cis-carotene biosynthesis, evolution and regulation in plants: The emergence of novel signaling metabolites

Accepted Manuscript cis-carotene biosynthesis, evolution and regulation in plants: The emergence of novel signaling metabolites Yagiz Alagoz, Pranjali Nayak, Namraj Dhami, Christopher I. Cazzonelli PII:

S0003-9861(18)30171-1

DOI:

10.1016/j.abb.2018.07.014

Reference:

YABBI 7777

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 2 March 2018 Revised Date:

11 July 2018

Accepted Date: 13 July 2018

Please cite this article as: Y. Alagoz, P. Nayak, N. Dhami, C.I. Cazzonelli, cis-carotene biosynthesis, evolution and regulation in plants: The emergence of novel signaling metabolites, Archives of Biochemistry and Biophysics (2018), doi: 10.1016/j.abb.2018.07.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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cis-carotene biosynthesis, evolution and regulation in plants: the emergence of novel signaling metabolites *

Yagiz Alagoz, *Pranjali Nayak, Namraj Dhami, Christopher I Cazzonelli

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Joint First authors

Affiliations:

Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797, Penrith

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NSW 2751, Australia

Corresponding Author: Christopher I Cazzonelli ([email protected])

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Telephone Number: 612-45701752

Address: Hawkesbury Institute for the Environment, Western Sydney University, Richmond NSW 2753, Australia

The author (s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors are: Christopher

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Cazzonelli ([email protected])

The authors declare no conflict of interest

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KEYWORDS

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Running Title: cis-carotene derived signaling metabolites

Carotenoid; cis-carotene; apocarotenoid; regulatory switch; photoswitch; photoisomerization

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Abstract

Carotenoids are isoprenoid pigments synthesised by plants, algae, photosynthetic bacteria as well as some non-photosynthetic bacteria, fungi and insects. Abundant carotenoids found in nature are synthesised via a linear route from phytoene to lycopene after which the pathway bifurcates into cyclised α- and β-carotenes. Plants evolved additional steps to generate a diversity of cis-carotene intermediates, which can accumulate in fruits or tissues exposed to an extended period of darkness.

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Enzymatic or oxidative cleavage, light-mediated photoisomerization and histone modifications can affect cis-carotene accumulation. cis-carotene accumulation has been linked to the production of signaling metabolites that feedback and forward to regulate nuclear gene expression. When ciscarotenes accumulate, plastid biogenesis and operational control can become impaired. Carotenoid

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derived metabolites and phytohormones such as abscisic acid and strigolactones, can fine-tune cellular homeostasis. There is a hunt to identify a novel a cis-carotene derived apocarotenoid signal

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and elucidate the molecular mechanism by which it facilitates communication between the plastid and nucleus. In this review, we describe the biosynthesis and evolution of cis-carotenes and their links to regulatory switches, as well as highlight how cis-carotene derived apocarotenoid signals might control organelle communication, physiological and developmental processes in response to environmental change.

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The essential functions of carotenoids in nature

Carotenoids are conjugated isoprenoids performing important biological functions in many organisms and are essential for life. There are a large number of carotenoids produced by a variety of organisms (http://carotenoiddb.jp/) [1]. Carotenoids are essential for all photosynthetic

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organisms, although occurrence is not restricted to plants, algae, and cyanobacteria, as some nonphotosynthetic bacteria, fungi, and a few creatures belonging to the animal kingdom can also

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synthesise carotenoids [2]. In plants, they function in the light capture during photosynthesis and protect photosystems by non-photochemical quenching (NPQ), a process that can dissipate excess excitation energy generated from high light and/or temperature stress [3]. Carotenoids are precursors for the biosynthesis of phytohormones (e.g. abscisic acid; ABA, and strigolactones; SL) that control abiotic stress acclimation, shoot and root branching, as well as apocarotenoid signaling metabolites that mediate chloroplast to nucleus communications and root-mycorrhizal interactions [4-6]. Regulation of carotenoid biosynthesis occurs throughout the life cycle of a plant, with the composition and levels of carotenoids finely tuned to developmental stages and changes in the environment [7]. Carotenoids also provide bright colours like yellow, orange and reddish-pink to flowers and fruits to help attract pollinators (reviewed by [8]). 2

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In animals, carotenoids provide a distinct colouration to flamingos, salmon and finches, in which they enhance ornament-based colour signaling and attraction thereby improving reproductive success [9, 10]. Critically, carotenoids provide essential functions in vision (lutein and zeaxanthin protect against age-related macular degeneration of the eye), vitamin A biosynthesis (β-carotene is the substrate for vitamin A production), boosting the immune response (e.g. canthaxanthin, α- and β-carotene, lutein and astaxanthin), antioxidation (e.g. β-carotene), behavioural characteristics,

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reducing cancer (e.g. lycopene), as well as many other reported human health benefits [11-16]. Vitamin A deficiency is a global health issue, especially in developing countries with limited access to carotenoid-rich foods (WHO ref: http://www.who.int/nutrition/topics/vad/en/). In recent years, several biotechnological and conventional breeding programs have focussed on biofortification of

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food and feed crops with promising achievements to enhance carotenoid supplementation to the food and live-stock industry. For example, to combat vitamin-A deficiency “GOLDEN” Rice and

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Bananas were engineered with enhanced levels of pro-vitamin A, thereby making major food crops consumed around the world more nutritious [17, 18]. Another breakthrough was the horizontal genome transfer of a synthetic operon for the biosynthesis of astaxanthin (mostly accumulates in microorganisms such as microalgae, red yeast and marine bacteria) into plants, which provides a highly valued colour and micronutrient supplement for artificial feeds used in poultry and aquaculture [19, 20].

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Over the last few decades, the genes and enzymes of carotenoid metabolic pathway have been well characterised and thoroughly studied in many plants [8, 21]. There are still gaps in our understanding of carotenoid regulation, sequestration and degradation (see the following reviews: [5, 7, 22]). One area of carotenoid biology that remains rather enigmatic and a current hot topic

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relates to identifying biological functions for cis-carotenes that have emerged as eukaryotic carotenoid signatures in the upper part of the carotenoid biosynthetic pathway in plants (Figure 1).

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Photosynthetic organisms can naturally generate cis-carotenes between phytoene and lycopene. cisisomers of lycopene are found to be more soluble than their trans-configured isomers in organic solvents [23]. There is an enhanced bioavailability and absorption of lycopene by living cells when consumed from foods rich in cis-isomer “bent” forms, as opposed to the rather long and “straight” all-trans isomer forms [14, 24]. Similarly, phytoene and phytofluene have a higher bioavailability, as cis-isomers appear to be more readily absorbed from fruits and juices when compared to their alltrans counterparts [25]. The direct benefits and bioactive roles of cis-carotenes in photosynthetic organisms and animals awaits further discovery. The biological reason behind why plants evolved additional enzymatic steps in the carotenoid biosynthetic pathway producing cis-carotenes in planta is rather enigmatic. In this 3

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review, we discuss the evolution of cis-carotene biosynthesis, as well as regulation, sequestration and degradation of these signatory molecules in plants. The developmental changes that control key catalytic steps of desaturation and isomerization, as well as where, when and in what tissues they accumulate are discussed. We describe how changes in the environmental conditions such as day length and light quality can switch the state of cis/trans-isomerization, in what is referred to as photoisomerization, a phenomenon awaiting molecular characterisation. How cis-carotenes are

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linked to feedback and feedforward regulation of transcriptional and translational mechanisms, as well as organelle and nucleus communications, are discussed. Finally, potential functions for cis-

their function(s) in planta.

Carotenoid biosynthesis in plants: from cis to trans

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carotene derived apocarotenoid signal(s) are highlighted with a mechanistic view to understanding

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Isoprenoid precursor substrates required for carotenoid biosynthesis are generated through the methylerythritol 4-phosphate (MEP) pathway by conversion of pyruvate and glyceraldehyde to the C5-compound, isopentenyl pyrophosphate (IPP). IPP is a precursor for the synthesis of isoprenoids, terpenes, quinones, sterols, chlorophylls, and carotenoids. Carotenoids generally consist of eight IPP units condensed by enzymatic catalysis [8]. Altering key steps in the MEP pathway can affect the availability of isoprenoid precursors and hence downstream carotenoid

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accumulation [26, 27].

