Carotenoid-derived bioactive metabolites shape plant root architecture to adapt to the rhizospheric environments.

Roots are important plant organs for the uptake of water and nutrient elements. Plant root development is finely regulated by endogenous signals and environmental cues, which shapes the root system architecture to optimize the plant growth and adapt to the rhizospheric environments. Carotenoids are precursors of plant hormones strigolactones (SLs) and ABA, as well as multiple bioactive molecules. Numerous studies have demonstrated SLs and ABA as essential regulators of plant root growth and development. In addition, a lot carotenoid-derived bioactive metabolites are recently identified as plant root growth regulators, such as anchorene, ß-cyclocitral, retinal and zaxinone. However, our knowledge on how these metabolites affect the root architecture to cope with various stressors and how they interact with each other during these processes is still quite limited. In the present review, we will briefly introduce the biosynthesis of carotenoid-derived root regulators and elaborate their biological functions on root development and architecture, focusing on their contribution to the rhizospheric environmental adaption of plants.

Screening for apocarotenoid plant growth regulators in Arabidopsis.

Apocarotenoids are bioactive metabolites found in animals, fungi and plants. Several carotenoid-derived compounds, apocarotenoids, were recently identified as new growth regulators in different plant species. Here, we introduce basic chemical screening methods, using a model plant, Arabidopsis thaliana, to elucidate the function of bioactive apocarotenoids in determining plant phenotypic traits. These short guidelines include essential practices, such as selecting the plant growth conditions and the type of treatment, as well as phenotyping methodologies for the initial screening of novel apocarotenoid plant growth regulators.

Identification and Characterization of Cinnamyl Alcohol Dehydrogenase Encoding Genes Involved in Lignin Biosynthesis and Resistance to Verticillium dahliae in Upland Cotton (Gossypium hirsutum L.).

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is one of the most devastating diseases in cotton (Gossypium spp.). Lignin in the cell wall forms a physical barrier to inhibit pathogen invasion, and defense-induced lignification reinforces secondary cell wall to prevent pathogens from further spreading. Cinnamyl alcohol dehydrogenases (CADs) catalyze the production of three main monolignols, p-coumaryl- (H), coniferyl- (G), and sinapyl-alcohols (S), which are the fundamental blocks of lignin. Here, we identified CAD genes in G. hirsutum, analyzed their expression profiles in cotton leaf, stem, and root from different developmental stages, and selected GhCAD35, GhCAD45, and GhCAD43, which were consistently induced by V. dahliae inoculation in G. hirsutum cultivars resistant or susceptible to V. dahliae. On the basis of confirmation of the in vitro enzymatic activity of the three proteins in generation of the three monolignols, we used virus-induced gene silencing (VIGS) to investigate the effects of silencing of GhCAD35, GhCAD45, or GhCAD43 on resistance to V. dahliae as well as on deposition and the composition of lignin. Silencing each of the three CADs impaired the defense-induced lignification and salicylic acid biosynthesis in stem, and compromised resistance to V. dahliae. Moreover, our study showed that silencing the three GhCADs severely affected the biosynthesis of S-lignin, leading to a decrease of the syringyl/guaiacyl (S/G) ratio. Heterogeneous overexpression of GhCAD35, GhCAD45, or GhCAD43 in Arabidopsis enhanced disease resistance. Taken together, our study demonstrates a role of the three GhCADs in defense-induced lignin biosynthesis and resistance to V. dahliae in G. hirsutum.

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.

Genome-wide identification, characterization and expression profiles of the CCD gene family in Gossypium species.

Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the selective oxidative cleavage steps from carotenoids to apocarotenoids, which are essential for the synthesis of biologically important molecules such as retinoids, and the phytohormones abscisic acid (ABA) and strigolactones. In addition, CCDs play important roles in plant biotic and abiotic stress responses. Till now, a comprehensive characterization of the CCD gene family in the economically important crop cotton (Gossypium spp.) is still missing. Here, we performed a genome-wide analysis and identified 33, 31, 16 and 15 CCD genes from two allotetraploid Gossypium species, G. hirsutum and G. barbadense, and two diploid Gossypium species, G. arboreum and G. raimondii, respectively. According to the phylogenetic tree analysis, cotton CCDs are classified as six subgroups including CCD1, CCD4, CCD7, CCD8, nine-cis-epoxycarotenoid dioxygenase (NCED) and zaxinone synthase (ZAS) sub-families. Evolutionary analysis shows that purifying selection dominated the evolution of these genes in G. hirsutum and G. barbadense. Predicted cis-acting elements in 2 kb promoters of CCDs in G. hirsutum are mainly involved in light, stress and hormone responses. The transcriptomic analysis of GhCCDs showed that different GhCCDs displayed diverse expression patterns and were ubiquitously expressed in most tissues; moreover, GhCCDs displayed specific inductions by different abiotic stresses. Quantitative reverse-transcriptional PCR (qRT-PCR) confirmed the induction of GhCCDs by heat stress, salinity, polyethylene glycol (PEG) and ABA application. In summary, the bioinformatics and expression analysis of CCD gene family provide evidence for the involvement in regulating abiotic stresses and useful information for in-depth studies of their biological functions in G. hirsutum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02805-9.

