Carotenoid oxygenases: cleave it or leave it - Cell Press

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TRENDS in Plant Science

researchers in other areas of arbuscular mycorrhizal research with an exciting opportunity to understand the genes involved in this common, but not always generalist, interaction. Acknowledgements Financial support from the Swiss National Science Foundation Professorial Fellowship programme (No. 631 – 058108.99) is gratefully acknowledged. I thank Martine Ehinger, Gerrit Kuhn and an anonymous reviewer for critically reading the manuscript.

References 1 Smith, S.E. and Read, D.J. (1997) Mycorrhizal Symbiosis, Academic Press 2 Newsham, K.K. et al. (1995) Multi-functionality and biodiversity in arbuscular mycorrhizas. Trends Ecol. Evol. 10, 407 – 411 3 Sanders, I.R. (2002) Specificity in the arbuscular mycorrhizal symbiosis. In Mycorrhizal Ecology (Van der Heijden, M.G.A. and Sanders, I.R., eds) pp. 415 – 437, Springer 4 Vandenkoornhuyse, P. et al. (2002) Arbuscular mycorrhizal community composition associated with two plant species in a grassland ecosystem. Mol. Ecol. 11, 1555 – 1564 5 Bidartondo, M.I. et al. (2002) Epiparasitic plants specialized on arbuscular mycorrhizal fungi. Nature 419, 389 – 392 6 Sanders, I.R. and Fitter, A.H. (1992) Evidence for differential responses between host-fungus combinations of vesicular-arbuscular mycorrhizas from a grassland. Mycol. Res. 96, 415 – 419

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7 Van der Heijden, M.G.A. et al. (1998) Mycorrhizal fungal diversity determines plant diversity, ecosystem variability and productivity. Nature 396, 69 – 72 8 Bever, J.D. et al. (1996) Host-dependent sporulation and species diversity of arbuscular mycorrhizal fungi in a mown grassland. J. Ecol. 84, 71 – 82 9 Robinson, D. and Fitter, A.H. (1999) The magnitude and control of carbon transfer between plants linked by a common mycorrhizal network. J. Exp. Bot. 50, 9 – 13 10 Pellmyr, O. et al. (1996) Non-mutualistic yucca moths and their evolutionary consequences. Nature 380, 155– 156 11 Sanders, I.R. et al. (1995) Identification of ribosomal DNA polymorphisms among and within spores of the Glomales: application to studies on the genetic diversity of arbuscular mycorrhizal fungal communities. New Phytol. 130, 419 – 427 12 Kuhn, G. et al. (2001) Evidence for the evolution of multiple genomes in arbuscular mycorrhizal fungi. Nature 414, 745 – 748 13 Helgason, T. et al. (1998) Ploughing up the wood-wide web? Nature 394, 431 – 432 14 Leake, J.R. (1994) The biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol. 127, 171– 216 15 Rausch, C. et al. (2001) A phosphate transporter expressed in arbuscule-containing cells in potato. Nature 414, 462 – 466

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Carotenoid oxygenases: cleave it or leave it Giovanni Giuliano1, Salim Al-Babili2 and Johannes von Lintig2 1 2

ENEA, Casaccia Research Centre, PO Box 2400, 00100AD Roma, Italy Centre for Applied Biosciences, University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Germany

Carotenoid cleavage products (apocarotenoids) are widespread in living organisms and exert key biological functions. In animals, retinoids function as vitamins, visual pigments and signalling molecules. In plants, apocarotenoids play roles as hormones, pigments, flavours, aromas and defence compounds. The first step in their biosynthesis is the oxidative cleavage of a carotenoid catalysed by a non-heme iron oxygenase. A novel family of enzymes, which can cleave different carotenoids at different positions, has been characterized. Carotenoids not only colour the world around us – their cleavage products also enable us to see it. Retinal, derived from the central cleavage of the 15,150 double bond of b-carotene (Fig. 1), is the chromophore in retinylidene proteins (rhodopsins) [1]. Rhodopsins are seven transmembrane helix proteins and are involved in light-driven ion transport and phototaxis signalling in microorganisms (including green algae) and various types of phototransduction systems in animals. The retinal derivatives, retinol (vitamin A) and retinoic acid, have important nutritional and signalling functions in animal development. Corresponding author: Giovanni Giuliano ([email protected]). http://plants.trends.com

