RESUMEN

Phytoene desaturase (PDS) is a key rate-limiting enzyme in the carotenoid biosynthesis pathway. Although commercial PDS inhibitors have been developed for decades, it remains necessary to develop novel PDS inhibitors with higher bioactivity. In this work, we used the scaffold hopping and linker modification approaches to design and synthesize a series of compounds (7a-7o, 8a-8l, and 14a-14d). The postemergence application assay demonstrated that 8e and 7e separately showed the best herbicidal activity at 750 g a.i./ha and lower doses (187.5 g, 375g a.i./ha) without no significant toxicity to maize and wheat. The surface plasmon resonance revealed strong binding affinity between 7e and Synechococcus PDS (SynPDS). The HPLC analysis confirmed that 8e at 750 g a.i./ha caused significant phytoene accumulation in Arabidopsis seedlings. This work demonstrates the efficacy of structure-guided optimization through scaffold hopping and linker modification to design potent PDS inhibitors with enhanced bioactivity and crop safety.

Asunto(s)

Inhibidores Enzimáticos , Herbicidas , Oxidorreductasas , Zea mays , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/antagonistas & inhibidores , Herbicidas/farmacología , Herbicidas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Zea mays/química , Relación Estructura-Actividad , Arabidopsis/enzimología , Arabidopsis/efectos de los fármacos , Arabidopsis/química , Arabidopsis/metabolismo , Triticum/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/antagonistas & inhibidores , Estructura Molecular , Triazoles/química , Triazoles/farmacología

RESUMEN

AIMS: Carotenoids are a class of hydrophobic substances that are important as food and feed colorants and as antioxidants. The pathway for ß-carotene synthesis has been expressed in various yeast species, albeit with rather low yields and titers. The inefficient conversion of phytoene to lycopene is often regarded as a bottleneck in the pathway. In this study, we aimed at the improvement of ß-carotene production in Saccharomyces cerevisiae by specifically engineering the enzymatic reactions producing and converting phytoene. METHODS AND RESULTS: We show that phytoene is stored in intracellular lipid droplets, whereas the enzyme responsible for its conversion, phytoene dehydrogenase, CrtI, is located at the endoplasmic reticulum, like the bifunctional enzyme CrtYB that catalyses the reaction before and after CrtI. To improve the accessibility of phytoene for CrtI and to delay its storage in lipid droplets, we tested the relocation of CrtI and CrtYB to mitochondria. However, only the retargeting of CrtYB resulted in an improvement of the ß-carotene content, whereas the mitochondrial variant of CrtI was not functional. Surprisingly, a cytosolic variant of this enzyme, which we obtained through the elimination of its carboxy-terminal membrane anchor, caused an increase in ß-carotene accumulation. Overexpression of this CrtI variant in an optimized medium resulted in a strain with a ß-carotene content of 79 mg g-1 cell dry weight, corresponding to a 76-fold improvement over the starting strain. CONCLUSIONS: The retargeting of heterologously expressed pathway enzymes improves ß-carotene production in S. cerevisiae, implicating extensive inter-organellar transport phenomena of carotenoid precursors. In addition, strong overexpression of carotenoid biosynthetic enzymes and the optimization of cultivation conditions are required for high contents.

Asunto(s)

Carotenoides , Saccharomyces cerevisiae , beta Caroteno , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta Caroteno/metabolismo , Carotenoides/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Ingeniería Metabólica , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Mitocondrias/enzimología

RESUMEN

Phytoene is a colourless carotenoid widely available from dietary sources and a precursor for the synthesis of other carotenoids. Although present at high concentrations across different tissues, phytoene is largely viewed as not having physiological activity. Here, we utilize the model organism C. elegans to show that phytoene is bioactive and has anti-ageing properties. Supplementation with phytoene protects against oxidative damage and amyloid-ß42 proteotoxicity (a major pathology of Alzheimer's disease), and extends lifespan. We also examine extracts from two microalgae, Chlorella sorokiniana and Dunaliella bardawil. We show that the extracts contain high levels of phytoene, and find that these phytoene-rich extracts have protective effects similar to pure phytoene. Our findings show that phytoene is a bioactive molecule with positive effects on ageing and longevity. Our work also suggests that phytoene-rich microalgae extracts can utilized to produce foods or supplements that promote healthy ageing and prevent the development of chronic age-related diseases.

