Apocarotenoids as Stress Signaling Molecules in Plants

Abstract

Apocarotenoids are ancient signaling molecules that have played crucial roles in biological communication and adaptation across evolutionary history. Originating in cyanobacteria, these molecules have diversified significantly in plants, where they contribute to stress perception, developmental regulation, and environmental responses. While some apocarotenoids, such as abscisic acid (ABA) and strigolactones (SLs), have been formally classified as plant hormones due to the identification of specific receptors, many others remain functionally enigmatic despite their profound effects on gene regulation and plant physiology. In this study, we focus on β-carotene-derived apocarotenoids that lack identified receptors, shedding light on their potential signaling roles beyond traditional hormone pathways. By synthesizing current knowledge, we highlight key gaps in understanding their biosynthesis, transport, perception, and downstream effects. Addressing these gaps is essential for unraveling the full scope of apocarotenoid-mediated signaling networks in plants. A deeper understanding of these molecules could not only redefine plant hormone classification but also open new avenues for improving crop resilience and stress adaptation in the face of climate change.

1. Introduction

In this study, we investigated the role of carotenoids, particularly their oxidative byproducts, the apocarotenoids, in plants’ ecophysiology. These molecules have coevolved with photosynthetic organisms and are believed to function not only in light capture, cellular redox regulation, and membranes protection but also as potential signaling compounds. Our focus was on the biosynthesis of apocarotenoids, with particular attention to those derived from β-carotene and lacking a currently identified receptor, and their involvement in plant stress responses.

2. Carotenoids

Carotenoids are a diverse class of molecules, with over 700 different structures found in nature. They were already present in archaea and bacteria before green photosynthesis [1,2]. These long lipophilic molecules, typically C30, C40, or C50, feature an extended conjugated double-bond system, which confers light absorption capabilities in the 400–500 nm range and is particularly sensitive to oxidation [3,4]. Although predominantly hydrophobic, carotenoids often have polar substituents at the extremities, α and β-carotene being almost an exception. This polarity influences their distribution in membranes and protein complexes [5,6,7]. Indeed, by spanning the membrane with their lipophilic core while fanchoring their polar regions to the aqueous interface, carotenoids have served as membrane stabilizers since early evolutionary stages [2,5].

Over billions of years of co-evolution with photosynthesis, carotenoid biosynthesis became confined to plastids, even though all the genes responsible for carotenogenesis were transferred to the nuclear genome [8]. During this time, significant modifications occurred in algae, expanding the diversity of carotenoids involved in light harvesting. In plants, the cyanobacterial pathway evolved further, leading to the hydroxylation of α-carotene into lutein and the epoxidation of zeaxanthin into violaxanthin, followed by additional modifications to neoxanthin [2]. At the same time, cyanobacteria possess characteristic carotenoids and biosynthetic pathways, including echinenone and canthaxanthin derived from β-carotene, nostoxanthin derived from zeaxanthin, as well as unique compounds such as mixol and myxoxanthophyll [9,10].

Now, the palette of carotenoids present in plants has specific roles in photosynthesis (Figure 1). Among them, β-carotene is both a structural component of the photosynthetic core complexes and a key player in photoprotection, a role held since cyanobacterial photosynthesis [3,11,12]. Additionally, oxygenated xanthophylls—namely, lutein, violaxanthin, antheraxanthin, zeaxanthin, and neoxanthin—stabilize the Light Harvesting Complex II (LHCII) antennae proteins, directly quench triplet-chlorophyll via energy transfer, and allow the xanthophyll cycle [13,14,15].

In Arabidopsis leaves under normal conditions, lutein and β-carotene are the most abundant carotenoids, accumulating at 67.4 and 62 μg g⁻¹ FW (Fresh Weight), respectively, followed by neoxanthin (32.8 μg g⁻¹ FW) and violaxanthin (13.5 μg g⁻¹ FW) [16]. Conversely, lycopene, α-carotene, and zeaxanthin are present in trace amounts or are below quantification limits [16].

Figure 1. Main carotenoids in Plantae derived from Cyanobacteria/like organisms. Biosynthetic pathways of the main carotenoids in Plants. Lutein and β-carotene and neoxanthin are the most abundant, while lycopene, α-carotene, and zeaxanthin are present in trace amounts in Arabidopsis leaves under control conditions. LCY: Lycopene cyclase beta (-b) or epsilon (-e), CHY: β-carotene hydroxylase (CYP97 and BCH), ZEP: Zeaxanthin epoxidase (ABA1), VDE: Violaxanthin de-epoxidase, NSY: Neoxanthin synthase (AtABA4, SlNXD1). Adapted from [2,17,18,19,20].

Figure 1. Main carotenoids in Plantae derived from Cyanobacteria/like organisms. Biosynthetic pathways of the main carotenoids in Plants. Lutein and β-carotene and neoxanthin are the most abundant, while lycopene, α-carotene, and zeaxanthin are present in trace amounts in Arabidopsis leaves under control conditions. LCY: Lycopene cyclase beta (-b) or epsilon (-e), CHY: β-carotene hydroxylase (CYP97 and BCH), ZEP: Zeaxanthin epoxidase (ABA1), VDE: Violaxanthin de-epoxidase, NSY: Neoxanthin synthase (AtABA4, SlNXD1). Adapted from [2,17,18,19,20].