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.

The Apocarotenoid Zaxinone Is a Positive Regulator of Strigolactone and Abscisic Acid Biosynthesis in Arabidopsis Roots.

Carotenoids are ubiquitous precursors of important metabolites including hormones, such as strigolactones (SLs) and abscisic acid (ABA), and signaling and regulatory molecules, such as the recently discovered zaxinone. Strigolactones and ABA are key regulators of plant growth and development, adaptation to environmental changes and response to biotic and abiotic stress. Previously, we have shown that zaxinone, an apocarotenoid produced in rice by the enzyme zaxinone synthase (ZAS) that is common in mycorrhizal plants, is required for normal rice growth and development, and a negative regulator of SL biosynthesis. Zaxinone is also formed in Arabidopsis, which lacks ZAS, via an unknown route. In the present study, we investigated the biological activity of zaxinone in Arabidopsis, focusing on its effect on SL and ABA biosynthesis. For this purpose, we quantified the content of both hormones and determined the levels of related transcripts in Arabidopsis (Arabidopsis thaliana), roots upon zaxinone treatment. For SL quantification, we also employed Striga seed germination bioassay. Results obtained show that zaxinone application to hydroponically grown Arabidopsis seedlings enhanced transcript levels of key biosynthetic genes of both hormones, led to higher root ABA and SL (methyl carlactonoate, MeCLA) content, and increased SL release, even under sufficient phosphate supply. Using the SL insensitive (max2-1) and the ABA deficient (aba1-6, aba2-1, and nced3) mutants, we also show that zaxinone application reduced hypocotyl growth and that this effect is caused by increasing ABA content. Our results suggest that zaxinone is a regulatory metabolite also in Arabidopsis, which triggers the biosynthesis of both carotenoid-derived hormones, SLs and ABA, in roots. In the non-mycotrophic plant Arabidopsis, zaxinone does not increase growth and may be perceived as a stress signal, while it acts as a growth-promoting metabolite and suppressor of SL biosynthesis in rice.

A New Series of Carlactonoic Acid Based Strigolactone Analogs for Fundamental and Applied Research.

Strigolactones (SLs) are a group of carotenoid derived plant hormones that play a key role in establishing plant architecture and adapting it to environmental changes, and are involved in plants response to biotic and abiotic stress. SLs are also released into the soil to serve as a chemical signal attracting beneficial mycorrhizal fungi. However, this signal also induces seed germination in root parasitic weeds that represent a major global threat for agriculture. This wide spectrum of biological functions has made SL research one of the most important current topics in fundamental and applied plant science. The availability of SLs is crucial for investigating SL biology as well as for agricultural application. However, natural SLs are produced in very low amounts, and their organic synthesis is quite difficult, which creates a need for efficient and easy-to-synthesize analogs and mimics. Recently, we have generated a set of SL analogs, Methyl Phenlactonoates (MPs), which resemble the non-canonical SL carlactonoic acid. In this paper, we describe the development and characterization of a new series of easy-to-synthesize MPs. The new analogs were assessed with respect to regulation of shoot branching, impact on leaf senescence, and induction of seed germination in different root parasitic plants species. Some of the new analogs showed higher efficiency in inhibiting shoot branching as well as in triggering parasitic seed germination, compared to the commonly used GR24. MP16 was the most outstanding analog showing high activity in different SL biological functions. In summary, our new analogs series contains very promising candidates for different applications, which include the usage in studies for understanding different aspects of SL biology as well as large scale field application for combating root parasitic weeds, such as Striga hermonthica that devastates cereal yields in sub-Saharan Africa.

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.

Anchorene is a carotenoid-derived regulatory metabolite required for anchor root formation in Arabidopsis.