In higher plants, many different apocarotenoids derive from the excentric cleavage of carotenoids. Probably the best-known example is the hormone abscisic acid (ABA) – a C15 compound derived from the cleavage of the 11,12 double bond of 9-cis-violaxanthin and 9-cis-neoxanthin (Fig. 1). ABA levels rise under stress conditions and during seed dehydration, and the cleavage reaction is the ratelimiting step in its biosynthesis [2]. At the onset of a mycorrhizal relationship, roots of several plants accumulate the C14 ‘yellow pigment’, also known as mycorradicin, and the cyclohexenone C13 compound, blumenin [3], which possesses antifungal properties [4]. A pathway for the synthesis of these compounds has been proposed, involving the oxidative cleavage of the 9,10 and 90 100 double bonds of a precursor xanthophyll, probably lutein [3] (Fig. 1). In addition, plants produce many volatiles, such as b-ionone, which probably act as insect attractants and are valued as flavour and aroma compounds. Plant-derived apocarotenoid pigments also have some economic value, for example, bixin (annatto) is commonly used as a natural food colourant and crocin is the main pigment in saffron (Crocus sativus). The production of carotenoid-derived volatile compounds is also common in cyanobacteria; for example, Microcystis aeruginosa blooms liberate b-ionone and b-cyclocitral.

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a) O HO

9 10 11 12

O HO

9 10 11 12

O

NCED

CHO

HO

Xanthoxin

9-cisneoxanthin

OH

HO

COOH

O

HO

O

9-cisviolaxanthin

ABA

OH

b) CHO

trans-retinal

15,15´ BCO

15 15´

β-carotene CH2OH

trans-retinol (vitamin A) c)

OR

OH 9

O

10´ 9´

+

CCD

10

Blumenin

Lutein

HO

COOR ROOC

Mycorradicin d)

OH 7 8

HO

8´

7´

ZCD

Zeaxanthin

CHO

OHC

CHO

+ HO

Hydroxy-βcyclocitral

Crocetin dialdehyde

CHO

OR

RO

Crocin

RO

Picrocrocin

CHO

Safranal TRENDS in Plant Science

Fig. 1. Proposed carotenoid oxidation pathways. (a) Abscisic acid biosynthesis. (b) Vitamin A biosynthesis. (c) Apocarotenoid biosynthesis in mycorrhizal roots. Lutein is in square parentheses because its role as a precursor is still hypothetical. (d) Zeaxanthin oxidation pathway in saffron styles. Abbreviations: BCO, b-carotene-15,150 -oxygenases; CCD, carotenoid cleavage dioxygenase; NCED, 9-cis-epoxycarotenoid dioxygenases; ZCD, zeaxanthin cleavage dioxygenase.

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viviparous 14 (vp14) of maize and notabilis (not) of tomato. The molecular cloning of vp14 using a transposon tagging approach revealed a resemblance to bacterial lignostilbene dioxygenase (LSDO) [5]. Recombinant purified Vp14 protein not only mediates the oxidative cleavage of 9-cis-violaxanthin and 9-cis-neoxanthin but also of 9-cis-zeaxanthin at the 11,12 double bond, suggesting that the enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage [6]. Vp14 and its

All these pathways have two characteristics in common: the starting compound is a C40 carotenoid and the first cleavage product is an aldehyde, suggesting that the enzymes mediating the cleavage reaction have a common reaction mechanism. ABA biosynthesis Several plant mutants are impaired in ABA synthesis without affecting carotenoid content. Two of these are

