Carotenoids are precursors of hormones and signaling molecules across all kingdoms of life. An increasing body of evidence suggests the presence of yet unidentified carotenoid-derived metabolites (apocarotenoids) with developmental and regulatory functions, besides the known plant hormones abscisic acid (ABA) and strigolactones (SLs). Generally, apocarotenoid synthesis is initiated by carotenoid cleavage dioxygenases (CCDs), which constitute a ubiquitous family of non-heme iron enzymes. In SL biosynthesis, an iron-binding cis/trans-isomerase, DWARF 27 (D27) converts all-trans-β-carotene into 9-cis-β-carotene. This reaction is followed by a double bond cleavage at 9, 10 position, mediated by the stereospecific CCD7. The cis-configured cleavage product of CCD7, 9-cis-β-apo-10’-carotenal, is simultaneously cleaved, triple-oxygenated and rearranged by CCD8, to produce carlactone (CL). CL is a central metabolite and the precursor of a wide range of SLs. The aim of this work is to investigate whether CCD8 synthesize CL-like compounds from other 9-cis-configured apocarotenoids to confirm their presence and synthesis in planta. We showed that CCD8 enzymes from different plants produce a hydroxylated carlactone (3-H-CL) from 9-cis-3-OH-β-apo-10’-carotenal in vitro. In addition, we detected 3-H-CL in Nicotiana benthamiana leaves transiently expressing the CL biosynthesis enzymes from rice and Arabidopsis. 3-H-CL is biologically active, as shown by Striga hermonthica seed germination assay and by its effect on the high-tillering phenotype of the rice d10 mutant. We also confirmed that 3-H-CL is a natural metabolite by detecting it in roots of the rice SL perception mutant d14. In a second project, we investigated the activity of three rice CCDs in vitro and showed that one of them (zaxinone synthase; ZAS) is an apocarotenoid cleavage enzyme with a clear preference for the substrate all-trans-3-OH-β-apo-10’-carotenal, as suggested by a kinetic study. ZAS produces two products, the C18 ketone zaxinone and an unstable C9 dialdehyde that could be identified by LC-MS after derivatization. Activity tests were performed with crude lysates of overexpressing Escherichia coli cells and with purified enzyme. We established that zaxinone is a natural metabolite present in planta. Investigations of a corresponding rice mutant (zas) and activity bioassays performed by our group demonstrate that zaxinone a novel signaling molecule required for normal rice growth and development.

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Carotenoids are the precursor of several metabolites with regulatory functions, which include the plant hormones abscisic acid (ABA) and strigolactones (SLs), and signaling molecules, such as β-cyclocitral. These carotenoid-derivatives originate from oxidative breakdown of the double bond resulting in carbonyl cleavage-products designated as apocarotenoids. The cleavage reaction causing apocarotenoid formation is catalyzed frequently by Carotenoid Cleavage Dioxygenases (CCDs). Several lines of evidence indicate the presence of yet unidentified apocarotenoids with regulatory or signaling function. Here, we first characterized the biological functions of the apocarotenoid zaxinone formed by ZAXINONE SYNTHASE (ZAS), a member of an overlooked, widely distributed plant CCD clade. The loss-of-function rice zas mutant contains less zaxinone, exhibiting retarded growth with elevated levels of SLs that determines plant architecture, mediates mycorrhization, and facilitates the germination of root parasitic seeds, such as Striga spp. The zaxinone treatment rescued zas phenotypes, down-regulated SL biosynthesis and release, and enhanced root growth in the wild-type rice seedlings. Next, we performed multi-omics analysis demonstrating zaxinone increased sugar metabolism and induced photosynthesis in a manner that led to phenotypical changes in rice roots. Besides, transcriptome analysis showed that zaxinone upregulated CYTOKININ GLUCOSYLTRANSFERASES expression in roots, which might explain the increase in the apex and meristem length, and in the number of cellular layers. Finally, the investigation of zaxinone biology and the utilization of its application is constrained by its laborious organic synthesis and low abundance in natural sources. Therefore, we developed easy-to-synthesize and highly efficient Mimic of Zaxinone (MiZax), based on the structure-activity-relationship study using a series of apocarotenoids. Activity-based experiments unraveled MiZax3 and MiZax5 were at least as active as zaxinone in rescuing root phenotypes of the zas mutant, promoting root growth in wild-type seedlings, and reducing SL biosynthesis and release. Taken together, zaxinone is a key regulator of rice growth and development, which regulates sugar metabolism, suppresses SL biosynthesis, fine-tunes cytokinins level, and modulates biotic interactions with arbuscular mycorrhizal (AM) fungi. Our work also provides easy-to-synthesize mimics for illuminating zaxinone biology and as a tool to improve crop growth and reduce the infestation by Striga hermonthica, a severe threat to food security worldwide.

