Disruption of the rice 4-DEOXYOROBANCHOL HYDROXYLASE unravels specific functions of canonical strigolactones.

Strigolactones (SLs) regulate many developmental processes, including shoot-branching/tillering, and mediate rhizospheric interactions. SLs originate from carlactone (CL) and are structurally diverse, divided into a canonical and a noncanonical subfamily. Rice contains two canonical SLs, 4-deoxyorobanchol (4DO) and orobanchol (Oro), which are common in different plant species. The cytochrome P450 OsMAX1-900 forms 4DO from CL through repeated oxygenation and ring closure, while the homologous enzyme OsMAX1-1400 hydroxylates 4DO into Oro. To better understand the biological function of 4DO and Oro, we generated CRISPR/Cas9 mutants disrupted in OsMAX1-1400 or in both OsMAX1-900 and OsMAX1-1400. The loss of OsMAX1-1400 activity led to a complete lack of Oro and an accumulation of its precursor 4DO. Moreover, Os1400 mutants showed shorter plant height, panicle and panicle base length, but no tillering phenotype. Hormone quantification and transcriptome analysis of Os1400 mutants revealed elevated auxin levels and changes in the expression of auxin-related, as well as of SL biosynthetic genes. Interestingly, the Os900/1400 double mutant lacking both Oro and 4DO did not show the observed Os1400 architectural phenotypes, indicating their being a result of 4DO accumulation. Treatment of wild-type plants with 4DO confirmed this assumption. A comparison of the Striga seed germinating activity and the mycorrhization of Os900, Os900/1400, and Os1400 loss-of-function mutants demonstrated that the germination activity positively correlates with 4DO content while disrupting OsMAX1-1400 has a negative impact on mycorrhizal symbiosis. Taken together, our paper deciphers the biological function of canonical SLs in rice and reveals their particular contributions to establishing architecture and rhizospheric communications.

The Arabidopsis D27-LIKE1 is a cis/cis/trans-ß-carotene isomerase that contributes to Strigolactone biosynthesis and negatively impacts ABA level.

The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-ß-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-ß-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-ß-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-ß-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.

ZAXINONE SYNTHASE 2 regulates growth and arbuscular mycorrhizal symbiosis in rice.

Carotenoid cleavage, catalyzed by CAROTENOID CLEAVAGE DIOXYGENASEs (CCDs), provides signaling molecules and precursors of plant hormones. Recently, we showed that zaxinone, a apocarotenoid metabolite formed by the CCD ZAXINONE SYNTHASE (ZAS), is a growth regulator required for normal rice (Oryza sativa) growth and development. The rice genome encodes three OsZAS homologs, called here OsZAS1b, OsZAS1c, and OsZAS2, with unknown functions. Here, we investigated the enzymatic activity, expression pattern, and subcellular localization of OsZAS2 and generated and characterized loss-of-function CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and associated protein 9)-Oszas2 mutants. We show that OsZAS2 formed zaxinone in vitro. OsZAS2 was predominantly localized in plastids and mainly expressed under phosphate starvation. Moreover, OsZAS2 expression increased during mycorrhization, specifically in arbuscule-containing cells. Oszas2 mutants contained lower zaxinone content in roots and exhibited reduced root and shoot biomass, fewer tillers, and higher strigolactone (SL) levels. Exogenous zaxinone application repressed SL biosynthesis and partially rescued the growth retardation of the Oszas2 mutant. Consistent with the OsZAS2 expression pattern, Oszas2 mutants displayed a lower frequency of arbuscular mycorrhizal colonization. In conclusion, OsZAS2 is a zaxinone-forming enzyme that, similar to the previously reported OsZAS, determines rice growth, architecture, and SL content, and is required for optimal mycorrhization.

Iso-anchorene is an endogenous metabolite that inhibits primary root growth in Arabidopsis.

