Anthocyanin biosynthesis and accumulation in blood oranges during postharvest storage at different low temperatures

[EN] Blood oranges require low temperature for anthocyanin production. We have investigated the activation of anthocyanin biosynthesis and accumulation in the pulp of Moro blood and Pera blond oranges (Citrus sinensis L. Osbeck) stored at either 4 or 9 degrees C after harvesting. Both temperatures stimulated anthocyanin accumulation in blood but not in blond oranges. Nonetheless, blood orange fruits stored at 9 degrees C reached a darker purple coloration, higher anthocyanin contents and enhanced upregulation of genes from the flavonoid pathway in the pulp and juice than those kept at 4 degrees C. Our results indicated that dihydroflavonol channeling toward anthocyanin production was boosted during the storage at 9 degrees C compared to 4 degrees C, providing more leucoanthocyanidins to enzymes downstream in the pathway. Finally, despite both low temperatures stimulated the expression of key transcription factors likely regulating the pathway, their expression profiles could not explain the differences observed at 9 and 4 degrees C. (C) 2017 Elsevier Ltd. All rights reserved.This work was support by the São Paulo Research Foundation (FAPESP, Brazil) project (FAPESP 2014/12616-9) and Fundecitrus. LC was funded by grant from FAPESP (2014/23447-3).Carmona-López, L.; Alquézar-García, B.; Marques, VV.; Peña, L. (2017). Anthocyanin biosynthesis and accumulation in blood oranges during postharvest storage at different low temperatures. Food Chemistry. 237:7-14. https://doi.org/10.1016/j.foodchem.2017.05.076S71423

Avances en el conocimiento de los mecanismos moleculares que regulan la síntesis y acumulación de carotenoides durante la maduración y conservación de frutos cítricos

El color de los frutos cítricos es un importante atributo de calidad y un factor decisivo en la aceptación por los consumidores. La coloración de los frutos cítricos se debe al contenido y composición en carotenoides, que constituyen una amplia familia de pigmentos isoprenoides con gran importancia nutricional y para la salud, ya que algunos de ellos son los precursores de la vitamina A. Los estudios sobre el contenido y la composición en carotenoides en diferentes especies y variedades de cítricos ha sido objeto de interés en el pasado. Sin embargo, se dispone de escasa información sobre los mecanismos moleculares que controlan la síntesis y acumulación de carotenoides en los frutos cítricos. La comprensión de estos procesos y entender las bases moleculares que determinan la pigmentación característica de los frutos de las diferentes variedades, así como los cambios durante su vida postcosecha, han sido objetivos prioritarios de nuestros proyectos de investigación. Para ello, en nuestro laboratorio hemos aislado cDNAs de longitud completa o parcial de 10 genes implicados en la biosíntesis de carotenoides de frutos cítricos y 3 genes de las etapas tempranas de la síntesis de isoprenoides en plastidios (ruta MEP), que son precursores de los carotenoides, cubriendo así la práctica totalidad de la ruta. Los genes aislados de la biosíntesis de carotenoides corresponden a las etapas tempranas de la ruta (fitoeno sintasa y desaturasa, z-caroteno desaturasa y una oxidasa terminal de plastidios), a la ciclación de licopeno (b- y elicopeno ciclasas), y a la biosíntesis de xantofilas (b- y e- caroteno hidroxilasas y zeaxantina epoxidasa), que son los carotenoides más abundantes en frutos coloreados de naranjas y mandarinas. El análisis de expresión de estos genes en el flavedo y la pulpa de frutos cítricos y su correlación con los cambios cuantitativos y cualitativos en carotenoides nos está permitido establecer las etapas reguladoras limitantes de la biosíntesis de carotenoides, así como identificar nuevos genes de la ruta que codifican enzimas con un papel clave en la composición de carotenoides. En esta comunicación haremos una revisión de los principales avances en estos estudios en los frutos cítricos, con especial atención a la comparación entre frutos de variedades con distinto grado de pigmentación (pomelos blancos y rojos, naranjas y mandarinas), así como a los cambios durante situaciones de interés en la postcosecha de los frutos cítricos, como el tratamiento con etileno o diferentes temperaturas de conservación