0.290

0.074

0.069

Sphingomonas sp. LSDO (AAC60447) 0.398

0.1 0.109

Synechococcus sp. (ZP00114682) 0.297 Synechocystis sp. (S76169) 0.377 Synechocystis sp. (S76206) 0.358 Drosophila 15,15′ BCO (CAB93141) 0.096 Mouse 9′,10′ 0.142 BCO(NP_573480) 0.068 0.098 Human 9′,10′ BCO (CAC27994) 0.239 Zebrafish BCO2 (NP_571874) 0.074 Mouse 15,15′ 0.085 BCO (NP_067461) 0.059 0.074 Human 15,15' BCO (NP_059125) 0.098 0.148 Chicken 15,15′ BCO (CAB90925) 0.212 Zebrafish BCO (NP_571873) 0.321 Human RPE65 (NP_000320) 0.411 Arabidopsis (CAB79998) 0.223 Trichodesmium (ZP_00072571) 0.142 Nostoc sp. 0.078 (NP_485149) 0.129 Nostoc sp. (ZP_00106997) 0.109 Arabidopsis CCD(T51734) 0.109

0.167

0.116 0.176

0.064 0.174 0.238 0.245 0.094

Bean CCD (AAK38744) Avocado CCD (AAK00622)

0.191 0.106

Saffron CCD (AJ132927)

0.200 0.165 0.164 0.183 0.150 0.140 0.164 0.182 0.166

Appletree (T17019)

Arabidopsis (NP_193652) Saffron ZCD (CAD33262) Arabidopsis (AAK38744) Maize Vp14 (AAB62181) Avocado NCED (AAK00623) Avocado NCED2 (AAK00632) Arabidopsis (NP_177960) Arabidopsis NCED3 (NP_188062) Tomato Not (T07123)

Arabidopsis (NP_174302) Arabidopsis (NP_193569) Bean NCED (AF190462_1) TRENDS in Plant Science

Fig. 2. ClustalW dendrogram of carotenoid oxygenases and related enzymes. Accession numbers are indicated in parentheses. Plant enzymes are indicated in green, cyanobacterial enzymes in blue and animal enzymes in red. http://plants.trends.com

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orthologues, such as the tomato Not gene product [7], have been collectively named NCEDs (9-cis-epoxycarotenoid dioxygenases) even though the epoxy group is not absolutely required, at least for Vp14 activity. NCED homologues are found in plants, cyanobacteria and animals: the Synechocystis, Arabidopsis and human genomes contain two, nine and three NCED homologues, respectively. This led to the hypothesis that some of these homologues also catalysed oxidative cleavage of carotenoids. Vitamin A biosynthesis RPE65, a protein expressed in the mammalian retinal pigment epithelium, was the first animal NCED homologue to be described. RPE65 seems to be involved in retinoid, rather than carotenoid metabolism, namely in the all-trans- to 11-cis-isomerization of retinal [8]. Direct proof of the existence in animals of carotenoid cleaving enzymes belonging to the NCED family was obtained by cloning and characterizing b-carotene-15,150 -oxygenases (BCOs) from various animal species [9,10]. In accordance with the hypothesis that these enzymes catalyze the first step in vitamin A synthesis, the Drosophila ninaB mutation, which inactivates BCO, leads to reduced retinoid and rhodopsin levels and blindness [11]. In vertebrates, an additional NCED homologue that cleaves the 9,10 double bond of b-carotene and lycopene has also been identified [12]. The demonstration of both centric and excentric cleavage pathways appears to resolve a longstanding debate concerning vitamin A biosynthesis in mammals. In addition, recent analyses of the mode of action of the animal BCO suggest that the oxidative cleavage is catalysed by a monooxygenase, rather than a dioxygenase, mechanism, via a transient carotene epoxide [13]. Emerging family of carotenoid cleaving enzymes in plants Several NCED family members are encoded in the Arabidopsis genome (Fig. 2). One of them, AtNCED3, is an orthologue of Vp14 and thus involved in the ABA biosynthetic pathway [14]. In addition, an enzyme cleaving the 9,10 (90 ,100 ) double bound of various carotenoids has been recently described in molecular detail [15]. Although the exact physiological role of this enzyme is unknown, b-ionone and its derivatives are essential components of the scents of several flowers. To distinguish it from NCEDs, the name CCD (carotenoid cleavage dioxygenase) has been suggested for this enzyme. Gardenia fruits and saffron styles contain mono- and diglycosides of the C20 polyene pigment crocetin, which have an intense red colour. Similar to mycorrhizal roots, saffron also accumulates cyclohexene aldehydes, such as picrocrocin and safranal, responsible for the bitter taste and aroma of this flavouring. The pathway for the biosynthesis of crocin, picrocrocin and safranal involves the cleavage of zeaxanthin at the 7,8 (70 ,80 ) positions [16] (Fig. 1). The enzyme responsible for this cleavage reaction was cloned using degenerated oligonucleotides, representing conserved regions present in carotenoid cleavage enzymes. Two different cDNAs were cloned from stylar tissues: an orthologue of the CCD from Arabidopsis and a http://plants.trends.com


