Making extra room for carotenoids in plant cells: new opportunities for biofortification

[EN] Plant carotenoids are essential for photosynthesis and photoprotection and provide colors in the yellow to red range to non-photosynthetic organs such as petals and ripe fruits. They are also the precursors of biologically active molecules not only in plants (including hormones and retrograde signals) but also in animals (including retinoids such as vitamin A). A carotenoid-rich diet has been associated with improved health and cognitive capacity in humans, whereas the use of carotenoids as natural pigments is widespread in the agrofood and cosmetic industries. The nutritional and economic relevance of carotenoids has spurred a large number of biotechnological strategies to enrich plant tissues with carotenoids. Most of such approaches to alter carotenoid contents in plants have been focused on manipulating their biosynthesis or degradation, whereas improving carotenoid sink capacity in plant tissues has received much less attention. Our knowledge on the molecular mechanisms influencing carotenoid storage in plants has substantially grown in the last years, opening new opportunities for carotenoid biofortification. Here we will review these advances with a particular focus on those creating extra room for carotenoids in plant cells either by promoting the differentiation of carotenoid-sequestering structures within plastids or by transferring carotenoid production to the cytosol.We greatly thank Carmen and Pilar Torres-Montilla for their collaboration and help in the design and execution of the figures. We also thank Luca Morelli and BioRender.com for some of the images. Work in our lab is funded by Spanish grants BIO2017-84041-P and PID2020-115810GB-I00 from the Agencia Estatal de Investigacion (AEI) and 202040E299 from Consejo Superior de Investigaciones Cientificas (CSIC) to MRC. STM was supported by PhD fellowship FPU16/04054 from the Spanish Ministerio de Educacion y CulturaTorres-Montilla, S.; Rodriguez-Concepcion, M. (2021). Making extra room for carotenoids in plant cells: new opportunities for biofortification. Progress in Lipid Research. 84:1-9. https://doi.org/10.1016/j.plipres.2021.101128S198

Interference with plastome gene expression and Clp protease activity in Arabidopsis triggers a chloroplast unfolded protein response to restore protein homeostasis

Disruption of protein homeostasis in chloroplasts impairs the correct functioning of essential metabolic pathways, including the methylerythritol 4-phosphate (MEP) pathway for the production of plastidial isoprenoids involved in photosynthesis and growth. We previously found that misfolded and aggregated forms of the first enzyme of the MEP pathway are degraded by the Clp protease with the involvement of Hsp70 and Hsp100/ClpC1 chaperones in Arabidopsis thaliana. By contrast, the combined unfolding and disaggregating actions of Hsp70 and Hsp100/ClpB3 chaperones allow solubilization and hence reactivation of the enzyme. The repair pathway is promoted when the levels of ClpB3 proteins increase upon reduction of Clp protease activity in mutants or wild-type plants treated with the chloroplast protein synthesis inhibitor lincomycin (LIN). Here we show that LIN treatment rapidly increases the levels of aggregated proteins in the chloroplast, unleashing a specific retrograde signaling pathway that up-regulates expression of ClpB3 and other nuclear genes encoding plastidial chaperones. As a consequence, folding capacity is increased to restore protein homeostasis. This sort of chloroplast unfolded protein response (cpUPR) mechanism appears to be mediated by the heat shock transcription factor HsfA2. Expression of HsfA2 and cpUPR-related target genes is independent of GUN1, a central integrator of retrograde signaling pathways. However, double mutants defective in both GUN1 and plastome gene expression (or Clp protease activity) are seedling lethal, confirming that the GUN1 protein is essential for protein homeostasis in chloroplasts

Nutritional Enrichment of Plant Leaves by Combining Genes Promoting Tocopherol Biosynthesis and Storage

The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually unexplored. We previously showed that leaf chloroplasts can be converted into artificial chromoplasts with a proliferation of plastoglobules by overexpression of the bacterial crtB gene. Here we combined coexpression of crtB with genes involved in tocopherol biosynthesis to investigate the potential of artificial leaf chromoplasts for vitamin E accumulation in Nicotiana benthamiana leaves. We show that this combination improves tocopherol levels compared to controls without crtB and confirm that VTE1, VTE5, VTE6 and tyrA genes are useful to increase the total tocopherol levels, while VTE4 further leads to enrichment in alpha-tocopherol (the tocochromanol showing highest vitamin E activity). Additionally, we show that treatments that further promote plastoglobule formation (e.g., exposure to intense light or dark-induced senescence) result in even higher improvements in the tocopherol content of the leaves. An added advantage of our strategy is that it also results in increased levels of other related plastidial isoprenoids such as carotenoids (provitamin A) and phylloquinones (vitamin K1)