Phytoene (C40) is the first carotenoid synthesised through the head-to-head condensation of two C20 geranylgeranyl pyrophosphate compounds by PHYTOENE SYNTHASE (PSY) [28] (Figure 1). PSY represents a rate-limiting enzyme acting as a bottleneck in carotenoid biosynthesis,

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and some species have multiple isoforms that function differently depending upon the tissue, developmental stage and environment [29-34]. PSY protein levels are upregulated by light and

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reduced under darkness, thereby limiting the availability of phytoene required for cis-carotene biosynthesis [35]. Recently, it was shown that PSY is a substrate for Clp-protease (CLP) mediated proteolysis and reveals a new post-translational mechanism controlling carotenogenic enzymes [36]. CLP degradation was counteracted by the ORANGE (OR) protein to maintain the PSY protein homeostasis and modulate carotenoid biosynthesis in plants. Furthermore, OR, a post-translational regulator of PSY with holdase chaperone activity, enhanced the stability of the PSY protein and enzymatic active proportion of PSY in clpc1 mutant, thereby counter balancing CLP proteolysis and helping to maintain PSY protein homeostasis [36, 37]. Finally, the direct targeting of a PSY upstream promoter cis-element (G-box) by the bZIP transcription factor LONG HYPOCOTYL 5 (HY5) and PHYTOCHROME INTERACTING FACTORS (PIFs) form a dynamic activation4

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suppression transcriptional complex responsive to light and temperature cues [38, 39]. Overall, PSY is a central regulatory hub under tight transcriptional (HY5 and PIF1) and post-translational (CLP and OR) control that limits the synthesis of downstream cis-carotenes and the biosynthesis of xanthophylls. The PHYTOENE DESATURASE (PDS) enzyme catalyses the desaturation of a 15-cisphytoene to phytofluene and 9,15,9′-tri-cis-ζ-carotene. PDS activity is critically important during

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early chloroplast development to interact with the PLASTID TERMINAL OXIDASE (PTOX), which acts as a key factor balancing the plastoquinol redox state of thylakoid membranes by transferring electrons from the plastoquinone pool (PQ) to molecular oxygen to maintain oxidation levels [40]. A lack of PTOX protein causes a light-dependent variegation phenotype developing as

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white sectors in leaves. PDS has been described to act as a rheostat most likely together with PTOX to facilitate retrograde metabolite signaling from the chloroplast to the nucleus as a crucial

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determinant of regulating a suite of photosynthesis associated genes required for chloroplast biogenesis and operational controls [41, 42].

Next, ζ-CAROTENE ISOMERASE (Z-ISO), a bona fide integral plastidial membrane enzyme catalyses the tri-cis-di-cis isomerization of the 15-15′ carbon-carbon double bond in 9,15,9′-tri-cis-ζ-carotene to form 9,9′-di-cis-ζ-carotene [43]. Isomerization is dependent on a unique heme cofactor carried by Z-ISO, that can undergo redox-dependent ligand switching [44].

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Photosynthetic activity in the light and non-photosynthetic activity in the dark can alter the redox state of plastids. Indeed, carotenoid biosynthesis is also under redox control, supported in part through evidence showing that Z-ISO mutants block the production of cis-carotene biosynthesis in a tissue-specific manner [45]. Changes in plastid redox state may directly influence Z-ISO activity

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and consequently alter carotenoid flux and accumulation. Redox tuning of Z-ISO activity could provide a gatekeeper for the dynamic control over cis-carotene biosynthesis and help to rapidly

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adjust the carotenoid pool in response to changes in photosynthesis and/or oxidative stress. ζ-CAROTENE DESATURASE (ZDS) converts 9,9′-di-cis-ζ-carotene to 7,9,7',9'-tetra-cislycopene (pro-lycopene, poly-cis-lycopene or tetra-cis-lycopene) [46, 47]. ZDS inserts additional cis double bonds at the 7,7′ positions and acts in concert with PDS to insert trans double bonds at the 11,11′ positions, in what becomes two separate symmetric catalysed dehydrogenation reactions [48]. Interestingly, the co-expression of PDS and ZDS enzymes in Escherichia coli cells producing phytoene yielded tetra-cis-lycopene (pro-lycopene) in the light, the final cis-carotene required for biosynthesis of all-trans-lycopene in plants. Whereas in the dark mainly ζ-carotene accumulated, demonstrating that it meets the stereospecific requirements necessary for ζ-carotene desaturation [48]. 5

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The CAROTENOID ISOMERASE (CRTISO) catalyses the production of all-translycopene from 7,9,7',9'-tetra-cis-lycopene [49, 50]. Mutations in CRTISO in melon, rice, Arabidopsis, tomato appear to impair plastid development exemplified by leaf virescent phenotypes and a delay in cotyledon greening. While the phenotypes were tentatively linked to seasonal changes in the photoperiod and/or extended periods of darkness, the mechanisms remain unclear [43, 44, 49-52]. CRTISO is strongly expressed in meristematic and reproductive tissues and

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expression is permissively maintained by a chromatin modifying enzyme, SET DOMAIN GROUP 8 (SDG8) [53, 54]. CRTISO can rate-limit the accumulation of downstream carotenoids by controlling flux through the branch in the pathway towards α- and β-carotenes [50].

The carotenoid pathway divides after the cis to trans isomerization of lycopene into α- and

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β-branches [55, 56]. The branch represents a key regulatory node involving a concerted interaction between ε- and β-LYCOPENE CYCLASE enzymes (εLCY & βLCY) [53], to synthesise α- and β-

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carotene, respectively [57-59]. A close physical association was proposed to occur between these two enzymes in order to channel substrates from εLCY and βLCY [60]. Next, a series of hydroxylation steps mediated by β- and ε-HYDROXYLASE enzymes convert α-carotene into lutein, the most abundant xanthophyll in plants [56, 61]. In the β-branch, hydroxylation of βcarotene produces violaxanthin via the epoxidation of zeaxanthin with antheraxanthin as an intermediate. Violaxanthin can undergo de-epoxidation back to zeaxanthin, a process known as the

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xanthophyll cycle, which helps to quench excess electrons generated during photosynthesis when light or temperature levels become stressful and dissipate as thermal energy [62-64]. Finally, neoxanthin, a direct substrate for ABA formation is synthesised by NEOXANTHIN SYNTHASE (NXS) and represents the last committed step in the carotenoid biosynthetic pathway as neoxanthin

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is found in all plants [65]. The xanthophylls can undergo further modifications in a tissue specific

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manner and have various physiological functions in different plant species (reviewed by [8]).

Carotenoid sequestration in plastids The sequestration of specific carotenoids depends upon the plastid type. Proplastids are undifferentiated plastids that do not synthesise carotenoids and are generally enriched in meristem and reproductive tissues. Depending upon the tissue and environmental conditions, a single colourless cytoplasmic body called a progenitor proplastid can differentiate into a range of plastid types having specialised functions in regard to carotenogenesis. Specialised plastids types include the chloroplast (leaves), etioplast (dark grown cotyledons), chromoplast (fruits), amyloplast (seeds) and leucoplast (roots) [22]. Carotenoids play an important role in the assembly and structural scaffolding of the plastid as well as control their biogenesis and operations [42, 66] (Figure 1). 6

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Different plastid types have evolved unique mechanisms to synthesise and sequester carotenoids. In embryonic leaves (cotyledons) emerging during skotomorphogenesis or true leaves developed under extended darkness, etioplasts differentiate from proplastids and are distinguished by having a prolamellar body (PLB), which is a tubular lattice of membranes with uniform geometry [67]. Etioplasts have a yellowish colour due to the accumulation of lutein. Upon exposure to light following de-etiolation, the etioplasts differentiate into chloroplasts. During this process, the

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PLB provides a scaffold to bind the carotenoids and form prothylakoids, which upon light exposure are converted into thylakoids and stacked into grana. In fruit and flower tissues, chromoplasts differentiate from chloroplasts or proplastids as colourful plastid types (usually red and orange in colour). Chromoplasts sequester carotenoids with lipoproteins in the form of crystals, globules,

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fibrils and tubules [68, 69]. They have roles during fruit ripening, leaf senescence and can attract pollinators [70-72]. In seeds, amyloplasts act as special plastids that store starch granules [73].