novel enzyme termed ZCD (zeaxanthin cleavage dioxygenase) catalysing the 7,8 (70 ,80 ) cleavage leading to the synthesis of crocin, picrocrocin and safranal (Fig. 1). The ZCD expression is restricted to the stylar tissue, which agrees with its role in the formation of the chromoplastderived apocarotenoids. Concluding remarks The different carotenoid cleavage enzymes identified to date exhibit variations in carotenoid substrates and position of cleavage, and different clades are recognizable within this enzyme family (Fig. 2). With increasing numbers of sequences becoming available in the public databases, this information can be used to define and narrow the catalytic domains of carotenoid cleavage enzymes and to identify their active sites as well as their physiological roles. Besides being a tool for increasing the levels of agronomically valuable substances such as colourants, scents and flavours, the identification of carotenoid cleavage enzymes enables ABA levels to be modified, which might lead to the development of crop plants with improved drought and stress tolerance [17] [14] as well as the metabolic engineering of retinol (vitamin A) production in plants and microorganisms. Acknowledgements We thank Jan D. Zeevaart and Dieter Strack for useful comments on the manuscript. Our work is supported by the EC (project ProVitA) and the Italian Ministry of Research and Education (special fund for basic research).

References 1 Spudich, J.L. et al. (2000) Retinylidene proteins: structures and functions from archaea to humans. Annu. Rev. Cell Dev. Biol. 16, 365 – 392 2 Cutler, A.J. and Krochko, J.E. (1999) Formation and breakdown of ABA. Trends Plant Sci. 4, 472– 478 3 Walter, M.H. et al. (2000) Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the ‘yellow pigment’ and other apocarotenoids. Plant J. 21, 571 – 578 4 Fester, T. et al. (1999) Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria. Mycorrhiza 8, 241 – 246 5 Tan, B.C. et al. (1997) Genetic control of abscisic acid biosynthesis in maize. Proc. Natl. Acad. Sci. U. S. A. 94, 12235 – 12240 6 Schwartz, S.H. et al. (1997) Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276, 1872– 1874 7 Burbidge, A. et al. (1999) Characterization of the ABA-deficient tomato mutant notabilis and its relationship with maize Vp14. Plant J. 17, 427 – 431 8 Redmond, T.M. et al. (1998) Rpe65 is necessary for production of 11-cisvitamin A in the retinal visual cycle. Nat. Genet. 20, 344– 351 9 von Lintig, J. and Vogt, K. (2000) Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving b-carotene to retinal. J. Biol. Chem. 275, 11915 – 11920 10 Wyss, A. et al. (2000) Cloning and expression of b,b-carotene 15,150 dioxygenase. Biochem. Biophys. Res. Commun. 271, 334 – 336 11 von Lintig, J. et al. (2001) Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo. Proc. Natl. Acad. Sci. U. S. A. 98, 1130 – 1135 12 Kiefer, C. et al. (2001) Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A. J. Biol. Chem. 276, 14110 – 14116

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13 Leuenberger, M.G. et al. (2001) The reaction mechanism of the enzyme-catalyzed central cleavage of b-carotene to retinal. Angew. Chem. Int. Ed. Engl. 40, 2613 – 2617 14 Iuchi, S. et al. (2001) Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 27, 325– 333 15 Schwartz, S.H. et al. (2001) Characterization of a novel carotenoid cleavage dioxygenase from plants. J. Biol. Chem. 276, 25208 – 25211 16 Bouvier, F. et al. (2003) Oxidative remodeling of chromoplast