A root specific induction of carotenoid biosynthesis contributes to ABA production upon salt stress in arabidopsis

Abscisic acid (ABA) is a hormone that plays a vital role in mediating abiotic stress responses in plants. Salt exposure induces the synthesis of ABA through the cleavage of carotenoid precursors (xanthophylls), which are found at very low levels in roots. Here we show that de novo ABA biosynthesis in salt-treated Arabidopsis thaliana roots involves an organ-specific induction of the carotenoid biosynthetic pathway. Upregulation of the genes encoding phytoene synthase (PSY) and other enzymes of the pathway producing ABA precursors was observed in roots but not in shoots after salt exposure. A pharmacological block of the carotenoid pathway substantially reduced ABA levels in stressed roots, confirming that an increase in carotenoid accumulation contributes to fuel hormone production after salt exposure. Treatment with exogenous ABA was also found to upregulate PSY expression only in roots, suggesting an organ-specific feedback regulation of the carotenoid pathway by ABA. Taken together, our results show that the presence of high concentrations of salt in the growth medium rapidly triggers a root-specific activation of the carotenoid pathway, probably to ensure a proper supply of ABA precursors required for a sustained production of the hormone

Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts

Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism

Carotenoids are isoprenoid compounds that are essential for plants to protect the photosynthetic apparatus against excess light. They also function as health-promoting natural pigments that provide colors to ripe fruit, promoting seed dispersal by animals. Work in Arabidopsis thaliana unveiled that transcription factors of the phytochrome-interacting factor (PIF) family regulate carotenoid gene expression in response to environmental signals (i.e. light and temperature), including those created when sunlight reflects from or passes though nearby vegetation or canopy (referred to as shade). Here we show that PIFs use a virtually identical mechanism to modulate carotenoid biosynthesis during fruit ripening in tomato (Solanum lycopersicum). However, instead of integrating environmental information, PIF-mediated signaling pathways appear to fulfill a completely new function in the fruit. As tomatoes ripen, they turn from green to red due to chlorophyll breakdown and carotenoid accumulation. When sunlight passes through the flesh of green fruit, a self-shading effect within the tissue maintains high levels of PIFs that directly repress the master gene of the fruit carotenoid pathway, preventing undue production of carotenoids. This effect is attenuated as chlorophyll degrades, causing degradation of PIF proteins and boosting carotenoid biosynthesis as ripening progresses. Thus, shade signaling components may have been co-opted in tomato fruit to provide information on the actual stage of ripening (based on the pigment profile of the fruit at each moment) and thus finely coordinate fruit color change. We show how this mechanism may be manipulated to obtain carotenoid-enriched fruits.Peer ReviewedPostprint (published version