Anchor roots (ANRs) arise at the root-shoot junction and are the least investigated type of Arabidopsis root. Here, we show that ANRs originate from pericycle cells in an auxin-dependent manner and a carotenogenic signal to emerge. By screening known and assumed carotenoid derivatives, we identified anchorene, a presumed carotenoid-derived dialdehyde (diapocarotenoid), as the specific signal needed for ANR formation. We demonstrate that anchorene is an Arabidopsis metabolite and that its exogenous application rescues the ANR phenotype in carotenoid-deficient plants and promotes the growth of normal seedlings. Nitrogen deficiency resulted in enhanced anchorene content and an increased number of ANRs, suggesting a role of this nutrient in determining anchorene content and ANR formation. Transcriptome analysis and treatment of auxin reporter lines indicate that anchorene triggers ANR formation by modulating auxin homeostasis. Together, our work reveals a growth regulator with potential application to agriculture and a new carotenoid-derived signaling molecule.

ß-Cyclocitral is a conserved root growth regulator.

Natural compounds capable of increasing root depth and branching are desirable tools for enhancing stress tolerance in crops. We devised a sensitized screen to identify natural metabolites capable of regulating root traits in Arabidopsis ß-Cyclocitral, an endogenous root compound, was found to promote cell divisions in root meristems and stimulate lateral root branching. ß-Cyclocitral rescued meristematic cell divisions in ccd1ccd4 biosynthesis mutants, and ß-cyclocitral-driven root growth was found to be independent of auxin, brassinosteroid, and reactive oxygen species signaling pathways. ß-Cyclocitral had a conserved effect on root growth in tomato and rice and generated significantly more compact crown root systems in rice. Moreover, ß-cyclocitral treatment enhanced plant vigor in rice plants exposed to salt-contaminated soil. These results indicate that ß-cyclocitral is a broadly effective root growth promoter in both monocots and eudicots and could be a valuable tool to enhance crop vigor under environmental stress.

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.

Methylation at the C-3' in D-Ring of Strigolactone Analogs Reduces Biological Activity in Root Parasitic Plants and Rice.

Strigolactones (SLs) regulate plant development and induce seed germination in obligate root parasitic weeds, e.g. Striga spp. Because organic synthesis of natural SLs is laborious, there is a large need for easy-to-synthesize and efficient analogs. Here, we investigated the effect of a structural modification of the D-ring, a conserved structural element in SLs. We synthesized and investigated the activity of two analogs, MP13 and MP26, which differ from previously published AR8 and AR36 only in the absence of methylation at C-3'. The de-methylated MP13 and MP26 were much more efficient in regulating plant development and inducing Striga seed germination, compared with AR8. Hydrolysis assays performed with purified Striga SL receptor and docking of AR8 and MP13 to the corresponding active site confirmed and explained the higher activity. Field trials performed in a naturally Striga-infested African farmer's field unraveled MP13 as a promising candidate for combating Striga by inducing germination in host's absence. Our findings demonstrate that methylation of the C-3' in D-ring in SL analogs has a negative impact on their activity and identify MP13 and, particularly, MP26 as potent SL analogs with simple structures, which can be employed to control Striga, a major threat to global food security.

The apocarotenoid metabolite zaxinone regulates growth and strigolactone biosynthesis in rice.

Carotenoid cleavage dioxygenases (CCDs) form hormones and signaling molecules. Here we show that a member of an overlooked plant CCD subfamily from rice, that we name Zaxinone Synthase (ZAS), can produce zaxinone, a novel apocarotenoid metabolite in vitro. Loss-of-function mutants (zas) contain less zaxinone, exhibit retarded growth and showed elevated levels of strigolactones (SLs), a hormone that determines plant architecture, mediates mycorrhization and facilitates infestation by root parasitic weeds, such as Striga spp. Application of zaxinone can rescue zas phenotypes, decrease SL content and release and promote root growth in wild-type seedlings. In conclusion, we show that zaxinone is a key regulator of rice development and biotic interactions and has potential for increasing crop growth and combating Striga, a severe threat to global food security.

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.

The Arabidopsis DWARF27 gene encodes an all-trans-/9-cis-ß-carotene isomerase and is induced by auxin, abscisic acid and phosphate deficiency.