RESUMEN

Two important plant enzymes are 4-hydroxyphenylpyruvate dioxygenase (HPPD; EC 1.13.11.27), which is necessary for biosynthesis of plastoquinone and tocopherols, and phytoene dehydrogenase (PDS; EC 1.3.99.26), which plays an important role in colour rendering. Dual-target proteins that inhibit pigment synthesis will prevent resistant weeds and improve the spectral characteristics of herbicides. This study introduces virtual screening of pharmacophores based on the complex structure of the two targets. A three-dimensional database was established by screening 1,492,858 compounds based on the Lipinski principle. HPPD&PDS dual-target receptor-ligand pharmacophore models were then constructed, and nine potential dual-target inhibitors were obtained through pharmacophore modeling, molecular docking, and molecular dynamics simulations. Ultimately, ADMET prediction software yielded three compounds with high potential as dual-target herbicides. The obtained nine inhibitors were stable when combined with both HPPD and PDS proteins. This study offers guidance for the development of HPPD&PDS dual-target inhibitors with novel skeletons.

Asunto(s)

4-Hidroxifenilpiruvato Dioxigenasa , Inhibidores Enzimáticos , Simulación del Acoplamiento Molecular , 4-Hidroxifenilpiruvato Dioxigenasa/antagonistas & inhibidores , 4-Hidroxifenilpiruvato Dioxigenasa/química , 4-Hidroxifenilpiruvato Dioxigenasa/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Herbicidas/química , Herbicidas/farmacología , Simulación de Dinámica Molecular , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Evaluación Preclínica de Medicamentos

RESUMEN

The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga Pyropia yezoensis-a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned PyPSY should encode a phytoene synthase; this was empirically confirmed by pigment complementation in E. coli. This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis.

Asunto(s)

Geranilgeranil-Difosfato Geranilgeraniltransferasa , Filogenia , Rhodophyta , Rhodophyta/genética , Rhodophyta/enzimología , Geranilgeranil-Difosfato Geranilgeraniltransferasa/genética , Geranilgeranil-Difosfato Geranilgeraniltransferasa/metabolismo , Carotenoides/metabolismo , Escherichia coli/genética , Clonación Molecular , Algas Comestibles , Porphyra

RESUMEN

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon-intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

RESUMEN

The extremely halophilic archaeon Haloarcula japonica accumulates the C50 carotenoid, bacterioruberin (BR). To reveal the BR biosynthetic pathway, unidentified phytoene desaturase candidates were functionally characterized in the present study. Two genes encoding the potential phytoene desaturases, c0507 and d1086, were found from the Ha. japonica genome sequence by a homology search using the Basic Local Align Search Tool. Disruption mutants of c0507 and d1086 and their complemented strains transformed with expression plasmids for c0507 and d1086 were subsequently constructed. High-performance liquid chromatography (HPLC) ana-lyses of carotenoids produced by these strains revealed that C0507 and D1086 were both bifunctional enzymes with the same activities as both phytoene desaturase (CrtI) and 3,4-desaturase (CrtD). C0507 and D1086 complemented each other during BR biosynthesis in Ha. japonica. This is the first study to identify two distinct enzymes with both CrtI and CrtD activities in an extremely halophilic archaeon.