Carotenoid-derived regulatory metabolites and hormones are generally known to arise through the oxidative cleavage of a single double bond in the carotenoid backbone, which yields mono-carbonyl products called apocarotenoids. However, the extended conjugated double bond system of these pigments predestines them also to repeated cleavage forming dialdehyde products, diapocarotenoids, which have been less investigated due to their instability and low abundance. Recently, we reported on the short diapocarotenoid anchorene as an endogenous Arabidopsis metabolite and specific signaling molecule that promotes anchor root formation. In this work, we investigated the biological activity of a synthetic isomer of anchorene, iso-anchorene, which can be derived from repeated carotenoid cleavage. We show that iso-anchorene is a growth inhibitor that specifically inhibits primary root growth by reducing cell division rates in the root apical meristem. Using auxin efflux transporter marker lines, we also show that the effect of iso-anchorene on primary root growth involves the modulation of auxin homeostasis. Moreover, by using liquid chromatography-mass spectrometry analysis, we demonstrate that iso-anchorene is a natural Arabidopsis metabolite. Chemical inhibition of carotenoid biosynthesis led to a significant decrease in the iso-anchorene level, indicating that it originates from this metabolic pathway. Taken together, our results reveal a novel carotenoid-derived regulatory metabolite with a specific biological function that affects root growth, manifesting the biological importance of diapocarotenoids.

Gardenia carotenoid cleavage dioxygenase 4a is an efficient tool for biotechnological production of crocins in green and non-green plant tissues.

Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the colour and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the carotenoid cleavage dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison with CsCCD2L, in citrus callus engineered by Agrobacterium-mediated supertransformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. We demonstrate that co-expression of GjCCD4a with phytoene synthase and ß-carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved ß-carotene, in planta, and produced crocetin dialdehyde via C30 ß-apocarotenoid intermediate. GjCCD4a also cleaved C27 ß-apocarotenoids, providing a new route for C17 -dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast-like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis-9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non-green crop tissues/organs.

An LC-MS profiling method reveals a route for apocarotene glycosylation and shows its induction by high light stress in Arabidopsis.

Apocarotenoid glycosylation serves as a valve regulating carotenoid homeostasis in plants and may contribute to their response to photo-oxidative stress. However, an analytical method that allows comprehensive and sensitive profiling of glycosylated apocarotenoids (GAPOs) is still missing. We developed an efficient ultra-high performance liquid chromatography-high resolution-mass spectrometry (UHPLC-HR-MS) method to analyze 25 GAPOs present in carotenoid-accumulating E. coli cells and plant tissues. Optimized HR-heated-electrospray ionization (HESI)-MS parameters enabled, based on HR MS and tandem mass spectrometry (MS/MS) data, the identification of yet undescribed GAPOs from Arabidopsis, which include Glc-apo-11-carotenal (GAPO11), Glc-apo-13-carotenone (GAPO13), and their isomers. The identity of these compounds was confirmed by the transformation of deuterium-labelled non-hydroxylated carotene cleavage products into the corresponding GAPOs in planta. Quantitative analysis of GAPOs in Arabidopsis showed that the levels of Glc-cyclocitral (GAPO7), Glc-cyclocitral isomer I (GAPO7I), Glc-ionone (GAPO9), Glc-ionone isomer I (GAPO9I), Glc-apo-11-carotenal isomer I (GAPO11I), Glc-apo-13-carotenone (GAPO13), and Glc-apo-13-carotenone isomers (GAPO13I, GAPO13II, and GAPO13III) significantly increase after high light (HL) treatment. This treatment also led to an obvious increase in the levels of most carotene- and all xanthophyll-derived apocarotenoids detected in our system. Our work demonstrates for the first time that HL stress induces apocarotenoid glycosylation in Arabidopsis and unravels a novel plant metabolic pathway that leads from carotene cleavage products to GAPOs that are identical to xanthophyll derived GAPOs. Thus, our new approach allows sensitive and reliable profiling of GAPOs, which is crucial for understanding the function of apocarotenoid glycosylation in plants and its role in the acclimation to HL stress.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

Characterization of a ß-carotene isomerase from the cyanobacterium Cyanobacteria aponinum.

