Effects of postharvest ethanol vapor treatment on activities and gene expression of chlorophyll catabolic enzymes in broccoli florets

Postharvest yellowing of broccoli (Brassica oleracea L. Italica Group) is an important indicator of quality deterioration and occurs with chlorophyll (Chl) degradation. Postharvest ethanol vapor treatment could delay the yellowing of broccoli florets, through the suppression of Chl degradation. As the first step in identifying the mechanism of this delay, the effects of postharvest ethanol vapor treatment on activities and gene expression of the Chl catabolic enzymes in broccoli were determined. Broccoli branchlets were placed in a perforated polyethylene bag with or without (control) an ethanol pad and stored at 20 °C in darkness. The Chl contents of the control broccoli florets decreased remarkably after three days in storage, whereas the contents of the ethanol-treated broccoli showed no significant changes except at day one. Changes in chlorophyllase activity in the ethanol-treated broccoli were similar to those of the control until three days in storage, but then the activity tended to decease. Mg-dechelatase and Chl-degrading peroxidase activities, which increased greatly with senescence in the controls, remained unchanged in the ethanol-treated broccoli. BoCLH1 expression showed changes almost similar to those of chlorophyllase activity. BoPAO expression in the control broccoli increased greatly at day one in storage and was maintained at a high level until three days. In contrast, this expression in ethanol-treated broccoli was suppressed until two days. BoRCCR expression in the control broccoli increased until two days in storage, while the expression in the ethanol-treated broccoli showed no change during storage. These results show that postharvest ethanol vapor treatment suppressed the activities and gene expression of Chl catabolic enzymes, resulting in delayed yellowing of broccoli florets.     

Pre-harvest calcium application increases biomass and delays senescence of broccoli microgreens

Microgreen consumption has been steadily increasing in recent years due to consumer awareness of their unique color, rich flavor, and concentrated bioactive compounds. However, industrial production and marketing is limited by their short shelf-life associated with rapid deterioration in product quality. This study investigated the effect of pre-harvest calcium application on the post-harvest quality and shelf-life of broccoli microgreens. Broccoli microgreen seedlings were sprayed daily with calcium chloride at concentrations of 1, 10 and 20 mM, or water (control) for 10 days. The fresh-cut microgreens were packaged in sealed polyethylene film bags. Package headspace atmospheric conditions, overall visual quality and tissue membrane integrity were evaluated on days 0, 7, 14, and 21, during 5 °C storage. Results indicated that the 10 mM calcium chloride treatment increased the biomass by more than 50%, and tripled the calcium content as compared to the water-treated controls. Microgreens treated with 10 mM calcium chloride spray exhibited higher superoxide dismutase and peroxidase activities, lower tissue electrolyte leakage, improved overall visual quality, and reduced microbial growth during storage. Furthermore, calcium treatment significantly affected expression of the senescence-associated genes BoSAG12, BoGPX6, BoCAT3 and BoSAG12. These results provide important information for commercial growers to enhance productivity and improve postharvest quality and shelf-life, potentially enabling a broadening of the retail marketing of broccoli microgreens.     

Expression of Stay-Green encoding gene (BoSGR) during postharvest senescence of broccoli

Degreening caused by chlorophyll degradation is the most important feature that determines postharvest loss of quality in broccoli. Chlorophyll molecules are assembled to several thylakoid proteins, from which chlorophylls must be released in order to be catabolized. Stay-Green (SGR), a chloroplast-located protein, specifically interacts with light harvesting complex subunits helping toward their destabilization and to the release of chlorophylls. In this work, a fragment of a gene encoding a SGR from broccoli (BoSGR) was cloned. The expression of BoSGR was analyzed and detected an important increment during postharvest senescence, simultaneously with chlorophyll degradation. In order to analyze the effect of different growth regulators, different groups of broccoli heads were treated with cytokinins, ethylene and 1-MCP. Cytokinins and 1-MCP delayed the increment of BoSGR expression whereas ethylene accelerated the process. In addition, several postharvest treatments that delay degreening in broccoli florets were applied to evaluate their effects on BoSGR expression. Samples treated with modified atmosphere, hot air, UV-C or white lights showed a delay in chlorophyll degradation and degreening. In most cases, the treatments also delayed the increment of BoSGR expression during senescence, reaffirming the relationship between the expression of this gene and chlorophyll degradation.     

