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A comprehensive study of the transcriptional regulation of jasmonate metabolism, perception and signaling by heat, cold stress, and heat-induced chilling tolerance in ‘Fortune’ mandarin was performed. Changes in the expression of a set of 22 relevant jasmonate-related genes were evaluated during fruit storage at a chilling (2 °C) and a non-chilling control temperature (12 °C); and during fruit storage at 2 °C after being heat-conditioned (37 °C for 3 d). The changes occurring in response to heat (37 °C) were also examined. The results indicated that the genes participating in cis-(+)− 12-oxo-phytodienoic (OPDA) (CsLOX1, CsAOS, CsAOC3) and jasmonic acid (JA) (CsOPR2) synthesis would be, among the cold-responsive genes, the best markers of chilling stress leading to peel damage in citrus fruit. Most jasmonate-related genes are regulated by heat, but the genes regulated by cold stress in the heated-fruit, respect to the unheated, may be especially relevant for heat-induced chilling tolerance. Major effects of heat+cold, as compared to cold, were found in the expression of genes participating in the biosynthesis of OPDA (CsLOX5 and CsAOC3), JA (CsOPR3 and CsKAT2) and jasmonoyl-isoleucine (JA-Ile) (CsJAR1, CsCYP94B3 and CsCYP94C); but also in the expression of genes involved in JA perception (CsCOI1) and signaling, including the jasmonate transcriptional activator CsMYC2 and abundant negative regulators (CsJAZs and CsTOPLESS).

Fruit nutritional value, plant growth, and yield can be compromised by deficient copper (Cu) bioavailability, which often appears in arable lands. This condition causes low Cu content and modifications in the ripening-associated processes in tomato fruit. This research studies the transcriptomic changes that occur in red ripe tomato fruit grown under suboptimal Cu conditions to shed light on the molecular mechanisms underlying this stress. Comparative RNA-sequencing and functional analyses revealed that Cu deficiency during cultivation activates signals for metal ion transport, cellular redox homeostasis, pyridoxal phosphate binding, and amino acid metabolism while repressing the response to phosphate starvation in harvested fruit. Transcriptomic analyses highlighted a number of novel Cu stress-responsive genes of unknown function and indicated that Cu homeostasis regulation in tomato fruit may involve additional components than those described in model plants. It also studied the regulation of high-affinity Cu transporters and a number of well-known Cu stress-responsive genes during tomato fruit ripening depending on Cu availability, which allowed potential candidates to be targeted for biotechnological improvements in reproductive tissues. We provide the first study characterizing the molecular responses of fruit to Cu deficiency stress for any fruit crop.

The involvement of jasmonic acid (JA), different JA-related metabolites, salicylic acid (SA), indole-3-acetic acid (IAA) and abscisic acid (ABA) in the heat-induced cross-adaptation to chilling has been investigated in citrus fruit. The effects of heating ‘Fortune’ mandarins at 37 ºC, and storing them at a chilling temperature (2 ºC) after being exposed, or not, to a heat-conditioning treatment (3 d at 37 ºC) on the levels of these signalling molecules were examined. Jasmonate metabolism activation at 37 ºC was followed by that of SA, and then by a rise in IAA and a drop in ABA. Storage at 2 ºC transiently increased the contents of IAA, of the JA precursor, cis-(+)− 12-oxo-phytodienoic acid (OPDA), and of the JA-derivatives jasmonoyl-isoleucine (JAIle) and methyl jasmonate (MeJA), and decreased ABA in the non-conditioned fruit. The results also indicated that fruit were protected from developing chilling symptoms by virtue of the heat-conditioning treatment having higher JAIle levels than the non-conditioned fruit for very long cold storage periods, while the heat-induced rises in SA, OPDA and MeJA noted in the cold-stored fruit were transient.

Copper (Cu) is an essential micronutrient for plants because it functions as a redox-active cofactor in vital processes inside the cells. Arable lands are often deficient in micronutrient contents and require the application of enriched fertilisers, whose overuse poses a high risk for human health, the environment and the food safety. Here, we aimed to decipher the effects of Cu deficiency during fruit growth on Cu and other micronutrients contents and on the fruit nutritional value and quality of tomato, the most consumed fruit worldwide, throughout the maturation process. Changes in the contents of important micronutrients for fruit physiology and human health, such as Fe and Mn, occurred in response to Cu deficient growing conditions at different fruit ripening stages, while lower Cu levels were detected in those fruit along the whole maturation process. Cu deficiency delayed changes in lycopene content and fruit colour, but increased acidity, and advanced the rise in antioxidant capacity and vitamin C content during fruit colour change from green to light red in the Moneymaker tomato; although this time lag eventually caught up in the most mature fruit stage. Cu deficiency also increased total phenolic and flavonoid contents only in green fruit.

