Proline accumulation and antioxidant response are crucial for citrus tolerance to UV-B light-induced stress.

Plants face a wide range of biotic and abiotic stress conditions, which are further intensified by climate change. Among these stressors, increased irradiation in terms of intensity and wavelength range can lead to detrimental effects, such as chlorophyll degradation, destruction of the PSII reaction center, generation of ROS, alterations to plant metabolism, and even plant death. Here, we investigated the responses of two citrus genotypes, Citrus macrophylla (CM), and Troyer citrange (TC) to UV-B light-induced stress, by growing plants of both genotypes under control and UV-B stress conditions for 5 days to evaluate their tolerance mechanisms. TC seedlings had higher sensitivity to UV-B light than CM seedlings, as they showed more damage and increased levels of oxidative harm (indicated by the accumulation of MDA). In contrast, CM seedlings exhibited specific adaptive mechanisms, including accumulation of higher levels of proline under stressful conditions, and enhanced antioxidant capacity, as evidenced by increased ascorbate peroxidase activity and upregulation of the CsAPX2 gene. Phytohormone accumulation patterns were similar in both genotypes, with a decrease in ABA content in response to UV-B light. Furthermore, expression of genes involved in light perception and response was specifically affected in the tolerant CM seedlings, which exhibited higher expression of CsHYH/CsHY5 and CsRUP1-2 genes. These findings underscore the importance of the antioxidant system in citrus plants subjected to UV-B light-induced stress and suggest that CsHYH/CsHY5 and CsRUP1-2 could be considered genes associated with tolerance to such challenging conditions.

Effect of Extraction Procedures with Ultrasound and Cellulolytic Enzymes on the Structural and Functional Properties of Citrus grandis Osbeck Seed Mucilage.

The structural and functional properties of Citrus grandis Osbeck (CGO) seed mucilage by different extraction practices, including conventional citrate buffer, ultrasonic-assisted (UAE), enzymatic-assisted extraction (EAE) with cellulase or Celluclast® 1.5 L and various ultrasonic-assisted enzymatic extraction (UAEE) procedures were investigated. It was found that CGO seed from agricultural and processing byproducts is an excellent new source of high methoxyl pectin with quite high intrinsic viscosity (about 108.64 dL/g) and molecular weight (about 1.9 × 106) as compared with other pectin sources. UAEE with Celluclast® 1.5 L enhanced the extraction yield most pronouncedly (about 2.3 times). Moreover, the monosaccharide composition of CGO seed mucilage is least affected by EAE with Celluclast® 1.5 L. In contrast, EAE with cellulase dramatically reduces the galacturonic acid (GalA) content to less than 60 molar%, and increases the glucose (Glc) content pronouncedly (to about 40 molar%), which may be considered as an adverse effect in terms of pectin purity. Though extraction procedures involved with ultrasound and cellulolytic enzymes generally show a decrease in GalA contents, weight average molar mass and intrinsic viscosity, EAE with Celluclast® 1.5 L is least affected, followed by UAE and UAEE with Celluclast® 1.5 L. These features can be leveraged in favor of diversified applications.

Application of accelerating energies to the maceration of sherry vinegar with citrus fruits.

BACKGROUND: In the present work we propose the use of accelerating energies (microwaves and ultrasounds) to the maceration process of sherry vinegar with citrus fruit peels (orange and lemon). For the application of microwaves, an experimental design has been developed in order to optimize the maceration conditions. To evaluate the effect of these energies on the maceration, the volatile and polyphenolic content of the samples has been analyzed, as well as their sensory characteristics. RESULTS: Orange peel provided a higher number of volatile and polyphenolic compounds to the vinegar, while lemon's yielded a greater amount of some of them. The multivariate analysis showed that the samples macerated using microwaves were the most similar to the samples macerated in the traditional way. This aspect was corroborated by the sensory analysis, which was more noticeable when orange peel was used in the macerations. CONCLUSION: Therefore, it seems that the use of microwaves to accelerate maceration is a good alternative to the traditional method of making sherry vinegars macerated with citrus peel, since it cuts down the maceration time from 3 days to just a few minutes. © 2020 Society of Chemical Industry.

