Grapefruit Flavedo Research Articles

Functionality of Semolina Pasta Supplemented with Grapefruit Flavedo Byproduct: Effect on Phytochemical, Functional, Textural, Cooking, and Sensorial Quality.

Pasta is consumed worldwide and can be an excellent food matrix for supplementation. Grapefruit (Citrus paradisi) flavedo byproduct is reported to contain considerable amounts of bioactive compounds, which represent a valuable and sustainable source of phenolics. So far, there is limited information available about the utilization of grapefruit flavedo byproducts for supplementation of pasta. The present study aimed to investigate the effect of freeze-dried grapefruit flavedo powder (GFP) incorporation at various concentrations (3, 6, 9, and 12%) on the phytochem-ical, functional, textural, microstructural, and sensory quality of the developed pasta. Fortification with GFP significantly (P<0.05) enhanced antioxidant activities rela-tive to DPPH and FRAP and increased the total phenolic and flavonoid content from 73.20-96.83 mg GAE/100g and 132.41-211.63 mg GAE/100g, respectively. Supplementation with GFP significantly (P<0.05) reduced the optimum cooking time of pasta, while cooking loss increased up to 6.32% with 12% GFP incorporation, which was still under the acceptable limits (<8%). The addition of GFP decreased the L* value, while the a* and b* values in-creased for the pasta. SEM analysis presented that GFP incorporation in pasta affected the structural integrity attributable to the enhanced fiber levels, inducing a weaker matrix, as ascertained by textural profile analysis (TPA) with a decline in hardness attribute with in-creasing GFP concentration. Sensory analysis divulged overall acceptability scores of 8.4 for the pasta with 6% GFP, while further increments in GFP levels led to decreased sensory scores. Grapefruit peels are typically discarded as waste, the outer layer of which, i.e., flavedo, could be used to enrich pasta. The present research divulged that pasta supplemen-tation with freeze-dried flavedo powder could provide total phenolic content, flavonoid con-tent, and antioxidant potential without compromising on the technological and sensory qual-ity of pasta. Utilization of this citrus waste as a bioactive food ingredient with promising effects represents a valuable byproduct valorization strategy.

Low-temperature storage regulates the expression of genes related to peel pigments of grapefruit

Grapefruit fruit (Citrus paradisi Macfad) are sensitive to postharvest temperature variations. In red grapefruit cultivars, peel color is a characteristic and important quality parameter that may undergo changes during storage due to alterations in the content of pigments. So far, it is not clear if the postharvest peel color variations in grapefruit are regulated at the transcriptional level due to storage conditions. The objective of this work was to investigate the effect of postharvest storage temperature (2 and 13 °C) on the relationships between peel color, chlorophyll (Chl) and carotenoid contents and the expression of genes related to the synthesis and degradation of these pigments in the flavedo of ‘Rio Red’ grapefruit grown in Northern Mexico. Fruit were harvested at breaker stage with a maturity index of 4.8 and stored for 42 days at 2 and 13 °C. Pigments, transcript levels, color index (CI) and ethylene production were periodically evaluated through storage. Pearson’s correlations between CI and Chl, lycopene, and β-carotene contents were evaluated. Storage at 13 °C increased lycopene content and CI and decreased Chl content. In fruit stored at 2 °C, the CI and content of β-carotenes remained almost unchanged. In contrast, the lycopene content increased at this temperature, but to a lesser extent than at 13 °C. The ANOVA showed the expression of ζ-carotene desaturase (ZDS), two types of lycopene cyclases (carotenoid biosynthesis pathway genes) and the pheophorbide a oxygenase gene (involved in Chl breakdown), was significantly influenced by the storage time-temperature interaction. The expression of the ZDS gene was significantly higher in fruit stored at 13 °C than in fruit stored at 2 °C, suggesting that lycopene biosynthesis is transcriptionally regulated by storage temperature. The lycopene levels showed a positive relation with the ZDS transcript levels and CI, indicating this pigment is implicated in the red coloration of the ‘Rio Red’ grapefruit flavedo during postharvest.