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Major carotenoids present in seed amyloplasts include lutein and zeaxanthin [74]. Likewise, leucoplasts differentiate in the roots as a colourless plastid type exemplified by the lack of colour in root tissues. Xanthophylls (e.g. violaxanthin, neoxanthin and lutein) can accumulate in leucoplasts of mature root cells at a trace levels, except in varieties having altered biosynthesis or regulations that promote colour in roots growing under the ground [75]. Leucoplasts can sequester higher levels of xanthophylls as crystals giving rise to a large array of colours. Some classic examples are the

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coloured roots of carrot and sweet potato [76, 77]. Further highlights describing advances in carotenoid related plastid biology have been reviewed elsewhere [22, 42].

cis-carotene accumulation and regulation in planta

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Cis-carotenes are synthesised by the upstream linear route of the carotenoid pathway and are unique to algae and plants. During the last decade, they have emerged to serve new functions in

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controlling plastid biogenesis and operational control during developmental programs, particularly in response to stress. There are a few studies reporting that cis-carotenes can accumulate in a tissue specific manner, depending upon the plant species. A list of species that accumulate cis-carotenes are tabled as a resource for the plant carotenoid community (Table 1). Cis-carotenes do not usually accumulate in photosynthetic tissues, however there are examples reporting their detection, albeit at low levels, in fruits of tomato [78-80], watermelon [81-85], sweet orange [86-89], pummelo [90], grapefruit [88] carrot [34] and few flower species like narcissus and tulip [91]. It is interesting to note that phytoene, phytofluene and to a lesser extent ζ-carotene, accumulate in many plant species examined (Table 1). Perhaps these cis-carotenes serve essential functions to enable physiological processes in planta, or their presence negates a lack of function in mediating physiological and/or 7

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developmental processes. Interestingly, ζ-carotene, neurosporene and pro-lycopene appear to accumulate under specific circumstances and could have essential functions to signal processes related to development and stress within or between organelles. It is noteworthy, that in “wild type” M82 tomato [78] and apricot (var. Pazza) [92], pro-lycopene was detected in addition to phytoene, phytofluene, and ζ-carotene. In varieties or tissues that accumulate ζ-carotene, neurosporene and/or pro-lycopene it seems likely that a perturbation/regulation in the activity of Z-ISO, ZDS or CRTISO

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would enable these cis-carotenes to accumulate. Finally, given that some cis-carotenes accumulate in a range of fruits, leaves and tubers it is possible that they provide health benefits for humans given their higher bioavailability [25].

The treatment of plant tissues with chemical inhibitors (e.g. norflurazon and fluridone), or

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by altering the quality/intensity of light and temperature, can enable the interrogation of ciscarotene accumulation and their functions in plants [93]. cis-carotene accumulation can be

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enhanced through the overexpression of upstream genes and/or by perturbing protein-protein interactions. For example, overexpression of; 1) PSY and its translational regulator OR [94, 95], 2) PDS and/or its cofactor PTOX [79] [96], and 3) upstream MEP pathway genes like 1-DEOXY-DXYLULOSE-5-PHOSPHATE SYNTHASE (DXS) and 1-DEOXY-D-XYLULOSE-5-PHOSPHATE REDUCTOISOMERASE (DXR) [97, 98] can all enhance carotenoid levels or force cis-carotene accumulation under certain environmental conditions. The perturbation (e.g. by loss-of-function,

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reduced enzymatic activity, impaired protein-protein interactions, or differential regulation) of key enzymatic steps of desaturation (PDS, ZDS) and/or isomerization (Z-ISO and CRTISO) are key targets to force the accumulation of cis-carotenes in plant tissues (reviewed by [8]). While PSY is rate-limiting for downstream carotenoid biosynthesis, it may also be a key

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regulator of cis-carotene accumulation. This could occur when PSY acts as a “bottleneck” to ratelimit phytoene biosynthesis and downstream carotenoid accumulation (Figure 1). In PSY

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overexpression lines of Arabidopsis At12 and At22, high accumulation of PSY proteins is associated with the enhanced production of cis-carotenes like phytoene and phytofluene in seed-derived callus, exemplifying that PSY can be rate-limiting for cis-carotene biosynthesis [93]. It is hypothetically possible that a mutation affecting the enzyme activity of PSY, or an interacting protein (e.g. OR or GGPPS11), could on the contrary reduce downstream cis-carotene biosynthesis. Although, in fruits of tomato, a PSY mutant line (e.g. yellow-flesh; r2997) showed an altered abundance of downstream xanthophylls, yet did not accumulate cis-carotenes [78]. A clear role for PSY in controlling downstream cis-carotene biosynthesis awaits better clarification. PDS and PTOX (immutants) mutants were also shown to accumulate phytoene and phytofluene in their leaves [40, 99]. Norflurazon inhibits PDS activity and chemically treated seedlings show a 8

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high accumulation of phytoene and to a lesser degree phytofluene [100]. The inhibition of PDS activity using norflurazon has served as an important strategy to study chloroplast-to-nucleus signal transduction (retrograde) and organelle communications. For example, classical retrograde signals (e.g. Heme; a product of tetrapyrrole biosynthesis and the chlorophyll precursor; Mgprotoporphyrin IX ) accumulate in plant cells treated with norflurazon, and repress photosynthesisassociated nuclear gene (PhANG) expression thereby perturbing chloroplast development [41]. The

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GENOMES UNCOUPLED (GUN) mutants defective in retrograde signaling can restore PhANG expression [101]. PDS and plastid terminal oxidase (PTOX) have evolved to sense the oxidation reduction (redox) state of photosynthetic electron transport by coupling carotene desaturation to the generation of ROS and together act like a “rheostat” to control retrograde signalling [40, 41, 102].

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While it seems unlikely that phytoene or phytofluene are substrates for a retrograde signal, a question remains about whether downstream cis-carotenes (e.g. ζ-carotene, neurosporene and pro-

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lycopene) are linked to the PDS-PTOX redox sensor and help mediate retrograde signaling (Figure 1).

Z-ISO has been referred to as the “gatekeeper” as it harbors a tetrapyrrole like structure resembling a nitrogenase enzyme and may function as a redox sensor based upon phylogenetic analysis (Figure 1) [45]. The accumulation of 9,9’-di-cis-ζ-carotene in the vp9 mutants of maize, blocked at the desaturation step by the loss-in-function of ZDS prompted the requirement of an

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isomerase that converts 9,15,9’-tri-cis-ζ-carotene to 9,9’-di-cis-ζ-carotene [43]. The isolation of dark grown maize y9 mutants having high accumulation levels of 9,15,9’-tri-cis-ζ-carotene enabled the discovery of Z-ISO [43]. Light-grown y9 leaves displayed a carotenoid profile similar to wild type due to photoisomerization. A similar study using Arabidopsis Z-ISO mutants (zic1-1, zic1-2)

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confirmed the existence of Z-ISO in a second species. Due to a lack of Z-ISO activity, etiolated leaves of Arabidopsis mutants accumulated 9,15,9’-tri-cis-ζ-carotene, while photoisomerization

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facilitated carotenogenesis in photosynthetic tissues [44]. zds mutants predominantly accumulate cis-phytoene, phytofluene and ζ-carotene albeit in different amounts depending upon the light. For example, maize zds mutant vp9 were shown to accumulate large amounts of ζ-carotene along with neurosporene and pro-lycopene in nonphotosynthetic tissues such as roots and etiolated seedlings [47]. The cotyledons of the sunflower nondormant-1 (nd-1) mutant accumulated ζ-carotene, and to a lesser extent phytoene and phytofluene [103]. The Arabidopsis spc1-1 mutant showed a spontaneous cell-death phenomenon along with a leaf bleaching phenotype revealing defects in chloroplast development [104]. Lastly, Arabidopsis chloroplast biogenesis 5 (clb5/zds) mutant accumulated 15-cis-phytoene and ζ-

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carotene, which were attributed as substrates for the production of an apocarotenoid signal that altered PhANG expression, cellular differentiation and leaf morphology (Figure 1) [105]. The loss-of-function mutation in CRTISO has been demonstrated as a major regulator of ciscarotene accumulation (especially neurosporene and pro-lycopene) in dark-grown etiolated tissues of Arabidopsis (ccr2) [50], chlorotic striped leaves from rice (zebra2) [106], orange tomato fruit (tangerine) [49], yellow-orange melon fruit (yofi) [52] and the orange inner leaves of Chinese

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cabbage (Br-or) [107] (Table 1). The accumulation of cis-carotenes in some of these crtiso mutants was linked to a perturbation in plastid development (Figure 1). Interestingly, the total levels of ciscarotenes accumulated in Arabidopsis ccr2 etiolated seedlings and seed-derived callus appear relatively high in comparison to the level of xanthophylls accumulated in wild type [50, 93]. An

under extended periods of darkness remains unclear.