Inducing mutations in Citrus spp.: Sensitivity of different sources of plant material to gamma radiation.

Gamma radiation represents an alternative for improving genotypes for which breeding through hybridization involves many difficulties because of their complex reproductive biology, as in the case of citrus. In this study in vitro and ex vitro mutagenesis was induced by gamma radiation in several citrus species ('Alemow' and sour orange as citrus rootstocks, lemon cv. 'Fino 49' and 'Verna 51', mandarin cv. 'Nova' and lime cv. 'Bearss'). Three different sources of materials - seeds, budwoods and nodal segments - from in vitro explants, were tested. Seeds and budwoods were germinated or induced to sprout, and de novo regeneration was obtained from in vitro nodal segments from which preexisting buds were previously removed. Budwoods were tested in summer and winter in order to assess their capacity for mutation and further sprouted in different seasons. Seeds were seen to be more resistant to gamma radiation (LD50 of 127 Gy in 'Alemow and 156 Gy in sour orange) than budwoods (LD50 about 50 Gy for all cultivars) and nodal segments (LD50 about 25 Gy for both lemon cultivars), the last being the most sensitive material tested. Similar LD50 were found for budwoods collected in winter and summer for all the genotypes. All the tested plant material can be considered suitable for gamma irradiation, although budwood is the most widely available and tissue culture material needs the highest degree of expertise.

Effects of acoustic power and pH on pectin-enriched extracts obtained from citrus by-products. Modelling of the extraction process.

BACKGROUND: The ultrasound-assisted extraction of pectic polysaccharides from orange by-products was investigated. Kinetics of mechanical agitation (0.2 × g) and acoustic (US1: 542 W L-1 and US2: 794 W L-1 ) extractions, were obtained and modelled at different pH values (1.5 and 2.0). All extractions were carried out at 25 °C, using citric acid as the extraction solvent. RESULTS: Higher pectic polysaccharides extraction yields were obtained with ultrasonic assistance, in comparison with the results obtained using mechanical agitation. Moreover, yield increases were significantly higher using the more acidic pH. Thus, at pH 1.5, pectin yield increased from ∼19%, obtained with agitation, to ∼47%, applying ultrasound; whereas, at pH 2.0, this increase was from ∼10%, with agitation, to ∼18%, applying ultrasound. A considerable decrease of the galacturonic acid proportion was observed on the extracts when ultrasound were applied for 60 min under pH 2.0. High methoxyl pectins were extracted at pH 1.5 whereas at pH 2.0, pectins exhibited a low methylation degree. Curves of acoustic and mechanical agitation extractions were properly represented by a second-order rate model (average mean relative error ≤ 7.4%). The extraction rate constant, initial extraction rate and maximum yield were determined for all experimental conditions. CONCLUSION: Overall, the results clearly indicated that the effect of ultrasound was highly dependent on the pH. Therefore, adequate acidic conditions must be applied in order to improve the efficiency of ultrasound on the pectin extraction process. © 2019 Society of Chemical Industry.

Investigating the involvement of ABA, ABA catabolites and cytokinins in the susceptibility of 'Nules Clementine' mandarin to rind breakdown disorder.

BACKGROUND: 'Nules Clementine' mandarin was used to investigate the potential involvement of endogenous plant hormones in mediating citrus fruit susceptibility to rind breakdown disorder (RBD). The effect of light exposure (namely canopy position and bagging treatments) on the endogenous concentration of ABA, 7'-hydroxy-abscisic acid (7-OH-ABA), ABA-glucose ester (ABA-GE) and dihydrophaseic acid (DPA), and t-zeatin was tested using four preharvest treatments: outside, outside bagged, inside and inside bagged. Phytohormones concentration was evaluated during nine weeks of postharvest storage at 8 °C. RESULTS: The shaded fruit inside the canopy had the highest RBD score (0.88) at the end of postharvest storage, while sun-exposed fruit had the lowest score (0.12). Before storage, ABA concentration was lowest (462.8 µg kg-1 ) for inside fruit, and highest in outside bagged fruit (680.5 µg kg-1 ). Although ABA concentration suddenly increased from the third week, reaching a maximum concentration of 580 µg kg-1 at week 6 in fruit from inside position, it generally reduced 1.6-fold ranging from 240.52 to 480.65 µg kg-1 throughout storage. The increase of 7-OH-ABA was more prominent in fruit from inside canopy. Overall, the concentration of ABA-GE increased three-fold with storage time. DPA concentration of bagged fruit from inside canopy position was significantly higher compared to outside fruit. The lower ABA-GE and higher DPA concentration in inside bagged fruit throughout storage also coincided with higher RBD. CONCLUSION: The strong positive correlations between 7-OH-ABA, DPA and RBD incidence demonstrated that these ABA catabolites could be used as biomarkers for fruit susceptibility to the disorder. © 2019 Society of Chemical Industry.