Pheophytinase activity and gene expression of chlorophyll-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets

Pheophytinase (PPH) activity and gene expression of chlorophyll (Chl)-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets were determined. PPH is involved in the dephytylation of Mg-free Chl a, pheophytin (Phy) a. However, in vitro chlorophyllase (Chlase, EC.3.1.1.14) also uses Phy a as a substrate to produce pheophorbide (Pheide) a by dephytylation. For an accurate determination of PPH activity, the PPH protein fraction was separated from Chlase protein by ammonium sulfate precipitation. The protein precipitated by 45-60% saturated ammonium sulfate included a little bit of Chlase activity and was suitable for PPH determination. PPH activity in broccoli florets treated with a UV-B dose of 19 kJ m⁻² was repressed for the first 2 d of storage at 15 °C, whereas it increased gradually with senescence of control broccoli florets. The expression level of BoCLH1 was reduced in broccoli florets on day 4 of storage, while BoCLH2 and BoCLH3 were up-regulated with UV-B treatment. A high BoPAO expression level was found in senescent broccoli florets, and the up-regulation of this gene was delayed by UV-B treatment. The highest expression level of BoPPH was found in the control, and its expression was clearly repressed by UV-B treatment on day 2 of storage. We suggest that the up-regulation of Chl-degrading enzyme genes could be delayed by UV-B treatment, resulting in the suppression of floret yellowing in stored broccoli.     

Expression of a lipoxygenase encoding gene (BoLOX1) during postharvest senescence of broccoli

Lipoxygenases (LOX) belong to a large family of plant enzymes that catalyze the hydroperoxidation of polyunsaturated fatty acids. Most of them are expressed during senescence and contribute to membrane deterioration and biosynthesis of jasmonic acid, a known senescence enhancer. In this work, we cloned a fragment of a gene encoding a LOX from broccoli (BoLOX1). The analysis of the sequence revealed that BoLOX1 is closely related to other LOX from higher plants. Furthermore, we analyzed the expression of BoLOX1 and detected a larger increase during postharvest senescence. A slight increase of total lipoxygenase activity was also found during senescence. In other sets of experiments, broccoli heads were treated with plant hormones, such as cytokinin and ethylene, as a way to assess the effect of such compounds on the expression of BoLOX1. Cytokinin treatment delayed the increase of BoLOX1 expression and lipoxygenase activity whereas ethylene accelerated both processes. Also, several postharvest treatments were applied in order to delay senescence in broccoli florets and to evaluate their effects on BoLOX1 expression. Samples treated with modified atmosphere, hot air, UV-C or white light showed a delay in chlorophyll degradation and degreening. In most cases, the treatments also delayed the increase of BoLOX1 expression, reaffirming the relationship between the expression of this gene and senescence. However, treatments like modified atmospheres and visible light markedly increased lipoxygenase activity, which suggests a lack of correlation between BoLOX1 expression and lipoxygenase activity.     

Effect of radiation intensity on the outcome of postharvest UV-C treatments

Studies on the use of UV-C radiation of fresh produce have focused on the selection of appropriate doses (energy per unit area) for different commodities, but little attention has been placed on the effect of radiation intensity (dose per unit time). In this study, tomatoes (Solanum lycopersicum cv. Elpida) and strawberries (Fragaria × ananassa cv. Camarosa), were harvested (breaker and 100% of surface red color respectively) and treated with 4 kJ m⁻² of UV-C, at low (3 W m⁻²) or high (33 W m⁻²) radiation intensities. Untreated fruits were used as controls. After the treatments and at different storage times the incidence of postharvest rots and the changes in fruit physical and chemical properties were determined. UV-C treatments reduced decay, with the effects being were more marked in fruit exposed to high intensities. Mold counts were unaffected by the treatments, suggesting that improved disease control did not result from greater germicide effect. In both fruit species exposure to UV-C radiation delayed ripening, evidenced as lower color development, pigment accumulation and softening. UV-C-treated fruit maintained better quality than the control. In strawberry, high intensity treatments were more effective to prevent deterioration than in tomato where the differences between UV-C treatments were subtler. Soluble solids, titratable acidity and ethanol soluble antioxidants were not affected regardless of the UV-C intensity. Consumer tests showed higher preference of fruit treated at high UV-C intensity. Results show that in addition to the applied dose, radiation intensity is a main factor determining the effectiveness of UV-C treatments and should not be over-sighted. For a given dose, increasing radiation intensity may in some cases maximize the benefits of UV-C on fruit quality, while significantly reducing the treatments time.     