3. Apocarotenoids

Carotenoids show an extensive system of conjugated double bonds formed by the fusion of multiple isoprene modules. This system is particularly prone to oxidation, which can lead to the oxidative breakdown of the carotenoids into smaller byproducts known as apocarotenoids [21,22,23]. Oxidation can occur either through direct attack by reactive oxygen species (ROS) or through enzymatic catalysis mediated by carotenoid cleavage dioxygenases (CCDs) and nine-cis-epoxycarotenoid dioxygenases (NCEDs) [21,24]. Indeed, among enzymatically derived apocarotenoids, the most well-known in plant biology are those that function as plant hormones, such as abscisic acid (ABA), derived from 9-cis-neoxanthin and 9-cis-violaxanthin, and strigolactones (SLs) from β-carotene (Figure 2) [25]. Both ABA and SLs have dedicated biosynthetic pathways and specific receptors: PYRABACTIN RESISTANCE/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) proteins for ABA and DWARF14 (D14) for SLs [26,27,28,29]. Interestingly, SLs biosynthesis also produces β-ionone in equimolar amounts as a byproduct, and it would be worth understanding the impact of this pathway on β-ionone accumulation (Figure 2) [30].

Recently, another apocarotenoid called zaxinone has emerged as a potential plant hormone [31]. Its biosynthesis originates from all-trans-zeaxanthin (or all-trans-lutein) and is likely catalyzed by CCD4 or CCD7 to apo-10′-zeaxanthinal (synonym: 3-OH-β-apo-10′-carotenal from zeaxanthin) and 3-OH-β-Ionone and the zaxinone synthase (ZAS), a CCD responsible for the C13-14 oxidation of apo-10′-zeaxanthinal. Zaxinone interacts with two receptors involved in the signaling of several apocarotenoids: D14 and Karrikin insensitive 2 (KAI2) (Figure 2) [32,33,34,35].

Even in the absence of identified receptors, several apocarotenoids exhibit signaling properties, including anchorene, β-cyclocitral, β-cyclogeraniol, β-ionone, and dihydroactinidiolide [3,36,37,38,39]. In this review, we focus on apocarotenoids without an identified receptor, as ABA, SLs, and zaxinone have been already carefully described in the literature [40].

Figure 2. Pathways of enzymatic biosynthesis of apocarotenoids with an already identified receptor. Strigolactones (Carlactone) biosynthesis from β-carotene mediated by the enzymes D27: β-carotene isomerase; CCD: Carotenoid Cleavage Dioxygenase 7 and 8. Zaxinone biosynthesis from zeaxanthin or lutein to apo-10′-zeaxanthinal mediated by CCD7 and then from apo-10′-zeaxanthinal to zaxinone mediated by the ZAS (zaxinone synthase) enzyme. Abscisic acid (ABA) biosynthesis from 9-cis-violaxanthin or 9-cis-neoxanthin to Xanthoxin mediated by NCED: Nine-Cis Epoxycarotenoid Dioxygenase; ABA2 (ABA DEFICIENT 2): short-chain dehydrogenase reductase; AAO: ABA aldehyde oxidase [31,41,42]. β-ionone is an equimolar side product of strigolactone biosynthesis with a not-identified receptor. 3-OH-β-ionone may be an equimolar side product of zaxinone biosynthesis. Every arrow indicates one biosynthetic step. Intermediates are not reported for these pathways.

Figure 2. Pathways of enzymatic biosynthesis of apocarotenoids with an already identified receptor. Strigolactones (Carlactone) biosynthesis from β-carotene mediated by the enzymes D27: β-carotene isomerase; CCD: Carotenoid Cleavage Dioxygenase 7 and 8. Zaxinone biosynthesis from zeaxanthin or lutein to apo-10′-zeaxanthinal mediated by CCD7 and then from apo-10′-zeaxanthinal to zaxinone mediated by the ZAS (zaxinone synthase) enzyme. Abscisic acid (ABA) biosynthesis from 9-cis-violaxanthin or 9-cis-neoxanthin to Xanthoxin mediated by NCED: Nine-Cis Epoxycarotenoid Dioxygenase; ABA2 (ABA DEFICIENT 2): short-chain dehydrogenase reductase; AAO: ABA aldehyde oxidase [31,41,42]. β-ionone is an equimolar side product of strigolactone biosynthesis with a not-identified receptor. 3-OH-β-ionone may be an equimolar side product of zaxinone biosynthesis. Every arrow indicates one biosynthetic step. Intermediates are not reported for these pathways.

4. Are ROS-Induced Apocarotenoids Singlet Oxygen Sensors?

Carotenoids biosynthesis and accumulation is confined to plastids. Consequently, apocarotenoids generated by direct ROS attack, which have signaling properties, have been described as indicators of the oxidative status of plastids [38,43]. While carotenoids accumulate in high concentrations in the chromoplast in yellow/red/orange-colored tissues, they are primarily found in the thylakoid membranes of chloroplasts in green tissues, which represents the main site of ROS production in plants [44,45,46].