The necrotrophic fungus Botrytis cinerea (B. cinerea) is one of the most aggressive and widespread pathogenic fungi, causing the gray mold disease in over 200 different plants species and severely impacting the quality of harvested fruits. Due to B. cinerea resistance to common synthetic fungicides and their ecological impact, there is a large demand for novel control approaches, such as bio-fungicides. The family of carotenoid-cleavage products, i.e. apocarotenoids, includes important compounds, such as hormones, signaling molecules and growth regulators. Previous studies indicated that the apocarotenoid B-ionone inhibits the growth of some fungal species. In this thesis, we unravel a novel role for B-ionone as a plant regulatory metabolite increasing the resistance against B. cinerea in several plant models. We achieved this by combining phenotypic, transcriptomic, and metabolomic analysis. We show that pretreatment of Arabidopsis plants with B-ionone significantly alleviated the symptoms of B. cinerea infection, modulated hormone homeostasis and affected the interactions between jasmonic acid (JA)/ethylene (ET) and abscisic acid (ABA) hormone signaling pathways β-ionone treatment stimulated JA/ET signaling pathways and repressed the synthesis of ABA upon B. cinerea infection, which reduced the susceptibility of Arabidopsis plants to B. cinerea. To get an overview on the effect of β-ionone on plants at transcript level, we performed an RNA-seq experiment that supported our hypothesis that B-ionone primes and enhances the Arabidopsis immune response to B. cinerea infection and raveled the effect of this volatile on the expression of several transcription factors involved in Arabidopsis immune response to B. cinerea and on transcripts related to cell wall biosynthesis, Map Kinase 3 signaling and hypoxia tolerance. Further experiments performed with transgenic tobacco and tomato plants confirmed the positive role of β-ionone in reducing the symptoms of B. cinerea in green tissues as well as in tomato fruits. The discovery of β-ionone as a new apocarotenoid signaling molecule that regulates plant hormone homeostasis opens up new possibility to control B. cinerea infection and to establish this natural volatile as an environmentally friendly and safe bio-fungicide.

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Tesis por compendio / [ES] La biotecnología de plantas actual, y la llamada agricultura molecular aspiran a convertir las plantas en "biofábricas" sostenibles para producir compuestos de valor como proteínas, metabolitos o nanopartículas de interés farmacéutico o industrial. Los virus de plantas constituyen una de las principales causas de enfermedades vegetales. Son capaces de secuestrar la maquinaria celular del huésped, y de ahí surgió la idea de reconvertir los virus de plantas en herramientas para la biotecnología de plantas como vectores de expresión transitoria y andamios para nanomateriales. Los carotenoides son metabolitos relevantes debido a sus propiedades nutricionales y beneficiosas para la salud. El primer objetivo fue manipular la ruta de biosíntesis de carotenoides para producir los muy apreciados apocarotenoides de azafrán, siendo estos los productos de la escisión de carotenoides. Para ello, se diseñó un vector derivado del virus del grabado del tabaco (TEV; género Potyvirus) manipulado para expresar unas enzimas específicas, dioxigenasas de escisión de carotenoides (CCD) de Crocus sativus y Buddleja davidii. Los análisis metabólicos de los tejidos infectados demostraron que, tras sólo dos semanas, se alcanzaron cantidades notables de crocinas y picrocrocina en plantas adultas de Nicotiana benthamiana. Sólo la expresión de CsCCD2L de C. sativus dio una acumulación en hoja de 0.2% de crocinas y 0.8% de picrocrocina en peso seco. La coexpresión de CsCCD2L con otra enzima carotenogénica, como la fitoeno sintasa de Pantoea ananatis (PaCrtB), usando el mismo vector aumentó la acumulación de crocinas al 0.35%. Pese a ser cantidades inferiores a las encontradas en fuentes naturales, este sistema mediado por virus representa el primer sistema heterólogo capaz de producir crocinas. Los compuestos fenólicos son otro amplio grupo de metabolitos secundarios en plantas muy apreciados también. Los curcuminoides son polifenoles con alta actividad antioxidante que se encuentran naturalmente en el rizoma de la cúrcuma (Curcuma longa). El segundo objetivo fue establecer un sistema para la producción heteróloga de curcuminoides utilizando vectores virales. Para ello, se desarrolló un sistema viral doble, basado en TEV y en el virus X de la patata (PVX; género Potexvirus), capaz de coexpresar diferentes enzimas biosintéticas en las mismas células. Este sistema se usó para expresar la dicétido-CoA sintasa 1 (DCS1) y la curcumina sintasa 3 (CURS3) de C. longa en plantas de N. benthamiana. El análisis metabólico confirmó