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explanation for continued flux into the pathway when xanthophyll accumulation has been blocked

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The sdg8 mutant (ccr1) has reduced CRTISO transcript levels and accumulated cis-carotenes in etiolated seedlings of Arabidopsis in a manner similar to that of ccr2 [54]. Interestingly, sdg8 mutants accelerate flowering time, cause partial male sterility, have altered hormone sensitivity, enhance shoot branching and altered root development, which could link cis-carotene accumulation to the control over one or more of these developmental functions [108]. SDG8 could control ciscarotene accumulation in actively dividing tissues that undergo cell division and/or reproduction

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[53]. This might implicate a function for cis-carotenes themselves or an apocarotenoid derived signal to mediate plastid to nucleus communication in order to facilitate epigenetic processes controlling development and/or stress acclimation. Further research is necessary to understand how cis-carotene biosynthesis is regulated and what additional biological functions cis-carotenes have

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acquired during the evolution of plants.

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The evolution of cis-carotenes

The carotenoid pathway originally emerged in prokaryotic bacteria as a single operon on a circular genome, which was subsequently transferred ~2.7 billion years ago to photosynthetic cyanobacteria, an endosymbiotic progenitor of the chloroplast [109]. Carotenoid pigments are also synthesised

in

some

non-photosynthetic

bacteria

(e.g.

Streptomyces,

Flavobacterium,

Mycobacterium, Brevibacterium, Rhodomicrobium and Erwinia) [110]. During evolution, carotenoid genes were transferred as larger operons (multiple carotenoid genes) due to a requirement for multiple enzymes to fulfil the biosynthesis of carotenoids, depending upon the function negated by the end product [111, 112]. For example, the marine bacterium Agrobacterium aurantiacum harbours a carotenoid biosynthesis gene cluster consisting of five carotenogenic genes 10

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with the same orientation, which together mediate the production of astaxanthin that protects against oxidative stress [113]. In fungi the enzymatic activities of phytoene synthase and lycopene cyclase can be encoded by a single gene fusion [114]. In fact, an extraordinary step in carotenoid evolution was the horizontal transfer of this single large carotenoid operon from fungi to the genomes of aphids [115], adelgids [116], spider mites [117], and gall midgets [118] providing a class of animals capable of producing their own carotenoids such as β-carotene.

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Another major event in the evolution of carotenogenesis was from bacteria to fungi to algae and plants. The addition of carotenoid desaturation and isomerization steps in higher organisms allowed cis-carotene intermediates to generate in between phytoene and all-trans-lycopene. There are two general pathways: (1) desaturation and cis-trans isomerization by a single enzyme,

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CAROTENOID ISOMERASE (CrtI; which can also produce all-trans-neurosporene in some microorganisms), or (2) desaturation to ζ-carotene by CrtP, desaturation of ζ-carotene to poly-cis-

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lycopene by CrtQ, and cis-trans isomerization by CrtH. The CrtI-dependent pathway can be found in most prokaryotes and fungi, whereas the CrtP/CrtQ/CrtH-dependent pathway specialised in evolution with photosynthetic organisms (cyanobacteria, algae, and plants) (Figure 2)[119]. CrtI encodes a multifunctional carotene desaturase that belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase that can produce alltrans-lycopene from 15-cis-phytoene in a single-step reaction [120, 121]. In planta overexpression

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of Erwinia uredovora CrtI (EuCrtI) was shown to produce all-trans-lycopene from phytoene, bypassing the need for PDS, ZDS, Z-ISO and CRTISO that evolved in higher plants [122]. Depending upon the organism and whether it is photosynthetic or not, different carotenoid isomerase enzyme variants evolved with overlapping, yet unique activities that generate different cis-carotene

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intermediates. That is, CrtI is not unspecific but rather a particular structural motif can recognize a variety of substrates and through evolution a capacity to generate unique end products emerged

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[119, 123]. For example, the CrtI isolated from photosynthetic Rhodobacter capsulatus bacterium catalyses the formation of neurosporene from phytoene [124]. In the plant and fungal kingdom, ζcarotene accumulates as an intermediate product from Al-1-mediated phytoene desaturation to 3,4didehydrolycopene in the ascomycete fungus, Neurospora crassa. Several of the desaturation intermediates, ζ-carotene, neurosporene, and pro-lycopene were also accepted as substrates by Al-1 [48, 125, 126]. Similarly, a recently identified CrtI from the red yeast Sporidiobolus pararoseus was shown to catalyse four- and five-step dehydrogenations from cis-phytoene through ζ-carotene, neurosporene-generated lycopene and lycopene-generated 3,4-didehydrolycopene [127]. Evidently, phytoene desaturases (e.g. CrtI) from bacterial and/or yeast origins can enable the in vitro biosynthesis of some cis-carotene intermediates. Therefore, if they can also accumulate in vivo, they 11

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might impart some function that was conserved through the evolution from microorganisms to plants that awaits discovery. Addition of extra enzymatic steps (PDS, Z-ISO, ZDS and CRTISO) for carotenoid production in plants advanced the diversity of cis-carotenes and their isomers in photosynthetic organisms [1]. The catalytic activity of enzymes involved in cis-carotene biosynthesis depends on the substrate and electron acceptor donors [128]. An evolvable pathway should consist of an

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evolvable core enzyme that can be coupled with locally specific downstream enzymes to produce new metabolites with limited genetic change [123]. In such an arrangement, mutations in a core enzyme (e.g. CrtI) can convert a single molecule (e.g. phytoene) into a greater diversity of new metabolites (e.g. cis-carotenes) [119, 121]. The core FAD-binding domain in the bacterial CrtI is

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well conserved in other cis-carotene modifying enzymes (e.g. PDS, ZDS, and CRTISO). This domain utilizes FAD as the sole redox-active cofactor and oxygen (replaceable by quinones in its

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absence) as the terminal electron acceptor to activate catalysis [121, 129, 130]. In contrast, Z-ISO may represent another core enzyme, which is a ferrous heme b cofactor discovered to mediate the redox-dependent isomerization of 9,15,9’-tri-cis-ζ-carotene to 9,9’-di-cis-ζ-carotene [45]. Previously, the only known enzyme that performs heme-dependent isomerization was the bacterial cis/trans fatty-acid isomerase (CTI) [131]. Even though their catalytic mechanisms are different, this may suggest an evolutionary origin for plant Z-ISO related homologues in bacteria. Another

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clue regarding the origin of Z-ISO was obtained from a comparative genomics study highlighting its ancestral relation with the NITRIC OXIDE REDUCTASE U (NnrU) gene required for the NO2 reduction in bacteria [44]. It’s suggested that Z-ISO evolved and acquired new functions from a progenitor that has a Z-ISO/NnrU-like gene clustered closely to other carotenoid and redox-related

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genes observed in cyanobacteria. In denitrifying bacteria, NnrU evolved to take function in denitrification, but does not appear to function in carotenogenesis. Whilst the acquisition of

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additional enzymes with homology to CrtI in plants appears evolvable, the reason for acquiring ZISO remains somewhat elusive. It is conceivable that for organisms ineffective in the photoisomerizion of poly-cis-carotenes or that cannot maintain carotenogenesis during extended periods of darkness, that Z-ISO acts like a “gatekeeper” to sense and/or control redox reactions in concert with PDS.