Until recently, apocarotenoids were believed to originate mainly within the Photosystem II (PSII) core, specifically in the grana stack of thylakoids [38]. This reasoning was based on their oxidative origin, particularly the chemical quenching of singlet oxygen by β-carotene [3,12,47]. Singlet oxygen derives from the charge recombination of excited triplet chlorophyll directly to molecular oxygen [48]. Intriguingly, in isolated thylakoids, only atmospheric molecular oxygen and not nascent oxygen from water splitting in the oxygen evolving complex (OEC) appears to contribute to singlet oxygen formation [49]. However, evidence for this statement is still under review, and its relevance in vivo might be limited, as PSII complexes are deeply buried in thylakoid grana, where high concentrations of oxygen from the external environment are unlikely.

Singlet oxygen formation happens with much higher probability in the isolated PSII reaction core (D1, D2, cytb559, and I proteins). In this configuration, β-carotene fails to physically quench the excited chlorophyll by energy transfer from the triplet P680 chlorophyll. However, it successfully dissipates excess energy in the core antennae (CP47 and CP43) [12,50]. Even less probable is the origin of singlet oxygen from peripheral or detached LHCII antennae, where the triplet wavefunction is shared between the carotenoids (lutein and zeaxanthin) and their adjacent chlorophylls, making the energy transfer almost instantaneous (in the range of sub-nanoseconds) and quenching around 95% of the excess energy via triplet carotenoid formation as heat [11,51,52]. Consistent with these mechanisms, singlet oxygen production has been measured in vitro in PSII cores, LHCII antennae, PSII-LHCII complexes, and PSI-LHCI complexes [45]. Indeed, PSII cores were the predominant singlet oxygen producers, showing around four times more signal of the singlet oxygen specific fluorescent probe (SOSG) than the PSII-LHCII complex or the LHCII alone under 100 μE m⁻² s⁻¹ photosynthetic photon flux density (PPFD) [45]. Furthermore, the Arabidopsis mutant line ch1 (Chlorophyll A Oxygenase, CAO), which lacks chlorophyll b and, consequently, the majority of LHCs, showed very enhanced photosensitivity and singlet oxygen production in vivo [53].

Singlet oxygen production from the illumination of the PSI complex is unlikely for structural reasons, and the excited triplet P700 half-life in PSI was unaffected by the presence of molecular oxygen [54,55]. Experimental evidence further confirms that PSI-LHCI complexes generate only low levels of singlet oxygen in vitro [45]. Moreover, the PSI is protected by multiple photoprotective mechanisms, including cyclic electron flow, state transitions, reduced energy absorption via antenna detachment from PSII, and non-photochemical quenching [11].

Since singlet oxygen is undoubtedly generated in vivo, it follows that energy quenching by β-carotene in PSII cores and complexes is insufficient, leading to the accumulation of apocarotenoids and peroxides of the molecules adjacent to the ROS source.

A novel interpretation of the interaction between singlet oxygen and β-carotene has recently emerged. Triplet β-carotene, an excited state that can form in the PSII extended core (core + CP47 and CP43) and in PSI, may directly react with molecular oxygen to produce the endoperoxides of β-carotene: β-carotene-5,8-EPO [56]. This endoperoxide previously identified as a specific marker of β-carotene oxidation by singlet oxygen accumulates during light exposure [57]. In the new model, β-carotene could bind up to eight oxygen atoms before breaking down, independently of singlet oxygen [56]. The oxidative breakdown of the endoperoxides (especially β-carotene-5,8-EPO and β-carotene-7,10-EPO) in the light would then generate not only apocarotenoids (such as β-cyclocitral, apo-8′-carotenal and apo-10-carotenal) but also a strong burst of free radicals and singlet oxygen. This phenomenon has also been linked to a Russell reaction involving lipid peroxides [58]. Thus, β-carotene endoperoxides may not merely be byproducts of singlet oxygen quenching but could instead act as precursors to singlet oxygen formation, originating from the β-carotene quenching of triplet chlorophyll. Anyway, these findings are in contrast with previous knowledge and with the quantification of singlet oxygen produced by isolated photosystems [45]. PSII was the major producer of singlet oxygen, and this was rather unaffected in mutants with reduced β-carotene (szl1), suggesting that β-carotene unlikely quenches triplet chlorophyll in the PSII [59]. Conversely, PSI was highly sensitive to β-carotene depletion, exhibiting nearly threefold higher singlet oxygen production in PSI complexes isolated from szl1 mutants, indicating that β-carotene can quench triplet chlorophyll in this configuration [45]. It would be particularly interesting to investigate triplet chlorophyll and singlet oxygen quenching in the recently developed mutant tobacco plants, in which β-carotene has been substituted by the xanthophyll astaxanthin [60].

