Transcriptional Analysis of C-Repeat Binding Factors in Fruit of Citrus Species with Differential Sensitivity to Chilling Injury during Postharvest Storage.

Citrus fruit are sensitive to chilling injury (CI) during cold storage, a peel disorder that causes economic losses. C-repeat binding factors (CBFs) are related to cold acclimation and tolerance in different plants. To explore the role of Citrus CBFs in fruit response to cold, an in silico study was performed, revealing three genes (CBF1, CBF2, and CBF3) whose expression in CI sensitive and tolerant cultivars was followed. Major changes occurred at the early stages of cold exposure (1-5 d). Interestingly, CBF1 was the most stimulated gene in the peel of CI-tolerant cultivars (Lisbon lemon, Star Ruby grapefruit, and Navelina orange), remaining unaltered in sensitive cultivars (Meyer lemon, Marsh grapefruit, and Salustiana orange). Results suggest a positive association of CBF1 expression with cold tolerance in Citrus cultivars (except for mandarins), whereas the expression of CBF2 or CBF3 genes did not reveal a clear relationship with the susceptibility to CI. Light avoidance during fruit growth reduced postharvest CI in most sensitive cultivars, associated with a rapid and transient enhance in the expression of the three CBFs. Results suggest that CBFs-dependent pathways mediate at least part of the cold tolerance responses in sensitive Citrus, indicating that CBF1 participates in the natural tolerance to CI.

Visualization of Carotenoid-Storage Structures in Fruits by Transmission Electron Microscopy.

Plastids are cell organelles that, beside other functions, have the capability to store carotenoids in specialized structures, which may vary among the different plant species, tissues or according to the carotenoid complement. Fruits are an important source of carotenoids, and during ripening, chloroplasts differentiate into chromoplasts that are able to accumulate large amounts of carotenoids, rendering then the characteristic fruit coloration. Whereas lycopene or ß-carotene may accumulate as crystal in the chromoplasts of some fruit, other xanthophyll-accumulating fruits differentiate plastoglobuli as a preferred system to enhance carotenoids stability and storage. Visualization of plastid ultrastructure and their transformation during ripening or in fruit of contrasting coloration are fundamental objectives within carotenoids research in fruits. Therefore, in this chapter, we describe a protocol for the visualization and analysis of plastid ultrastructure by transmission electron microscopy (TEM), specially designed and adapted to fruit tissues.

Dietary Carotenoid Roles in Redox Homeostasis and Human Health.

Classic nutrition believed that healthy diets should simply provide sufficient antioxidant loads to organisms, to hamper free radical processes and avoid oxidative stress. Current redox biology was proven much more intricate. Carotenoids are bioactive compounds in the human diet with a multifaceted role in redox metabolism. This perspective discusses the participation of α/ß-carotene, lutein, zeaxanthin, lycopene, ß-cryptoxanthin, astaxanthin, and derivatives in redox homeostasis focusing on (i) their antioxidant/pro-oxidant activities, (ii) control of gene expression via Nrf2-Keap1 and NF-κB pathways, and (iii) their link with (sub)cellular redox circuits, as part of the "redox code" that orchestrates physiological processes and health in humans.

Effect of ethylene and 1-methylcyclopropene on the postharvest behavior of cape gooseberry fruits (Physalis peruviana L.).

Cape gooseberry (Physalis peruviana L.) fruits are highly perishable berries that exhibit a climacteric respiratory behavior. The objective of this study was to evaluate the effect of ethylene and the ethylene action inhibitor 1-methylcyclopropene on the postharvest behavior of cape gooseberry fruits (ecotype Colombia). Fruits were treated with ethylene, in an ethephon application (1000 µL L-1), and pretreated with 1-methylcyclopropene (1 µL L-1), 1-methylcyclopropene+ethylene, and results compared with a control without application. Subsequently, the fruits were maintained at room temperature (20 ℃, 75% RH) for up to 11 days. The pretreatment of the cape gooseberry fruits with 1-methylcyclopropene delayed most of the ripening-associated parameters, with a reduction in the respiration rate and ethylene production, skin color development, total soluble solids, total carotenoid content, loss of firmness, loss of total titratable acidity and emission of volatile compounds such as ethyl octanoate, ethyl butanoate, ethyl decanoate, and hexyl decanoate. Conversely, application of ethephon accelerated most of these physiological changes and also overcame most of the effects prevented by the ethylene action inhibitor. Altogether, the results supported the idea of a climacteric-like behavior for cape gooseberry fruits and pointing out that the pretreatment with 1-methylcyclopropene may be a promising and efficient postharvest treatment to delay maturity and extend the postharvest period.

Novel Bread Wheat Lines Enriched in Carotenoids Carrying Hordeum chilense Chromosome Arms in the ph1b Background.