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change

Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development

[EN] Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.We greatly thank Jaume F. Martinez-Garcia and Ralf Welsch for fruitful discussions on the manuscript; Ralf Welsch and Li Li for providing seeds of the Arabidopsis ccd1 ccd4 and ator atorl mutants, respectively; Juan Jose Lopez-Moya and Maria Luisa Domingo-Calap for the gift of the HcProWMV-pGWB702 vector; and M. Rosa Rodriguez-Goberna for excellent technical support. The help of Marti Bernardo, Fidel Lozano, Lidia Jimenez, and members of the CRAG core facilities is also appreciated. This work was funded by the European Regional Development Fund and Spanish Agencia Estatal de Investigacion Grants BIO2017-84041-P, BIO2017-83184-R, BIO2017-90877-REDT, BES-2017-080652, and AGL2017-85563-C2-1-R; Ministry of Education, Culture and Sports Grants AP2012-3751 and FPU16/04054; and Generalitat de Catalunya Grant 2017SGR-710. We also thank the financial support of the European Union's Horizon 2020 (EU-H2020) COST Action CA15136 (EuroCaroten) and Marie S. Curie Action (MSCA) 753301 (Arcatom), the Severo Ochoa Programme for Centres of Excellence in R&D 2016-2019 Grant SEV-2015-0533 and the Generalitat de Catalunya CERCA Programme (to CRAG). B.L. is supported by grants from the CSIRO Synthetic Biology Future Science Platform and Macquarie University. L.M. is supported by La Caixa Foundation PhD INPhINIT Fellowship LCF/BQ/IN18/11660004, which received funding from the EU-H2020 through MSCA Grant 713673. A.R.F. is supported by Deutsche Forschungsgemeinschaft Grant DFG TRR 175.Llorente, B.; Torres-Montilla, S.; Morelli, L.; Florez-Sarasa, I.; Matus, JT.; Ezquerro, M.; D'andrea, L.... (2020). Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development. Proceedings of the National Academy of Sciences of the United States of America (Online). 117(35):21796-21803. https://doi.org/10.1073/pnas.2004405117S21796218031173

Comparative transcriptional profiling analysis of developing melon (Cucumis melo L.) fruit from climacteric and non-climacteric varieties