Asunto(s)

Carotenoides , Haloarcula , Oxidorreductasas , Carotenoides/metabolismo , Haloarcula/genética , Haloarcula/enzimología , Haloarcula/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Vías Biosintéticas/genética , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Prueba de Complementación Genética , Filogenia

RESUMEN

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 µg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

Asunto(s)

Echinochloa , Herbicidas , Oxidorreductasas , Proteínas de Plantas , Malezas , Piridazinas , Herbicidas/farmacología , Herbicidas/química , Piridazinas/farmacología , Piridazinas/química , Echinochloa/efectos de los fármacos , Echinochloa/enzimología , Echinochloa/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/antagonistas & inhibidores , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Malezas/efectos de los fármacos , Malezas/enzimología , Malezas/genética , Relación Estructura-Actividad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Raíces de Plantas/química , Raíces de Plantas/efectos de los fármacos , Estructura Molecular

RESUMEN

Mango is a good source of carotenoids for use in food, cosmetic, and pharmaceutical products because of their organoleptic and health-promoting properties. Safe and sustainable methods for their extraction is required. The present investigation was aimed to study concentration and carotenoid profile of 'Kent' mango pulp through a conventional extraction (CE) and ultrasound-assisted extraction (UAE) using traditional solvents (tetrahydrofuran-THF and diethyl ether: petroleum ether-DE:PE) and green solvents (GS) (2-metiltetrahydrofuran, 2 m-THF; cyclopentyl methyl ether, CPME). Mango showed (µg/g d.w.) ß-carotene (29.4), zeaxanthin (1.28), ß-cryptoxanthin (2.8), phytoene (18.68) and phytofluene (7.45) in a CE using DE:PE. Similar results were obtained applying DE:PE in UAE and GS in a CE, so CPME and 2-mTHF seem suitable solvents to replace DE:PE in CE. The yield of total carotenes, xanthophylls and carotenoids using GS combined with UAE was lower than with CE, but important enough to be used as a sustainable procedure for obtaining carotenoids from mango pulp.

Asunto(s)

Carotenoides , Frutas , Mangifera , Extractos Vegetales , Solventes , Mangifera/química , Carotenoides/química , Carotenoides/aislamiento & purificación , Carotenoides/análisis , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Frutas/química , Solventes/química , Tecnología Química Verde , Fraccionamiento Químico/métodos , Ultrasonido

RESUMEN

The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak (Quercus ilex) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 106 protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at phytoene desaturase (pds) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

RESUMEN

The structure and phylogeny of the Solanum tuberosum L. phytoene synthase genes StPSY1, StPSY2, and StPSY3 were characterized. Their expression was studied in potato seedlings exposed to cold stress in the dark phase of the diurnal cycle to simulate night cooling. All of the three genes were activated as the temperature decreased, and the greatest response was observed for StPSY1. StPSY3 was for the first time shown to respond to cold stress and photoperiod. A search for cis-regulatory elements was carried out in the promoter regions and 5'-UTRs of the StPSY genes, and the regulation of all three genes proved associated with the response to light. A high level of cold-induced activation of StPSY1 was tentatively attributed to the presence of cis elements associated with sensitivity to cold and ABA.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Solanum tuberosum , Solanum tuberosum/genética , Solanum tuberosum/enzimología , Geranilgeranil-Difosfato Geranilgeraniltransferasa/genética , Geranilgeranil-Difosfato Geranilgeraniltransferasa/metabolismo , Frío , Respuesta al Choque por Frío/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Filogenia , Estrés Fisiológico/genética

RESUMEN

Modern tomatoes produce colorful mature fruits, but many wild tomato ancestors form green or gray green ripe fruits. Here, tomato cultivar 'Lvbaoshi' (LBS) that produces green ripe fruits was found to contain three recessive loci responsible for fruit development. The colorless peel of LBS fruits was caused by a 603 bp deletion in the promoter of SlMYB12. The candidate genes of the remaining two loci were identified as STAY-GREEN 1 (SlSGR1) and PHYTOENE SYNTHASE 1 (SlPSY1). SGR1 and PSY1 co-suppression by RNAi converted the pink fruits into green ripe fruits in transgenic plants. An amino acid change in PSY1 and a deletion in the promoter of SGR1 were also identified in several wild tomatoes bearing green or gray ripe fruits. Overexpression of PSY1 from green ripe fruit wild tomatoes in LBS plants could only partially rescue the green ripe fruit phenotype of LBS, and transgenic lines expressing ProSGR1::SGR1 from Solanum pennellii also failed to convert purple-flesh into red-flesh fruits. This work uncovers a novel regulatory mechanism by which SlMYB12, SlPSY1, and SlSGR1 control fruit color in cultivated and some wild tomato species.