Expression of antioxidant-related genes in flavedo of cold-stored grapefruit (Citrus paradisi Macfad cv. Rio Red) treated with pectic oligosaccharides

Cold storage is one of the main postharvest technologies for fruit preservation, however, cold-sensitive citrus fruits develop peel physiological alterations affecting external quality known as chilling injury (CI). An efficient antioxidant system has been associated to an enhanced cold stress tolerance in citrus fruits. The application of pectic oligosaccharides (POs) stimulates the enzymatic antioxidant system in plants and reduces CI development in grapefruit fruit (Citrus paradisi) during cold storage. The aim of this study was to contribute to the basic understanding of peel physiological disorders in cold-stressed ‘Rio Red’ grapefruit fruit at ultrastructural level and to determine whether the POs are involved in the CI reduction by modulating the antioxidant enzymatic system at the transcriptional level. Peel morphology in grapefruit fruit stored at chilling (2 °C) and non-chilling (13 °C) temperatures was analyzed by electronic microscopy and the effect of POs treatment on manganese superoxide dismutase (MnSOD), ascorbate peroxidase (APX1), catalase (CAT1) and glutathione reductase (GR2) gene expression was investigated by RT-qPCR. Results indicate that a prolonged cold storage promoted the incidence of CI symptoms in 'Rio Red' grapefruit and altered the ultrastructural morphology of flavedo epidermal tissue. POs significantly modulated the MnSOD, APX1 and CAT1 expression levels mainly in a storage time- and temperature-dependent manner with regards to controls. By contrast, POs only significantly affected the GR2 gene expression when grapefruit were stored at non-chilling temperatures. Our results revealed a possible involvement of the MnSOD, APX1 and CAT1 genes in the CI susceptibility reduction induced by POs in ‘Rio Red’ grapefruit stored at chilling temperatures.

Involvement of lycopene in the induction of tolerance to chilling injury in grapefruit

Grapefruit are among the more sensitive Citrus varieties likely to develop chilling injury (CI) symptoms during postharvest storage at low temperatures. Comparative observations of the incidence of CI in fruit of white Marsh (MSH) and red Star Ruby (SR) grapefruit during postharvest storage at 2°C plus 7 days at 20°C to simulate shelf-life revealed that (1) the former was consistently more sensitive to CI, developing cold damage uniformly throughout the whole rind surface, and (2) more strikingly, CI symptoms in fruit of SR grapefruit were restricted to the yellow areas of the rind and the red-colored zones were almost absent of cold damage. This tolerance to CI in red flavedo was associated with high carotenoid (×2) and lycopene (×14) contents, as compared with yellow-colored flavedo. Absence of chilling damage in red areas of SR grapefruit rind was confirmed by cellular ultrastructure observations, in which these epidermal cells were intact, with a well-defined structure and compact vacuoles filled with content. Cells of yellow-colored tissue developing CI, were collapsed, with a contracted vacuole and shrinking organelles. To explore whether the tolerance to CI in red areas of grapefruit rind was due to an elevated lycopene concentration, chemical and environmental stimulation of this carotenoid was performed in fruit of both grapefruit varieties. Application of the inhibitor of the lycopene cyclase activity, CPTA (2-(4-chlorophenylthio) triethylamine hydrochloride) induced red coloration, increased lycopene accumulation (×32) and significantly delayed development of CI symptoms in the rind the CI-sensitive MSH. Bagging of SR grapefruit enhanced a homogenous red coloration and substantially induced lycopene accumulation (×75). CI symptoms in bagged fruit were notably delayed and reduced, as compared with non-bagged yellow fruit, upon subsequent storage at 2°C for up to 58 days and 7 days at 20°C. Analysis of the expression of ethylene biosynthetic genes (ACS1, ACS2 and ACO) revealed a significant induction in chilling-damaged tissue of both varieties that was almost absent in red chilling-tolerant tissue. Similarly, accumulation of transcripts of the ethylene receptors ETR1 and ETR3 were also associated with chilling damage, but a cold factor appears to also mediate the expression of these genes. Taken together, our results indicate that high lycopene concentration appears to be responsible for the induction of tolerance to chilling in the red-colored areas of the flavedo of grapefruit during postharvest storage at low temperatures.