Epicuticular waxes are important natural compounds that influence cuticle properties and can protect fruit from factors that harm its external quality. We demonstrated that, at a dose that reduces postharvest citrus fruit quality loss (4 d 2 µL L−1), ethylene redirected epicuticular wax metabolism towards the synthesis of primary alcohols, mostly behenyl alcohol, by favouring the acyl-reduction pathway. This treatment also reduced the synthesis of terpenoids by redirecting the mevalonate pathway towards farnesol accumulation to the detriment of the accumulation of most triterpenoids, but not of their precursor squalene. Moreover, the 4 d ethylene treatment sharply increased the synthesis of docosane and lignoceric acid and lowered that of cerotic acid. Longer ethylene exposure (8 d) reversed some of these effects by lowering the contents of most alcohols, lignoceric acid and squalene, while increasing that of its derivative sitosterol. The 8 d ethylene treatment also increased farnesol and docosane contents.

The phytohormone abscisic acid (ABA) is a major regulator of fruit response to water stress, and may influence cuticle properties and wax layer composition during fruit ripening. This study investigates the effects of ABA on epicuticular wax metabolism regulation in a citrus fruit cultivar with low ABA levels, called Pinalate (Citrus sinensis L. Osbeck), and how this relationship is influenced by water stress after detachment. Harvested ABA-treated fruit were exposed to water stress by storing them at low (30–35%) relative humidity. The total epicuticular wax load rose after fruit detachment, which ABA application decreased earlier and more markedly during fruit-dehydrating storage. ABA treatment changed the abundance of the separated wax fractions and the contents of most individual components, which reveals dependence on the exposure to postharvest water stress and different trends depending on storage duration. A correlation analysis supported these responses, which mostly fitted the expression patterns of the key genes involved in wax biosynthesis and transport. A cluster analysis indicated that storage duration is an important factor for the exogenous ABA influence and the postharvest environment on epicuticular wax composition, cuticle properties and fruit physiology. Dynamic ABA-mediated reconfiguration of wax metabolism is influenced by fruit exposure to water stress conditions.

Penicillium digitatum is the main postharvest pathogen of citrus fruit. Although the inner fruit peel part (albedo) is less resistant than the outer part (flavedo) to P. digitatum, the global mechanisms involved in their different susceptibility remain unknown. Here, we examine transcriptome differences between both tissues at fruit harvest and in their early responses to infection. At harvest, not only was secondary metabolism, involving phenylpropanoids, waxes, and terpenoids, generally induced in flavedo vs. albedo, but also energy metabolism, transcription factors (TFs), and biotic stress-related hormones and proteins too. Flavedo-specific induced responses to infection might be regulated in part by ERF1 TF, and are related to structural plant cell wall reinforcement. Other induced responses may be related to H2O2, the synthesis of phenylpropanoids, and the stress-related proteins required to maintain basal defense responses against virulent pathogens, whereas P. digitatum represses some hydrolase-encoding genes that play different functions and auxin-responsive genes in this peel tissue. In infected albedo, the repression of transport and signal transduction prevail, as does the induction of not only the processes related to the synthesis of flavonoids, indole glucosinolates, cutin, and oxylipins, but also the specific genes that elicit plant immunity against pathogens.

Water stress is the most important environmental agent that contributes to the crop productivity and quality losses globally. In citrus, water stress is the main driver of the fruit peel disorders that impact the quality and market ability. An increasingly present post-harvest peel disorder is non-chilling peel pitting (NCPP). Non-chilling peel pitting is manifested as collapsed areas of flavedo randomly scattered on the fruit and its incidence increases due to abrupt increases in the environmental relative humidity (RH) during post-harvest fruit manipulation. In this study, we have used a custom-made cDNA microarray containing 44k unigenes from Citrus sinensis (L. Osbeck), covering for the first time the whole genome from this species, to study transcriptomic responses of mature citrus fruit to water stress. In the study, the global gene expression profiles of flavedo from Navelate oranges subjected to severe water stress are compared with those fruits subjected to rehydration stress provoked by changes in the RH during post-harvest, which enhances the development of NCPP. The study results show that NCPP is a complex physiological process that shares molecular responses with those from prolonged dehydration in fruit, but the damage associated with NCPP may be explained by unique features of rehydration stress at the molecular level, such as membrane disorganization, cell wall modification, and proteolysis.