Apart from the above major components of carotenoid pathway, the PTOX regulatory protein was also inherited from prokaryotic systems to function in synchronisation with PDS to keep the plastoquinone pool in balance [132]. Moreover, phylogenetic analysis based upon mature protein sequence information showed a linkage between the eukaryotic and prokaryotic origins [133]. Interestingly, mitochondrial alternative oxidase (AOX; ubiquinol oxidase) was shown to be 12

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functional when substituted for the PTOX (plastoquinol oxidase) activity in the Arabidopsis chloroplast. That is, in Arabidopsis IMMUTANTS (IM), a chloroplast localized and reengineered AOX-like protein replaced the function of PTOX, even though AOX is normally localised to function in the mitochondria [100]. Despite having little similarity, PTOX and AOX were proposed to originate from a common ancestor. Moreover, the high similarity among photosynthetic organisms reveals that PTOX may be well conserved without significant change throughout

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eukaryote evolution [40]. In conclusion, the specialisation and co-evolution of additional ciscarotene generating enzymes together with PTOX have led to the generation of a diverse abundance of cis-carotenes and their isomers in higher plants. It is intriguing to wonder if cis-carotenes evolved to provide substrates for the biosynthesis of signalling metabolites. Alternatively, the evolution of

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additional genes encoding evolvable enzymatic activities could have imparted new functions in

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higher photosynthetic organisms, like plants.

Photoisomerization of cis-carotenes: a light-mediated regulatory switch The loss in function of desaturases (e.g. PDS and ZDS) blocks the production of downstream xanthophyll carotenoids necessary for photosynthesis, thereby triggering an albino phenotype and eventually cell death, unless mutant seedlings are maintained on artificial media containing sucrose [99, 104]. This is in striking contrast to the loss-of-function in enzymes acting

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downstream of lycopene to generate α-carotene (e.g. εLCY) and β-carotene (e.g. βLCY), which can continue to produce one or more xanthophylls and facilitate photosynthesis under reduced lighting conditions [56, 134]. cis-carotene isomerases (e.g. Z-ISO and CRTISO) do not cause seedling lethality when light is sufficient to compensate for the isomerization of tri- to di-cis-ζ-carotene and

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tetra-cis to all-trans-lycopene, normally performed by Z-ISO and CRTISO, respectively [135]. The light-mediated conversion of cis-carotenes to their geometric isomers is a phenomenon referred to

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as photoisomerization.

The event of carotenoid photoisomerization was first reported in a study revealing that polycis-carotenes could change their configurations when associated with chlorophyll in algae [136]. Chlorella and cyanobacterium mutants, which accumulate poly-cis-carotenes in the dark, were isomerized into their all-trans-forms when exposed to light [137]. The photoisomerization of 15cis-ζ-carotene into all-trans-ζ-carotene was also demonstrated by shifting dark-grown cells of Euglena gracilis (W3BUL) into the light [138]. Another report demonstrated that light can isomerise tetra-cis-lycopene to all-trans-lycopene in dark-grown C-6D mutant of green algae, Scenedesmus obliquus, which was later demonstrated to have a mutation affecting phytoene desaturase activity

13

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[139, 140]. These early reports highlighted that light can influence the biosynthesis of lycopene from phytoene. More recently, light-mediated photoisomerization was shown to compensate the loss-infunction of CRTISO and Z-ISO in plants [43, 49, 106]. The accumulation of cis-carotenes in Z-ISO and CRTISO mutant tissues depends upon the period of darkness and/or seasonal environment [44, 49, 52, 141]. cis-carotene photoisomerization occurs mostly in photosynthetic tissues (e.g. leaves,

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but not in roots and fruits) as there is a requirement for a chlorophyll related photosensitizer to facilitate the cis/trans-photoisomerization [142]. Photoisomerization has been thoroughly demonstrated in-vitro [143] (Figure 1). In fact, the in-vitro photoisomerization of tetra-cis-lycopene to its all-trans-lycopene can occur within minutes (≃5 min) with the addition of meso-

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tetraphenylporphyrin (TPP) photosensitizer in the media [143]. What more, cis-carotene photoisomerization in the Br-or mutant of Chinese cabbage can occur within 10 minutes of light

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exposure to inner leaves [107]. It appears that photoisomerization of cis-carotenes can occur rapidly, depending upon the cellular and light environment. As such, specific cis-carotenes amenable to photoisomerization may behave like a photoswitch, enabling light to mediate control over downstream xanthophyll biosynthesis when Z-ISO/CRTISO protein levels/activity become impaired (Figure 1). In fact, the conversion of pro-lycopene to all-trans-lycopene has been described as a rate-limiting step for the production of xanthophylls (mostly lutein) that drive

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photosynthetic and photoprotective functions [49, 50]. The function of a cis-carotene photoswitch would depend upon the plastid type, developmental age and environmental factors (e.g. light and temperature) known to affect cis/trans-isomerization. As an example, leaf and plant age are critical factors affecting photoprotection in Arabidopsis [144]. Young leaves of Arabidopsis accumulate

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substantially higher levels of carotenoids in comparison to the mature leaves, and even more so under elevated CO2 [145]. It is not surprising that young leaves of z-iso and tangerine mutants

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accumulate traces of cis-carotenes and leaf virescence phenotypes reminiscent of a perturbation in plastid development [49]. These evidences highlight that photoisomerization can be rate-limited in younger tissues and places a requirement for Z-ISO and CRTISO enzyme activity in newly emerging tissues.

There are many questions that remain to be answered about exactly how cis-carotene photoisomerization is sensitized in plastids, and how does it work in concert with enzymatic activities driven by Z-ISO and CRTISO. How does light enable cis-carotene photoisomerization? A cis-carotene photoswitch could represent a logical cis/trans chemical switch by which plants sense light and alter cis-carotene isoform abundance. Such a mechanism could enable the rapid lightmediated control over feedback or feedforward regulation within the carotenoid pathway. Future 14

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research is needed to shed light on the enigmatic phenomenon of photoisomerization and decipher what function it might have in controlling plant physiological processes.

Feedback and feedforward regulation are linked to cis-carotene accumulation Cis-carotenes and/or their derivatives have been implicated in feedback and feedforward regulation of the upstream MEP and downstream carotenoid pathways, respectively (Figure 1)

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[146]. Plastid derived retrograde signals can accumulate in response to environmental stress and act as mobile messengers to control PhANG expression [147] (Figure 1). Perturbing the carotenoid pathway using chemicals such as D15 (aryl-C3N hydroxamic acid inhibits CCD activity), CPTA (4Chlorophenyl-thio-triethylamine hydrochloride inhibits ε/β-lycopene cyclase activity), norflurazon

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(NFZ; blocks PDS activity), and fosmidomycin (FSM; impairs DXR in MEP pathway) have notable effects on the levels of nuclear gene expression [97, 148-150]. Studies using carotenoid loss-of-

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function mutants and virus-induced gene silencing (VIGS) in Arabidopsis, rice and tomato have also revealed changes in nuclear gene expression [80, 105, 107, 151, 152]. Plastids from these mutant tissues or tissues exposed to some of the aforementioned chemicals can accumulate specific cis-carotenes.

A number of studies have shown that nuclear gene expression and protein levels change in response to the accumulation of cis-carotenes, yet the underlying molecular mechanism remains

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unknown (reviewed by [8, 153]). For example, the EPSILON LYCOPENE CYCLASE (εLCY) is a key regulatory enzyme required for the production of lutein [55] and was reported to be subject to feedforward regulation (Figure 1). εLCY mRNA expression was reduced in the Arabidopsis dark grown cotyledons (ccr2/crtiso), tomato fruits (tangerine/crtiso) and new emerged leaves

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(ccr1/sdg8) that accumulate cis-carotenes [54, 151, 152]. Interestingly, the overexpression of bacterial CrtI in the tomato Ailsa Craig (AC) wild type background and in tangerine3183 reduced

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cis-carotene levels and enhanced εLCY expression [152]. How the nucleus perceives the presence of cis-carotenes that signal the downstream regulation of εLCY gene expression awaits discovery. Another example of feedback regulation linked cis-carotene accumulation in tomato tissues to an epistatic interaction between mutations affecting CRTISO and PSY1 (Figure 1). A recessive mutation in yellow-flesh (r2997) reduced carotenoid biosynthesis and eliminated transcription of PSY1. In tangerine (t3002 or tmic), the loss-of-function in crtiso caused cis-carotenes to accumulate in tomato fruits and enhanced PSY expression [49, 78]. Generation of a double mutant (r2997/t3002) caused an epistatic interaction and a partial recovery of PSY1 transcript levels in r2997/t3002 in comparison to t2997. The up-regulation of PSY expression was linked to the accumulation of prolycopene and neurosporene isomers [78]. It should be noted that during the breaker stage of fruit 15

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development in tangerine3183 negative feedback regulation of PSY and DXS mRNA expression levels was reported [152]. Therefore, depending upon the CRTISO mutant locus, variety of tomato, and fruit ripening stage, the accumulation of cis-carotenes can be associated with varying effects on PSY expression that require further investigation. Nonetheless, the evidence is strong to suggest that an unknown cis-carotene derived apocarotenoid signal is likely to mediate the aforementioned regulation of PSY [78].