In summary, the in vivo formation of apocarotenoids is most likely linked to singlet oxygen quenching. However, there is also the possibility that apocarotenoids originate directly from overexcited photosystems (Figure 3). Anyway, we can safely assume that apocarotenoids serve as a signal of the overexcited photosynthetic chain.

5. Origin of Apocarotenoids

Apocarotenoids can originate either from chemical oxidation by ROS or through enzymatic oxidation. Certain apocarotenoids, such as ABA, SLs, and zaxinone (hydroxy-β-apo-13-carotenone), follow specific biosynthetic pathways. Smaller apocarotenoids may result from the enzymatic oxidation of β-carotene by CCD enzymes or by direct oxidation.

In Arabidopsis, typical apocarotenoids are β-cyclocitral, the APOcarotenoid deriving from the oxidation of β-carotene at C7/8 (APO7), β-ionone (APO9) and hydroxy- β-ionone (OH-APO9) deriving from the oxidation of β-carotene or zeaxanthin at C9/10, β-apo-11-carotenal (APO-11) and hydroxy-β-apo-11-carotenal (OH-APO-11) deriving from the oxidation of β-carotene or zeaxanthin at C11/12, β-apo-13-carotenone (APO13, d’orenone) deriving from the oxidation of β-carotene at C13/14, and β-apo-15-carotenal (APO15) and hydroxy-β-apo-15-carotenal (OH-APO15, retinal) deriving from the oxidation of β-carotene or zeaxanthin at C15/15′. Additional apocarotenoids include β-apo-14′-carotenal (APO14′) and hydroxy-β-apo-14′-carotenal (OH-APO14′) deriving from the oxidation of β-carotene or zeaxanthin at C13′/14′, β-apo-12′-carotenal (APO12′) and hydroxy-β-apo-12′-carotenal (OH-APO12′) deriving from the oxidation of β-carotene or zeaxanthin at C11′/12′, β-apo-10′-carotenal (APO10′) and hydroxy-β-apo-10′-carotenal (OH-APO10′) deriving from the oxidation of β-carotene or zeaxanthin at C9′/10′, and β-apo-8′-carotenal (APO8′) and hydroxy-β-apo-8′-carotenal (OH-APO8′) deriving from the oxidation of β-carotene or zeaxanthin at C7′/8′ [61]. Furthermore, other apocarotenoids include α-ionone deriving from the oxidation of α-carotene at C9/10, blumenols and mycorradicins from zeaxanthin, α-ionene and anchorene (12,12′-diapocarotene-12,12′-dial) from β-carotene, dihydroactinidiolide (resulting from the secondary oxidation of β-ionone through the intermediate 5,6-epoxy-β-ionone), and β-cyclogeranic acid (deriving from the oxidation of β-cyclocitral in water) (Figure 4) (

Table 1. List of cyclic apocarotenoids derived from a specific oxidative cleavage site from β-carotene and zeaxanthin.

Name	Symbol	Cleavage Site	Precursor
β-cyclocitral hydroxy- β-cyclocitral	APO7 OH-APO7	C7/8 C7/8	β-carotene zeaxanthin
β-ionone	APO9	C9/10	β-carotene
hydroxy-β-ionone	OH-APO9	C9/10	zeaxanthin
β-apo-11-carotenal	APO11	C11/12	β-carotene
hydroxy-β-apo-11-carotenal	OH-APO11	C11/12	zeaxanthin
β-apo-13-carotenone (d’orenone)	APO13	C13/14	β-carotene
hydroxy-β-apo-13-carotenone (zaxinone)	OH-APO13	C13/14	zeaxanthin
β-apo-15-carotenal (retinal)	APO15	C15/15′	β-carotene
hydroxy-β-apo-15-carotenal	OH-APO15	C15/15′	zeaxanthin
β-apo-14′-carotenal	APO14′	C13′/14′	β-carotene
hydroxy-β-apo-14′-carotenal	OH-APO14′	C13′/14′	zeaxanthin
β-apo-12′-carotenal	APO12′	C11′/12′	β-carotene
hydroxy-β-apo-12′-carotenal	OH-APO12′	C11′/12′	zeaxanthin
β-apo-10′-carotenal	APO10′	C9′/10′	β-carotene
hydroxy-β-apo-10′-carotenal	OH-APO10′	C9′/10′	zeaxanthin
β-apo-8′-carotenal	APO8′	C7′/8′	β-carotene
hydroxy-β-apo-8′-carotenal	OH-APO8′	C7′/8′	zeaxanthin

) [32,36,62].

Among these, β-cyclocitral is one of the most abundant apocarotenoids under control conditions, showing values of 40–60 ng g⁻¹ FW or 600 ng g⁻¹ dry weight (DW) [3,61]. Its oxidized form, β-cyclogeranic acid, is even more present, reaching about 120 ng g⁻¹ (FW) [63]. β-apo-10′-carotenal (APO10′) also accumulates at around 600 ng g⁻¹ (DW), but it was not extensively studied, due to the lack of a response to stress conditions [61]. β-ionone is very interesting as a comparison, being present at around 40 ng g⁻¹ (FW) or 350 ng g⁻¹ (DW) in its unconjugated form, similar to the other apocarotenoids [3,61]. In contrast, β-ionone concentrations are affected by its transformation to dihydroactinidiolide, around 5 ng g⁻¹ (FW), and it is extensively metabolized together with hydroxy-β-ionone to glycosyl-β-ionone (GAPO9). Notably, GAPO9 reaches extraordinarily high levels compared to other glycosylated apocarotenoids, around 2500 ng g⁻¹ (DW), far surpassing the aglycone form [61].

