Molecular Responses of Red Ripe Tomato Fruit to Copper Deficiency Stress.

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.

Deficient copper availability on organoleptic and nutritional quality of tomato fruit.

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.

Relative humidity regimes modify epicuticular wax metabolism and fruit properties during Navelate orange conservation in an ABA-dependent manner.

Relative humidity (RH) during conservation and the chemical composition of epicuticular wax layer are factors that determine fruit quality and weight loss. This study investigates the influence of RH on the epicuticular wax metabolism during citrus fruit storage, and how it is affected by abscisic acid (ABA). Low RH conditions increased alcohols and fatty acids abundance, mainly due to accumulation of docosanol and lignoceric and cerotic acids. Low RH also decreased terpenoids and nonacosane and hentriacontane contents, the most abundant alkanes. Consequently, the alkane/terpenoid ratio was decreased concomitantly with fruit weight loss and cuticle permeability increments. ABA treatment differently mediated wax compositional changes at high or low RH. At low RH, ABA attenuated the increase in fatty acids and enhanced the decrease in alcohols and the accumulation of terpenoids, mainly affecting lignoceric and cerotic acids, docosanol, α-amyrin, sitosterol, friedelin and friedelanone contents. These trends were inversed under high RH conditions.

Ethylene-driven changes in epicuticular wax metabolism in citrus 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 Combination of Abscisic Acid (ABA) and Water Stress Regulates the Epicuticular Wax Metabolism and Cuticle Properties of Detached Citrus Fruit.

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.

Identification and molecular characterization of the high-affinity copper transporters family in Solanum lycopersicum.

Copper (Cu) plays a key role as cofactor in the plant proteins participating in essential cellular processes, such as electron transport and free radical scavenging. Despite high-affinity Cu transporters (COPTs) being key participants in Cu homeostasis maintenance, very little is known about COPTs in tomato (Solanum lycopersicum) even though it is the most consumed fruit worldwide and this crop is susceptible to suboptimal Cu conditions. In this study, a six-member family of COPT (SlCOPT1-6) was identified and characterized. SlCOPTs have a conserved architecture consisting of three transmembrane domains and ß-strains. However, the presence of essential methionine residues, a methionine-enriched amino-terminal region, an Mx3Mx12Gx3G Cu-binding motif and a cysteine rich carboxy-terminal region, all required for their functionality, is more variable among members. Accordingly, functional complementation assays in yeast indicate that SlCOPT1 and SlCOPT2 are able to transport Cu inside the cell, while SlCOPT3 and SlCOPT5 are only partially functional. In addition, protein interaction network analyses reveal the connection between SlCOPTs and Cu PIB-type ATPases, other metal transporters, and proteins related to the peroxisome. Gene expression analyses uncover organ-dependency, fruit vasculature tissue specialization and ripening-dependent gene expression profiles, as well as different response to Cu deficiency or toxicity in an organ-dependent manner.

Differential Transcriptomic Regulation in Sweet Orange Fruit (Citrus sinensis L. Osbeck) Following Dehydration and Rehydration Conditions Leading to Peel Damage.

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.

Albedo- and Flavedo-Specific Transcriptome Profiling Related to Penicillium digitatum Infection in Citrus Fruit.

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.

Abscisic Acid Deficiency Alters Epicuticular Wax Metabolism and Morphology That Leads to Increased Cuticle Permeability During Sweet Orange (Citrus sinensis) Fruit Ripening.

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.

A sweet orange mutant impaired in carotenoid biosynthesis and reduced ABA levels results in altered molecular responses along peel ripening.

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.

High-temperature conditioning induces chilling tolerance in mandarin fruit: a cell wall approach.

BACKGROUND: High-temperature conditioning (3 days at 37 °C and 95% relative humidity), which protects 'Fortune' mandarins from chilling injury (CI), manifested as pitting in the outer part of the peel (flavedo), was applied prior to cold storage (2 °C) in order to investigate the involvement of cell wall composition in the chilling tolerance of mandarins. RESULTS: Both low-temperature storage and high-temperature conditioning barely modified the alcohol-insoluble substance (AIS) content or the degree of pectin esterification in the flavedo. Water-soluble pectins (WSP) were higher in heat-conditioned than in non-conditioned fruits at the onset of CI. In addition, the heat-conditioning treatment was able to increase chelator-soluble pectins (CSP) after short cold storage periods. Covalently bound polyuronides in alkali-soluble pectins (ASP) increased only in fruits with high incidence of CI. Cellulose and hemicellulose increased at 2 °C in both conditioned and non-conditioned fruits, indicating that these polysaccharides may be altered by low temperature but are not related to chilling-induced damage. CONCLUSION: High-temperature conditioning may reduce chilling-induced flavedo pitting in 'Fortune' mandarin fruit by maintaining normal levels of WSP and increasing putative sites for calcium bridge formation within the cell wall, but not by inducing changes in other matrix cell wall components.

Dehydrin from citrus, which confers in vitro dehydration and freezing protection activity, is constitutive and highly expressed in the flavedo of fruit but responsive to cold and water stress in leaves.

A cDNA encoding a dehydrin was isolated from the flavedo of the chilling-sensitive Fortune mandarin fruit (Citrus clementina Hort. Ex Tanaka x Citrus reticulata Blanco) and designed as Crcor15. The predicted CrCOR15 protein is a K2S member of a closely related dehydrin family from Citrus, since it contains two tandem repeats of the unusual Citrus K-segment and one S-segment (serine cluster) at an unusual C-terminal position. Crcor15 mRNA is consistently and highly expressed in the flavedo during fruit development and maturation. The relative abundance of Crcor15 mRNA in the flavedo was estimated to be higher than 1% of total RNA. The high mRNA level remained unchanged during fruit storage at chilling (2 degrees C) and nonchilling (12 degrees C) temperatures, and it was depressed by a conditioning treatment (3 days at 37 degrees C) that induced chilling tolerance. Therefore, the expression of Crcor15 appears not to be related to the acquisition of chilling tolerance in mandarin fruits. However, Crcor15, which was barely detected in unstressed mandarin leaves, was rapidly induced in response to both low temperature and water stress. COR15 protein was expressed in Escherichia coli, and the purified protein conferred in vitro protection against freezing and dehydration inactivation. The potential role of Citrus COR15 is discussed.

A survey of genes differentially expressed during long-term heat-induced chilling tolerance in citrus fruit.

Long-term storage at low, non-freezing, temperature (1.5 degrees C) induces chilling injury in fruit of Fortune mandarin (Citrus clementina Hort. Ex Tanaka x Citrus reticulata, Blanco), manifested as pitting and brown depressed areas that may end up with local cell death. Pre-conditioning of fruit for 3 days at 37 degrees C prevented chilling injury. The use of suppression subtractive hybridization permitted the isolation of genes differentially expressed in heat-conditioned fruit exposed to chilling conditions, which may be candidates for heat-induced chilling tolerance. Northern blot analysis revealed that some genes were up-regulated by prolonged heat (3 days/37 degrees C) and their expression persisted in fruit cells upon subsequent chilling exposure. The expression of other genes was specifically induced by the combination of heat and cold. Among the putative tolerance-associated genes, we identified two transcription factors of the WRKY family and one TFIIB factor. Heat conditioning also altered the expression of genes encoding proteins involved in secondary metabolism, cell wall modification, oxidative damage and other stress-responsive proteins. These results illustrate the complexity of molecular mechanisms operating during heat-induced chilling tolerance in citrus fruit.