Resistance of True Citrus species to Diaphorina citri

This is the peer reviewed version of the following article: Eduardo, W. I., Miranda, M. P., Volpe, H. X. L., Garcia, R. B., Girardi, E. A., Alquezar, B., ... & Peña, L. (2022). Resistance of True Citrus species to Diaphorina citri. Pest management science, 78(11), 4783-4792, which has been published in final form at [https://doi.org/10.1002/ps.7098]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] BACKGROUND Host genetic resistance is a promising strategy for the management of Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and consequently Huanglongbing (HLB). To date, no study has investigated the resistance to D. citri in the clonal and vegetatively propagated plants of the Microcitrus, Eremocitrus, and Atalantia genera. This study assesses Near and True Citrus genotype antixenosis and antibiosis against D. citri, with trichome density and volatile emission as possible mechanisms of resistance. RESULTS All genotypes were oviposited by D. citri, however, 8 of 14 genotypes were less oviposited than Citrus x sinensis 'Valencia' (susceptible control). Diaphorina citri nymphs had lower nymphal viability in E. glauca (31%) and M. warburgiana (58%) than that in Citrus x sinensis (77%). The behavioral assay showed that 30% of D. citri nymphs in the last instars evaded E. glauca shoots, whereas no nymphs evaded Citrus x sinensis shoots. A higher trichome density was observed in E. glauca shoots compared to the other genotypes. Chemical analysis revealed differences in the volatile profiles of E. glauca and Citrus x sinensis. CONCLUSION Eremocitrus glauca and M. warburgiana genotypes were more resistant to D. citri than Citrus x sinensis. Higher trichome density in the shoots may negatively influence the development of D. citri nymphs. Eremocitrus glauca volatiles may also be involved in their resistance to D. citri.This work was funded by the Fund for Citrus Protection (Fundecitrus), the European Union H2020 Innovation Action Program (grant #817526), and the project PID2019-104569RB-I00 from AEI-Spain. WIE received a postdoctoral fellowship (Proc. 2019/19649-3) from SAo Paulo Research Foundation (FAPESP). MPM received a research fellowship (Proc. 301805/2018-0) from Council for Scientific and Technological Development (CNPq), Brazil. The authors thank the Metabolomic service from Instituto de Biologia Molecular y Celular de Plantas, CSIC/UPV (IBMCP), Valencia, Spain for their help with the volatile emission analysis. The authors also acknowledge Fundecitrus and EMBRAPA Cassava & Fruits for providing plant materials.Eduardo, WI.; Miranda, MP.; Linhares Volpe, HX.; Garcia, RB.; Alquézar-García, B.; Girardi, EA.; Espinosa Ruiz, A.... (2022). Resistance of True Citrus species to Diaphorina citri. Pest Management Science. 78(11):4783-4792. https://doi.org/10.1002/ps.709847834792781

Engineered Orange Ectopically Expressing the Arabidopsis beta-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing

[EN] Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-beta-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.This work was funded by the Fundo de Defesa da Citricultura (Fundecitrus), Sao Paulo Research Foundation (FAPESP, grant #2015/07011-3), and EU H2020 Innovation Action Program (grant #817526). Consent for research and field trial of genetically modified organisms was granted by the National Technical Biosafety Commission from Brazil (CTNBio) to Fundecitrus.Alquézar-García, B.; Linhares Volpe, HX.; Facchini Magnani, R.; Pedreira De Miranda, M.; Almeida Santos, M.; Vieira Marques, V.; Rodrigues De Almeida, M.... (2021). Engineered Orange Ectopically Expressing the Arabidopsis beta-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing. Frontiers in Plant Science. 12:1-15. https://doi.org/10.3389/fpls.2021.6414571151

Caracterización bioquímica y molecular de la carotenogénesis en frutos cítricos

Tesis doctoral. Universidad de Valencia, Depto de Medicina Preventiva y Alimentación; Instituto de Agroquímica y Tecnología de Alimentos (IATA CSIC), 2008Peer reviewe

Cultural Management of Huanglongbing: Current Status and Ongoing Research

[EN] Huanglongbing (HLB), formerly known as greening, is a bacterial disease restricted to some Asian and African regions until two decades ago. Nowadays, associated bacteria and their vectors have spread to almost all citrus-producing regions, and it is currently considered the most devastating citrus disease. HLB management can be approached in terms of prevention, limiting or avoiding pathogen and associated vectors to reach an area, or in terms of control, trying to reduce the impact of the disease by adopting different cultural strategies depending on infestation/infection levels. In both cases, control of psyllid populations is currently the best way to stop HLB spread. Best cultural actions (CHMAs, TPS system) to attain this goal and, thus, able to limit HLB spread, and ongoing research in this regard is summarized in this review.This work was funded by the EU H2020 Innovation Action Program (grant 817526) and by the Fundo de Defensa da Citricultura (Fundecitrus).Alquézar-García, B.; Carmona, L.; Bennici, S.; Miranda, MP.; Bassanezi, RB.; Peña, L. (2022). Cultural Management of Huanglongbing: Current Status and Ongoing Research. Phytopathology. 112(1):11-25. https://doi.org/10.1094/PHYTO-08-21-0358-IA1125112