Effects of gamma and UV-C irradiation on the postharvest control of papaya anthracnose

Anthracnose is the main postharvest disease in papaya fruit. Today, there is considerable interest on alternative methods of control to promote resistance against pathogens and supplement or replace the use of fungicides. The goal of this work was to evaluate the effects of gamma and UV-C irradiation on Colletotrichum gloeosporioides, the causal agent of anthracnose. Mycelial growth, sporulation, and conidial germination were evaluated in vitro after fungal exposition to different irradiation doses. In the in vivo assays, ‘Golden’ papaya fruit were inoculated through subcuticular injections of a conidial suspension or mycelium discs. Next, fruit were submitted to different irradiation doses (0, 0.12, 0.25, 0.5, 0.75, and 1 kGy), using Co⁶⁰ as source, or UV-C (0, 0.2, 0.4, 0.84, 1.3, and 2.4 kJ m⁻²). To check the possibility of resistance induction by irradiation, papayas were also inoculated 24, 48, or 72 h after the treatments. The fruit were stored at 25 °C/80% RH for 7 days and evaluated for incidence and rot severity. The results showed that the 0.75 and 1 kGy doses inhibited conidial germination and mycelial growth in vitro. All doses increased fungal sporulation. The 0.75 and 1 kGy doses reduced anthracnose incidence and severity, but did not reduce them when the fruit were inoculated after irradiation. All UV-C doses inhibited conidial germination and those higher than 0.84 kJ m⁻² inhibited mycelial growth. The 0.4, 0.84, and 1.3 kJ m⁻² UV-C doses reduced fungal sporulation in vitro. There was no effect of UV-C doses and time intervals between treatment and inoculation on anthracnose control and fungal sporulation in fruit lesions. Moreover, all UV-C doses caused scald on the fruit. Thus, gamma irradiation can contribute for the reduction of postharvest losses caused by anthracnose and reduce the use or doses of fungicides on disease control.     

Combination of electrolysed water,UV-C and superatmospheric O2 packaging for improving fresh-cut broccoli quality

The effects of neutral electrolysed water (NEW), ultraviolet light C (UV-C) and superatmospheric O₂ packaging (HO), single or combined, on the quality of fresh-cut kailan-hybrid broccoli for 19 days at 5 °C were studied. As controls, washing with water and sanitation with NaClO were both used. Electrolyte leakage, sensory, microbial and nutritional quality changes throughout shelf-life were studied. At day 15, the combined treatments achieved lower mesophilic and psychrophilic growth compared to the single ones. Single treatments produced higher ascorbate peroxidase (APX) reductions just after its application, while superoxide dismutase (SOD) showed the opposite behaviour. After 5 days at 5 °C, a great increase of APX and guaiacol peroxidase (GPX) activity was observed, NEW + UV-C + HO and HO-including treatments achieving the highest and the lowest APX increases, respectively. UV-C-including treatments produced the highest α-linolenic acid (ALA) decreases ranging 35–38% over control contents on the processing day. NEW-including treatments greatly reduced, throughout shelf-life, ALA and stearic acid (SA) content by 27–44% and 31–61%, respectively. Total phenolic content and antioxidant capacity (1415 mg ChAE kg⁻¹ fw and 287 mg AAE kg⁻¹ fw, respectively) remained quite constant during shelf-life. In general, the treatments and their possible combinations seem to be promising techniques to preserve, or even enhance, the quality of fresh-cut kailan-hybrid broccoli and, probably, other vegetables.     