Asunto(s)

Transferasas Alquil y Aril , Frutas , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Proteínas de Plantas , Solanum lycopersicum , Solanum lycopersicum/genética , Frutas/genética , Frutas/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Geranilgeranil-Difosfato Geranilgeraniltransferasa/genética , Geranilgeranil-Difosfato Geranilgeraniltransferasa/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Mutación , Plantas Modificadas Genéticamente/genética , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

Asunto(s)

Sistemas CRISPR-Cas , Fabaceae , Oxidorreductasas , Edición Génica/métodos , Mutagénesis , Fabaceae/genética , Plantas Modificadas Genéticamente/genética

RESUMEN

Fruit color affects its commercial value. ß-carotene is the pigment that provides color for many fruits and vegetables. However, the molecular mechanism of ß-carotene metabolism during apricot ripening is largely unknown. Here, we investigated whether ß-carotene content affects apricot fruit color. First, the differences in ß-carotene content between orange apricot 'JTY' and white apricot 'X15' during nine developmental stages (S1-S9) were compared. ß-carotene contents highly significantly differed between 'JTY' and 'X15' from S5 (color transition stage) onwards. Whole-transcriptome analysis showed that the ß-carotene synthesis genes 15-cis-phytoene desaturase (PaPDS) and 15-cis-phytoene synthase (PaPSY) significantly differed between the two cultivars during the color transition stage. There was a 5 bp deletion in exon 11 of PaPDS in 'X15', which led to early termination of amino acid translation. Gene overexpression and virus-induced silencing analysis showed that truncated PaPDS disrupted the ß-carotene biosynthesis pathway in apricot pulp, resulting in decreased ß-carotene content and a white phenotype. Furthermore, virus-induced silencing analysis showed that PaPSY was also a key gene in ß-carotene biosynthesis. These findings provide new insights into the molecular regulation of apricot carotenoids and provide a theoretical reference for breeding new cultivars of apricot.

RESUMEN

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130, and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or ß-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Geranilgeranil-Difosfato Geranilgeraniltransferasa/genética , Arabidopsis/metabolismo , Carotenoides/metabolismo , Plastidios/genética , Plastidios/metabolismo , Clorofila/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo

RESUMEN

Gene editing using clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease is an excellent tool for assessing gene function in plants. However, delivery of CRISPR/Cas-editing components into plant cells is still a major bottleneck and requires tissue culture-based approaches and regeneration of plants. To overcome this limitation, several plant viral vectors have recently been engineered to deliver single-guide RNA (sgRNA) targets into SpCas9-expressing plants. Here, we describe an optimized, step-by-step protocol based on the tobacco rattle virus (TRV)-based vector system to deliver sgRNAs fused to mobile tRNA sequences for efficient heritable editing in Nicotiana benthamiana and Arabidopsis thaliana model systems. The protocol described here could be adopted to study the function of any gene of interest.

Asunto(s)

Arabidopsis , ARN Guía de Sistemas CRISPR-Cas , Edición Génica , Nicotiana/genética , Arabidopsis/genética , Endonucleasas

RESUMEN

As an important mangrove species, Kandelia obovata plays an irreplaceable role in the coastal ecosystem. However, due to a lack of genetic technology, there is limited research on its functional genes. As such, establishing an efficient and rapid functional verification system is particularly important. In this study,tobacco rattle virus (TRV) and the phytoene desaturase gene KoPDS were used as the vector and target gene, respectively, to establish a virus-induced gene silencing system (VIGS) in K. obovata. Besides, the system was also used to verify the role of a Chlorophyll a/b binding protein (Cab) gene KoCAB in leaf carbon sequestration of K. obovata. RNA-Seq and qRT-PCR showed that the highest gene-silencing efficiency could reach 90% after 10 days of inoculation and maintain above 80% after 15 days, which was achieved with resuspension buffer at pH 5.8 and Agrobacterium culture at OD600 of 0.4-0.6. Taken together, the TRV-mediated VIGS system established herein is the first genetic analysis tool for mangroves, which may greatly impel functional genomics studies in mangrove plants.