Temperature conditioning alters transcript abundance of genes related to chilling stress in ‘Marsh’ grapefruit flavedo

Grapefruit ( Citrus × paradisi) develop symptoms of chilling injury (CI) if held at temperatures below about 10 °C. Conditioning grapefruit at a low, but non-chilling (16 °C) temperature prior to storage at a chilling temperature reduces the development of CI symptoms. Changes in transcript abundance for a number of genes have been correlated with chilling stress in citrus fruit. We tested the hypothesis that conditioning affects transcript abundance of genes related to chilling stress in grapefruit. Grapefruit were harvested from a commercial grove in Florida in September and divided into two groups; one group was placed immediately at 5 °C (non-conditioned, NC); the second group was placed at 16 °C for 1 week (conditioned, C) and then transferred to 5 °C. Symptoms of CI were visible on NC and C fruit following 14 d at 5 °C, but were consistently more severe on NC than C fruit. Storage at 5 °C caused increases in abundance of transcripts for 1-aminocyclopropane-1-carboxylate oxidase (ACO), galactinol synthase, a chilling-induced oxygenase, and a temperature-induced lipocalin, consistent with previous reports. Levels of these transcripts were lower in C than in NC fruit during storage at 5 °C, but this pattern did not persist following transfer to 20 °C. Levels of transcripts for catalase, a metallothionein-like protein, a lipid transfer protein, a stress-responsive zinc finger protein, and a citrus low temperature inducible protein were consistently higher in C than NC fruit during storage at 5 °C. Our results show that conditioning increases chilling tolerance in grapefruit and demonstrates that abundance of transcripts of a number of genes related to chilling stress is affected by conditioning. This suggests a potential quantitative relationship between gene expression and conditioning induced chilling tolerance. We cannot say, however, that these changes are related to chilling tolerance per se; they may only reflect the difference between chilling-sensitive and chilling-tolerant fruit.

Transcriptome profiling of grapefruit flavedo following exposure to low temperature and conditioning treatments uncovers principal molecular components involved in chilling tolerance and susceptibility

A pre-storage conditioning (CD) treatment of 16 degrees C for 7 d enhanced chilling tolerance of grapefruit and reduced the development of chilling injuries during storage at 5 degrees C. To gain a better understanding of the molecular mechanisms involved in the responses of citrus fruit to low temperatures, we performed genome-wide transcriptional profiling analysis of RNA isolated from grapefruit flavedo using the newly developed Affymetrix Citrus GeneChip microarray. Utilizing very restrictive cut-off criteria, including pair-wise anova comparisons significantly different at P < or = 0.05 and induction or repression of transcript levels by at least fourfold, we found that out of 30 171 probe sets on the microarray, 1345 probe sets were significantly affected by chilling in both control and CD-treated fruits, 509 probe sets were affected by chilling specifically in the CD-treated fruits, and 417 probe sets were specifically expressed in chilling-sensitive control fruits. Overall, exposure to chilling led to expression arrest of general cellular metabolic activity, including concretive down-regulation of cell wall, pathogen defence, photosynthesis, respiration, and protein, nucleic acid and secondary metabolism. On the other hand, chilling enhanced adaptation processes that involve changes in the expression of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.

Ethylene modulates transcription of the phosphoenolpyruvate carboxykinase gene in grapefruit flavedo