la producción exitosa de curcuminoides usando dos vectores virales. Posteriormente se analizó la coexpresión de DCS1 y CURS3 usando un solo vector viral derivado de TEV, obteniendo una producción más eficiente, aumentando al doble la acumulación de curcumina. Tras un análisis temporal usando el vector TEVΔN-DCS1-CURS3, se vio que a los 11 días se lograba una acumulación máxima de 22 ± 4 µg/g peso seco. Las nanopartículas virales (VNP) también han atraído la atención en biotecnología por su uso potencial como componentes básicos para nuevos materiales en nanotecnología y medicina. Los nanoanticuerpos son los dominios variables de los anticuerpos de camélidos de sólo cadena pesada (VHH) que han ganado interés como moléculas terapéuticas por su estructura simple, tamaño pequeño y alta especificidad. El último objetivo de este trabajo fue producir VNPs decoradas con un nanoanticuerpo codificadas genéticamente. El virus del mosaico amarillo del calabacín (ZYMV; género Potyvirus) y TEV se utilizaron como andamios para producir VNPs decoradas con un nanoanticuerpo contra la proteína verde fluorescente en plantas de calabacín y N. benthamiana, respectivamente. Confirmándose el ensamblaje y unión de ambas VNPs contra GFP. En conjunto, el trabajo presentado en esta tesis contribuye al concepto de que los virus de plantas, convenientemente manipulados, pueden convertirse en poderosas herramientas en biotecnología vegetal y agricultura molecular. / [CA] La biotecnologia de plantes actual i la anomenada agricultura molecular aspiren a convertir les plantes en "biofàbriques" sostenibles per a produir compostos de valor com a proteïnes, metabòlits o nanopartícules d'interès farmacèutic o industrial. Els virus de plantes constitueixen una de les principals causes de malalties vegetals. Son capaços de segrestar la maquinària cel·lular de l'hoste, i d'ací va sorgir la idea de reconvertir els virus en eines per la biotecnologia de plantes com a vectors d'expressió transitòria i bastides per a nanomaterials. Els carotenoides són metabòlits rellevants a causa de les seues propietats nutricionals i beneficioses per a la salut. El primer objectiu va ser manipular la ruta de biosíntesi de carotenoides per a produir els valuosos apocarotenoides de safrà, sent aquests els productes de l'escissió de carotenoides. Per a això, es va dissenyar un vector derivat del virus del gravat del tabac (TEV; gènere Potyvirus) manipulat per a expressar uns enzims específics, dioxigenases d'escissió de carotenoides (CCD) de Crocus sativus i Buddleja davidii. Les anàlisis metabòliques dels teixits infectats van demostrar que, després de només dues setmanes, es van aconseguir quantitats notables de crocines i picrocrocina en plantes adultes de Nicotiana benthamiana. Només l'expressió de CsCCD2L de C. sativus va donar com a resultat una acumulació en fulla de 0.2% de crocines i 0.8% de picrocrocina en pes sec. La coexpressió de CsCCD2L amb un altre enzim carotenogènic, com la fitoé sintasa de Pantoea ananatis (PaCrtB), usant el mateix vector viral va augmentar l'acumulació de crocines al 0.35%. Malgrat ser quantitats inferiors a les trobades en fonts naturals, aquest sistema mediat per virus representa el primer sistema heteròleg capaç de produir crocines. Els compostos fenòlics són un altre ampli grup de metabòlits secundaris en plantes, també molt valuosos. Els curcuminoides són polifenols amb alta activitat antioxidant que es troben naturalment en el rizoma de la cúrcuma (Curcuma longa). El segon objectiu va ser establir un sistema per a la producció heteròloga de curcuminoides utilitzant vectors virals. Per a això, es va desenvolupar un sistema viral doble, basat en TEV i en el virus X de la creïlla (PVX; gènere Potexvirus, família Alphaflexiviridae), capaç de coexpressar diferents enzims biosintètics en les mateixes cèl·lules. Aquest sistema es va usar per a expressar la dicétid-CoA sintasa 1 (DCS1) i la curcumina sintasa 3 (CURS3) de C. longa en plantes de N. benthamiana. L'anàlisi metabòlica va confirmar la producció reeixida de curcuminoides. Posteriorment es va analitzar la coexpresió de DCS1 i CURS3 usant un sol vector viral derivat de TEV, obtenint una producció més eficient, augmentant al doble l'acumulació de curcumina. Una anàlisi temporal usant el vector TEVΔN-DCS1-CURS3 va mostrar que als 11 dies s'aconseguia una acumulació màxima de 22 ± 4 µg/g pes sec. Les nanopartícules virals (VNP) també han atret l'atenció en biotecnologia pel seu ús potencial com a components bàsics per a nous materials en nanotecnologia i medicina. Els nanoanticossos són els dominis variables dels anticossos de camèlids de només cadena pesada (VHH) que han guanyat interès com a molècules terapèutiques per la seua estructura simple, grandària xicoteta i alta especificitat. L'últim objectiu d'aquest treball va ser produir VNPs decorades amb un nanoanticos codificades genèticament. El