Recent studies suggest a number of uncharacterised ACSs modulate plant developmental and/or stress responses. This is in addition to phytohormones such as ABA and strigolactones, new aspects of which are still being discovered (Fan et al., 2009; Seto and Yamaguchi, 2014). In fact, the structure of apocarotenoids suggests that they can play signaling roles. Most apocarotenoids contain an α,β-unsaturated carbonyl moiety, which can easily react with the nucleophilic moieties of biological molecules (Farmer and Mueller, 2013). Reactive electrophilic species (RES) can react with thiols on transcription factors, altering gene expression (Levonen et al., 2004); this is how apocarotenoids mediate apoptosis (Liu et al., 2008) and retinoid signaling (Eroglu et al., 2012) in mammals. The relationships between apocarotenoid structure and signalling activity however, remain unclear (Linnewiel et al., 2009)
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in most instances. Indeed, most plant ACSs remain unidentified.
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Figure 1.3 Apocarotenoid biosynthesis. Many carotenoids are cleaved to form apocarotenoids through enzymatic reactions. Nine-cis-epoxy-dioxygenase (NCED) cleaves 9-
cis-violaxanthin and 9-cis-neoxanthin to yield the precursor of ABA. The Carotenoid Cleavage Dioxygenase (CCD) enzymes cleave carotenoids to yield various apocarotenoids. CCD7 and CCD8, for example, contribute to strigolactone synthesis following β-carotene isomerisation by D27. The CCD8 product carlactone is further elaborated by the P450 enzyme MAX1, and possibly other unknown enzymes before yielding strigolactones (5-deoxystrigol is depicted as an example). CCD7 and CCD1 contribute to the formation of mycorradicin and blumenol derivatives, which accumulate in AM-colonised roots. CCD7 cleaves an unknown carotenoid substrate (possibly lutein or an ε,ε-xanthophyll) to yield a C27 apocarotenoid which is further cleaved by CCD1. The end products mycorradicin and blumenol are often further glycosylated. One ACS, β-cyclocitral, is formed via non-enzymatic cleavage of β-carotene. cis-carotene- derived apocarotenoids may also be important plant development signals. We propose at least two groups of unidentified Apocarotenoid Signals, ACS1 and ACS2, function in a range of processes, including feedback regulation, based on evidence from mutants in the cis-carotene pathway (i.e. Arabidopsis mutants psy, pds3, ziso, clb5 and ccr2, and S. lycopersicum mutants
yellow-flesh, z2803and tangerine). Another unidentified signal, ACS3, seems to be derived from
β-carotene cleavage and functions in root development. Some apocarotenoids are flavour, fragrance and pigment apocarotenoids (mainly in carotenoid sink tissues); these are depicted in the top-right panel. CCD cleavage activities have been denoted by coloured circles next to the carotenoid substrates (see colour code). Given the importance of chemical inhibitors in the study of apocarotenoid biosynthesis, the enzymatic targets of norflurazon, fluridone, CPTA, D15 and abamine-SG have been annotated with coloured stars (see colour code).
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Figure 1.4 Apocarotenoid signal generation and perception take place across several subcellular compartments. In this scheme, the biosynthesis of some known (ABA, SL, ‘Yellow Pigment’ products, β-cyclocitral) and novel (ACS1) ACSs are placed in the context of a plant cell, and the various chloroplastic compartments. The carotenoid precursors are typically found in chloroplast membranes, such as PSII-associated β-carotene. The 9-cis-
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epoxyxanthophyll ABA precursors are believed to be inner membrane-embedded, given the necessary carotenogenic enzymes are also localised here. ζ-carotene and phytofluene, putative ACS1 precursors, are believed to accumulate in the plastoglobules. It is not known where the precursors for strigolactones (9-cis-β-carotene) and the yellow-pigment products (unknown) are localised. These substrates are cleaved by CCD and NCED enzymes, save for β- carotene, which is cleaved by singlet-oxygen to yield β-cyclocitral. CCD4 is localised to the plastoglobules, consistent with its putative role in ACS1 generation. CCD7 and CCD8 are localised to the stroma, as are the NCEDs involved in ABA biosynthesis (although transient association with the stroma-facing thylakoid membrane has also been observed). Subsequent modification of ACS precursors may occur in the cytoplasm or other organelles, as depicted for ABA, SL and yellow pigment products. Unknown mechanisms must export these precursors from the chloroplast. Notably, elaboration of carlactone to strigolactones may occur in the endoplasmic reticulum, based on localisation predictions for MAX1, a strigolactone biosynthetic enzyme downstream of CCD8. CCD1, localised to the cytosol (but also known to associate with the cytoplasm-facing outer chloroplast membrane), further cleaves C27- apocarotenoid substrates to yield the yellow pigment products. ACSs may then interact with receptors, such as for ABA, which binds to PYR/PYL/RCARs (denoted here as PYR) and SL to MAX2, to effect biological response(s). It is not known how, or where, the yellow pigment products, ACS1-3 and β-cyclocitral exert their effects. Abbreviations: AAO3, Arabidopsis
aldehyde oxidase 3; ABA, abscisic acid; ABA2, short-chain dehydrogenase/reductase ABA2; BETA, β-carotene; β-CYC, β-cyclocitral; ER, endoplasmic reticulum; MAX1, more axillary growth 1; MAX2, more axillary growth 2; PHYTF, phytofluene; PYR1, pyrabactin resistance 1; ZETA, ζ-carotene.