Citrus fruit ripening is coupled with the synthesis and deposition of epicuticular waxes, which reduces water loss during fruit postharvest storage. Although abscisic acid (ABA) is a major regulator of citrus fruit ripening, whether ABA mediates epicuticular wax formation during this process remains poorly understood. We investigated the implication of ABA in cuticle properties and epicuticular wax metabolism, composition, and morphology by comparing the Navelate orange [Citrus sinensis (L.) Osbeck] and its ABA biosynthesis-impaired mutant Pinalate in four ripening stages. ABA deficiency had minor effects on cuticle thickness and epicuticular wax load, but correlated with cuticle permeability. ABA content aligned with mostly fatty acids accumulation in both cultivars, and also with specific alkane, terpenoid, and aldehyde constituents in the parental fruit. In turn, cuticle permeability correlated with the fatty acid profile during fruit ripening in the Navelate and Pinalate, and with primary alcohols, terpenoids, and aldehydes, but only in the mutant fruit. Low ABA levels increased the susceptibility of waxes to crack and were lost from the epicuticular layer. The RNA-seq analysis highlighted the differential regulation of a list of 87 cuticle-related genes between genotypes and ripening stages. Changes in the gene expression of the selected genes in both cultivars were consistent with the content of the aliphatics and terpenoid fractions during ripening. The results suggest a role for ABA in the regulation of fatty acid content and primary alcohol composition, and point out the importance of alkane and triterpenoid for controlling water permeance through fruit cuticles.

The biochemical changes induced by LED Blue Light (LBL) (450 nm) in Lane Late oranges were investigated. The selected quantum flux (60 µmol m−2 s−1, 2 days) was associated with resistance against Penicillium digitatum, the main postharvest pathogen of citrus fruit. A holistic overview was obtained by a comparative transcriptome profile analysis, which revealed that LBL favored energy metabolism and redirected metabolic pathways toward the synthesis of diverse primary and secondary metabolism products. LBL favored reactive oxygen species homeostasis and metabolic activities involving lipid metabolism, specifically the synthesis of pigments and oxylipins, and the metabolism of carbohydrates, amino acids and indol- and alkaloid-derivatives. LBL also repressed limonene catabolism and triggered phenylpropanoid derivatives-related changes, which increased content in total flavonoids. Transferring fruit from LBL to darkness favored those processes involving amino acids, different phenylpropanoid, alkaloid and terpenoid classes, and ferrochelatase activity.

Harvested fruit undergo carbon and energy deprivation. However, the events underlying this energy-related stress in detached fruit and their involvement in cell damage have not yet been elucidated. We showed that supplementing detached sweet oranges with additional carbon or energy sources reduced peel damage, while inhibitors of energy metabolism increased it. We investigated the effect of an exogenous source of carbon (glycerol), energy (ATP), and an inhibitor of energy metabolism 2-deoxy-D-glucose (DeOGlc) + sodium iodoacetate (IAc), on the transcriptome of harvested fruit flavedo (outer peel part). ATP and Gly induced common, but also specific, alternative modes of energy metabolism by reducing the stress caused by energy shortage. They also induced shifts in energy metabolism that led to the production of the intermediates required for plant defense secondary metabolites to form. ATP and Gly triggered changes in the expression of the genes involved in cell lesion containment through a defined pathway involving hormones and redox-mediated signaling. DeOGlc + IAc had a contrasting effect on some of these mechanisms. These chemicals altered the biological processes related to membrane integrity and molecular mechanisms involving reactive oxygen species (ROS) production, and lipid and protein degradation.

Copper (Cu) deficiency affects iron (Fe) homeostasis in several plant processes, including the increased Fe requirements due to cuproprotein substitutions for the corresponding Fe counterpart. Loss-of-function mutants from Arabidopsis thaliana high affinity copper transporter COPT5 and Fe transporters NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 3/4 (NRAMP3 and NRAMP4) were used to study the interaction between metals internal pools. A physiological characterisation showed that the copt5 mutant is sensitive to Fe deficiency, and that nramp3nramp4 mutant growth was severely affected under limiting Cu. By a transcriptomic analysis, we observed that NRAMP4 expression was highly induced in the copt5 mutant under Cu deficiency, while COPT5 was overexpressed in the nramp3nramp4 mutant. As a result, an enhanced mobilisation of the vacuolar Cu or Fe pools, when the other metal export through the tonoplast is impaired in the mutants, has been postulated. However, metals coming from internal pools are not used to accomplish the increased requirements that derive from metalloprotein substitution under metal deficiencies. Instead, the metal concentrations present in aerial parts of the copt5 and nramp3nramp4 mutants conversely show compensated levels of these two metals. Together, our data uncover an interconnection between Cu and Fe vacuolar pools, whose aim is to fulfil interorgan metal translocation.