virus del mosaic groc de la carabasseta (ZYMV; gènere Potyvirus) i TEV es van utilitzar com a bastides per a produir VNPs decorades amb un nanocos contra la proteïna verda fluorescent en plantes de carabasseta i N. benthamiana, respectivamente. Confirmant-se l'assemblatge i unió de les VNPs contra GFP. En conjunt, el treball presentat en aquesta tesi contribueix al concepte que els virus de plantes, convenientment manipulats, poden convertir-se en poderoses eines en biotecnologia vegetal i agricultura molecular. / [EN] Modern plant biotechnology and molecular farming aim to convert plants into sustainable 'biofactories' to produce valuable compounds as proteins, metabolites or nanoparticles of pharmaceutical or industrial interest. Plant viruses, constitute a major cause of plant diseases inducing devastating crop losses. Based on their ability to hijack the host cell machinery, it arose the idea of repurposing plant viruses from foes to friends into tools for plant biotechnology as transient expression vectors and scaffolds for nanomaterials. Carotenoids are relevant metabolites based on their nutritional and health-promoting properties. The first goal of this work was to manipulate the carotenoid biosynthesis pathway to produce highly appreciated saffron apocarotenoids. For this purpose, a vector derived from Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) was engineered to express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that, after only two weeks, remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants were reached. The sole virus-driven expression of C. sativus CsCCD2L resulted in an accumulation of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight (DW). Co-expression of CsCCD2L with another carotenogenic enzyme, such as Pantoea ananatis phytoene synthase (PaCrtB), using the same viral vector increased crocin accumulation to 0.35%. Although these amounts are still far from those accumulating in natural sources, such as saffron stigma, this virus-driven system represents the first heterologous system able to produce crocins. Phenolic compounds represent another broad group of plant secondary metabolites highly appreciated for their health promoting properties. Curcuminoids are polyphenols with high antioxidant activity that are naturally found in turmeric (Curcuma longa) rhizome. The second goal of this work was to establish a system for the heterologous production of curcuminoids using viral vectors. To this aim, a double-virus vector system, based on TEV and Potato virus X (PVX; genus Potexvirus, family Alphaflexiviridae), able to co-express different biosynthetic enzymes in the same cells was developed. This system was used to express C. longa diketide-CoA synthase 1 (DCS1) and curcumin synthase 3 (CURS3) in N. benthamiana plants. Metabolic analysis confirmed the successful production of curcuminoids. Curcumin quantification indicated that sequential inoculation of both viral vectors was more efficient than co-inoculation. Co-expression of DCS1 and CURS3 was next analysed using a single viral vector derived from TEV (TEVΔN-DCS1-CURS3). This resulted in a more efficient approach as it led to a 2-fold increase in curcumin accumulation (11.7 ± 1.5 µg/g DW). A time-course analysis using the TEVΔN-DCS1-CURS3 vector showed that a maximum accumulation of 22 ± 4 µg/g DW was achieved at 11 days post-inoculation. Viral nanoparticles (VNPs) have also attracted attention in biotechnology for their potential use as building blocks for novel materials in nanotechnology and medicine. Nanobodies are the variable domains of heavy-chain (VHH) camelid antibodies that have sparked interest as therapeutic molecules due to their simple structure, small size and high specificity. The last goal of this work was to produce genetically encoded VNPs decorated with a nanobody. Zucchini yellow mosaic virus (ZYMV; genus Potyvirus, family Potyviridae) and TEV were used as scaffolds to produce VNPs decorated with a nanobody against the green fluorescent protein in zucchini (Cucurbita pepo) and N. benthamiana plants, respectively. Assembly and binding functionality of both VNPs against GFP was confirmed. Altogether, the work presented in this thesis contribute to the concept that plant viruses, conveniently manipulated, can turn into powerful tools in plant biotechnology and molecular farming. / This work was supported by grants BIO2016-77000-R, PID2020-114691RB-I00 and BIO2017-83184-R from the Spanish Ministerio de Ciencia, Innovación y Universidades (co-financed European Union FEDER funds). M.M. was the recipient of a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/05294). / Martí Botella, MC. (2022). Plant-Based Production of Metabolites and Nanoparticles Using Potyvirus Vectors [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/187155 / TESIS / Compendio

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