Sensitivity to Ethephon Degreening Treatment Is Altered by Blue LED Light Irradiation in Mandarin Fruit.

Although citrus fruits are not climacteric, exogenous ethylene is widely used in the degreening treatment of citrus fruits. Irradiation with blue light-emitting diode (LED) light (450 nm) for 10 h can promote the formation of good coloration of ethephon-degreened fruit. This study evaluated the effect of blue LED light irradiation on the pigments contents of ethephon-degreened fruit and evaluated whether the blue LED light irradiation could influence the sensitivity of mandarin fruit to ethylene. The results indicated that blue light can accelerate the color change of ethephon-degreened fruit, accompanied by changes in plastid ultrastructure and chlorophyll and carotenoid contents. Ethephon-induced expressions of CitACS1, CitACO, CitETR1, CitEIN2, CitEIL1, and CitERF2 were enhanced by blue LED light irradiation, which increased the sensitivity to ethylene in ethephon-degreened fruits. These results indicate that blue LED light-induced changes in sensitivity to ethylene in mandarin fruit may be responsible for the improved coloration of ethephon-degreened mandarin fruits.

A process to preserve valuable compounds and acquire essential oils from pomelo flavedo using a microwave irradiation treatment.

A microwave pretreatment method was developed to preserve pectin, naringin, and limonin contents in pomelo flavedo to allow for longer storage times and subsequent extraction of pomelo essential oil. In terms of the essential oil, microwave pretreatment performed better than hydrodistillation with respect to extraction efficiency (1.88±0.06% in 24min versus 1.91±0.08% in 240min), oxygenation fraction (48.59±1.32% versus 29.63±1.02%), energy consumption (0.15kWh versus 1.54kWh), and environmental impact (123.20g CO2 versus 1232g CO2). Microwave-pretreated samples retained higher amounts of pectin, naringin, and limonin compared with non-pretreated samples. No obvious change in the degree of pectin esterification was observed. This study shows that the proposed process is a promising methodology for both preserving valuable compounds in pomelo flavedo during storage and acquiring essential oils.

LED Blue Light-induced changes in phenolics and ethylene in citrus fruit: Implication in elicited resistance against Penicillium digitatum infection.

The objective was to investigate whether LED Blue Light (LBL) induces changes in phenolics and ethylene production of sweet oranges, and whether they participate in LBL-elicited resistance against the most important postharvest pathogen (Penicillium digitatum) of citrus fruit. The expression of relevant genes of the phenylpropanoid and ethylene biosynthetic pathways during elicitation of resistance was also determined. Different LBL (wavelength 450nm) quantum fluxes were used within the 60-630µmolm-2s-1 range. The HPLC analysis showed that the most relevant increase in phenylpropanoids occurred in scoparone, which markedly increased 3days after exposing fruits to a very high quantum flux (630µmolm-2s-1) for 18h. However, phenylpropanoids, including scoparone, were not critical factors in LBL-induced resistance. The genes involved in ethylene biosynthesis were differentially regulated by LBL. Ethylene is not involved in elicited resistance, although high LBL levels increased ethylene production in only 1h.

Improving the antioxidant functionality of Citrus junos Tanaka (yuzu) fruit juice by underwater shockwave pretreatment.