Various enzymes responsible for carotenoid oxidation have been identified across plants, animals, fungi, and bacteria [64]. In plants, CCD enzymes are very active in flowers, where they contribute to scent production [64]. CCD1 and CCD2 exhibit broad substrate specificity, cleaving lycopene, β-carotene, and zeaxanthin at multiple sites. Their activity results in both linear apocarotenoids —6-methyl-5-hepten-2-one (MHO, C5/6) and pseudo-ionone (C9/10) from lycopene, C14-dyaldehyde (2 × C9/10) from β-carotene, crocetin (2 × C7/8) from zeaxanthin— and cyclic apocarotenoids hydroxy-β-cyclocitral (C7/8) and hydroxy-β-ionone (C9/10) from zeaxanthin [64]. CCD4 catalyzes a broad range of reactions mediating mainly the C9/10 oxidation of epoxy-β-xanthophylls, such as violaxanthin, and of β-apo-8′-carotenal to β-ionone in Arabidopsis, but also C7/8 oxidation in Citrus, Crocus, and rice and C5/6 oxidation in grapevine [64,65,66,67,68,69,70]. Meanwhile, CCD7 oxidizes 9-cis-β-carotene at C9′/10′, yielding β-ionone and 9-cis-β-apo-10′-carotenal, while CCD8 further converts this intermediate into carlactone, two dedicated steps in strigolactone biosynthesis [71,72]. Belonging to the same family of enzymes, plants also possess NCEDs that can catalyze the oxidation of 9′-cis-violaxanthin and 9′-cis-neoxanthin to cis-xanthoxin in the abscisic acid biosynthesis [73].

A surprising recent discovery is the involvement of lipoxygenase 2 (LOX2) in apocarotenoid biosynthesis. LOX2 is a 13-lipoxygenase necessary for the normal accumulation of β-cyclocitral, β-ionone, and dihydroactinidiolide in Arabidopsis leaves [74]. Even more volatile apocarotenoids were affected in the Solanum lycopersicum TomLoxC antisense line. Arabidopsis LOX2 is known to participate in the biosynthesis of jasmonic acid and C5 and C6 green leaf volatiles from linoleic and linolenic acids [75,76]. LOX enzymes catalyze the oxygenation of these unsaturated fatty acids producing either C5 and C6 volatiles (via 13-LOX) or C9 volatiles (via 9-LOX) [77]. Though the precise mechanism remains unclear, LOX2 involvement in apocarotenoid formation may be linked to the generation of aggressive peroxyl radicals, which in turn attack carotenoid polyene chains, leading to apocarotenoid production [74,78]. Further research into LOX2 role under excessive light exposure could clarify whether this enzyme directly contributes to apocarotenoid biosynthesis or if its activity is merely a byproduct of lipid peroxidation, which is a well-documented ROS-driven process under high light stress [79,80].

Among apocarotenoids, β-ionone and hydroxy-β-ionone are particularly abundant, likely due to their formation by multiple enzymatic pathways. This could explain the exceptionally high levels of glycosylated β-ionone detected in leaves [61]. Conversely, other apocarotenoids, which accumulate in lower concentrations, may be primarily ROS-induced or subjected to rapid detoxification.

Indeed, β-ionone is converted by Alkenal Reductase (AER) to dihydro-β-ionone, and by Aldehyde dehydrogenase (ALDH3H1 and I1) to β-ionol [81]. At the same time, β-apo-13-carotenone and β-apo-14′-carotenal are also substrates of AER; β-apo-11-carotenal, β-apo-10′-carotenal, β-apo-12′-carotenal, and β-apo-14′-carotenal are substrates of ALDH3H1 and I1; β-apo-8′-carotenal is a substrate of ALDH3I1; and β-apo-11-carotenal is a substrate of Aldo-keto-reductase (AKR4C8 and C9) and of chloroplastic aldehyde reductase (ChlADR) [81]. On the contrary, β-cyclocitral was not metabolized by these enzymes, but it showed non-enzymatical metabolization to β-cyclogeranic acid via a Baeyer–Villiger-type oxidation in water [63,82].

To conclude, apocarotenoids serve as valuable indicators of photosynthetic overexcitation, yet their detection can be challenging, not due to limitations in analytical techniques but rather because of their complex metabolism.

