisic acid (ABA) is a plant growth regulator involved in the induction of seed dormancy and in adaptation to various stresses, such as drought (10). Elevated levels of ABA induced by stress are, in part, responsible for stomatal closure, changes in gene expression, and other plant adaptations to stress.

Since the elucidation of the structure of ABA, its biosynthetic derivation from carotenoids has been proposed (11). Labeling experiments with 18O2 suggest that ABA is synthesized from a large precursor pool that already contains two of the four oxygens found in the molecule (12). Oxygen derived from the hydroxyl and epoxide of neoxanthin or violaxanthin could account for the observed 18O labeling pattern. In addition, when etiolated bean seedlings were stressed, there was a decrease in the level of these xanthophylls and a corresponding increase in ABA and its catabolites (13).

Direct evidence for a cleavage enzyme in ABA biosynthesis is lacking because of the apparent low abundance and lability of the enzyme. However, several features of the cleavage reaction have been inferred by analysis of the later steps in ABA biosynthesis (Fig.1). The initial C15 cleavage product xanthoxin is rapidly converted to ABA in vivo and in vitro (14). In cell-free extracts, trans-xanthoxin is converted to trans-ABA, which indicates that there is no cis/trans isomerization after cleavage (14). Thus, the xanthophyll precursor must have a 9-cis configuration to produce cis-xanthoxin and subsequently ABA, which is biologically active only in the cis form.

Figure 1

Proposed pathway of ABA biosynthesis in higher plants.

ABA biosynthetic mutants have been identified in a variety of plant species (15). To date, no mutants impaired in the cleavage reaction have been identified. A new ABA-deficient viviparous mutant in maize, vp14, has been isolated and the corresponding gene has been cloned (16). This mutant is not impaired in carotenoid biosynthesis or in the conversion of xanthoxin to ABA. The derived amino acid sequence of VP14 shows significant sequence similarity to lignostilbene dioxygenases (LSDs) fromPseudomonas paucimobilis (17). These LSDs catalyze a reaction similar to the proposed cleavage reaction in ABA biosynthesis. Specifically, a double bond is oxidatively cleaved, yielding two products with aldehyde groups at the site of cleavage.

Using 9-cis-violaxanthin as a substrate, the recombinant VP14 protein was tested for cleavage activity (18) and the products were analyzed by high-performance liquid chromatography (HPLC) (Fig. 2). The expected cleavage products, xanthoxin and the C25 epoxy apo-aldehyde, were identified by their ultraviolet/visible absorption spectra and mass spectra (19).

Figure 2

HPLC chromatogram (31) of the cleavage reaction products with the use of 9-cis-violaxanthin as a substrate (32). Absorbance was measured at 280 nm and 436 nm with a photodiode array detector.

Molecular oxygen, ferrous iron, and a detergent were necessary for the cleavage activity (Table 1). A number of organic cofactors were initially added to the assays but none had any effect on the activity (20). The cleavage reaction was totally inhibited by EDTA, a chelator of divalent cations (21). The removal of EDTA and addition of ferrous iron were sufficient to restore activity. Ascorbate was added to the assays to maintain iron in the proper redox state (21).

Table 1

The requirements for cleavage activity in vitro. This is the standard reaction (18) minus the indicated cofactors and 6 μg of VP14 protein.

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With increasing VP14 concentration, there was a decrease in 9-cis-violaxanthin and an equimolar increase in xanthoxin and the C25 epoxy apo-aldehyde (Fig. 3). Whereas photo- or chemical oxidation would result in random cleavage, this stoichiometric conversion of 9-cis-violaxanthin to the two products illustrates the specificity of cleavage between the 11 and 12 positions of the polyene chain.

Figure 3

Decrease in 9-cis-violaxanthin and the concomitant increase in xanthoxin and the C25 epoxy apo-aldehyde as a function of the VP14 protein concentration. Assays were incubated for 10 min at 22° to 24°C, extracted, and quantified (31).

To determine the substrate specificity of the cleavage reaction, the all-trans and the 9-cis isomers of neoxanthin and violaxanthin were tested. The reaction products were separated on thin-layer chromatography plates and sprayed with 2,4-dinitrophenyl hydrazine to detect aldehydes (Fig. 4). Xanthoxin and the predicted C25 products were present only in reactions containing the 9-cis isomers. The 9-cis isomer of zeaxanthin, formed by iodine isomerization of the all-trans zeaxanthin (22), was cleaved at the 11-12 position by the VP14 protein (23) (Fig. 4). Therefore, it appears that the 9-cis configuration was the primary determinant of cleavage specificity for the in vitro assays. Cleavage of 9-cis-epoxy-carotenoids results in the production ofcis-xanthoxin, which will subsequently be converted to the biologically active isomer of ABA in vivo.

Figure 4

Thin-layer chromatography of assays with (+) and without (−) VP14 protein. Assays contained approximately 5 μg of the indicated substrate (32): the all-trans isomers (at) and the 9-cis isomers (9c) of zeaxanthin (Z), violaxanthin (V), neoxanthin (N), and standard xanthoxin (Std. Xan). Substrate and products were separated on a silica gel 60 plate (EM Separations) developed with 10%iso-propanol in hexane. The plates were sprayed with 2,4-dinitro-phenylhydrazine to detect xanthoxin and other aldehydes. The C25 products are indicated by a dagger and the C15 products are indicated by an asterisk. The unlabeled spots are the carotenoid precursors.

The LSDs from Pseudomonas (17) and VP14 compose a novel class of dioxygenases that catalyze similar double-bond cleavage reactions. The conserved sequences have also been identified in several plant expressed sequence tags, two open reading frames in the genomic sequence of Synechocystis (24), and a protein expressed in the retinal pigment epithelium of mammals, RPE65 (25). The function of these gene products has not yet been determined, but their existence indicates the presence of this class of enzymes in a diverse range of species.

The environment of the carotenoids in the thylakoid and envelope membranes (26) is very different from in vitro assays in which the carotenoid substrates are solubilized by detergent. However, the characteristics of the cleavage reaction in substrate specificity and position of cleavage are consistent with the proposed pathway (27). Current evidence suggests that this cleavage reaction is the key regulatory step in ABA biosynthesis (27). Further characterization of the cleavage reaction and its regulation may allow the manipulation of ABA levels in plants, which would affect such processes as seed dormancy, drought tolerance, and cold hardening.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: zeevaart{at}pilot.msu.edu

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