Role of carotenoid cleavage dioxygenase 1 (CCD1) in apocarotenoid biogenesis revisited

Abstract

Oxidative tailoring of C₄₀ carotenoids by double bond-specific cleavage enzymes (carotenoid cleavage dioxygenases, CCDs) gives rise to various apocarotenoids. AtCCD1 generating C₁₃ and C₁₄ apocarotenoids and orthologous enzymes in other plants are the only CCDs acting in the cytosol, while the hitherto presumed C₄₀ substrate is localized in the plastid. A new model for CCD1 action arising from a RNAi-mediated CCD1 gene silencing study in mycorrhizal hairy roots of Medicago truncatula may solve this contradiction. This approach unexpectedly resulted in the accumulation of C₂₇ apocarotenoids but not C₄₀ carotenoids suggesting C₂₇ as the main substrates for CCD1 in planta. It further implies a consecutive two-step cleavage process, in which another CCD performs the primary cleavage of C₄₀ to C₂₇ in the plastid followed by C₂₇ export and further cleavage by CCD1 in the cytosol. We compare the specificities and subcellular locations of the various CCDs and propose the plastidial CCD7 to be the first player in mycorrhizal apocarotenoid biogenesis.

Carotenoids are isoprenoids (mostly C₄₀) synthesized by bacteria, algae, fungi and plants. Coordinated tailoring by specific enzymes is thought to be the main principle for generation of defined carotenoid cleavage products (apocarotenoids), which have important functions in their own right.¹ These enzymes, called carotenoid cleavage dioxygenases (CCDs), exhibit a high degree of regio- and stereospecificity for certain double bond positions as opposed to their frequent promiscuity towards substrates. In Arabidopsis, the CCD family consists of nine members forming the basis for CCD classification in plants. Five of them exhibit specificity for 9-cis-epoxycarotenoids and are designated nine-cis-epoxycarotenoid dioxygenases (NCEDs). They are involved in ABA biosynthesis.^1–3 The remaining 4 CCDs (AtCCD1, AtCCD4, AtCCD7, AtCCD8) cleave a variety of transcarotenoid substrates.^1,4 The majority of CCDs/NCEDs has been shown to reside in plastids. The only exception is AtCCD1 and orthologous enzymes in other plants, which act in the cytosol to generate C₁₃ and C₁₄ apocarotenoids.^1,4 This contribution will compare current knowledge on enzymatic carotenoid cleavage pathways with recently obtained new insights resulting from silencing the expression of a CCD1 gene in plant roots colonized by arbuscular mycorrhizal (AM) fungi.⁵

Known Pathways of Carotenoid Cleavage Leading to C₁₅ and C₁₈ Apocarotenoids

The phytohormone ABA is the best-studied member of plant apocarotenoids. Single-step cleavage of the 11,12 double bond of 9-cis violaxanthin and 9′-cis neoxanthin by NCEDs results in formation of C₁₅ xanthoxin inside plastids (Fig. 1A).² The next steps of xanthoxin conversion to abscisic aldehyde and abscisic acid are known to take place in the cytosol. Thus, considering the plastidial location of NCEDs, a transport of xanthoxin into the cytosol must be postulated but the mechanisms by which this occurs are still unknown.⁶

Figure 1.

Comparison of substrates, enzymes and their compartmentation in three carotenoid cleavage pathways. (A) ABA biosynthesis involves cleavage of cis-carotenoid substrates by NCEDs in the plastid, C₁₅ xanthoxin export to the cytosol followed by further metabolization steps and transport.⁶ (B) Strigolactone biosynthesis is assumed to start from β-carotene and to proceed via two consecutive cleavage steps (CCD7 and CCD8) inside the plastid as exemplified by the MAX3 and MAX4 proteins of Arabidopsis.⁴ The C₁₈ cleavage product of CCD8 or a derivative of it is predicted to serve as mobile strigolactone precursor undergoing export to the cytosol, further modification steps, transport and eventually perception as a regulator of shoot branching.⁷ (C) Proposed organization of local C₁₃ and C₁₄ apocarotenoid biosynthesis in a mycorrhizal root cell. Lactucaxanthin as the tentatively proposed C₄₀ carotenoid precursor containing two α-ionone rings is tailored by two consecutive cleavage steps in the plastid (CCD7) and subsequently, following export of the C₂₇ intermediate, in the cytosol (CCD1). The C₂₇ intermediate has only been detected upon silencing CCD1 expression.⁵ Additional modification steps in the cytosol lead to the various C₁₃ cyclohexenone and C₁₄ mycorradicin derivatives accumulating in mycorrhizal roots. Abbreviations: MAX, more axillary branching; Gly, glycoside.

In addition to ABA, another carotenoid-derived phytohormone exists, whose long-sought chemical nature was recently identified as strigolactone.^7–9 Mutants in its biosynthesis or its perception display a striking increase in shoot branching.⁷ One CCD involved is CCD7 converting C₄₀ trans-carotenoids to C₂₇ apocarotenoids (Fig. 1B).^10,11 A second cleavage activity is contributed by CCD8. Several lines of evidence argue for a consecutive action of these CCDs with CCD8 converting C₂₇ to C₁₈ and C₉ (Fig. 1B).^7,11 Both CCDs have transit peptides indicative of their action in plastids.⁴ The C₁₈ reaction product of CCD8 and strigolactone precursor subsequently undergoes still uncharacterized steps of export from the plastid, further metabolization and transport to the shoot (Fig. 1B).