Generation of Transfer-DNA-Free Base-Edited Citrus Plants

[EN] To recover transgenic citrus plants in the most efficient manner, the use of selection marker genes is essential. In this work, it was shown that the mutated forms of the acetolactate synthase (ALS) gene in combination with the herbicide selection agent imazapyr (IMZ) added to the selection medium may be used to achieve this goal. This approach enables the development of cisgenic regenerants, namely, plants without the incorporation of those bacterial genes currently employed for transgenic selection, and additionally it allows the generation of edited, non-transgenic plants with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus mutants, in which ALS has been converted into IMZ-resistant forms using a base editor system, were recovered after cocultivation of the explants with Agrobacterium tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free plants indicated that the transient expression of the T-DNA genes was sufficient to induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency. To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although further optimization is required to increase edition efficiency, this methodology will allow generating new citrus varieties with improved organoleptic/agronomic features without the need to use foreign genes.Funding This work was funded by the EU H2020 Innovation Action Program (grant #817526) and by the Fundo de Defensa da Citricultura (Fundecitrus).Alquézar-García, B.; Bennici, S.; Carmona, L.; Gentile, A.; Peña, L. (2022). Generation of Transfer-DNA-Free Base-Edited Citrus Plants. Frontiers in Plant Science. 13:1-12. https://doi.org/10.3389/fpls.2022.8352821121

Effect of low temperature-storage on the proteome of Moro blood orange flesh

[EN] Anthocyanins are a subclass of flavonoid pigments with important therapeutic properties beneficial to human health. Blood oranges are rich in these pigments, which present synergistic effects (i.e., anti-inflammatory and anti-obesity) with other orange juice phytonutrients. Anthocyanin biosynthesis is cold induced in blood citrus cultivars, requiring a broad day/night thermal range to get purple pigmentation in fruit. This cold-dependency limits geographically a reliable quality for commercial production to only a few regions worldwide. For example, cultivation of blood oranges under tropical/subtropical climates, as those of Brazil, yields fruit with very low level or lack of anthocyanins. A feasible alternative in tropical countries to enhance anthocyanin content is cold postharvest storage, as it has been shown that anthocyanin synthesis is induced when fruits are kept below 10°C for a few weeks after harvesting. Blood oranges kept at 9°C showed higher expression levels of anthocyanin biosynthetic genes, increased anthocyanin content and reached a darker purple coloration than those stored at 4°C. In order to gain insight on the effect of storage temperature on activation of anthocyanin biosynthesis and accumulation as well as on purple orange coloration, we have investigated the proteome of 'Moro' sweet orange [Citrus sinensis (L.) Osbeck] fruit stored either at 4°C (low temperature) or at 9°C (moderate temperature). Results on increased or reduced accumulation of specific proteins in the 'Moro' orange pulp upon storage at different temperatures reinforces that postharvest storage at 9°C could be better to enhance anthocyanin biosynthesis and accumulation on blood orange juices.This work was support by the Sao Paulo Research Foundation (FAPESP, Brazil) project (FAPESP 2014/12616-9) and Fundecitrus. LC was funded by grants from FAPESP (2014/23447-3).Carmona-López, L.; Alquézar-García, B.; Tarraga Herrero, S.; Peña Garcia, L. (2019). Effect of low temperature-storage on the proteome of Moro blood orange flesh. Acta Horticulturae. 1230:49-56. https://doi.org/10.17660/ActaHortic.2019.1230.7S4956123

A mutant allele of ζ-carotene isomerase (Z-ISO) is associated with the yellow pigmentation of the “Pinalate” sweet orange mutant and reveals new insights into its role in fruit carotenogenesis