6. Signaling of β-Carotene-Derived Apocarotenoids

6.1. β-cyclocitral (APO7, β-cc, Bcc, β-cyc)

From an evolutionary perspective, β-cyclocitral was first identified in cyanobacteria, such as Microcystis, where it plays a role in quorum sensing during eutrophication events in water blooms [82]. Under these circumstances, in these unicellular organisms, there is a characteristic oxidation of β-carotene, which first generates β-cyclocitral and then β-cyclocitric acid by its successive oxidation. Unlike plants, in cyanobacteria, β-cyclocitric acid accumulation induces cell death through the acidification of the growth medium. Over evolutionary timescales, these molecules have been repurposed as stress signals in plants (Figure 5) [38].

While the predominant pathway leading to β-cyclocitral accumulation in plants is the oxidation of β-carotene by reactive oxygen species, namely, singlet oxygen, some exceptions have been observed in specific plants. Enzymatic production has been observed in specific plant species, such as CCD4-mediated cleavage in Citrus and Crocus and the JA-responsive Oryza sativa CCD4b, which produces β-cyclocitral both in vitro and in planta [70].

A key player in β-cyclocitral signaling is SCARECROW-like 14 (SCL14), a member of the 33 GIBBERELLIC-ACID INSENSITIVE, REPRESSOR of GAI and SCARECROW (GRAS) regulatory proteins in Arabidopsis [83,84], and, in particular, of the LISCL (Lilium longiflorum Scarecrow-like) subfamily [85]. SCL14 was first identified in a yeast two-hybrid screen using TGA2 as bait [86]. Like all GRAS proteins, SCL14 is characterized by a conserved GRAS domain at its C-terminal region, consisting of two leucine-rich regions (LHRI and LHRII) and three conserved motifs (VHIID, PFYRE, and SAW) [87]. Its N-terminal region, characteristic of the LISCL subfamily, contains an activation domain (motif I) and a DEDED domain (motif II) involved in protein–protein interactions [86]. SCL14 cellular localization was studied by fluorescence microscopy, using a SCL14-GFP fusion protein. SCL14 has a predominant nuclear localization, although partial cytoplasmic distribution was observed due to exportin-mediated transport [86]. SCL14 is weakly expressed in all plant tissues but shows expression values 10-fold higher in dry seeds and high expression values in the conductive tissues of young leaves [86,88]. It interacts with TGAII transcription factors (TGA2, TGA5, and TGA6) to activate genes containing the as-1-like cis-regulatory element, forming a pre-assembled transcriptional complex that is activated under stress conditions [86].

Under unfavorable environmental conditions, plants absorb light in excess to their photosynthetic capacity (excessive light) and thus produce ROS leading to photooxidative stress [89,90,91]. This results in the formation of lipid peroxides that naturally break to reactive carbonyl species (RCS), which, at high levels, trigger programmed cell death (PCD) [53,92,93,94]. β-cyclocitral mitigates these effects by activating an acclimation response through the SCL14-mediated detoxification of peroxides and carbonyl compounds (Figure 5) [88]. SCL14/TGA2 activation induces the transcription factor ANAC102, which, in turn, regulates ANAC002, ANAC032, and ANAC081 [88,95]. The ANAC transcription factors orchestrate a three-phase detoxification response. Phase I is the modification of the xenobiotic, which occurs through the induction of P450 monooxygenases, several reductases (AER, AKR, SDR), dehydrogenases (ALDH), and peroxidases; phase II is the conjugation of toxic molecules with glucose or glutathione by UDP and GST enzymes to produce less toxic and water-soluble molecules; this is followed by phase III, storage in the vacuole or apoplast. Several phase I and II genes are targets of SCL14 [38,86].

Notably, SCL14-mediated detoxification targets many enzymes that catabolize lipid peroxides and RCS, as well as apocarotenoids, except for β-cyclocitral itself [81,96].

Additionally, β-cyclocitral induces markers of the jasmonic acid (JA) pathway, such as MYELOCITOMATOSIS 2 (MYC2), without triggering JA biosynthesis [88]. This was observed in Solanum lycopersicum, where β-cyclocitral treatment upregulated JA-dependent transcription factors (MYC2, MYB44, ERFs) without eliciting JA or ABA biosynthesis [97]. A strict relation between β-cc and JA was also observed in Oryza sativa where JA induced β-cyclocitral accumulation [70]. Indeed, both β-cyclocitral and JA signaling are required for a strong induction of the Phase I gene CYP81D11, whose regulation is under the control of both TGAII and MYC transcription factors [98].

As expected from molecules which had a two-billion-year co-evolution, β-cyclocitral elicits several pathways. Surely, in addition to the already described processes, β-cyclocitral activates the signaling of METHYLENE BLUE SENSITIVITY 1 (MBS1), a protein required for acclimation of Arabidopsis to singlet oxygen and induces its mobilization to the nucleus [99,100]. However, many molecular players in apocarotenoid sensing remain unidentified, underscoring gaps in our understanding of phototolerance mechanisms [37].