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Feedback and feedforward regulations can occur simultaneously within the carotenoid pathway [79, 152]. CrtI and PDS enzymes utilise phytoene as their substrate, yet have profoundly different effects on pathway gene expression and carotenoid accumulation in tomato fruit when missregulated. The overexpression of bacterial CrtI in tangerine and the old gold crimson (ogc) tomato

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mutants demonstrated the presence of a negative feedback mechanism controlling DXS and PSY1 expression, as well as feedforward mechanisms positively controlling the transcription of εLCY and

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βLCY genes depending upon the genotype [152]. However, the overexpression of heterologous Arabidopsis PDS (AtPDS) in tomato fruits did not impact carotenoid gene expression, except for a slight reduction in CAROTENOID ISOMERASE-LIKE 1 (CRTIL1) gene expression [79]. While higher PDS transcript levels slightly increased lycopene accumulation, overexpression of CrtI dramatically enhanced β-carotene. Both enzymes reduced the level of phytoene and phytofluene, but oppositely effected ζ-carotene levels highlighting that the abundance of specific cis-carotene

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isoforms can have opposing effects on regulatory processes. The presence of regulatory loops that regulate carotenoid gene expression were also reported to occur in tomato fruits subjected to TRV-mediated VIGS of carotenoid pathway genes [80]. The carotenoid phenotypes of PSY1 and CRTISO silenced fruits resembled those of yellow-flesh and

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tangerine mutants respectively, thereby validating VIGS in fruits as an effective system to study feedback/forward regulation. Silencing of PDS and ZDS in tomato fruit caused the over-

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accumulation of cis-carotenes and increased relative expression of Z-ISO and CRTISO, respectively. All-trans-ζ-carotene increased in ZDS silenced fruits and disappeared in CRTISO-like silenced fruit, revealing a metabolic branch and centre at ζ-carotene that involves feedback and forward metabolic regulatory loops that sense cis-carotene and/or carotenoid enzyme levels [80]. The Arabidopsis clb5 mutant lacks ZDS activity and accumulates cis-carotenes upstream of ZDS [44] (Figure 1). Genetic and biochemical analyses demonstrated that transcriptional changes in photosynthesis associated genes (e.g. DXS, ClpP1) in clb5 were due to cleavage of phytofluene and/or ζ-carotene isomers [105]. Interestingly, blocking PDS activity using norflurazon or knocking out ccd4 activity in the clb5 mutant background, both restored ClpP1 and DXS gene expression [105]. These data provide solid evidence for the existence of a novel cis-carotene derived 16

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apocarotenoid produced through perturbation in zds, which alters nuclear gene expression by a retrograde signaling process (Figure 1). The physiological basis and identity of an apocarotenoid signal generated by clb5 awaits discovery. A recent study described a regulatory role for alternative splicing of the PSY 5’UTR in controlling protein translation [154]. In this work, two PSY splice variants were identified and their presence or absence was proposed to be responsive to de-etiolation and sudden changes in the light

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intensity. The authors identified a putative RNA regulatory switch in the PSY mRNA 5’UTR domain that could enhance translation of a promoter-reporter gene fusion. The 5’UTR showed some similarities with that of bacterial riboswitches in that it harboured two alternative structures and hence proposed it could behave like a ligand-mediated switch in controlling PSY splicing in

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response to cis-carotene accumulation [154]. This was the first study that suggested the idea that a carotenoid derived signal might mediate control over carotenoid feedback regulation in plants via a

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5’UTR regulatory switch. Indeed, the identity of the ligand and mechanism by which the PSY 5’UTR might control splicing are areas of research that require further validation. Overall, the evidence is strong to link the regulation of genes and proteins upstream, within, as well as downstream of cis-carotene biosynthesis in fruits (chromoplasts: CRTISO), emerging leaves (chloroplasts: ZDS) and etiolated cotyledons (etioplasts: CRTISO) that accumulate cis-carotenes. The carotenoid signaling pathways involving feedback and/or feedforward regulations are complex.

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Exploring if such regulatory mechanisms can occur in different plastid and tissue types and whether they are species specific or not, are essential steps towards deciphering the function of ciscarotenes. How the plant perceives the presence of cis-carotenes themselves or an apocarotenoid

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derived signal awaits discovery.

The hunt for novel cis-carotene derived apocarotenoid signaling metabolites

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Carotenoids are large molecules unable to pass through the plastid membrane and require a more hydrophobic environment. Carotenoids can change their three dimensional configuration depending upon light or temperature, and some isoforms of carotenoids are amenable for enzymatic or non-enzymatic cleavage into apocarotenoids. Apocarotenoids can be relatively small, soluble and in some cases volatile, that help facilitate transport and/or diffusion across plastid membranes [5]. Secondary metabolites generated by an energy organelle such as the chloroplast can in some cases translocate to the nucleus where they exert an effect on gene regulation, a process known as retrograde signalling. Retrograde signalling in plants allows the rheostatic control over nuclear gene expression and acts to coordinate intrinsic developmental cues and instantaneous physiological 17

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changes to prevailing environmental stress [147]. Some apocarotenoids have been shown to have retrograde signalling properties, while others exert their effect like that of a hormone. In Arabidopsis, there are five 9-cis-epoxy-carotenoid dioxygenases (NCED2, 3, 5, 6, 9) and four carotenoid cleavage dioxygenases (CCD1, 4, 7, 8), some of which are required to produce abscisic acid (ABA; NCEDs) and strigolactone (SL: CCD7/8) hormones from neoxanthin and β-carotene, respectively [155-158]. The CCD enzymes are responsible for generating apocarotenoids. CCD1

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was shown to have high cleavage activity in Arabidopsis seeds, where the loss-of-function caused significantly higher carotenoid levels, in particularly β-carotene, and facilitated the synthesis of apocarotenoid flavour and aroma volatiles [159]. CCD4 was shown to have a major role in βcarotene degradation in drying seeds and senescing leaves [160]. Both ccd1 and ccd4 were further

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demonstrated to enhance α/β-carotene levels using an Arabidopsis seed-derived callus assay [93]. Therefore, the activities of CCD enzymes on cleaving β-carotene as a substrate are rather consistent, regardless of the tissue or the plastid type. Non-enzymatic oxidative cleavage can also generate

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various apocarotenoids [93]. Singlet oxygen (1O2) was shown to cause the oxidative cleavage of βcarotene into β-cyclocitral and β-ionone [161-163]. These apocarotenoid signals accumulate in Arabidopsis within minutes of intense light exposure and alter stress-related gene expression that helps to facilitate tolerance to photooxidative stress [164].