Citrus junos Tanaka (yuzu) has a strong characteristic aroma, and hence, yuzu juice is used in a number of Japanese foods. We herein evaluated the functional compounds of yuzu juice to investigate whether underwater shockwave pretreatment affects its functionality. Employing the shockwave pretreatment at an increased discharge and energy of 3.5kV and 4.9kJ, respectively, resulted in an increase in the flavanone glycoside content and oxygen radical absorbance capacity (ORAC). The ORAC value of yuzu juice cultivated in Rikuzentakata increased approximately 1.7 times upon underwater shockwave pretreatment. The treatment method proposed herein exhibited reliable and good performance for the extraction of functional and antioxidant chemicals in yuzu fruits, and was comparable with traditional squeezing methods. The high applicability and reliability of this technique for improving the antioxidant functionality of yuzu fruit juice was demonstrated, confirming the potential for application to a wide range of food extraction processes.

Inhibitory effect of gamma irradiation and its application for control of postharvest green mold decay of Satsuma mandarins.

Gamma irradiation has been shown to be effective for the control of postharvest fungi in vitro, but little is known regarding antifungal action, responses to gamma irradiation, and its application to fresh produce. Gamma irradiation was evaluated for its in vitro and in vivo antifungal activity against Penicillium digitatum on Satsuma mandarin fruits. Green mold was inhibited in a dose-dependent manner. Gamma irradiation showed a complete inhibition of spore germination, germ tube elongation, and mycelial growth of P. digitatum, particularly at 1.0kGy. To further investigate the mechanisms by which gamma irradiation inhibits fungal growth, the membrane integrity and cellular leakage of conidia were tested, indicating that gamma irradiation results in the loss of plasma membrane integrity, causing the release of intracellular contents such as soluble proteins. In vivo assays demonstrated that established doses can completely inhibit the growth of fungal pathogens, but such high doses cause severe fruit damage. Thus, to eliminate the negative impact on fruit quality, gamma irradiation at lower doses was evaluated for inhibition of P. digitatum, in combination with a chlorine donor, sodium dichloro-s-triazinetrione (NaDCC). Interestingly, only a combined treatment with 0.4kGy of gamma irradiation and 10ppm of NaDCC exhibited significant synergistic antifungal activity against green mold decay. The mechanisms by which the combined treatment decreased the green mold decay of mandarin fruits can be directly associated with the disruption of cell membrane of the fungal pathogen, which resulted in a loss of cytoplasmic material from the hyphae. These findings suggest that a synergistic effect of combining treatment with gamma irradiation with NaDCC has potential as an antifungal approach to reduce the severity of green mold in mandarin fruits.

Characterization of the Aldehydes and Their Transformations Induced by UV Irradiation and Air Exposure of White Guanxi Honey Pummelo (Citrus Grandis (L.) Osbeck) Essential Oil.

Aldehydes are key aroma contributors of citrus essential oils. White Guanxi honey pummelo essential oil (WPEO) was investigated in its aldehyde constituents and their transformations induced by UV irradiation and air exposure by GC-MS, GC-O, and sensory evaluation. Nine aldehydes, i.e., octanal, nonanal, citronellal, decanal, trans-citral, cis-citral, perilla aldehyde, dodecanal, and dodecenal, were detected in WPEO. After treatment, the content of citronellal increased, but the concentrations of other aldehydes decreased. The aliphatic aldehydes were transformed to organic acids. Citral was transformed to neric acid, geranic acid, and cyclocitral. Aldehyde transformation caused a remarkable decrease in the minty, herbaceous, and lemon notes of WPEO. In fresh WPEO, ß-myrcene, d-limonene, octanal, decanal, cis-citral, trans-citral, and dodecenal had the highest odor dilution folds. After the treatment, the dilution folds of decanal, cis-citral, trans-citral, and dodecenal decreased dramatically. This result provides information for the production and storage of aldehyde-containing products.

Short-term UV-B exposure induces metabolic and anatomical changes in peel of harvested lemons contributing in fruit protection against green mold.