SummaryThe role of ethylene in citrus fruit physiology is poorly understood. Gene expression studies with ethylene and ethylene inhibitors suggest a regulatory role for the low basal levels of endogenous ethylene in citrus fruits; however, only a few ethylene-modulated genes have been reported. 1-Methylcyclopropene (1-MCP), an ethylene antagonist that inhibits ethylene signal transduction by preventing ethylene from binding to its receptors, is a useful tool to further identify genes associated with the regulatory roles of ethylene. We determined the effects of ethylene and 1-MCP on gene expression in grapefruit flavedo. Total RNA from fruit treated with air, 5 µl l–l ethylene, or 0.3 µl l–l 1-MCP for 48 h was used in differential-display reverse transcription-polymerase chain reaction (DD-RT-PCR) analysis. Our hypothesis was that genes whose expression is induced by ethylene would be up-regulated in response to ethylene, whereas genes whose expression is inhibited by ethylene would be up-regulated in response to 1-MCP. A 440 bp cDNA, induced by ethylene, was used as a probe to screen a cDNA library of flavedo, and obtain a clone with a deduced amino acid sequence 88% similar to the enzyme phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.49) from both Arabidopsis and Flaveria trinervia. We designated this gene CpPEPCK (Citrus paradisi phosphoenolpyruvate carboxykinase). Northern blot and RT-PCR analyses revealed increased expression of CpPEPCK in grapefruit flavedo after exposure to ethylene, and with increased exposure time. In contrast, treatment with 1-MCP inhibited the accumulation of CpPEPCK transcripts in the same tissue. Although CpPEPCK is expressed in leaves, roots, and petals, higher transcript abundance was found in the flavedo of ethylene-treated fruit than in other tissues. This study provides additional evidence for a role for ethylene in citrus fruit physiology.

Isolation of a thioredoxin h cDNA from grapefruit peel tissue that is induced upon infection by Penicillium digitatum and elicitation of pathogen resistance

Thioredoxins are ubiquitous small proteins with a redox-active disulfide bridge; they participate in redox regulation of selected target proteins, such as transcription factors, receptors and various metabolic and regulatory enzymes. In the present study, we isolated by PCR cDNA subtraction analysis a grapefruit 135-bp polymerase chain reaction (PCR) fragment whose expression was enhanced in the fruit peel tissue following the application of Candida oleophila yeast cells, an elicitation treatment that induces resistance towards the green mold pathogen Penicillium digitatum (Pers.:Fr.) Sacc., and that showed high sequence homology with various thioredoxin h ( TRXh) genes. The full-length cDNA sequence of cTRXh (for citrus TRXh) was further isolated by screening a grapefruit flavedo cDNA library; it had a total length of 785 bp with an open reading frame of 369 bp, and encoded a predicted polypeptide of 123 amino acids with a molecular mass of 13.4 kDa. Expression analysis studies revealed that cTRXh mRNA levels increased in grapefruit peel tissue upon infection by P. digitatum, and following the elicitation of fruit disease resistance by application of C. oleophila yeast cells, wounding, exposure to ethylene, and a hot water rinsing and brushing treatment. These results indicate that cTRXh may be part of the molecular mechanisms involved in the induction of pathogen defense responses in grapefruit, and may be involved in the signaling cascades leading to enhanced resistance.

Hot Water and Elevated CO2 Effects on Proline and Other Compositional Changes in Relation to Postharvest Chilling Injury of `Marsh' Grapefruit

Heat treatments and exposure to elevated CO2 are known to reduce the incidence of chilling injury on grapefruit. In the current study, `Marsh' grapefruit (Citrus paradisi Macf.) were harvested on 17 Jan. or 22 Mar. 1996 and exposed to hot water (HW) dips (48 °C for 120 minutes) or exposed to controlled atmosphere (CA) of 10% or 16% CO2 during the first 3 weeks of an 8-week cold storage period (4.5 °C) to test their effects on the development of peel pitting (i.e., chilling injury) and proline and other compositional changes of the peel and juice. All HW and CA treatments from both harvests greatly reduced the development of peel pitting compared to the control. These treatments were also associated with lower average proline levels in the flavedo during storage. This suggests that HW and elevated CO2 may reduce chilling-induced peel pitting by facilitating proline metabolism in grapefruit flavedo tissue. HW and CA treatments resulted in higher peel total soluble and nonreducing sugar levels, but effects on peel reducing sugar and free amino acid concentrations were not consistent. In the juice, HW reduced titratable acidity (TA) concentrations while CA tended to increase both TA and ascorbic acid concentrations. Compared to the control, CA resulted in a slight decrease in total soluble solids during storage, while the effect of HW was inconsistent.

Isolation of a dehydrin cDNA from orange and grapefruit citrus fruit that is specifically induced by the combination of heat followed by chilling temperatures.