The big question is whether the CCDs or non-enzymatic oxidation are capable of cleaving a cis-

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carotene produced in the upper part of the pathway between phytoene and lycopene. A recent study using a seed-derived callus assay revealed that cis-carotenes are less susceptible to oxidative cleavage and rather stable intermediates that favour them as substrates for cleavage by a CCD [93]. CCDs appear to require specific cis-carotene substrates in order to undertake oxidative catalytic

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cleavage in vitro [159, 165]. CCD1 has been reported to cleave phytoene in vitro [166]. However, an in vivo seed-derived callus assay provided evidence to exclude phytoene as an in vivo substrate

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for CCD1 and CCD4-catalyzed cleavage [93]. The enzymatic activity of AtCCD4 and AtCCD7 and substrate specificity towards various cis-carotene substrates (9,15,9'-tri-cis-ζ-carotene, 9,9'-di-cis-ζcarotene,

9,15-di-cis-phytofluene,

15-cis-phytofluene,

7,9,9'-tri-cis-neurosporene;

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neurosporene, 7,9,9',7'-tetra-cis-lycopene; pro-lycopene) was tested using an in-vitro bacterial assay system. Only CCD7 converted 9-cis-configured acyclic carotenes (e.g. 9-cis-ζ-carotene, 9'-cisneurosporene, and 9-cis-lycopene) to yield 9-cis-configured carotenes. This indicated that AtCCD7, rather than AtCCD4 is a better candidate for forming acyclic apocarotenoid retrograde signals in vitro [167]. This finding contrasts to how an uncharacterised apocarotenoid derived from phytofluene or ζ-carotene isomers that accumulated in clb5/zds mutants of Arabidopsis, required CCD4 activity [105]. The zds/clb5 mutant alleles displayed a needle-like leaf morphology, had 18

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altered cellular differentiation, plastid biogenesis and changes in photosynthesis-associated nuclear gene expression. The inhibition of cis-carotene biosynthesis using fluridone to block PDS activity or the loss-in-function of ccd4, were sufficient to restore leaf development and photosynthesis associated nuclear gene expression in clb5 [105]. It is interesting that proteomic and GFPlocalisation reveal that CCD4 localized with ZDS in chloroplastic plastoglobules revealing a potential site for an apocarotenoid signal to accumulate under stressful conditions. Overall, the

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evidence for an apocarotenoid signal requiring CCD4 activity in planta is strong and further research is necessary to resolve the controversial substrate specificity of CCD4.

Finally, when ccr2 seedlings are grown in the dark, the etiolated cotyledons do not form a prolamellar body, which was linked to cis-carotene accumulation [50]. Treatment of the seedlings

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with NFZ restored PLB formation, and it was proposed that the cis-carotenes caused a structural perturbation during PLB formation [50, 151]. In light of the evidence discussed above, it seems also

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plausible that perhaps Arabidopsis CRTISO mutants (ccr2) generate a cis-carotene derived apocarotenoid signal that controls PLB formation during skotomorphogenesis and perturbs plastid biogenesis and seedling greening following photomorphogenesis (Figure 1). The mechanism of how cis-carotenes generated in ccr2 dark grown tissues during skotomorphogenesis affect PLB formation remains elusive.

Clearly, cis-carotenes are rather stable intermediates and can be cleaved by some CCDs as

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observed in vitro. The next big step will be to demonstrate CCD cleavage of a cis-carotene substrate in-vivo and identify the apocarotenoid derivative. A great deal of research will be necessary to couple CCD activity in-vivo with physiological functions in planta. There may be an undiscovered carotenoid oxygenase or other CCD/NCED-like proteins awaiting characterisation. Moreover, CCD

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enzyme activity could require other interacting cofactor proteins in combination with multifactorial metabolon complex (including free radicals and reactive oxygen species) to localise at specific

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plastid membrane sites. The CCDs and cis-carotene substrates required to generate an apocarotenoid signal continue to be an unresolved area of carotenoid biology. Future perspectives for cis-carotenes in plant biology The next frontier for the carotenoid research field is to identify a cis-carotene derived apocarotenoid signal and show how it can fine-tune secondary metabolism in plastids and/or contribute to maintaining cellular homeostasis during plant development. Uncovering the ciscarotene substrate required to generate a novel apocarotenoid is imperative to meet this challenge. Can a cis-carotene derived apocarotenoid behave as a mobile retrograde signal to control nuclear gene expression and if so, how? Can a structural switch in the 5’UTR be one new player on the operon for sensing and control metabolic feedback and feedforward regulation in plants [154, 168]? 19

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Intronic regions within the 5’UTR sequence can cause alternative transcription initiation and splicing depending upon the environmental stress conditions [169]. It is intriguing to think that a cis-carotene derived apocarotenoid could act as a ligand to exert a structural effect on RNA, disrupt the heterodimerization of transcription factors, change coactivator concentrations, or interact directly with the ligand binding site of receptors, in a manner similar to how β-apo-13-carotenone affects retinoic acid receptors in mammals [170, 171].

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A change in light quantity and quality can affect the rate of photoisomerization. A physiological basis for plants evolving a more complex cis-carotene-mediated signalling pathway could be linked to seasonal shifts in our environment and day length. The energy of light can switch cis/trans configurations of both ζ-carotene and pro-lycopene representing a potential metabolite

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photoswitch that could control upstream cis-carotene and downstream xanthophyll levels. It is curious that plants evolved two additional enzymatic steps in cis-carotene biosynthesis that can be

redefine how plants perceive light.

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substituted by light. Discovering a molecular mechanism for a cis-carotene photoswitch could

A greater insight into how plants acquired a complex cis-carotene pathway will undoubtedly shed light on our understanding of carotenoid biosynthesis in all organisms. For now, it seems that PSY is still a “bottleneck” for cis-carotene biosynthesis. Will PDS emerge as the “redox sensor”, ZISO the “gatekeeper”, ZDS the “developer” and CRTISO as “epigenetic” (Figure 2)? Only time will

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tell how colourful the research into cis-carotene biology can evolve. Identifying a cis-carotene derived apocarotenoid and confirming how it controls transcriptional and/or translational mechanisms to enable physiological acclimation to environmental change will undoubtedly deliver

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the next holy grail of carotenoid biology.

C.I.C).

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Acknowledgements We acknowledge the support of Australian Research Council Discovery Grant DP130102593 (to

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Figure 1. cis-carotene biosynthesis, regulation and signaling in plants. 15-cis-phytoene is synthesised from the condensation of geranylgeranyl pyrophosphate by PSY. This step is referred to as a “regulatory bottleneck” that is rate-limiting for carotenoid biosynthesis. The PSY mRNA harbors a putative regulatory switch in the 5’Untranslated Region (5’UTR) that might be responsive to an unknown cis-carotene derived apocarotenoid signal. Next, 15-cis-phytoene and 9,15-di-cisphytofluene undergo desaturation by PDS to generate 9,15,9’-tri-cis-ζ-carotene. The enzymatic 20

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activity of PDS can be blocked by NFZ. PDS can act like a redox sensor mediating plastid control and operations. Inhibition of PDS triggers a retrograde signal in plastids that reduces photosynthesis associated gene expression. Isomerization of 9,15,9’-tri-cis-ζ-carotene to 9,9’-di-cis-ζ-carotene is catalysed by Z-ISO, a reaction also facilitated by photoisomerization. ZDS catalyses the production of 7,9,9’,7’-tetra-cis-lycopene (pro-lycopene) from 7,9,9’-tri-cis-neurosporene. The clb5/zds mutant alters leaf development in comparison to wild type (WT). CRTISO catalyses the final isomerization

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of 7,9,9’,7’-tetra-cis-lycopene to all-trans-lycopene, a reaction also fulfilled by photoisomerization. Null mutants of crtiso (tangerine, ccr2, zebra2) do not contain a prolamellar body (PLB) in their etioplast and display impaired plastid biogenesis causing a delay in cotyledon greening and leaf virescence in multiple plant species. cis-carotenes can be cleaved by CCDs to generate an unknown

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apocarotenoid signal that can feedback and/or forward to regulate PSY and εLCY gene expression, respectively. The pathway bifurcates thereafter to produce α/β-carotenes. Abbreviations: PSY,

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phytoene synthase; PDS, phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase; CCD, carotenoid cleavage dioxygenase; εLCY, lycopene ε-cyclase; βLCY, lycopene β-cyclase; ABA, abscisic acid; SL, strigolactones; PLB, prolamellar body; NFZ, norflurazon

Figure 2. Evolution of cis-carotene biosynthesis from bacteria to algae to plants. In bacteria,

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the biosynthesis of lycopene from phytoene is regulated by a multifunctional enzyme, CrtI. During evolution, the pathway evolved additional enzymes such as CrtP, CrtQ and CrtH, that can produce different cis-carotene end products. Plants evolved two desaturases (PDS and ZDS) and two isomerases (Z-ISO and CRTISO). PSY represents the “bottleneck” for carotenoid biosynthesis.