UV-B radiation (UVBR) is a small fraction of the solar spectrum from 280 to 315nm. UVBR produces photomorphogenic acclimation responses in plants, modulating their cellular structure and physiology. Here, changes in the peel of harvested lemons after short time exposure to UVBR were analyzed and its potential effects against fungal infection were studied. In the flavedo, UVBR treatment induced variations in the respiratory profiles and increased the phenolic compound contents. Final products of the flavonoid pathway (flavones, flavonols and anthocyanins) increased more markedly than their precursors (flavanones and dihydroflavonols). The increased accumulation of soluble phenolics in the flavedo of treated lemons is associated with the high antioxidant activity found in the flavedo of these samples. Supporting the biochemical determinations, anatomical observations showed abundant intravacuolar deposits of phenolic compounds and an increase in the cell wall thickness in UVBR-treated samples. Metabolic and anatomical modifications associated to UVBR improved natural defenses against Penicillium digitatum, the causal agent of green mold disease. Our results suggest that mature postharvest lemons exposed to the artificial radiation showed phenotypic plasticity, allowing an acclimation response to UVBR which confers fruit resistance to pathogens. Thus, combination of UVBR with other treatments could represent an important improvement to control postharvest diseases on citrus.

Lower leaf gas-exchange and higher photorespiration of treated wastewater irrigated Citrus trees is modulated by soil type and climate.

Water quality, soil and climate can interact to limit photosynthesis and to increase photooxidative damage in sensitive plants. This research compared diffusive and non-diffusive limitations to photosynthesis as well as photorespiration of leaves of grapefruit trees in heavy clay and sandy soils having a previous history of treated wastewater (TWW) irrigation for >10 years, with different water qualities [fresh water (FW) vs TWW and sodium amended treated wastewater (TWW + Na)] in two arid climates (summer vs winter) and in orchard and lysimeter experiments. TWW irrigation increased salts (Na(+) and Cl(-) ), membrane leakage, proline and soluble sugar content, and decreased osmotic potentials in leaves of all experiments. Reduced leaf growth and higher stomatal and non-stomatal (i.e. mesophyll) limitations were found in summer and on clay soil for TWW and TWW + Na treatments in comparison to winter, sandy soil and FW irrigation, respectively. Stomatal closure, lower chlorophyll content and altered Rubisco activity are probable causes of higher limitations. On the other hand, non-photochemical quenching, an alternative energy dissipation pathway, was only influenced by water quality, independent of soil type and season. Furthermore, light and CO2 response curves were investigated for other possible causes of higher non-stomatal limitation. A higher proportion of non-cyclic electrons were directed to the O2 dependent pathway, and a higher proportion of electrons were diverted to photorespiration in summer than in winter. In conclusion, both diffusive and non-diffusive limitations contribute to the lower photosynthetic performance of leaves following TWW irrigation, and the response depends on soil type and environmental factors.

Quantification of applied dose in irradiated citrus fruits by DNA Comet Assay together with image analysis.

The experiments were conducted for quantification of applied dose for quarantine control in irradiated citrus fruits. Citrus fruits exposed to doses of 0.1 to 1.5 kGy and analyzed by DNA Comet Assay. Observed comets were evaluated by image analysis. The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Irradiated citrus fruits showed the separated tails from the head of the comet by increasing applied doses from 0.1 to 1.5 kGy. The mean tail length and mean tail moment% levels of irradiated citrus fruits at all doses are significantly different (p < 0.01) from control even for the lowest dose at 0.1 kGy. Thus, DNA Comet Assay may be a practical quarantine control method for irradiated citrus fruits since it has been possible to estimate the applied low doses as small as 0.1 kGy when it is combined with image analysis.

Effect of blue LED light intensity on carotenoid accumulation in citrus juice sacs.