β-cyclocitral and Salicylic Acid

A special role for salicylic acid (SA) in β-cyclocitral signaling was described in 2015 [101]. β-cyclocitral treatments were able to induce SA accumulation under 12 h of excessive light, and SA was essential for β-cyclocitral-induced tolerance to excessive light (Figure 5) [101]. Specifically, β-cyclocitral induced the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent expression of ISOCHORISMATE SYNTHASE 1 (ICS1), the primary enzyme in SA biosynthesis in Arabidopsis. Furthermore, SA accumulation and NPR1 expression were responsible for the induction of GST3 and GST13 expression and total GST activity in β-cyclocitral and excessive light-treated samples.

These results suggest that the role of SA in β-cyclocitral and excessive light signaling is more complex than that present in the literature, and SA may exert different roles at different moments of stress. In fact, we described an opposite negative role for SA under excessive light [37,102]. In our experiments, SA accumulation was induced at later stages of stress response (28 h), induced downstream of jasmonic acid accumulation, and responsible for inducing programmed cell death (PCD) [102]. A clear example was the treatment of sid2 (mutant lines in the ICS1 gene) with methyl-jasmonate, which did not proceed toward PCD, in opposition to wild type plants which extremely suffered from the treatment. Similarly, sid2 lines showed much lower lipid peroxidation and PCD under excessive light conditions.

In addition, several GST enzymes, such as GST7 and GST9, are part of the phase II of the SCL14-controlled detoxification via TGAII transcription factors, and SA seems to induce this family of enzymes through SCL14 interaction with TGAII rather than NPR1 interaction with the same transcription factors [86]. NPR1 was clearly demonstrated to interfere with the activation of GST7 by SA [86]. Competition between SCL14, NPR1, and glutaredoxin GRX480/ROXY19 for TGA transcription factor binding further complicates the regulatory network [103]. Moreover, TGAII factors are implicated in SA metabolism via SCL14-mediated detoxification [86,104].

Finally, the intersection of β-cyclocitral and SA signaling remains unclear. Further research is needed to shed light on the role of SA at different timepoints of excessive light (6–12–24 h), as SA seems to be accumulating only in a part of the literature.

6.2. β-Cyclogeranic Acid (β-Cyclocitric Acid, β-cca, Bcca, APO7-OOH)

β-cyclocitral is an aldehyde that is uniquely reactive in water and naturally transforms into the water-soluble β-cyclogeranic carboxylic acid (β-cca) [82]. This conversion occurs more rapidly inside plants, where β-cca induces protective mechanisms against drought stress (Figure 5) [63]. The protective effect of β-cca is a conserved mechanism, observed in various plant species, like Arabidopsis, Capsicum annuum, Viola tricolor, Solanum lycopersicum, and Prunus persica [63,105,106], both annual plants and perennial fruit crops.

Although β-cca derives directly from the oxidation of β-cyclocitral, it induces only a subset of β-cyclocitral signaling responses [63,107]. Interestingly, the overexpression of SCL14 enhances drought tolerance and amplifies the protective effect of β-cca, suggesting an involvement of SCL14-dependent detoxification in drought resistance. In P. persica, treatment with β-cca under drought stress improved photosynthetic performance while also enhancing superoxide dismutase and peroxidase activity and reducing ROS levels [105].

β-cca accumulates under drought conditions, yet SCL14 induction is not the sole mechanism triggered by β-cca. Several Arabidopsis mutant lines have been tested, all of which retained β-cca-induced drought tolerance, suggesting that β-cca activates multiple pathways simultaneously and/or exploits an unidentified signaling mechanism. Indeed, β-cca functions independently of ABA and does not induce stomatal closure, adjustments in water potential, or jasmonate signaling [63,106]. Recent findings suggest β-cca plays a role in root development, reducing primary root length, revealing a novel link between root growth, SMR5 proteins, suberin deposition, and drought tolerance (Figure 5) [108]. Interestingly, while investigating the role of β-cca in root development, the authors also examined the effects of β-cyclocitral. Contrary to previously published data, they found that 25 µM β-cyclocitral reduced root length, whereas 750 nM β-cyclocitral had no significant effect [108,109].

6.3. β-Ionone (β-I, APO9)

β-Ionone is the most abundant cyclic apocarotenoid, primarily found in its glycosylated form (GAPO9). Its accumulation is largely determined by the presence of biosynthetic enzymes, with CCD1, CCD2, CCD4, and CCD7 cleaving β-carotene to produce β-Ionone [110].

Despite its structural similarity to β-cyclocitral and β-cca, β-Ionone does not enhance plant tolerance to excessive light or drought stress [3]. Instead, β-Ionone functions predominantly in biotic stress responses (Figure 5). For example, exogenous application at micromolar concentrations induces global transcriptional reprogramming in Arabidopsis, ultimately enhancing resistance to the necrotrophic fungus Botrytis cinerea [111]. Also, the pre-treatment of tobacco and tomato plants with β-Ionone, followed by inoculation with B. cinerea, confirmed its effect in increasing the resistance to this pathogen in crop plants [111].