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to environmental stresses such as water stress, salinity and freezing or which occur during the late stages of embryogenesis. Previously, it was reported that citrus contains a small gene family encoding a unique class of dehydrins that differs from most other plant dehydrins in various respects, such as having an unusual K-segment similar to that of gymnosperms. In the present study, we identified by cDNA differential display analysis a 'Navel' orange 202-bp polymerase chain reaction (PCR) fragment, which encoded the typical plant angiosperm-type K-segment consensus sequence, and of which the expression was down-regulated by exposure to low oxygen levels. The full-length cDNA sequence of the orange DHN, designated csDHN (for Citrus sinensis DHN), was further isolated by 5'-and 3'-RACE; it had a total length of 933 bp and encoded a predicted polypeptide of 235 amino acids. In addition, the same 202-bp 'Navel' dehydrin PCR fragment was used to screen a 'Star Ruby' grapefruit flavedo cDNA library, and its full-length grapefruit homologue, designated cpDHN (for C. paradisi DHN) was isolated and found to have a total length of 1024 bp and to encode a predicted polypeptide of 234 amino acids. The defined orange and grapefruit DHN proteins were completely identical in the 196 amino acids of their N-terminus but differed in their C-terminus region. Overall, the csDHN and cpDHN proteins share 84% identity and contain the conserved dehydrin serine cluster (S-segment) and a putative nuclear localization signal, but csDHN has one conserved dehydrin K-segment consensus sequence, whereas cpDHN contains two dehydrin K-segments. Both csDHN and cpDHN represent single copy genes, in 'Navel' orange and 'Star Ruby' grapefruit genomes, respectively. We found that the cpDHN gene was consistently expressed in the fruit peel tissue at harvest, but that its message levels dramatically decreased during storage at either ambient or low temperatures. However, a pre-storage hot water treatment, given to enhance fruit-chilling tolerance, increased cpDHN mRNA levels during the first 3 weeks of cold storage at 2 degrees C, and enabled the message levels to be retained for up to a further 8 weeks of cold storage at 2 degrees C. The hot water treatment by itself had no inductive effect on cpDHN gene expression when the fruits were held at non-chilling temperatures. Other stresses applied to the fruit, such as wounding, UV irradiation, water stress, low oxygen and exposure to the stress hormone ethylene decreased DHN mRNA levels, whereas abscisic acid had no effect at all.

A hot water treatment induces resistance to Penicillium digitatum and promotes the accumulation of heat shock and pathogenesis‐related proteins in grapefruit flavedo

A new postharvest hot water brushing (HWB) treatment, which sprays hot water on fruit as they move along a belt of brush‐rollers, induced resistance against green mould decay caused by Penicillium digitatum (Pers. Fr.) Sacc in ‘Star Ruby’ grapefruit. The HWB treatment (62°C for 20 s) was most effective in inducing disease resistance when the fruit were inoculated after 1 and 3 days, but was less effective when the fruit were inoculated on the same day or 7 days later. The HWB treatment induced the accumulation of a 105‐kDa protein that cross‐reacted with an antibody raised against a bovine heat shock protein 70 (HSP70) and 18‐ and 21‐kDa proteins that cross‐reacted with pea HSP18 and HSP21 antibodies. The accumulation of a grapefruit 17‐kDa protein that cross‐reacted with a pea HSP17 antibody was not affected by the HWB treatment. HWB also induced the accumulation of 21‐, 22‐ and 25‐kDa proteins that cross‐reacted with citrus and tobacco chitinase antibodies and 38‐, 42‐ and 43‐kDa proteins that cross‐reacted with citrus and tobacco β‐1,3‐glucanase antibodies. The induction of the 105‐, 18‐ and 21‐kDa HSPs by the HWB treatment indicates that it was sufficient to provide a heat stress and, thus, was able to induce biochemical changes in the fruit peel tissue. Nevertheless, the accumulation of these HSPs was probably not related to the induction of fruit resistance against P. digitatum, since their accumulation could not be induced neither by direct inoculation with the pathogen nor following exposure to other treatments, such as UV irradiation, which also induce disease resistance. On the other hand, the increases in the accumulation of the 21‐, 22‐ and 25‐kDa chitinase proteins and of the 38‐ and 43‐kDa β‐1,3‐glucanases proteins, which were observed 1 and 3 days after the HWB treatment when the fruit appeared to be more resistant to P. digitatum, may be part of the complex fruit disease resistance mechanisms induced by the heat treatment.