In the present study, the effects of blue LED light intensity on carotenoid accumulation and expression of genes related to carotenoid biosynthesis were investigated in the juice sacs of Satsuma mandarin (Citrus unshiu Marc.) and Valencia orange (Citrus sinensis Osbeck) in vitro. The results showed that 100 µmol m(-2)s(-1) blue LED light (100B) was effective for increasing carotenoid content, especially ß-cryptoxanthin, in Satsuma mandarin after cultured in vitro for four weeks. In Valencia orange, in contrast, 50 µmol m(-2)s(-1) blue LED light (50B) treatment was effective for inducing carotenoid accumulation through increasing the contents of two major carotenoids, all-trans-violaxanthin and 9-cis-violaxanthin. In addition, gene expression results showed that the simultaneous increases in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb2, and CitHYb) involved in producing ß,ß-xanthophylls were well consistent with the accumulation of ß-cryptoxanthin in Satsuma mandarin under 100B, and violaxanthin in Valencia orange under 50B. The results presented herein contribute to further elucidating the regulatory mechanism of carotenoid accumulation by blue LED light.

Synthesis of amphiphilic polysuccinimide star copolymers for responsive delivery in plants.

While polymeric nanocarriers are widely used in medicine for controlled release and site-specific delivery, few reports have applied such delivery methods within agriculture, despite the urgent need for specific delivery of pesticides and nutrients. We report the synthesis of stimuli-responsive and biodegradable polymeric nanocarriers designed for delivery to the phloem of plants and describe methods employed to evaluate their toxicity in plant cells.

Regulation of ascorbic acid metabolism by blue LED light irradiation in citrus juice sacs.

In the present study, the effects of red and blue LED lights on the accumulation of ascorbic acid (AsA) were investigated in the juice sacs of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon. The results showed that the blue LED light treatment effectively increased the AsA content in the juice sacs of the three citrus varieties, whereas the red LED light treatment did not. By increasing the blue LED light intensity, the juice sacs of the three citrus varieties accumulated more AsA. Moreover, continuous irradiation with blue LED light was more effective than pulsed irradiation for increasing the AsA content in the juice sacs of the three citrus varieties. Gene expression results showed that the modulation of AsA accumulation by blue LED light was highly regulated at the transcription level. The up-regulation of AsA biosynthetic genes (CitVTC1, CitVTC2, CitVTC4, and CitGLDH), AsA regeneration genes (CitMDAR1, CitMDAR2, and CitDHAR) and two GSH-producing genes (CitGR and CitchGR) contributed to these increases in the AsA content in the three citrus varieties.

Investigation of sunlight-induced deterioration of aroma of pummelo (Citrus maxima) essential oil.

Deterioration of aromas of pummelo essential oil (EO) induced by sunlight was compared to those induced by heat and oxygen exposure using the techniques of sensory evaluation and GC-MS analysis. The sunlight-exposed EO was found to possess an oily off-flavor odor, which was significantly different from its counterparts induced by oxygen and heat. The strong oily note of the sunlight-exposed EO was attributed to the existence of linalool oxides and limonene oxides, as well as the lack of neral and geranial, for which UV sunlight was revealed to be the critical contributor causing the chemical reactions for the aroma changes. The results demonstrated that UV sunlight could significantly affect the aroma of the pummelo EO, providing valuable information that will benefit the production and storage of EO-based aromatic products.

Optimization of culture conditions (sucrose, pH, and photoperiod) for in vitro regeneration and early detection of somaclonal variation in ginger lime (Citrus assamensis).

Various explants (stem, leaf, and root) of Citrus assamensis were cultured on MS media supplemented with various combinations and concentrations (0.5-2.0 mg L(-1)) of NAA and BAP. Optimum shoot and root regeneration were obtained from stem cultures supplemented with 1.5 mg L(-1) NAA and 2.0 mg L(-1) BAP, respectively. Explant type affects the success of tissue culture of this species, whereby stem explants were observed to be the most responsive. Addition of 30 gL(-1) sucrose and pH of 5.8 was most optimum for in vitro regeneration of this species. Photoperiod of 16 hours of light and 8 hours of darkness was most optimum for shoot regeneration, but photoperiod of 24 hours of darkness was beneficial for production of callus. The morphology (macro and micro) and anatomy of in vivo and in vitro/ex vitro Citrus assamensis were also observed to elucidate any irregularities (or somaclonal variation) that may arise due to tissue culture protocols. Several minor micromorphological and anatomical differences were observed, possibly due to stress of tissue culture, but in vitro plantlets are expected to revert back to normal phenotype following full adaptation to the natural environment.