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Box 1.1 Environmental and developmental stimuli regulate apocarotenoid formation
Many examples exist of changes in apocarotenoid accumulation in response to environmental and/or developmental cues. This, together with genetic and biochemical studies demonstrate that some apocarotenoids have regulatory roles in planta. The nature of the environmental and developmental stimuli that alter apocarotenoid formation may in some cases also reflect the ecological roles of apocarotenoids (e.g. as pollinator attractants). Examples of stimuli that affect apocarotenoid formation are summarised in Table 1.1.
Environmental and developmental stimuli also alter the expression of many carotenoid biosynthetic genes, altering carotenoid composition, which may in turn promote production of specific apocarotenoids (Tuan et al., 2013a; Tuan et al., 2013b; Lao et al., 2014). However, the relationship between carotenoid and apocarotenoid levels is not always straightforward as expression and localisation of substrates and enzymes will affect apocarotenoid production.
Table 1.1 Environmental stimuli that regulate apocarotenoid formation and CCD enzymes involved
Environmental
stimulus Species CCD enzyme involved Apocarotenoids produced Refs.
Light Petunia hybrida CCD1 β-ionone (Simkin et al.,
2004b)
Light Phaseolus vulgaris Unknown 3-hydroxy-β-ionone (Kato-Noguchi, 1996)
Seed dehiscence and dark-induced leaf senescence
Arabidopsis thaliana CCD4 Possibly β-carotene-
derived apocarotenoids (Gonzalez-Jorge et al., 2013) Wounding, heat,
cold, osmotic stress Crocus sativus CCD4c Possibly β-cyclocitral β-ionone and (Rubio-Moraga et al., 2014) Abbreviations: CCD, carotenoid cleavage dioxygenase.
biogenesis (Avendano-Vazquez et al., 2014). The Arabidopsischloroplast biogenesis 5 (clb5) mutant, lacking ζ-carotene desaturase (ZDS) function, accumulates cis-carotenes upstream of ZDS (Figure 1.3). Clb5 plants also exhibits needle-like, translucent leaves and strong suppression of nuclear- and plastid-encoded genes required for chloroplast biogenesis, photosynthetic activity and carotenoid biosynthesis. Genetic and biochemical analyses demonstrated the clb5 leaf and transcriptional phenotypes were due to cleavage of
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phytofluene and/or ζ–carotene isomers to produce ACS1 (Figure 1.3). That is, blocking accumulation or cleavage by CCD4 of these carotenes rescues leaf development and gene expression patterns (Avendano-Vazquez et al., 2014). Curiously, past in vitro studies suggest
cis-ζ-carotene isomers are not CCD4 substrates (Huang et al., 2009), but this does not preclude phytofluene or cis-ζ-carotene cleavage in planta and demonstrates the need for multiple lines of evidence when determining in vivo carotenoid substrates for CCDs.
What is ACS1 and where might it be produced? Proteomics and GFP-tagging data indicate CCD4 is localised to plastoglobules (Figure 1.4) (Ytterberg et al., 2006; Lundquist et al., 2012). In chromoplasts, these plastidic structures accumulate high levels of carotenoids (Rubio et al., 2008; Frusciante et al., 2014) and chloroplastic plastoglobules may also contain carotenoids. Thus, it is likely the first step in ACS1 biosynthesis might be in these suborganellar structures, but the structure, the number of steps in its biosynthesis and its mechanism of action are all unknown.
cis-carotenoids in tomato (Solanum lycopersicum) fruit may yield ACS2. SlPSY1 transcription, eliminated in the yellow-flesh mutant r2997, is partially recovered in the double mutant
r2997/t3002 (Figure 1.3). PSY1 transcription recovery seems linked to accumulation of tetra-cis-
lycopene and various neurosporene isomers present in the t3002 tomato, which lacks a
functional CRTISO. Either of these two cis-carotenoids might be precursors of ACS2. Consistent with our theory, expression of PSY1 is not rescued in the loss-of-function PSY/ZISO double mutant r2997/z2803that lacks the aforementioned cis-carotenoids (Figure 1.3) (Kachanovsky et
al., 2012).
ACS3, derived from the β,β-branch of the carotenoid pathway, regulates periodic root branching and lateral root (LR) capacity in Arabidopsis in conjunction with the oscillatory LR- clock gene expression network (Van Norman et al., 2014). Treatment of Arabidopsis seedlings with the carotenoid cleavage inhibitor D15 reduced LR capacity, similarly to that seen upon carotenoid biosynthesis inhibition (i.e. via Norflurazon and CPTA treatment) and in carotenogenic mutants (clb6 and psy, Figure 1.3). Genetic analysis suggests that the reduced LR-capacity was not related to ABA or strigolactone signaling, demonstrating the existence of a yet to be identified ACS3. Analysis of the lut mutants revealed ACS3 was not derived from the ε,β-carotenoid pathway branch, indicating ACS3 is derived from a β,β-carotenoid (Van Norman et al., 2014).
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Are there other ACSs? Metabolic feedback within the carotenoid and MEP pathways at transcriptional and/or post-transcriptional levels is essential for regulating carotenoid biosynthesis in planta (Qin et al., 2007; Bai et al., 2009; Rodriguez-Villalon et al., 2009a; Ruiz- Sola and Rodriguez-Concepcion, 2012) and feedback metabolites may originate from the plant poly-cis carotenoid pathway, in which phytoene is converted to lycopene. In bacteria, CrtI converts 15-cis-phytoene to all-trans-lycopene, whereas plants, algae and cynobacteria require two desaturases and two isomerases that are highly conserved (Sandmann, 2009). The cis-configured intermediates produced by these enzymes may be susceptible to cleavage and modification to form plant-specific regulatory signals, thereby fine-tuning the carotenoid pathway in response to stimuli (Kachanovsky et al., 2012).