Furthermore, a positive effect of β-ionone on mycorrhization has been observed in rice. In this model, it accumulates in roots during arbuscular mycorrhizal (AM) symbiosis, and CCD1 and CCD7, key enzymes in β-Ionone biosynthesis, are upregulated in mycorrhizal roots [112]. β-Ionone-treated plants exhibit increased AM colonization and arbuscule abundance. Moreover, it enhances the expression of OsPT11 and OsLysM, two AM marker genes in rice [113]. Notably, β-Ionone influences AM symbiosis in a strigolactone (SL)-independent manner, as CCD8, a key gene in SL biosynthesis, remains unaffected by β-Ionone treatment at 50 nM [112].

Regarding its hydroxylated derivative (hydroxy-β-ionone, OH-APO9), studies indicate diverse biological functions. OH-APO9 inhibits hypocotyl growth in Phaseolus vulgaris under light exposure [114] and exhibits allelopathic activity in plant extracts, suggesting a role in interspecies interactions [115,116,117,118].

6.4. Dihydroactinidiolide (dhA)

Dihydroactinidiolide (dhA) is a lactone (cyclic ester) derived from the secondary oxidation of β-ionone via 5,6-epoxy-β-ionone [36]. It accumulates in Arabidopsis leaves under excessive light and, like β-cc, it triggers genetic responses that enhance tolerance to photooxidative stress (Figure 5) [3]. Specifically, dhA treatment upregulates ¹O₂-responsive genes (AGC2, AtRD20, AtGSTU5, UGT73B4, LTI30), also induced by β-cyclocitral, while inhibiting lipid peroxidation and improving PSII photochemistry (Fv/Fm) in leaves under excess light [36]. Interestingly, dhA has been identified in cyanobacteria, alongside β-cyclocitral and β-cyclogeranic acid, as a growth inhibitor [119,120]. Moreover, it is the primary apocarotenoid produced from the thermal degradation of β-carotene, indicating that its formation may occur independently of singlet oxygen stress [121].

6.5. β-Apo-11-carotenoids

These compounds result from the oxidative cleavage of β-carotene at the C11/12 position for β-apo-11-carotenal (APO-11) and of zeaxanthin for hydroxy-β-apo-11-carotenal (OH-APO-11), along with their isomers 9-cis-β-apo-11-carotenal and 9-cis-3-OH-β-apo-11-carotenal. These molecules are particularly intriguing because they can be converted to xanthoxin in Arabidopsis, downstream of the zeaxanthin epoxidase ABA1 (ABA DEFICIENT 1), and likely through an epoxidase, and then from xanthoxin to ABA using the ABA biosynthetic pathway [122]. In the conventional ABA biosynthetic pathway, zeaxanthin is converted into violaxanthin via ABA1 (ZEP) and then into neoxanthin via neoxanthin synthase (NSY/ABA4). An unidentified isomerase then modifies these intermediates before NCED enzymes cleave them at the C11–C12 double bond, generating xanthoxin. Xanthoxin is subsequently transported to the cytoplasm, where ABA DEFICIENT 2 (ABA2), ABA DEFICIENT 3 (ABA3), and ABSCISIC ALDEHYDE OXIDASE 3 (AAO3) convert it into ABA [42]. When applied exogenously, β-apo-11-carotenoids mimic ABA activity by promoting seed dormancy and upregulating ABA-responsive genes in Arabidopsis (Figure 5) [122].

6.6. Anchorene and Iso-Anchorene (12,12′-Diapocarotene-12,12′-dial)

Anchorene is a linear apocarotenoid derived from C11/12 oxidative cleavage, forming alongside β-apo-11-carotenoids. However, in vitro studies indicate that Arabidopsis CCDs do not catalyze both C11/12 and C11′/C12′ cleavages, leaving its biosynthetic pathway unresolved [39]. Evidence suggests that anchorene is produced downstream of NCED oxidation of zeaxanthin, which generates 3-OH-β-apo-12′-carotenal. The treatment of Arabidopsis seedlings with 25 μM 3-OH-β-apo-12′-carotenal induces anchorene accumulation, whereas 3-OH-β-apo-10′-carotenal does not. Unlike β-cyclocitral and β-ionone, the further oxidation of anchorene into its di-alcohol, di-acid, or diethyl ester forms yields inactive compounds [39]. Functionally, anchorene promotes the development of anchor roots from the collar region at the hypocotyl–root junction in Arabidopsis (Figure 5). This phenotype is caused by the modulation of auxin distribution by suppressing GH3-mediated auxin conjugation, and it is triggered by nitrogen deficiency [39,40,123]. Interestingly, an isomer of anchorene, deriving from oxidations of the C7/8 and C15/15 double bonds (8,15-diapocarotene-8,15-dial) and the longer apocarotenoid 8,12′-diapocarotene-8,12′-dial, showed root growth inhibition properties and did not induce the development of anchor roots [124].

7. Conclusions

By analyzing and elaborating a part of the existing literature, we show that apocarotenoids possess intricate metabolic pathways and diverse signaling functions. While only a subset has been formally classified as plant hormones due to the identification of specific receptors, our findings highlight that many more of these ancient molecules play pivotal roles in regulating gene expression and plant physiology. Furthermore, we identify key knowledge gaps that must be addressed to elucidate the origins and mechanisms of apocarotenoid action in plants.