[Background] Fruit coloration is one of the main quality parameters of Citrus fruit primarily determined by genetic factors. The fruit of ordinary sweet orange (Citrus sinensis) displays a pleasant orange tint due to accumulation of carotenoids, representing β,β-xanthophylls more than 80% of the total content. ‘Pinalate’ is a spontaneous bud mutant, or somatic mutation, derived from sweet orange ‘Navelate’, characterized by yellow fruits due to elevated proportions of upstream carotenes and reduced β,β-xanthophylls, which suggests a biosynthetic blockage at early steps of the carotenoid pathway.[Results] To identify the molecular basis of ‘Pinalate’ yellow fruit, a complete characterization of carotenoids profile together with transcriptional changes in carotenoid biosynthetic genes were performed in mutant and parental fruits during development and ripening. ‘Pinalate’ fruit showed a distinctive carotenoid profile at all ripening stages, accumulating phytoene, phytofluene and unusual proportions of 9,15,9′-tri-cis- and 9,9′-di-cis-ζ-carotene, while content of downstream carotenoids was significantly decreased. Transcript levels for most of the carotenoid biosynthetic genes showed no alterations in ‘Pinalate’; however, the steady-state level mRNA of ζ-carotene isomerase (Z-ISO), which catalyses the conversion of 9,15,9′-tri-cis- to 9,9′-di-cis-ζ-carotene, was significantly reduced both in ‘Pinalate’ fruit and leaf tissues. Isolation of the ‘Pinalate’ Z-ISO genomic sequence identified a new allele with a single nucleotide insertion at the second exon, which generates an alternative splicing site that alters Z-ISO transcripts encoding non-functional enzyme. Moreover, functional assays of citrus Z-ISO in E.coli showed that light is able to enhance a non-enzymatic isomerization of tri-cis to di-cis-ζ-carotene, which is in agreement with the partial rescue of mutant phenotype when ‘Pinalate’ fruits are highly exposed to light during ripening.[Conclusion] A single nucleotide insertion has been identified in ‘Pinalate’ Z-ISO gene that results in truncated proteins. This causes a bottleneck in the carotenoid pathway with an unbalanced content of carotenes upstream to β,β-xanthophylls in fruit tissues. In chloroplastic tissues, the effects of Z-ISO alteration are mainly manifested as a reduction in total carotenoid content. Taken together, our results indicate that the spontaneous single nucleotide insertion in Z-ISO is the molecular basis of the yellow pigmentation in ‘Pinalate’ sweet orange and points this isomerase as an essential activity for carotenogenesis in citrus fruits.Financial support of the research grants AGL2012–34576 and AGL2015–70218 (Ministerio Economía y Competitividad, Spain), and RTI2018-095131-B-I00 (Ministerio Ciencia, Innovacion y Universidades, Spain). Work in the group of JH was supported by Israel Science Foundation Grant 850/13. MJR, LZ, JH and OD are members of Eurocaroten (COST_Action CA15136). MJR and LZ belong to CaRed (Spanish Carotenoid Network BIO2017–90877-REDT, Ministerio de Ciencia, Innovación y Universidades, Spain). Authors acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Curing and low-temperature combined post-harvest storage enhances anthocyanin biosynthesis in blood oranges

[EN] Anthocyanins are pigments present in blood oranges which can be enriched by post-harvest cold storage. Additionally, citrus fruits contain appreciable levels of other flavonoids, whose content increases under postharvest heat treatments. Here, we investigated the effects of curing (37 degrees C for 3 days) and storage at lowtemperature (9 degrees C) during 15, 30 and 45 days on accumulation of anthocyanins and other flavonoids in Moro and Sanguinelli Polidori blood oranges (Citrus sinensis L. Osbeck). Cured fruits reached up to 191.4 +/- 1.4 mg/L of anthocyanins in their juice after cold storage and a 3-fold enrichment of other flavonoids such as flavones and flavanones, compared to 85.7 +/- 3.3 mg/L anthocyanins from fruits with cold storage alone. Concomitantly, qPCR analysis showed that curing enhanced upregulation of the main structural and transcription factor genes regulating the flavonoid pathway. GC-MS analysis showed that no unpleasant compounds were generated in the cured plus cold-stored juice volatilome.This work was supported by the Sao Paulo Research Foundation (FAPESP, Brazil) project FAPESP 2014/12616-9 and Fundecitrus. LC was funded by FAPESP grant (2014/23447-3). FS acknowledges a Ph.D. fellowship from the University of Naples Federico II. We appreciate the help of LF Girotto and the Faro group for providing blood orange fruits. We also appreciate the careful review of the MS by Dr. James H. Graham (University of Florida, Lake Alfred, USA).Carmona, L.; Alquézar-García, B.; Diretto, G.; Sevi, F.; Malara, T.; Lafuente, MT.; Peña Garcia, L. (2021). Curing and low-temperature combined post-harvest storage enhances anthocyanin biosynthesis in blood oranges. Food Chemistry. 342:1-12. https://doi.org/10.1016/j.foodchem.2020.128334S112342Allan, A. C., & Espley, R. V. (2018). MYBs Drive Novel Consumer Traits in Fruits and Vegetables. 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Acta Horticulturae, (1230), 51-58. doi:10.17660/actahortic.2019.1230.7Cheong, M.-W., Liu, S.-Q., Yeo, J., Chionh, H.-K., Pramudya, K., Curran, P., & Yu, B. (2011). Identification of Aroma-Active Compounds in Malaysian Pomelo (Citrus grandis(L.) Osbeck) Peel by Gas Chromatography-Olfactometry. Journal of Essential Oil Research, 23(6), 34-42. doi:10.1080/10412905.2011.9712279Crifò, T., Petrone, G., Lo Cicero, L., & Lo Piero, A. R. (2011). Short Cold Storage Enhances the Anthocyanin Contents and Level of Transcripts Related to Their Biosynthesis in Blood Oranges. Journal of Agricultural and Food Chemistry, 60(1), 476-481. doi:10.1021/jf203891eDiretto, G., Jin, X., Capell, T., Zhu, C., & Gomez-Gomez, L. (2019). Differential accumulation of pelargonidin glycosides in petals at three different developmental stages of the orange-flowered gentian (Gentiana lutea L. var. aurantiaca). 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