Citrus fruit ripening is a complex process involving biochemical, physiological and molecular events that differ between the flesh and the peel of the fruit. We characterized sweet orange peel maturation by means of a comparative transcriptomic analysis between Navelate orange (Citrus sinensis L. Osbeck) and its mutant fruit Pinalate, which presents a severe blockage at early steps of the carotenoid biosynthetic pathway and consequently reduced ABA levels. Peel ripening involved the decrease of the photosynthetic activity and the transmembrane transport processes, as well as the buildup of starch and cuticular waxes and the cell wall modification. In addition, a number of biotic and abiotic stress responses, including the defense response, and the response to blue light, water deprivation and abscisic acid stimulus were modulated in a ripening-stage specific manner. The regulation of energy-related processes and secondary metabolism pathways was attenuated in Pinalate, while the molecular mechanisms underlying stress responses displayed dependency on ABA levels. These results indicate that ABA is a key signal inducing stress responses along orange peel ripening, which might determine the fruit postharvest performance.

The ethylene perception inhibitor 1-methylcyclopropene (1-MCP) has been critical in understanding the hormone's mode of action. However, 1-MCP may trigger other processes that could vary the interpretation of results related until now to ethylene, which we aim to understand by using transcriptomic analysis. Transcriptomic changes in ethylene and 1-MCP-treated ‘Navelate’ (Citrus sinensis L. Osbeck) oranges were studied in parallel with changes in ethylene production, respiration and peel damage. The effects of compounds modifying the levels of the ethylene co-product cyanide and nitric oxide (NO) on fruit physiology were also studied. Results suggested that: 1) The ethylene treatment caused sub-lethal stress since it induced stress-related responses and reduced peel damage; 2) 1-MCP induced ethylenedependent and ethylene-independent responsive networks; 3) 1-MCP triggered ethylene overproduction, stress-related responses and metabolic shifts aimed to cope with cell toxicity, which mostly affected to the inner part of the peel (albedo); 4) 1-MCP increased respiration and drove metabolism reconfiguration for favoring energy conservation but up-regulated genes related to lipid and protein degradation and triggered the over-expression of genes associated with the plasma membrane cellular component; 5) Xenobiotics and/or reactive oxygen species (ROS) might act as signals for defense responses in the ethylene-treated fruit, while their uncontrolled generation would induce processes mimicking cell death and damage in 1-MCP-treated fruit; 6) ROS, the ethylene co-product cyanide and NO may converge in the toxic effects of 1-MCP.

Water stress affects many agronomic traits that may be regulated by the phytohormone abscisic acid (ABA). Within these traits, loss of fruit quality becomes important in many citrus cultivars that develop peel damage in response to dehydration. To study peel dehydration transcriptional responsiveness in harvested citrus fruit and the putative role of ABA in this process, this study performed a comparative large-scale transcriptional analysis of water-stressed fruits of the wild-type Navelate orange (Citrus sinesis L. Osbeck) and its spontaneous ABA-deficient mutant Pinalate, which is more prone to dehydration and to developing peel damage. Major changes in gene expression occurring in the wild-type line were impaired in the mutant fruit. Gene ontology analysis revealed the ability of Navelate fruits to induce the response to water deprivation and di-, tri-valent inorganic cation transport biological processes, as well as repression of the carbohydrate biosynthesis process in the mutant. Exogenous ABA triggered relevant transcriptional changes and repressed the protein ubiquitination process, although it could not fully rescue the physiological behaviour of the mutant. Overall, the results indicated that dehydration responsiveness requires ABA-dependent and-independent signals, and highlight that the ability of citrus fruits to trigger molecular responses against dehydration is an important factor in reducing their susceptibility to developing peel damage.

The aim of this work has been to unravel the influence of the phytohormone ABA in the molecular mechanisms underlying the postharvest dehydration response and the development and ripening of citrus fruit, taking advantage of the spontaneous fruit-specific ABA-deficient 'Pinalate' mutant, which is more prone to dehydration and to develop non-chilling peel pitting (NCPP) than its wild-type 'Navelate' orange. A comparative transcriptomic analysis highlighted the ability of parental fruit to induce early molecular responses aimed to reduce water loss and its detrimental effects. ABA application to mutant fruit modulated relevant transcriptomic changes but did not substantially modify either dehydration rate or NCPP incidence, which suggested that 'Pinalate' mutant could be insensitive to ABA. Therefore, the ABA perception system components in Citrus were identified and their regulation under developmental and stressful conditions was investigated. Minor differences between varieties were found in the CsPYR/PYL/RCAR ABA receptors and the CsSnRK2s downstream protein kinases transcript levels, whereas CsPP2CAs negative regulators accumulation was lower in the mutant fruit. ABA receptors and CsSnRK2s gene expression patterns depended on the tissue, the stress severity and the source of the ABA signal from a developmental or stressful stimulus, whilst CsPP2CAs displayed a consistent pattern. Overall, this work suggest that the ABA-deficient 'Pinalate' fruit may sense ABA although the hormone signal could be impaired because reduced CsPP2CAs levels causing altered water stress response and higher NCPP susceptibility.