Strigolactones (SLs) are carotenoid-derived plant hormones that influence various aspects of plant growth and development in response to environmental conditions, especially nutrients deficiency. SLs are synthesized via a strict stereo-specific core pathway that leads to the intermediate carlactone, requiring the iron-containing polypeptide DWARF27 (D27) and the carotenoid cleavage dioxygenases 7 (CCD7) and 8 (CCD8). It has been shown that the rice OsD27 is a ß-carotene isomerase catalyzing the interconversion of all-trans- into 9-cis-ß -carotene. However, data about the enzymatic activity of D27 from other species are missing. Here, we investigated the activity and substrate specificity of the Arabidopsis AtD27 by testing a broad range of carotenoid substrates. Both in vivo and in vitro assays show that AtD27 catalyzes the reverse isomerization of all-trans-/9-cis-ß-carotene. AtD27 did not isomerize 13-cis- or 15-cis-ß-carotene, indicating high specificity for the C9-C10 double bond. The isomerization reaction was inhibited in the presence of silver acetate, pointing to the involvement of an iron-sulfur cluster. We further investigated the expression of AtD27, using Arabidopsis transgenic lines expressing ß-glucuronidase (GUS) under the control of AtD27 native promoter. AtD27 is ubiquitously expressed throughout the plant with the highest expression in immature flowers. In lateral roots, AtD27 expression was induced by treatment with auxin and ABA, while the application of SL analogs did not show an effect. Lower ABA levels in atd27 mutant indicated an interference with the ABA pathway. Quantitative real-time RT-PCR showed that transcript levels of AtD27 and other SL biosynthetic genes in roots are induced upon phosphate starvation. Taken together, our study on AtD27 confirms the postulated enzymatic function of this enzyme, shows its strict substrate- and regio-specificity and indicates an important role in response to multiple plant hormones and phosphate deficiency.

A rapid LC-MS method for qualitative and quantitative profiling of plant apocarotenoids.

Carotenoid cleavage products (apocarotenoids; APOs) exert important biological functions in light perception and as vitamin A source, signaling molecules, hormone precursors, pigments and volatiles. However, an analytical method that allows simultaneous profiling of these diverse compounds is still missing. We developed an efficient method to analyze APOs present in plant tissues, which is based on ultra-high performance liquid chromatographic separation and high-resolution hybrid quadrupole-Orbitrap (Q-Orbitrap) mass spectrometry (MS). Our approach allowed unambiguous identification and quantification of volatile and non-volatile APOs in a single run. Modified sample preparation and optimized ultra-high performance liquid chromatography (UHPLC)-MS parameters permitted the measurement of APOs in Oryza sativa seedlings and Spinacia oleracea leaves, unraveling 20 endogenous APOs with chain lengths ranging from C10 to C30, confirmed by high-resolution MS, MS/MS data and using synthetic standards. Our experimentation demonstrates that the usage of methanol with 0.1% butylated hydroxytoluene facilitates the extraction of both short-chain and long-chain APOs from plant materials. In addition, our validated analytical method allows the quantitative analysis of APOs with a wide content range from 2.5 pg/mg to 10 ng/mg dried weight. The adoption of the analytical protocol, as described in this study, realizes the measurement of volatile APOs by using a LC-MS method, hence, allowing informative and reliable profiling of APOs, which is important for determining the content of these compounds in food and crucial for understanding their function and metabolism in plants.

Methyl phenlactonoates are efficient strigolactone analogs with simple structure.

Strigolactones (SLs) are a new class of phytohormones that also act as germination stimulants for root parasitic plants, such as Striga spp., and as branching factors for symbiotic arbuscular mycorrhizal fungi. Sources for natural SLs are very limited. Hence, efficient and simple SL analogs are needed for elucidating SL-related biological processes as well as for agricultural applications. Based on the structure of the non-canonical SL methyl carlactonoate, we developed a new, easy to synthesize series of analogs, termed methyl phenlactonoates (MPs), evaluated their efficacy in exerting different SL functions, and determined their affinity for SL receptors from rice and Striga hermonthica. Most of the MPs showed considerable activity in regulating plant architecture, triggering leaf senescence, and inducing parasitic seed germination. Moreover, some MPs outperformed GR24, a widely used SL analog with a complex structure, in exerting particular SL functions, such as modulating Arabidopsis roots architecture and inhibiting rice tillering. Thus, MPs will help in elucidating the functions of SLs and are promising candidates for agricultural applications. Moreover, MPs demonstrate that slight structural modifications clearly impact the efficiency in exerting particular SL functions, indicating that structural diversity of natural SLs may mirror a functional specificity.