Carotenoids are essential components of the photosynthetic apparatus and precursors of plant hormones, such as strigolactones (SLs). SLs are involved in various aspects of plant development and stress-response processes, including the establishment of root and shoot architecture. SL biosynthesis begins with the reversible isomerization of all-trans-carotene into 9-cis-ß-carotene, catalysed by DWARF27 ß-carotene isomerase (D27). Sequence comparisons have revealed the presence of D27-related proteins in photosynthetic eukaryotes and cyanobacteria lacking SLs. To gain insight into the evolution of SL biosynthesis, we characterized the activity of a cyanobacterial D27 protein (CaD27) from Cyanobacterim aponinum, using carotenoid-accumulating Escherichia coli cells and in vitro enzymatic assays. Our results demonstrate that CaD27 is an all-trans/cis and cis/cis-ß-carotene isomerase, with a cis/cis conversion preference. CaD27 catalysed 13-cis/15-cis-, all-trans/9-cis-ß-carotene, and neurosporene isomerization. Compared with plant enzymes, it exhibited a lower 9-cis-/all-trans-ß-carotene conversion ratio. A comprehensive genome survey revealed the presence of D27 as a single-copy gene in the genomes of 20 out of 200 cyanobacteria species analysed. Phylogenetic and enzymatic analysis of CaD27 indicated that cyanobacterial D27 genes form a single orthologous group, which is considered an ancestral type of those found in photosynthetic eukaryotes. This article is part of the theme issue 'The evolution of plant meta|bolism'.

Strigolactone biosynthesis in rice can occur via a 9-cis-3-OH-10'-apo-ß-carotenal intermediate.

Strigolactones (SLs) play a crucial role in regulating plant architecture and mediating rhizosphere interactions. They are synthesized from all-trans-ß-carotene converted into the intermediate carlactone (CL) via the intermediate 9-cis-ß-apo-10'-carotenal. Recent studies indicate that plants can also synthesize 3-OH-CL from all-trans-ß-zeaxanthin via the intermediate 9-cis-3-OH-ß-apo-10'-carotenal. However, the question of whether plants can form bioactive SLs from 9-cis-3-OH-ß-apo-10'-carotenal remains elusive. In this study, we supplied the 13 C-labeled 9-cis-3-OH-ß-apo-10'-carotenal to rice seedlings and monitored the synthesis of SLs using liquid chromatography-mass spectrometry (LC-MS) and Striga bioassay. We further validated the biological activity of 9-cis-3-OH-ß-apo-10'-carotenal-derived SLs using the ccd7/d17 SL-deficient mutant, which demonstrated increased Striga seed-germinating activity and partial rescue of tiller numbers and plant height. Our results establish 9-cis-3-OH-ß-apo-10'-carotenal as a significant SL biosynthetic intermediate with implications for understanding plant hormonal functions and potential applications in agriculture.

Chromosome-scale pearl millet genomes reveal CLAMT1b as key determinant of strigolactone pattern and Striga susceptibility.

The yield of pearl millet, a resilient cereal crop crucial for African food security, is severely impacted by the root parasitic weed Striga hermonthica, which requires host-released hormones, called strigolactones (SLs), for seed germination. Herein, we identify four SLs present in the Striga-susceptible line SOSAT-C88-P10 (P10) but absent in the resistant 29Aw (Aw). We generate chromosome-scale genome assemblies, including four gapless chromosomes for each line. The Striga-resistant Aw lacks a 0.7 Mb genome segment containing two putative CARLACTONOIC ACID METHYLTRANSFERASE1 (CLAMT1) genes, which may contribute to SL biosynthesis. Functional assays show that P10CLAMT1b produces the SL-biosynthesis intermediate methyl carlactonoate (MeCLA) and that MeCLA is the precursor of P10-specific SLs. Screening a diverse pearl millet panel confirms the pivotal role of the CLAMT1 section for SL diversity and Striga susceptibility. Our results reveal a reason for Striga susceptibility in pearl millet and pave the way for generating resistant lines through marker-assisted breeding or direct genetic modification.

Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets.

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including ß-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and ß-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, ß-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial ß-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of ß-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops.

Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system.

Cytochrome P450 enzymes (CYPs) are involved in metabolic steps that provide structural diversity during the biosynthesis of carotenoids and their oxidative cleavage products called apocarotenoids. Recent studies on bioactive apocarotenoids in plants revealed the necessity of performing further research to uncover the function of novel CYP enzymes that might be involved in apocarotenoid metabolism. We describe a series of in-vitro methods to characterize plant CYPs that metabolize apocarotenoids, using a specific Saccharomyces cerevisiae strain, WAT11, engineered to express a CYP redox partner, Arabidopsis thaliana NADPH-P450 reductase 1 (ATR1). This chapter provides protocols for construction and transformation of plasmids that express CYPs in yeast, isolation of yeast microsomes, and in-vitro enzymatic assays to validate the final metabolic products using LC-MS.

Protocol for characterizing strigolactones released by plant roots.

The plant hormone strigolactones (SLs) are secreted by plant roots to act as rhizospheric signals. Here, we present a protocol for characterizing plant-released SLs. We first outline all necessary steps required for collection, processing, and analysis of plant root exudates using the C18 column for SL extraction, followed by liquid chromatography-mass spectrometry (LC-MS) for SL quantification. We then describe image processing by SeedQuant, an open-source artificial-intelligence-based software, for measuring the biological activity of SLs in inducing root parasitic plant seed germination. For complete details on the use and execution of this protocol, please refer to Wang et al. (2019) and Braguy et al. (2021).

Perspectives on the metabolism of strigolactone rhizospheric signals.

Strigolactones (SLs) are a plant hormone regulating different processes in plant development and adjusting plant's architecture to nutrition availability. Moreover, SLs are released by plants to communicate with beneficial fungi in the rhizosphere where they are, however, abused as chemical cues inducing seed germination of root parasitic weeds, e.g. Striga spp., and guiding them towards host plants in their vicinity. Based on their structure, SLs are divided into canonical and non-canonical SLs. In this perspective, we describe the metabolism of root-released SLs and SL pattern in rice max1-900 mutants, which are affected in the biosynthesis of canonical SLs, and show the accumulation of two putative non-canonical SLs, CL+30 and CL+14. Using max1-900 and SL-deficient d17 rice mutants, we further investigated the metabolism of non-canonical SLs and their possible biological roles. Our results show that the presence and further metabolism of canonical and non-canonical SLs are particularly important for their role in rhizospheric interactions, such as that with root parasitic plants. Hence, we proposed that the root-released SLs are mainly responsible for rhizospheric communications and have low impact on plant architecture, which makes targeted manipulation of root-released SLs an option for rhizospheric engineering.

Ultra-high performance liquid chromatography-mass spectrometry analysis of plant apocarotenoids.

Apocarotenoids (APOs) are a class of carotenoid oxidation products with high structural and functional diversity. Apart from serving as precursors of phytohormones, fungal pheromones and vitamin A, several APOs act as signaling molecules involved in stress response and growth as regulators in plants. To comprehensively profile plant APOs, we established an improved ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap MS) analytical platform. The improved APO analytical platform consists of an optimized sequential APO sample preparation and multiple UHPLC-MS detection methods and was successfully used to identify and quantify multiple subclasses of APOs from tomato fruits. By integrating ultrasound-assisted extraction, solid phase extraction, and chemical derivatization, the improved sequential APOs sample preparation facilitates the simultaneous preparation of different subclasses of APOs from plant materials. In addition, multiple UHPLC-MS detection methods enables high throughput analysis of APOs. Application of this analytical strategy can make important contributions to understanding the function of these compounds and significantly facilitate the elucidation of plant APO metabolism.