RESUMEN

Carotenoids are essential and beneficial substances for both plant and human health. Identifying the regulatory network of these pigments is necessary for improving fruit quality and commodity value. In this study, we performed integrative analyses of transcriptome data from two different type fruits, ripening peel color at green ('Neelum' mango) and red ('Irwin' mango). Specifically, we found that MiMYB10 transcription level was highly associated with mango peel color. Further, silencing MiMYB10 homologous gene in tomato fruits resulted in lower carotenoid and anthocyanin content. Electrophoretic mobility shift assays and dual-luciferase clarified that MiMYB10 regulates the carotenoid biosynthesis gene MiPDS (phytoene desaturase gene) in a direct manner. On the other hand, MiMYB10 activates the expression of carotenoid biosynthesis genes (PSY, Z-ISO, CRTISO, LCYE) and chlorophyll degradation gene (SGR1), promoting the accumulation of carotenoid, accelerating chlorophyll degradation, and controlling peel color. In summary, this study identified important roles of MiMYB10 in pigment regulatory and provided new options for breeding strategies aiming to improve fruit quality.

Asunto(s)

Mangifera , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Frutas/metabolismo , Mangifera/genética , Regulación de la Expresión Génica de las Plantas , Fitomejoramiento , Carotenoides/metabolismo , Clorofila/genética , Clorofila/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo

RESUMEN

Background: Carotenoids, which are secondary metabolites derived from isoprenoids, play a crucial role in photo-protection and photosynthesis, and act as precursors for abscisic acid, a hormone that plays a significant role in plant abiotic stress responses. The biosynthesis of carotenoids in higher plants initiates with the production of phytoene from two geranylgeranyl pyrophosphate molecules. Phytoene synthase (PSY), an essential catalytic enzyme in the process, regulates this crucial step in the pathway. In Daucus carota L. (carrot), two PSY genes (DcPSY1 and DcPSY2) have been identified but only DcPSY2 expression is induced by ABA. Here we show that the ectopic expression of DcPSY2 in Nicotiana tabacum L. (tobacco) produces in L3 and L6 a significant increase in total carotenoids and chlorophyll a, and a significant increment in phytoene in the T1L6 line. Tobacco transgenic T1L3 and T1L6 lines subjected to chronic NaCl stress showed an increase of between 2 and 3- and 6-fold in survival rate relative to control lines, which correlates directly with an increase in the expression of endogenous carotenogenic and abiotic-related genes, and with ABA levels. Conclusions: These results provide evidence of the functionality of DcPSY2 in conferring salt stress tolerance in transgenic tobacco T1L3 and T1L6 lines.

RESUMEN

The recent availability of a number of tea plant genomes has sparked substantial interest in using reverse genetics to explore gene function in tea (Camellia sinensis). However, a hurdle to this is the absence of an efficient transformation system, and virus-induced gene silencing (VIGS), a transient transformation system, could be an optimal choice for validating gene function in the tea plant. In this study, phytoene desaturase (PDS), a carotenoid biosynthesis gene, was used as a reporter to evaluate the VIGS system. The injection sites of the leaves (leaf back, petiole, and stem) for infiltration were tested, and the results showed that petiole injection had the most effective injection, without leading to necrotic lesions that cause the leaves to drop. Tea leaves were inoculated with Agrobacterium harboring a tobacco rattle virus plasmid (pTRV2) containing a CsPDS silencing fragment. The tea leaves exhibited chlorosis symptoms 7-14 days after inoculation, depending on the cultivar. In the chlorosis plants, the coat protein (CP) of tobacco rattle virus (TRV) was detected and coincided with the lower transcription of CsPDS and reduced chlorophyll content compared with the empty vector control, with 81.82% and 54.55% silencing efficiency of 'LTDC' and 'YSX', respectively. These results indicate that the VIGS system with petiole injection could quickly and effectively silence a gene in tea plants.