Ethylene: A regulator of stress‐induced ACC synthase activity in nonclimacteric fruit

In spite of precautions, harvesting, packing and shimpping of grapefruit (Citrus paradisi) can often result in injury to the peel, which in turn leads to an increased evolution of the ripening hormone ethylene by the fruit. The present study is an attempt to determine how the stress‐induced ethylene pathway is regulated using the 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase (EC 4.4.1.14) action inhibitor aminoethoxyvinylglycine (AVG). Incubation of mature grapefruit flavedo discs in the presence of AVG created a super‐induction of ACC synthase (ACS) activity. Maximum enzyme activity was achieved following an incubation period of between 24 and 36 h. From AVG‐treated flavedo discs, cDNA encoding ACS was isolated, employing reverse transcriptase‐polymerase chain reaction (RT‐PCR), and the accumulation of ACS transcripts, which peaked after 36 h incubation, was found to complement the recorded enzyme activity. Saturation of ACS activity was achieved with 100 μM AVG. Incubation of flavedo with AVG significantly decreased both the conversion of S‐adenosyl‐l‐methionine (AdoMet) to ACC and the evolution of ethylene from the treated tissue, and in vitro incubation of extracted ACS enzyme with AVG led to repression of the action of the enzyme. Treatment of flavedo discs with ACC and ethylene inhibited the AVG‐induced ACS activity. Northern blot analysis showed that ethylene sumppressed the accumulation of ACS mRNA. It is suggested that in nonclimacteric fruit, the synthesis of ethylene can be regulated through a process of autoinhibition working at the level of gene expression.

Supercritical carbon dioxide (SC‐CO 2 ) extraction of grapefruit flavedo

Grapefruit flavedo (Citrus paradisi Macf.) was extracted by different methods: hydrodistillation; and solvent extraction using pentane, ethanol and supercritical carbon dioxide (SC-CO2) at two fluid densities. The composition of the distillates and oleoresins were compared. Monoterpene hydrocarbons decrease in SC-CO2 extracts at 87–90% with respect to their quantity in pentane extracts (95%) and in hydrodistillates (97%); these levels in monoterpene hydrocarbons were related to the limonene content, the most representative compound in grapefruit essence. Sesquiterpenes, aldehydes, alcohols and esters increased their GC area percentage in SC-CO2 extract at a high density of the solvent, with respect to the hydrodistillate and the pentane extract. Very interesting was the high increase in nootkatone concentration in the SC-CO2 extracts; this grapefruit essence component was 4.72% in the extract at 8 MPa, and it was 5.19% in that at 25 MPa; the ketone was very low in the hydrodistillates (0.22–0.26%). The monoterpene hydrocarbon concentrations, expressed as g of component in 100 g of extract, showed a decrease in SC-CO2 oleoresins. Oxygenated, sesquiterpenes and particularly nootkatone increase their amount in SC-CO2 extracts. © 1998 John Wiley & Sons, Ltd.

Purification and Characterization of a Polygalacturonase-inhibiting Protein from Grapefruit Flavedo

Polygalacturonase-inhibiting proteins (PGIPs) are believed to be one component of plants inherent defense mechanisms against fungal pathogens. We have purified a PGIP from mature grapefruit (Citrus paradisi cv. Marsh) flavedo using ammonium sulfate precipitation, preparative isoelectric focusing and ion exchange chromatography. Two peaks of PGIP activity were separated by isoelectric focusing, one at pH 6–7 and one at pH 9–10. The basic protein was more abundant than the neutral protein and was selected for further purification. The basic protein binds to S Sepharose at pH 6.1 and has an apparent Mr of ≈43,000 based on SDS-PAGE analysis. The protein is glycosylated as revealed by binding to ConA sepharose and is serologically similar to PGIPs from bean hypocotyl and pear fruit. Two dimensional PAGE analysis revealed the presence of two bands of similar Mr but with slightly different pIs (≈9.0–9.5). The N-terminal amino acid sequence of grapefruit PGIP shows high homology with PGIPs from fruit of other species and with a cDNA clone of PGIP that was isolated from a Citrus sinensis cv. Hamlin expression library. Grapefruit PGIP inhibits polygalacturonases from Aspergillus niger, and the citrus pathogen Penicillium italicum. We are interested in the role of PGIP in resistance of citrus fruit to postharvest decay fungi.