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PDS has often been referred to as a “redox sensor” through interactions with PTOX. Z-ISO was termed as the “gatekeeper” in that it evolutionarily resembles a heme-like protein structure. We

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refer to ZDS as the “developer”, since mutants display an altered leaf morphology. Here we dub CRTISO as “epigenetic” since it is regulated by chromatin modifying enzyme. Abbreviations: CrtI, bacterial carotenoid isomerase; CrtP, algae phytoene desaturase: CrtQ, algae ζ-carotene desaturase; CrtH, algae carotenoid isomerase; PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, ζcarotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, plant carotenoid isomerase.

Tables Table 1. cis-carotenes accumulate in photosynthetic tissues, and various fruits, vegetables and crops species. Abbreviations: MG, mutated gene; OG, overexpressed gene; Phy, Phytoene; Phyf, Phytofluene; ζ-Car, ζ-Carotene; Neu, neurosporene; Plyc, pro-lycopene; Ref, references; “+”, 21

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present; “-“, absent; “N”, not specified/not detected; “T”, trace amount; “n/a”, not available/unknown References

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Table 1. cis-carotenes accumulate in photosynthetic tissues, and various fruits, vegetables and crops species.

Genotype

Tissue

Tomato

M82 Rutgers Ailsa Craig Moneymaker Ailsa Craig tangerine tangerine t3002 x r2997 Beta old gold old gold crimson old gold crimson hp2 fcd1-1; fcd1-2 apricot (fcd1at) hp3 zeta (z2803) Dumara Candy Red Zaohua Hongyihao 307Chaofeng Sanbai CN66

fruit fruit fruit fruit fruit fruit juice fruit fruit fruit fruit fruit fruit fruit pericarp fruit, leaves, flower fruit, leaves, flower fruit, leaves fruit ripe fruit fruit pulp fruit fruit fruit fruit fruit

CRTISO CRTISO CRTISO & PSY1 CYC-B CYC-B CYC-B CYC-B DET1 IDI1 IDI2 ZEP Z-ISO -

M AN U

TE D

EP

AC C

Watermelon

Gene OE

Phy

Phyf

ζ- Car

Neu

Plyc

Ref

CrtI CrtI CrtI -

+ + + + + + + + + + + + + T T + + + + N N N N +

+ + + + + + + N N + + + + + + + + +

+ + + + + + + N N N + + + + + + +

+ N N N N N T + N N N N N

+ + + + N N N N T T N N N N N

[78] [78] [79] [80] [152] [78] [152] [78] [85, 172] [85] [152] [152] [173] [174] [174] [175] [78] [81] [82] [83] [83] [83] [83] [84]

SC

Species

Genetic Locus Impaired

RI PT

Abbreviations: Overexpressed, OE; Phy, Phytoene; Phyf, Phytofluene; ζ-Car, ζ- Carotene; Neu, neurosporene; Plyc, pro-lycopene; Ref, references; “+”, present; “-“, absent; “N”, not specified/not detected; “T”, trace levels; “n/a”, not available/unknown

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Peach

Sweet orange

Redhaven Bianca (RHB) Pera Valencia Sanguinelli Newhall

fruit fruit juice fruit juice fruit juice fruit

-

+ + + T + T T T + N N N N N N + + + + + + + + N

+ + + T T + + + + + + T + + + + + + + +

+ + + T + + T + + + + + + + + + + + T + N N -

N N N + N N N N N N + N N N N N N N N

N N N + N N N N N N + + N N N N N N N

[84] [84] [84] [85] [85] [85] [85] [85] [52] [176] [176] [176] [176] [176] [176] [52] [92] [92] [92] [92] [92] [177] [177] [178]

-

N + + + +

+ + + + +

+ N N N N

N N N N N

N N N N N

[178] [86] [86] [86] [87]

RI PT

M AN U

SC

CRTISO unknown (bud mutant of RH) -

TE D

EP

Apricot

fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit fruit

AC C

Melon

CN62 ZXG381 ZXG507 Calsweet Orangelo Crimson sweet Malali NY162003 CEZ Iroquois Blenheim Orange Birde Red Quincy Tiffany Hale's Best yofI Pazza Real Fino Amal P.A.803-304 Orange Gold Goldrich Moniqui Redhaven (RH)

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Mandarin

Star Ruby Satsuma Wilking

Carrot

Potato

Sunburst Bolero Nutired Chatenay Red Cored Nantaise 2

Queen Anne's Lace Berolina pP-YBI 17;30 (Desiree background) L29;55;88 (Desiree

+ + + + + + + + +

+ + + + + + T + -

N + + N N N + + +

N N N N N N N N N N

N N N N N N N N N N

[87, 88] [89] [90] [90] [90] [90] [179] [89] [89] [89]

-

-

+

+

N

N

N

[88]

-

-

+ + +

+ -

N + +

N N N

N N N

[88] [179] [179]

bacterial PSY (crtB) CrtB, CrtI, CrtY OR

+ + +

+ + +

+ + N N

N N N N N

N N N N N

[88] [180] [180] [34] [34]

+ N

+ +

T N

N N

N N

[34] [181]

+ +

N +

N +

N N

N N

[77] [182]

RI PT

-

SC

-

M AN U

Pink Marsh

TE D

Grapefruit

EP

Lemon

AC C

Pummelo

Cara Cara Shamouti Yuhuan Yuhuan Chuhou Early Red Chuhou Early Red Lisbon Eureka Frost Volkamer Meyer

fruit, dark-grown callus, flavedos fruit juice peel juice vesicle peel, juice vesicle juice vesicle fruit fruit juice fruit juice fruit juice dark-grown callus & falvedos dark-grown callus & falvedos fruit fruit dark-grown callus & falvedos root root root root

-

root tubers

-

tubers tubers

-

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Sunflower Chinese cabbage Tangor Carambola Cashew apple Daffodil Narcissus Tulip

-

+ T

T + T

+ + + +

D (EYI105 background)

orange seed endosperm

-

L (EYI105 background) zebra2 nd-1

orange seed endosperm yellow seed endosperm etiolated tissue, stem cotyledons

+

+

+

+

N

N

+

N

CRTISO ZDS

AtDXS, ZmPSY1, PaCRTI AtOR, ZmPSY1, PaCRTI ZmPSY1 , PaCRTI -

+ + +

Br-or Kiyomi Golden Star

head leaves fruit fruit

CRTISO -

-

n/a King Alfred Scarlet Elegance Golden Harvest

fruit corona & perianths corona perianths

-

-

SC

-

-

[183] [43] [43] [43]

-

[43]

N

N

[184]

N

N

N

[184]

N + +

N + +

N + -

N + -

[184] [51] [103]

N + -

N +

N + +

N N +

+ N N

[107] [179] [185]

N + + +

+ + +

+ + +

N N N N

N N N N

[186] [91] [91] [91]

RI PT

Z-ISO

O (EYI105 background)

ZDS ZDS ZDS ZDS

TE D

y9

endosperm endosperm etiolated leaf root endosperm, etiolated leaf, root

EP

Rice

vp-wl2 vp9 vp9 vp9

AC C

Maize

M AN U

background) -

Regulatory Bottleneck Geranylgeranyl Pyrophosphate

Promoter

5’UTR

Epistasis

Enhancer

PSY 15-cis-phytoene

Plastid Control

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Feedback

PDS

Plastids

9,15-di-cis-phytofluene

NFZ

PDS Signal

CCD

RI PT

9,15,9’-tri-cis-ζ-carotene Z-ISO

Nucleus

9,9’-di-cis-ζ-carotene Feedforward

ZDS

clb5

M AN U

7,9,9’-tri-cis-neurosporene

SC

Leaf Development

ZDS

7,9,9’,7’-tetra-cis-lycopene

AC C

α-carotene

all-trans-lycopene βLCY βLCY

EP

εLCY

lutein

WT

Plastid Biogenesis

tangerine ccr2, zebra2

TE D

CRTISO

clb5

β-carotene

neoxathin

psuedo

no PLB

CCD

NCED

SL

ABA

Bacteria

Algae

Plants

GGPP PSY “Bottleneck”

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15-cis-phytoene

CrtP (PDS) CrtI (PDS)

PDS “Redox Sensor” Z-ISO “Gatekeeper” ZDS “Developer” CRTISO “Epigenetic”

CrtQ (ZDS)

AC C

EP

TE D

M AN U

SC

all-trans-lycopene

RI PT

CrtH (CRTISO)