This review focuses on plant carotenoids, but it also includes progress made on microbial and animal carotenoid metabolism to better understand the functions and the evolution of these structurally diverse compounds with a common backbone. Plants have evolved isogenes for specific key steps of carotenoid biosynthesis with differential expression profiles, whose characteristic features will be compared. Perhaps the most exciting progress has been made in studies of carotenoid cleavage products (apocarotenoids) with an ever-expanding variety of novel functions being discovered. This review therefore covers structural, molecular genetic and functional aspects of carotenoids and apocarotenoids alike. Apocarotenoids are specifically tailored from carotenoids by a family of oxidative cleavage enzymes, but whether there are contributions to their generation from chemical oxidation, photooxidation or other mechanisms is largely unknown. Control of carotenoid homeostasis is discussed in the context of biosynthetic and degradative reactions but also in the context of subcellular environments for deposition and sequestration within and outside of plastids. Other aspects of carotenoid research, including metabolic engineering and synthetic biology approaches, will only be covered briefly.

Apocarotenoids are tailored from carotenoids by oxidative enzymes [carotenoid cleavage oxygenases (CCOs)], cleaving specific double bonds of the polyene chain. The cleavage products can act as hormones, signaling compounds, chromophores and scent/aroma constituents. Recent advances were the identification of strigolactones as apocarotenoids and the description of their novel role as shoot branching inhibitor hormones. Strigolactones are also involved in plant signaling to both harmful (parasitic weeds) and beneficial [arbuscular mycorrhizal (AM) fungi] rhizosphere residents. This review describes the progress in the characterization of CCOs, termed CCDs and NCEDs, in plants. It highlights the importance of sequential cleavage reactions of C(40) carotenoid precursors, the apocarotenoid cleavage oxygenase (ACO) nature of several CCOs and the topic of compartmentation. Work on the biosynthesis of abundant C(13) cyclohexenone and C(14) mycorradicin apocarotenoids in mycorrhizal roots has revealed a new role of CCD1 as an ACO of C(27) apocarotenoid intermediates, following their predicted export from plastid to cytosol. Manipulation of the AM-induced apocarotenoid pathway further suggests novel roles of C(13) apocarotenoids in controlling arbuscule turnover in the AM symbiosis. CCD7 has been established as a biosynthetic crosspoint, controlling both strigolactone and AM-induced C(13) apocarotenoid biosynthesis. Interdependence of the two apocarotenoid pathways may thus play a role in AM-mediated reduction of parasitic weed infestations. Potential scenarios of C(13) scent/aroma volatile biogenesis are discussed, including the novel mechanism revealed from mycorrhizal roots. The recent progress in apocarotenoid research opens up new perspectives for fundamental work, but has also great application potential for the horticulture, food and fragrance industries.

SUMMARYThe regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C 13 cyclohexenone and C 14 mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source-sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids.

SummaryIsopentenyl diphosphate, the universal precursor of isoprenoids, is synthesized by two separate routes, one in the cytosol and the other in plastids. The initial step of the plastidial pathway is catalysed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which was previously thought to be encoded by a singlecopy gene. We have identi®ed two distinct classes of DXS-like cDNAs from the model legume Medicago truncatula. The deduced mature MtDXS1 and MtDXS2 proteins, excluding the predicted plastid-targeting peptides, are similar in size (72.7 and 71.2 kDa) yet share only 70% identity in their amino acid sequences, and both encode functional DXS proteins as shown by heterologous expression in Escherichia coli. Available DXS sequences from other plants can easily be assigned to either class 1 or class 2. Partial sequences of multiple DXS genes in a single genome may be found in the databases of several monocot and dicot plants. Blot analyses of RNA from M. truncatula, maize, tomato and tobacco demonstrate preferential expression of DXS1 genes in many developing plant tissues except roots. By contrast, DXS2 transcript levels are low in most tissues but are strongly stimulated in roots upon colonization by mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids. Monoterpene-synthesizing gland cells of leaf trichomes appear to be another site of DXS2 gene activity. The potential importance of DXS1 in many housekeeping functions and a still hypothetical role of DXS2 in the biosynthesis of secondary isoprenoids is discussed.

SummaryPlants and certain bacteria use a non-mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids. This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway. We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). This induction is temporarily and spatially correlated with speci®c and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C 13 cyclohexenone derivatives and mycorradicin (C 14 ) conjugates, the latter being a major component of the long-knowǹ yellow pigment'. A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots. Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identi®ed from mycorrhizal wheat roots after alkaline treatment of an`apocarotenoid complex' of yellow root constituents. We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor. This is the ®rst report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus.

We have searched for induced transcripts in a cDNA library derived from bean cell supension cultures treated with an elicitor from Colletotrichum lindemuthianum. Six independently isolated cDNAs corresponding to rapidly induced small mRNAs have been classified by their DNA sequence and slightly different induction behaviour into two groups. 5'- and 3'-untranslated regions exhibit little similarity, but the deduced small acidic proteins designated PvPR1 and PvPR2 are 89% identical. No relationship was found with the well-characterized PR1 proteins from tobacco. However, the PvPR proteins are closely related to pI49 in pea (64% identity), pSTH2 in potato (41% identity) and PcPR1-1 in parsley (39% identity), which are also induced in response to elicitor or microbial attack. Moreover, a major pollen allergen in birch (BetvI) has a 44% identity with PvPR1 proteins. These similarities establish a ubiquitous class of conserved defense-related proteins and suggest a common yet still unknown function. Southern blot analysis indicates that PvPR protein gene organization is highly complex with an estimated copy number of more than 12 genes.

Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C 40 carotenoid substrates at 9,10 and 9#,10# positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C 13 cyclohexenone and C 14 mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C 27 apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C 27 apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C 27 apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C 27 / C 14 + C 13 ), while the first step (C 40 / C 27 + C 13 ) may be catalyzed by CCD7 and/or CCD4 inside plastids.

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