Characterization of chitinases and β‐1,3‐glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β‐1,3‐glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit (Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β‐1,3‐glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS‐PAGE were probed with antibodies raised against purified citrus chitinase and β‐1,3‐glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β‐1,3‐glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β‐1,3 linkages for activity, were active against both soluble and insoluble β‐1,3 glucans and generated similar products.

Characterization of chitinases and beta-1,3-glucanases in grapefruit flavedo during fruit development

Characterization of chitinases and beta-1,3-glucanases in grapefruit flavedo during fruit development

Characterization of Chitinases and β-1,3-glucanases in Grapefruit Flavedo during Fruit Development

Chitinases (EC 3.2.1.14) and β-1,3-endoglucanase (EC 3.2.1.39) are enzymes that are believed to be components of a plant's natural defense against fungal pathogens. We are interested in the potential role of these enzymes in citrus decay resistance. In our preliminary work, we have determined the activities of chitinases and β-1,3-endoglucanases in the flavedo of grapefruit (Citrus paradisi cv. Marsh) at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant, throughout the remainder of fruit development. In contrast to chitinase, β-1,3-endoglucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS-PAGE were probed with antibodies raised against purified citrus chitinase and glucanase, and results revealed that changes in the activities of chitinase and β-1,3-endoglucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Partial purification of flavedo chitinases and glucanases revealed that acidic and basic forms of both enzymes were present in the extracts, although the basic forms were predominant.

Glycosidase activities in grapefruit flavedo, albedo and juice vesicles during maturation and senescence

Glycosidase activities in grapefruit flavedo, albedo and juice vesicles during maturation and senescence

Glycosidase activities in grapefruit flavedo, albedo and juice vesicles during maturation and senescence

Glycosidases were extracted from grapefruit (Citrus paradisi Macf. cv. Ruby Red) flavedo, albedo, and juice vesicles harvested at five periods throughout the season. Flavedo β‐galactosidase activity was high at the September harvest and then significantly declined by November. Thereafter, no further changes occurred in β‐galactosidase activity. Flavedo α‐galactosidase activity was low and unchanged throughout the study. α‐Mannosidase, initially low in flavedo, steadily increased with advanced maturity. Trends in glycosidase activities of albedo were similar but attenuated. Juice vesicle β‐galactosidase did not change through the study period, whereas α‐galactosidase activity decreased 70% after the initial harvest period. α‐Mannosidase was initially high and then decreased to 50% of the original activity. A second peak of activity was measured in March, followed by a second decline. Extractability differences of the glycosidases suggest differences in compartmentation and function. Two isozymes of α‐mannosidase were separated in flavedo and one in juice vesicles, and characteristics were determined at an early and late harvest period. The results suggest that changes in the three glycosidases could be used to further define maturity and senescence in grapefruit.

Changes in grapefruit flavedo cell wall noncellulosic neutral sugar composition

Analysis of grapefruit flavedo cell walls detected seven neutral sugars. Arabinosyl and galactosyl residues were most abundant, followed by xylosyl, mannosyl, glucosyl and fucosyl residues. The percentage of the cell wall composed of neutral sugars (38%) decreased during development and ripening to 27%. There was a change in cell wall neutral sugar composition from June to April with a reduced percentage of flavedo cell wall galactosyl and arabinosyl residues as the season progressed. Degreening of fruit accelerated the decrease in flavedo cell wall galactosyl and arabinosyl residues, and a single preharvest spray with gibberellic acid, three months prior to harvest, reduced the decrease in flavedo cell wall galactosyl and arabinosyl residues, as well as reducing the rate of peel softening and loss of green colour.