[EN] Background: In climacteric fruit-bearing species, the onset of fruit ripening is marked by a transient rise in respiration rate and autocatalytic ethylene production, followed by rapid deterioration in fruit quality. In non-climacteric species, there is no increase in respiration or ethylene production at the beginning or during fruit ripening. Melon is unusual in having climacteric and non-climacteric varieties, providing an interesting model system to compare both ripening types. Transcriptomic analysis of developing melon fruits from Védrantais and Dulce (climacteric) and Piel de sapo and PI 161375 (non-climacteric) varieties was performed to understand the molecular mechanisms that differentiate the two fruit ripening types. Results: Fruits were harvested at 15, 25, 35 days after pollination and at fruit maturity. Transcript profiling was performed using an oligo-based microarray with 75 K probes. Genes linked to characteristic traits of fruit ripening were differentially expressed between climacteric and non-climacteric types, as well as several transcription factor genes and genes encoding enzymes involved in sucrose catabolism. The expression patterns of some genes in PI 161375 fruits were either intermediate between. Piel de sapo and the climacteric varieties, or more similar to the latter. PI 161375 fruits also accumulated some carotenoids, a characteristic trait of climacteric varieties. Conclusions: Simultaneous changes in transcript abundance indicate that there is coordinated reprogramming of gene expression during fruit development and at the onset of ripening in both climacteric and non-climacteric fruits. The expression patterns of genes related to ethylene metabolism, carotenoid accumulation, cell wall integrity and transcriptional regulation varied between genotypes and was consistent with the differences in their fruit ripening characteristics. There were differences between climacteric and non-climacteric varieties in the expression of genes related to sugar metabolism suggesting that they may be potential determinants of sucrose content and post-harvest stability of sucrose levels in fruit. Several transcription factor genes were also identified that were differentially expressed in both types, implicating them in regulation of ripening behaviour. The intermediate nature of PI 161375 suggested that classification of melon fruit ripening behaviour into just two distinct types is an over-simplification, and that in reality there is a continuous spectrum of fruit ripening behaviourWe wish to thank Marta Casado for assistance with qRT-PCR analysis, Anna Orozco and Rosa Rodriguez for help with carotenoid measurements, Kostas Alexiou for uploading the microarray data at GEO and Walter Sanseverino for obtaining the correspondence of the microarray contigs with the melon annotated genes. We thank Beatrice Encke and Nicole Krohn for technical help with the sugar analysis. We also thank Laura Pascual for a critical reading of the manuscript. MSa was supported by a JAE-Doc grant from the Spanish Ministry of Science. This work was supported by the Spanish Ministry of Science and Innovation (MICINN project GEN2006-27773-C2-1-E to JGM) and the German Federal Ministry of Education and Research (BMBF project 0313987 to MSt) within the framework of the EU Framework 6 ERA-NET Plant Genomics programme.Saladie, M.; Cañizares Sales, J.; Michael A. Phillips; Rodriguez Concepcion, M.; Larrigaudière, C.; Gibon, Y.; Stitt, M.... (2015). Comparative transcriptional profiling analysis of developing melon (Cucumis melo L.) fruit from climacteric and non-climacteric varieties. BMC Genomics. 16(440):1-20. https://doi.org/10.1186/s12864-015-1649-3S12016440McMurchie EJ, McGlasson WB, Eaks IL. Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature. 1972;237:235–6.Giovannoni JJ. Genetic regulation of fruit development and ripening. Plant Cell. 2004;16:S170–80.Yamaguchi M, Hughes DL, Yabumoto K, Jennings WG. Quality of cantaloupe muskmelons: Variability and attributes. Scientia Hort. 1977;6:59–70.Seymour GB, McGlasson WB. Melons. In: Seymour GB, Taylor JE, Tucker GA, editors. Biochemistry of fruit ripening. London: Chapman & Hall; 1993. p. 273-290.Pech JC, Balague C, Latché A, Bouzayen M. Post-harvest physiology of climacteric fruits: recent developments in the biosynthesis and action of ethylene. Sci Aliments. 1994;14:3–15.Latché A, Ayub RA, Martinez G, Guis M, Ben Amor M, Rombaldi CV, et al. Biosynthèse et mode d’action de l’hormone végétale éthylène. Fruits. 