Fernando Alférez

Unravelling molecular responses to moderate dehydration in harvested fruit of sweet orange (Citrus sinensis L. Osbeck) using a fruit-specific ABA-deficient mutant

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.

Effect of LED Blue Light on Penicillium digitatum and Penicillium italicum Strains

Studies on the antimicrobial properties of light have considerably increased due in part to the development of resistance to actual control methods. This study investigates the potential of light‐emitting diodes (LED) blue light for controlling Penicillium digitatum and Penicillium italicum. These fungi are the most devastating postharvest pathogens of citrus fruit and cause important losses due to contaminations and the development of resistant strains against fungicides. The effect of different periods and quantum fluxes, delaying light application on the growth and morphology of P. digitatum strains resistant and sensitive to fungicides, and P. italicum cultured at 20°C was examined. Results showed that blue light controls the growth of all strains and that its efficacy increases with the quantum flux. Spore germination was always avoided by exposing the cultures to high quantum flux (700 μmol m−2 s−1) for 18 h. Continuous light had an important impact on the fungus morphology and a fungicidal effect when applied at a lower quantum flux (120 μmol m−2 s−1) to a growing fungus. Sensitivity to light increased with mycelium age. Results show that blue light may be a tool for P. digitatum and P. italicum infection prevention during handling of citrus fruits.

A transcriptional approach to unravel the connection between phospholipases A2 and D and ABA signal in citrus under water stress

The effect of water stress on the interplay between phospholipases (PL) A2 and D and ABA signalling was investigated in fruit and leaves from the sweet orange Navelate and its fruit-specific ABA-deficient mutant Pinalate by studying simultaneously expression of 5 PLD and 3 PLA2-encoding genes. In general, expression levels of PLD-encoding genes were higher at harvest in the flavedo (coloured outer part of the peel) from Pinalate. Moreover, a higher and transient increase in expression of CsPLDα, CsPLDβ, CsPLDδ and CsPLDζ was observed in the mutant as compared to Navelate fruit under water stress, which may reflect a mechanism of acclimation to water stress influenced by ABA deficiency. An early induction in CsPLDγ gene expression, when increase in peel damage during fruit storage was most evident, suggested a role for this gene in membrane degradation processes during water stress. Exogenous ABA on mutant fruit modified the expression of all PLD genes and reduced the expression of CsPLDα and CsPLDβ by 1 week to levels similar to those of Navelate, suggesting a repressor role of ABA on these genes. In general, CssPLA2α and β transcript levels were lower in flavedo from Pinalate than from Navelate fruit during the first 3 weeks of storage, suggesting that expression of these genes also depends at least partially on ABA levels. Patterns of expression of PLD and PLA2-encoding genes were very similar in Navelate and Pinalate leaves, which have similar ABA levels, when comparing both RH conditions. Results comparison with other from previous works in the same experimental systems helped to decipher the effect of the stress severity on the differential response of some of these genes under dehydration conditions and pointed out the interplay between PLA2 and PLD families and their connection with ABA signalling in citrus.

Influence of fruit maturity in the susceptibility of Navelina oranges to develop postharvest non-chilling peel pitting

Peel pitting is a disorder occurring mostly during postharvest storage at non-chilling temperatures in different varieties of citrus fruit and consists in collapse of flavedo and albedo tissues that may affect oil glands. It has been demonstrated that during postharvest, sharp variations in water potential of cells from flavedo and albedo are sufficient to provoke fractures in cell walls from external albedo resulting in tissue collapse. However, morphology and composition of cells and cell walls in flavedo and albedo varies during fruit maturation and this may affect water flow through the different fruit peel layers and susceptibility of fruit to develop peel pitting. In this paper, we have studied the influence of the stage of maturation in the susceptibility of Navelina orange to develop peel pitting. Except in mature-green fruit, peel pitting increased with maturation after transferring fruit from 45% to 95% relative humidity and was also more severe as more dehydrated was the tissue before transference. Also, differences in water potential of fruit maintained at 45 or 95% relative humidity increased as fruit matured, suggesting that tissue reduces the ability of water adjustment during maturation. In this sense, only mature-green fruit flavedo was able to recover water potential when transferred from 45 to 95% relative humidity. Ethylene production upon transfer from low to high relative humidity increased only in mature tissue and was rapid and transient, and before initial symptoms of peel pitting. Flavedo and albedo water potential (ψw) was substantially reduced during fruit maturation. As lower was the ψw of freshly harvested fruit, minor variations were observed by changes in the storage relative humidity and higher the induced damage. Therefore, the increasing susceptibility of Navelina fruits to develop peel pitting with fruit maturation may be related to a reduced ability to regulate peel evapotranspiration and osmotic adjustment during postharvest storage.

Light-emitting Diode Blue Light Alters the Ability of Penicillium digitatum to Infect Citrus Fruits

Penicillium digitatum (Pers.:Fr.) Sacc. is the main fungus causing postharvest losses in citrus fruits. Previous work showed the potential of LED blue light (LBL) in controlling P. digitatum growth. Here, we have investigated whether LBL alters the ability of this fungus to infect citrus fruits. Before fruit infection, Petri plates inoculated with the same conidia concentration were held under darkness (control) or LBL (100 μmol m−2 s−1) for 8 d (continuous light), or were treated with the same LBL for 3 d and then shifted to darkness for 5 d (non‐continuous light). Spores from cultures exposed to continuous light showed very low capacity to germinate (1.8% respect to control) but a high viability and a similar morphology and ability to infect the fruits than spores from control cultures. The number of spores produced in plates exposed to non‐continuous light was slightly lower than in control plates, but they showed much lower viability and lower capacity to infect the fruits. This effect was more likely related to aberrant morphology of spores, which formed aggregates, than to its metabolic activity or its ability to produce ethylene that might contribute to destroy natural defense barriers from the fruit.

Regulation by Carbon Dioxide of Wound-induced Ethylene Biosynthesis in the Peel of Citrus Fruit

The effect of carbon dioxide (CO2) on wound-induced ethylene biosynthesis in flavedo discs of mature orange fruits (Citrus sinensis L. Osbeck) is investigated. Wounding induced a marked and rapid increase on the rate of ethylene production, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) and on the in vivo ACC oxidase (ACO) activity. Incubation of flavedo discs in a 15% CO2 atmosphere suppressed activation of these processes. Wound-induced ethylene production was inhibited by CO2 in a concentration-dependent manner but ACO activity was enhanced at concentrations between 1% and 5%. Kinetic analysis of the interaction between CO2 and ACO activity indicated that high CO2 acted as a noncompetitive inhibitor. Removal of CO2 after 24 h incubation did not restore normal rates of ethylene production. CO2 partially counteracted the increase in ethylene production and ACO activity induced by a pretreatment with an ethylene action inhibitor (STS, silver thiosulfate). This suggested that part of CO2 action on ethylene biosynthesis might be due to interfering ethylene action. Collectively, the results indicated that ACS activity appeared to be the major regulatory step by which CO2 suppresses wound-induced ethylene production. ACO was differentially modulated by CO2, which is being stimulated at low concentrations and inhibited at high concentrations.