The use of crop wild relative species to improve major crops performance is well established. Hordeum chilense has a high potential as a genetic donor to increase the carotenoid content of wheat. Crosses between the 7Hch H. chilense substitution lines in wheat and the wheat pairing homoeologous1b (ph1b) mutant allowed the development of wheat-H. chilense translocation lines for both 7Hchα and 7Hchß chromosome arms in the wheat background. These translocation lines were characterized by in situ hybridization and using molecular markers. In addition, reverse phase chromatography (HPLC) analysis was carried out to evaluate the carotenoid content and both 7Hchα∙7AL and 7AS∙7Hchß disomic translocation lines. The carotenoid content in 7Hchα∙7AL and 7AS∙7Hchß disomic translocation lines was higher than the wheat-7Hch addition line and double amount of carotenoids than the wheat itself. A proteomic analysis confirmed that the presence of chromosome 7Hch introgressions in wheat scarcely altered the proteomic profile of the wheat flour. The Psy1 (Phytoene Synthase1) gene, which is the first committed step in the carotenoid biosynthetic pathway, was also cytogenetically mapped on the 7Hchα chromosome arm. These new wheat-H. chilense translocation lines can be used as a powerful tool in wheat breeding programs to enrich the diet in bioactive compounds.

Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands.

Volatile organic compounds (VOCs) are secondary metabolites acting as a language for the communication of plants with the environment. In orange fruits, the monoterpene D-limonene accumulates at very high levels in oil glands from the peel. Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges harboring a D-limonene synthase transgene in antisense (AS) configuration altered the monoterpene profile in oil glands, mainly resulting in reduced accumulation of D-limonene. This led to fruit resistance against Penicillium digitatum (Pd), Xanthomonas citri subsp. citri (Xcc) and other specialized pathogens. Here, we analyze resistance to pathogens in independent AS and empty vector (EV) lines, which have low, medium or high D-limonene concentrations and show that the level of resistance is inversely related to the accumulation of D-limonene in orange peels, thus explaining the need of high D-limonene accumulation in mature oranges in nature for the efficient attraction of specialized microorganism frugivores.

Light avoidance reduces ascorbic acid accumulation in the peel of Citrus fruit.

Citrus fruits are highly consumed worldwide and represent one of the most important sources of ascorbic acid (AsA). However, information about the molecular mechanisms regulating AsA accumulation in Citrus fruit and the effects of environmental factors is scarce. In this study we have investigated the effect of fruit shading on AsA content and the expression of AsA biosynthetic, degrading and recycling genes in fruits of different Citrus species. Immature-green fruits were covered at the end of the cell enlargement phase and AsA concentration in the flavedo declined and remained at low levels as compared with light-exposed fruits. Fruit shading marginally altered the expression of genes from the l-galactose pathway and this effect was variable in the four Citrus species. However, specific isoforms (GalUR8 or GalUR12) from the l-galacturonic acid pathway were significantly repressed paralleling the reduction in AsA concentration. No significant effect of shading was detected in transcription of genes of the myo-inositol and l-gulose pathways as well as recycling and degradation. Collectively, results indicate that light avoidance inhibited accumulation of AsA in the flavedo of Citrus fruits and suggest that the l-galacturonic acid pathway has a relevant contribution to AsA content in this tissue.

Analysis of ethylene biosynthesis and perception during postharvest cold storage of Marsh and Star Ruby grapefruits.

Grapefruits are among the citrus species more sensitive to cold and develop chilling injury symptoms during prolonged postharvest storage at temperatures lower than 8 ℃-10 ℃. The plant hormone ethylene has been described either to protect or potentiate chilling injury development in citrus whereas little is known about transcriptional regulation of ethylene biosynthesis, perception and response during cold storage and how the hormone is regulating its own perception and signaling cascade. Then, the objective of the present study was to explore the transcriptional changes in the expression of ethylene biosynthesis, receptors and response genes during cold storage of the white Marsh and the red Star Ruby grapefruits. The effect of the ethylene action inhibitor, 1-MCP, was evaluated to investigate the involvement of ethylene in the regulation of the genes of its own biosynthesis and perception pathway. Ethylene production was very low at the harvest time in fruits of both varieties and experienced only minor changes during storage. By contrast, inhibition of ethylene perception by 1-MCP markedly induced ethylene production, and this increase was highly stimulated during shelf-life at 20 ℃, as well as transcription of ACS and ACO. These results support the auto-inhibitory regulation of ethylene in grapefruits, which acts mainly at the transcriptional level of ACS and ACO genes. Moreover, ethylene receptor1 and ethylene receptor3 were induced by cold while no clear role of ethylene was observed in the induction of ethylene receptors. However, ethylene appears to be implicated in the transcriptional regulation of ERFs both under cold storage and shelf-life.