1995;50:379–96.Moore S, Payton P, Wright M, Tanksley S, Giovannoni J. Utilization of tomato microarrays for comparative gene expression analysis in the Solanaceae. J Exp Bot. 2005;56:2885–95.Giovannoni JJ. Fruit ripening mutants yield insights into ripening control. Curr Opin Plant Biol. 2007;10:283–9.Saladié M, Rose JKC, Cosgrove DJ, Catalá C. Characterization of a new xyloglucan endotransglucosylase/hydrolase (XTH) from ripening tomato fruit and implications for the diverse modes of enzymic action. Plant J. 2006;47:282–95.Stepanova AN, Alonso JM. Ethylene signalling and response: where different regulatory modules meet. Curr Opin Plant Sci. 2009;12:548–55.Alexander L, Grierson D. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. J Exp Bot. 2002;53:2039–55.Klee HJ. Ethylene signal transduction. Moving beyond Arabidopsis. Plant Physiol. 2004;135:660–7.Adams-Phillips L, Barry C, Giovannoni J. Signal transduction systems regulating fruit ripening. Trends Plant Sci. 2004;9:331–8.Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D, Drake R, et al. A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus. Science. 2002;296:343–6.Giovannoni JJ, Tanksley S, Vrebalov J, Noensie F. NOR gene compositions and methods for use thereof. US Patent 6762347_B1. July 13, 2004.Manning K, Tor M, Poole M, Hong Y, Thompson AJ, King GJ, et al. A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet. 2006;38:948–52.Itkin M, Seybold H, Breitel D, Rogachev I, Meir S, Aharoni A. TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network. Plant J. 2009;60:1081–95.Lin Z, Hong Y, Yin M, Li C, Zhang K, Grierson D. A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening. Plant J. 2008;55:301–10.Chung M-Y, Vrebalov J, Alba R, Lee JM, McQuinn R, Chung J-D, et al. A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening. Plant J. 2011;64:936–47.Karlova R, Rosin FM, Busscher-Lange J, Parapunova V, Do PT, Fernie AR, et al. Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. Plant Cell. 2011;23:923–41.Seymour GB, Chapman NH, Chew BL, Rose JKC. Regulation of ripening and opportunities for control in tomato and other fruits. Plant Biotechnol J. 2013;11:269–78.Klee HJ, Giovannoni JJ. Genetics and control of tomato fruit ripening and quality attributes. Annu Rev Genet. 2011;45:41–59.Bemer M, Karlova R, Ballester AR, Tikunov YM, Bovy AG, Wolters-Arts M, et al. The tomato FRUITFUL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-Independent aspects of fruit ripening. Plant Cell. 2012;24:4437–51.Fujisawa M, Shima Y, Higuchi N, Nakano T, Koyama Y, Kasumi T, et al. Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses. Planta. 2012;235:1107–22.Kumar R, Sharma MK, Kapoor S, Tyagi AK, Sharma A. Transcriptome analysis of rin mutant fruit and in silico analysis of promoters of differentially regulated genes provides insight into LeMADS-RIN-regulated ethylene-dependent as well as ethylene-independent aspects of ripening in tomato. Mol Genet Genom. 2012;287:189–203.Zhong S, Fei Z, Chen Y-R, Zheng Y, Huang M, Vrebalov J, et al. Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat Biotechnol. 2013;31:154–9.Wilkinson JQ, Lanahan MB, Conner TW, Klee HJ. Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display. Plant Mol Biol. 1995;27:1097–108.Manning K. Isolation of a set of ripening-related genes from strawberry: Their identification and possible relationship to fruit quality traits. Planta. 1998;205:622–31.Aharoni A, Keizer LC, Bouwmeester HJ, Sun Z, Alvarez-Huerta M, Verhoeven HA, et al. Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell. 2000;12:647–62.Osorio S, Alba R, Nikoloski Z, Kochevenko A, Fernie AR, Giovannoni JJ. Integrative comparative analyses of transcript and metabolite profiles from pepper and tomato ripening and development stages uncovers species-specific patterns of network regulatory behavior. Plant Physiol. 2012;159:1713–29.Ezura H, Owino WO. Melon ripening and ethylene. Plant Sci. 2008;175:121–9.Pech JC, Bouzayen M, Latché A. Climacteric fruit ripening: ethylene-dependent and independent regulation of ripening pathways in melon fruit. Plant Sci. 2008;175:114–20.Vegas J, Garcia-Mas J, Monforte AJ. Interaction between QTLs induces an advance in ethylene biosynthesis during melon fruit ripening. Theor Appl Genet. 2013;126:1531–44.Lyons JM, Pratt HK, McGlasson WB. Ethylene production, respiration, and internal gas concentrations in cantaloupe fruits at various stages of maturity. Plant Physiol. 1962;37:31–26.Hadfield KA, Dang T, Guis M, Pech JC, Bouzayen M, Bennett AB. Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed Charentais melon fruit. Plant Physiol. 2000;122:977–83.Ayub R, Guis M, Ben Amor M, Gillot L, Roustan JP, Latché A, et al. Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nat Biotechnol. 