Strigolactones were previously known as germination stimulants for parasitic weeds and as signaling molecules to promote hyphal branching of AM fungi.^12,13 Most recent data might point to additional roles of strigolactones in roots. A C₁₈ β-apo-13-carotenone called “D'orenone” blocks the growth of root hairs by interfering with PIN2-mediated auxin transport.¹⁴ The synthetic compound “D'orenone” is structurally identical to the proposed C₁₈ apocarotenoid precursor of strigolactone biosynthesis (Fig. 1B). The effects observed might therefore be strigolactone-related.

C₁₃ and C₁₄ Apocarotenoid Biogenesis via CCD1: Single-Step or Stepwise Cleavage and the Importance of Compartmentation

Strigolactones exert their signaling functions in low amounts and can act in early stages of the AM symbiosis. Conversely, two other classes of apocarotenoids with unknown functions accumulate in large amounts in mycorrhizal roots and at later stages of the AM interaction.^15,16 These AM-induced colorless C₁₃ cyclohexenone and yellow linear polyene derivatives have been C₁₄ identified independently but probably originate from a common precursor (Fig. 1C).^15–17 Both types of compounds accumulate locally in cells harbouring arbuscules, which are the symbiotic organs of the AM symbiosis mediating nutrient exchange between plant and fungus.¹⁸ To identify a function for these apocarotenoids in the symbiosis, both a CCD1 gene and an AM-induced MEP pathway isogene were targeted by gene silencing approaches.^5,19

CCD1 is, next to the NCEDs, the best-studied CCD due to its involvement in C₁₃ apocarotenoid-based flower scent as well as fruit and wine aroma biosynthesis.^20–22 Recombinant CCD1 enzymes from several plants have been shown to preferentially catalyze a single-step symmetrical cleavage at the 9,10 and the 9′,10′ double bonds of various C₄₀ carotenoids.^1,4,22,23 Cleavage activity on 5,6 (5′,6′) double bonds in vitro has also been reported.²⁴ However, being able to convert a substrate in vitro does not mean that this activity must be the main in vivo functional role of the enzyme. Indeed, in planta studies with CCD1 mutants or gene silencing transgenics have raised doubts in an exclusive role of CCD1 in C₁₃ apocarotenoid generation.^4,20,21 Strong suppression of CCD1 transcript accumulation resulted in near 50% C₁₃ apocarotenoid levels compared to wild type plants implicating additional players in C₁₃ apocarotenoid biogenesis.^20,21

Given this background knowledge of presumed single-step symmetrical CCD1 action on C₄₀ carotenoid substrates we performed a CCD1 RNAi knock-down approach in hairy roots of Medicago truncatula. Surprisingly, HPLC analyses of mycorrhizal hairy roots clearly indicated a differential reduction of C₁₃ and C₁₄ apocarotenoids. C₁₄ mycorradicin derivatives were strongly reduced (3–6% residual amounts relative to mycorrhizal empty vector controls) but C₁₃ cycohexenone derivatives exhibited an actual decrease to only 30–47% of empy vector controls.⁵ This result is incompatible with the assumption of a symmetrical cleavage action of CCD1 in planta. Moreover, a striking color change to yellow-orange was observed in mycorrhizal RNAi roots. Analysis of the corresponding chromophore indicated that its chemical nature is a C₂₇ apocarotenoid. This suggests that C₂₇ but not C₄₀ derivatives as previously thought¹⁷ are the main substrates for CCD1 in mycorrhizal roots. Based on these data a new scheme of carotenoid cleavage and CCD1 action in mycorrhizal roots is proposed, in which CCD1 catalyzes only the second of at least two carotenoid cleavage steps from C₄₀ carotenoids to the C₁₃/C₁₄ apocarotenoid end-products (Fig. 1C). As in the case of strigolactone biosynthesis a consecutive action of two CCDs on C₄₀ carotenoids and on the primary cleavage product, respectively, is predicted. Interestingly, the two pathways have a C₂₇ intermediate in common (Fig. 1B and C). In strigolactone biosynthesis the generation of C₂₇ apocarotenoids is due to CCD7 activity and it is tempting to speculate that CCD7 is the first cleavage enzyme in AM-induced C₁₃/C₁₄ apocarotenoid biosynthesis as well. Preliminary evidence for such an involvement has been obtained.⁵ CCD7 might thus constitute a crosspoint where the two pathways meet but subsequently branch into different directions towards CCD8 or CCD1, respectively (Fig. 1B and C). This is supported by the wide substrate specificity of CCD7,¹⁰ as opposed to the narrow specificity reported for CCD8.²⁵

However, compartmentation of the second cleavage step in both pathways is different. The C₂₇ compound tentatively identified in mycorrhizal CCD1-RNAi roots was a 3-hydroxy-α-apo-10′carotenoic acid glycosylated by two hexose moieties at the 3-hydroxyl position.⁵ Glycosylation is a modification usually carried out by cytosolic enzymes. Therefore, the C₂₇ compound is most likely exported from the plastid to the cytosol, where it is further cleaved by CCD1. In this new view of carotenoid cleavage and its compartmentation (Fig. 1A–C) the cytosolic location of CCD1 finally makes sense.

In conclusion, we have presented here a new scheme of C₁₃ and C₁₄ apocarotenoid biogenesis and of the role of CCD1 in this process, which illustrates both the importance of consecutive cleavage steps and the decisive role of compartmentation. The pivotal question is now, whether this scheme is also applicable to the biosynthesis of C₁₃ apocarotenoids (also referred to as “C₁₃ norisoprenoids”²²) involved in flower scent, fruit aroma and wine bouquet.