An alternative, zeaxanthin epoxidase-independent abscisic acid biosynthetic pathway in plants.

Abscisic acid (ABA) is an important carotenoid-derived phytohormone that plays essential roles in plant response to biotic and abiotic stresses as well as in various physiological and developmental processes. In Arabidopsis, ABA biosynthesis starts with the epoxidation of zeaxanthin by the ABA DEFICIENT 1 (ABA1) enzyme, leading to epoxycarotenoids; e.g., violaxanthin. The oxidative cleavage of 9-cis-epoxycarotenoids, a key regulatory step catalyzed by 9-CIS-EPOXYCAROTENOID DIOXYGENASE, forms xanthoxin, which is converted in further reactions mediated by ABA DEFICIENT 2 (ABA2), ABA DEFICIENT 3 (ABA3), and ABSCISIC ALDEHYDE OXIDASE 3 (AAO3) into ABA. By combining genetic and biochemical approaches, we unravel here an ABA1-independent ABA biosynthetic pathway starting upstream of zeaxanthin. We identified the carotenoid cleavage products (i.e., apocarotenoids, ß-apo-11-carotenal, 9-cis-ß-apo-11-carotenal, 3-OH-ß-apo-11-carotenal, and 9-cis-3-OH-ß-apo-11-carotenal) as intermediates of this ABA1-independent ABA biosynthetic pathway. Using labeled compounds, we showed that ß-apo-11-carotenal, 9-cis-ß-apo-11-carotenal, and 3-OH-ß-apo-11-carotenal are successively converted into 9-cis-3-OH-ß-apo-11-carotenal, xanthoxin, and finally into ABA in both Arabidopsis and rice. When applied to Arabidopsis, these ß-apo-11-carotenoids exert ABA biological functions, such as maintaining seed dormancy and inducing the expression of ABA-responsive genes. Moreover, the transcriptomic analysis revealed a high overlap of differentially expressed genes regulated by ß-apo-11-carotenoids and ABA, suggesting that ß-apo-11-carotenoids exert ABA-independent regulatory activities. Taken together, our study identifies a biological function for the common plant metabolites, ß-apo-11-carotenoids, extends our knowledge about ABA biosynthesis, and provides new insights into plant apocarotenoid metabolic networks.

Canonical strigolactones are not the major determinant of tillering but important rhizospheric signals in rice.

Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga-resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed-germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.

A Method for Extraction and LC-MS-Based Identification of Carotenoid-Derived Dialdehydes in Plants.

We developed a chemical derivatization based ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap MS) analytical method to identify low-abundant and instable carotenoid-derived dialdehydes (DIALs, diapocarotenoids) from plants. Application of this method enhances the MS response signal of DIALs, enabling the detection of diapocarotenoids, which is crucial for understanding the function of these compounds and for elucidating the carotenoid oxidative metabolic pathway in plants.

A Highly Sensitive SPE Derivatization-UHPLC-MS Approach for Quantitative Profiling of Carotenoid-Derived Dialdehydes from Vegetables.

Oxidative cleavage of carotenoids leads to dialdehydes (diapocarotenoids, DIALs) in addition to the widely known apocarotenoids. DIALs are biologically active compounds that presumably impact human health and play different roles in plant development and carotenoid metabolism. However, detection of DIALs in plants is challenging due to their instability, low abundance, and poor ionization efficiency in mass spectrometry. Here, we developed a solid-phase extraction and derivatization protocol coupled with ultrahigh performance liquid chromatography-mass spectrometry for quantitative profiling of DIALs. Our method significantly enhances the sensitivity of DIAL detection with a detection limit of 0.05 pg/mg of dried food materials, allowing unambiguous profiling of 30 endogenous DIALs with C5 to C24 from vegetables. Our work provides a new and efficient approach for determining the content of DIALs from various complex matrices, paving the way for uncovering the functions of DIALs in human health and plant growth and development.