1996;14:862–6.Monforte AJ, Oliver M, Gonzalo MJ, Alvarez JM, Dolcet-Sanjuan R, Arus P. Identification of quantitative trait loci involved in fruit quality traits in melon (Cucumis melo L.). Theor Appl Genet. 2004;108:750–8.Cuevas HE, Staub JE, Simon PW, Zalapa JE. A consensus linkage map identifies genomic regions controlling fruit maturity and beta-carotene-associated flesh color in melon (Cucumis melo L.). Theor Appl Genet. 2009;119:741–56.Bian W, Barsan C, Egea I, Purgatto E, Chervin C, Zouine M, et al. Metabolic and molecular events occurring during chromoplast biogenesis. J Bot. 2011;10:1–13.Pratt HK, Goeschl JD, Martin FW. Fruit growth and development, ripening, and role of ethylene in honey dew muskmelon. J Am Soc Hortic Sci. 1977;102:203–10.Lester GE, Dunlap JR. Physiological-changes during development and ripening of perlita muskmelon fruits. Sci Hortic. 1985;26:323–31.Rose JKC, Hadfield KA, Labavitch JM, Bennett AB. Temporal sequence of cell wall disassembly in rapidly ripening melon fruit. Plant Physiol. 1998;117:345–61.Nishiyama K, Guis M, Rose JK, Kubo Y, Bennett KA, Wangjin L, et al. Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon. J Exp Bot. 2007;58:1281–90.Portnoy V, Diber A, Pollock S, Karchi H, Lev S, Tzuri G, et al. Use of non-normalized, non-amplified cDNA for 454-based RNA sequencing of fleshy melon fruit. Plant Genome. 2011;4:1.Dai N, Cohen S, Portnoy V, Tzuri G, Harel-Beja R, Pompan-Lotan M, et al. Metabolism of soluble sugars in developing melon fruit: a global transcriptional view of the metabolic transition to sucrose accumulation. Plant Mol Biol. 2011;76:1–18.Corbacho J, Romojaro F, Pech JC, Latché A, Gomez-Jimenez MC. Transcriptomic events involved in melon mature-fruit abscission comprise the sequential induction of cell-wall degrading genes coupled to a stimulation of endo and exocytosis. PLoS One. 2013;8:e58363.Mascarell-Creus A, Cañizares J, Vilarrasa-Blasi J, Mora-García S, Blanca J, Gonzalez-Ibeas D, et al. An oligo-based microarray offers novel transcriptomic approaches for the analysis of pathogen resistance and fruit quality traits in melon (Cucumis melo L). BMC Genomics. 2009;10:e467.Garcia-Mas J, Benjak A, Sanseverino W, Bourgeois M, Mir G, Gonzalez VM, et al. The genome of melon (Cucumis melo L.). Proc Natl Acad Sci U S A. 2012;109:11872–7.Gonzalez-Ibeas D, Cañizares J, Aranda MA. Microarray analysis shows that recessive resistance to Watermelon mosaic virus in melon is associated with the induction of defense response genes. Mol Plant Microbe Interact. 2012;25:107–18.Roig C, Fita A, Ríos G, Hammond JP, Nuez F, Picó B. Root transcriptional responses of two melon genotypes with contrasting resistance to Monosporascus cannonballus (Pollack et Uecker) infection. BMC Genomics. 2012;13:601–12.Conesa A, Nueda MJ, Ferrer A, Talón M. MaSigPro: a method to identify significantly differential expression profiles in time-course microarray experiments. Bioinformatics. 2006;22:1096–102.Al-Shahrour F, Díaz-Uriarte R, Dopazo J. FatiGO: a web tool for finding significant associations of Gene Ontology terms with groups of genes. Bioinformatics. 2004;20:578–80.Pilati S, Perazzolli M, Malossini A, Cestaro A, Demattè L, Fontana P, et al. Genome-wide transcriptional analysis of grapevine berry ripening reveals a set of genes similarly modulated during three seasons and the occurrence of an oxidative burst at vèraison. BMC Genomics. 2007;8:428.Miki T, Yamamoto M, Nakagawa H, Ogura N, Mori H, Imaseki H, et al. Nucleotide sequence of a cDNA for 1-aminocyclopropane-1-carboxylate synthase from melon fruits. Plant Physiol. 1995;107:297–8.Yamamoto M, Miki T, Ishiki Y, Fujinami K, Yanagisawa Y, Nakagawa H, et al. The synthesis of ethylene in melon fruit during the early stage of ripening. Plant Cell Physiol. 1995;36:591–59.Lehman A, Black R, Ecker JR. HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl. Cell. 1996;85:183–94.Elitzur T, Vrebalov J, Giovannoni JJ, Goldschmidt EE, Friedman H. The regulation of MADS-box gene expression during ripening of banana and their regulatory interaction with ethylene. J Exp Bot. 2010;61(5):1523–35.Yang Y, Wu Y, Pirrello J, Regad F, Bouzayen M, Deng W, et al. Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response phenotype, accelerated plant senescence, and fruit ripening in tomato. J Exp Bot. 2010;61(3):697–708.Tanaka R, Oster U, Kruse E, Rudiger W, Grimm B. Reduced activity of geranylgeranyl reductase leads to loss of chlorophyll and tocopherol and to partially geranylgeranylated chlorophyll in transgenic tobacco plants expressing antisense RNA for geranylgeranyl reductase. Plant Physiol. 