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carotenoids: identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in crocus secondary metabolite biogenesis. Plant Cell 15, 47 – 62 17 Thompson, A.J. et al. (2000) Ectopic expression of a tomato 9-cisepoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J. 23, 363 – 374 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00053-0

A novel gene for rust resistance Richard C. Staples Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, NY 14853, USA

The Rpg1 gene, which has provided North American cultivars of barley with resistance to the stem rust fungus Puccinia graminis f.sp. tritici for more than 60 years, has been cloned. A single copy of the gene can confer resistance to a susceptible barley variety. Although unexplained, the progeny are consistently more resistant than the variety from which the gene was obtained. The gene might represent a new class of plant resistance genes. Stem rust caused by the fungus Puccinia graminis f.sp. tritici (PGT) historically has been the most devastating disease of barley in the USA. The rust fungi are obligately biotrophic pathogens; they grow and reproduce only on living host plants [1]. Stem rust is heteroecious: five types of spores are borne on two alternate hosts, the asexual stage on the cereal host and the sexual stage on barberry (Berberis spp.). Although stem rust is caused by a single species of fungus, Puccinia graminis, there is considerable genetic variation within the species and there are special forms of the fungus, forma specialis (f.sp.), that differ from each other in host range. As well as causing stem rust on wheat, Puccinia graminis f.sp. tritici also causes stem rust on barley. Elvin C. Stakman and his associates in Minnesota found that even Puccinia graminis f.sp. tritici was not homogenous, consisting instead of a multitude of physiological forms (i.e. pathotypes or races) that differed from each other in ability to attack particular cultivars of the cereal host [2,3]. The recognition of races within the stem rust species and the development of a simple method for their identity vastly improved the breeding of rustresistant crops. New races can arise through mutations that occur at a sufficiently high rate that race-specific resistance mediated by single R genes is easily broken, and even completely resistant varieties do not remain so forever. The continuous production of mutants and hybrids in PGT leads, sooner or later, to the appearance of pathotypes that can infect previously resistant varieties. Corresponding author: Richard C. Staples ([email protected]). http://plants.trends.com

In 1947, the recognition that PGTconsists of physiological pathotypes enabled Harold H. Flor [4] to show that resistance in flax to the flax rust fungus Melampsora lini is governed by pairs of genes: the resistance (R) genes of the plant and the avirulence (Avr) genes of the pathogen. Generally R and Avr genes are dominant. To gain resistance, the R and Avr genes must interact, but it is not clear yet how this happens. Today, several resistance genes, including the flax rust resistance gene L6, have been cloned, and molecular studies have confirmed Flor’s gene-for-gene model [5]. Rpg1 resistance gene In barley, the Rpg1 gene has provided resistance to most pathotypes of the stem rust fungus since 1942. It has been incorporated into North American barley cultivars to breed agronomically useful resistant varieties of barley to control the stem rust epidemics that plagued the Northern Great Plains of the USA during the first half of the 20th century, a remarkable span of time [6]. The Rpg1 gene can be traced to a gene source from Switzerland that yielded the resistant cultivars Chevron and Peatland [7]. In contrast with experience that resistance mediated by single R genes is unstable, Rpg1 has provided durable protection against stem rust losses in widely grown barley cultivars, but the gene had not been cloned. Now, using map-based cloning, Robert Brueggeman and colleagues [7] have cloned the gene from the barley cultivar Morex, a derivative of one of the original cultivars, and Henriette Horvath et al. [8] have genetically engineered the gene into a susceptible variety of barley, showing that a stem-rust-susceptible barley cultivar can be made resistant by transformation with the cloned Rpg1 gene. The identity of the cloned Rpg1 gene was confirmed by high-resolution genetic and physical mapping and other techniques [7]. Rpg1 is located on the short arm of chromosome 1, and the gene contains 14 exons in a total sequence of 4466 bp coding for an 837 amino acid protein [7]. Translation of the sequence showed amino acid homology to the S-receptor kinase gene SRK protein family [8]. The SRK protein family encodes a plasma membrane-spanning receptor serine/threonine kinase