1999;120:695–704.Kim J, DellaPenna D. Defining the primary route for lutein synthesis in plants: the role of Arabidopsis carotenoid beta-ring hydroxylase CYP97A3. Proc Natl Acad Sci U S A. 2006;103:3474–9.Welsch R, Maass D, Voegel T, Dellapenna D, Beyer P. Transcription factor RAP2.2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves. Plant Physiol. 2007;145:1073–85.Neta-Sharir I, Isaacson T, Lurie S, Weiss D. Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation. Plant Cell. 2005;17:1829–38.Li L, Paolillo DJ, Parthasarathy MV, Dimuzio EM, Garvin DF. A novel gene mutation that confers abnormal patterns of beta-carotene accumulation in cauliflower (Brassica oleracea var. botrytis). Plant J. 2001;26:59–67.Lu S, Van Eck J, Zhou X, Lopez AB, Halloran DM, Cosman KM, et al. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation. Plant Cell. 2006;18:3594–605.Akhtar MS, Goldschmidt EE, John I, Rodoni S, Matile P, Grierson D. Altered patterns of senescence and ripening in gf, a stay-green mutant of tomato. J Exp Bot. 1999;50(336):1115–22.Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003;34:374–8.Lohse M, Nunes-Nesi A, Krüger P, Nagel A, Hannemann J, Giorgi FM, et al. Robin: an intuitive wizard application for R-based expression microarray quality assessment and analysis. Plant Physiol. 2010;153:642–51.Thimm O, Bläsing O, Gibon Y, Nagel A, Meyer S, Krüger P, et al. MapMan: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J. 2004;37:914–39.Usadel B, Poree F, Nagel A, Lohse M, Czedik-Eysenberg A, Stitt M. A guide to using MapMan to visualize and compare omics data in plants: a case study in the crop species, maize. Plant Cell Environ. 2009;32:1211–29.Costa F, Alba R, Schouten H, Soglio V, Gianfranceschi L, Serra S, et al. Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening. BMC Plant Biol. 2010;10:229.Zhang C, Yu X, Ayre BG, Turgeon R. The origin and composition of cucurbit “phloem” exudate. Plant Physiol. 2012;158:1873–82.Rose JKC, Saladié M, Catalá C. The plot thickens: new perspectives of primary cell wall modification. Current Opin Plant Biol. 2004;7:296–301.Manning K. Changes in gene-expression during strawberry fruit ripening and their regulation by auxin. Planta. 1994;194:62–8.Saladié M, Wright LP, Garcia-Mas J, Rodriguez-Concepcion M, Phillips MA. The 2-C-methylerithritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps. J Exp Bot. 2014;65:5077–92.Périn C, Gomez-Jimenez M, Hagen L, Dogimont C, Pech JC, Latché A, et al. Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit. Plant Physiol. 2002;129:300–9.Eduardo I, Arús P, Monforte AJ. Development of a genomic library of near isogenic lines (NILs) in melon (Cucumis melo L.) from the exotic accession PI161375. Theor Appl Genet. 2005;112:139–48.Stitt M, McLilley R, Gerhardt R, Heldt HW. Metabolite levels in specific cells and subcellular compartments of plant leaves. Meth Enzymol. 1989;174:518–52.Ibdah M, Azulay Y, Portnoy V, Wasserman B, Bar E, Meir A, et al. Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon. Phytochemistry. 2006;67:1579–89.Rodriguez-Concepcion M, Fores O, Martinez-Garcia JF, Gonzalez V, Phillips MA, Ferrer A, et al. Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell. 2004;16:144–56.Fraser PD, Pinto MES, Holloway DE, Bramley PM. Application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant J. 2000;24:551–8.Burns J, Fraser PD, Bramley PM. Identification and quantification of carotenoids, tocopherols and chlorophylls in commonly consumed fruits and vegetables. Phytochemistry. 2003;62:939–47.Taylor KL, Brackenridge AE, Vivier MA, Oberholster A. High-performance liquid chromatography profiling of the major carotenoids in Arabidopsis thaliana leaf tissue. J Chromatogr A. 2006;1121:83–91.Hartigan JA, Wong MA. A k-means clustering algorithm. Appl Stat. 1979;28:100–8.Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:0034.1.Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26:509–15.Gonzalez-Ibeas D, Blanca J, Roig C, González-To M, Picó B, Truniger V, et al. MELOGEN: An EST database for melon functional genomics. BMC Genomics. 2007;8:306.Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45.Masoudi-Nejad A, Goto S, Jauregui R, Ito M, Kawashima S, Moriya Y, et al. EGENES: transcriptome-based plant database of genes with metabolic pathway information and expressed sequence tag indices in KEGG. Plant Physiol. 2007;144:857–66.Usadel B, Nagel A, Thimm O, Redestig H, Blaesing OE, Palacios-Rojas N, et al. Extension of the visualisation tool MapMan to allow statistical analysis of arrays, display of corresponding genes and comparison with known responses. Plant Physiol. 2005;138:1195–204.Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Stat Methodol. 1995;57:289–300