Influence of UV-C treatment on antioxidant capacity,antioxidant enzyme activity and texture of postharvest shiitake (Lentinus edodes) mushrooms during storage

Shiitake (Lentinus edodes) mushrooms were exposed to UV-C light (4 kJ/m²) and stored in modified atmosphere packaging (MAP) prior to 15 days at 1 ± 1 °C and 95% relative humidity plus 3 days at 20 °C. Mushroom firmness, total phenolics, total flavonoids, ascorbic acid and H₂O₂ contents, superoxide anion (O₂⁻) production rate and activities of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) were measured. UV-C treatment resulted in maintenance of a high level of firmness during 15 days at low temperature and reduced the decrease in firmness during shelf-life storage. Furthermore, treated samples showed higher total flavonoids, ascorbic acid, and delayed the increases in both O₂⁻ production rate and H₂O₂ contents. However, no clear treatment effects were seen in total phenolics contents. The treatment also increased the antioxidant enzyme activities of CAT, SOD, APX and GR throughout the storage period. These results indicate that postharvest application of UV-C radiation can delay softening and enhance antioxidant capacity in shiitake mushrooms.     

Application of low intensity light pulses to delay postharvest senescence of Ocimum basilicum leaves

Fresh basil (Ocimum basilicum L.) is a highly perishable leafy green vegetable with a storage life of 4–5 d at room temperature. Exposure of basil leaves to temperatures below 12 °C during storage results in chilling injury; therefore, refrigeration cannot be used to extend postharvest life of basil. Typically, leafy vegetables are stored in darkness or extremely low irradiance. Darkness is known to induce senescence, and the initial phase of senescence is reversible by exposure to light. In this work, we studied the effects of low-intensity white light pulses at room temperature on postharvest senescence of basil leaves. Daily exposure for 2 h to 30–37 μmol m⁻² s⁻¹ of light was effective to delay postharvest senescence of basil leaves. Chlorophyll and protein levels decreased, ammonium accumulated and leaves developed visual symptoms of deterioration (darkening) during storage in darkness. Light pulses reduced the intensity of these senescence symptoms. The photosynthesis light compensation point of basil leaves was 50 μmol m⁻² s⁻¹ i.e., higher than the intensity used in this study, and the effect of treatment with red light was the same as with white light, while far red light was ineffective. Light pulses exerted a local effect on chlorophyll loss, but the effect on protein degradation was systemic (i.e., spreading beyond the illuminated parts of the leaf blade). The results of this study indicate that daily treatment for 2 h with low intensity light (30–37 μmol m⁻² s⁻¹ every day) during storage at 20 °C is an effective treatment to delay postharvest senescence of basil leaves. The delay of postharvest senescence by low intensity light pulses seems to be mediated by phytochromes, and it is systemic for protein, and partially systemic for chlorophyll degradation.     

Optimization of postharvest ultrasonic treatment of strawberry fruit

Freshly harvested strawberry fruit were treated at ultrasonic powers from 250 to 450 W at a constant frequency of 40 kHz for different times (5–15 min). Response surface methodology (RSM) based on a two factors three level central composite design was applied to optimize ultrasonic treatments on decay incidence, microbial population and quality maintenance of strawberries. According to response surface analysis, the optimal treatment parameters were an ultrasonic power of 250 W and treatment time of 9.8 min. Decay incidence and quality parameters of strawberries treated at the determined optimum conditions were compared with a water treatment during storage for 8 d at 5 °C. An ultrasonic treatment was found to be effective in inhibiting decay incidence and preserving quality in strawberries, and these results suggest that such a treatment may provide an alternative for extending shelf-life and maintaining quality of strawberry fruit.     

Expression of ROP/RAC GTPase genes in postharvest loquat fruit in association with senescence and cold regulated lignification

ROP/RAC GTPases regulate various development processes and play important roles in plant defense responses. Recently, lignification or secondary cell wall formation related ROP members were reported in rice, zinnia, cotton and Eucalyptus. The present study aimed to investigate the possible association of loquat ROPs with flesh lignification under different temperatures. Four ROP cDNA fragments, EjROP1.1, EjROP1.2, EjROP2 and EjROP3, were isolated from ‘Luoyangqing’ (LYQ) loquat fruit, and all of them shared over 80% nucleotide identity with known ROPs from other plants. Sequence analysis revealed that EjROP1.1, EjROP2 and EjROP3 might be functional while EjROP1.2, with mutated C-terminal resulted from a 65 bp deletion in the corresponding nucleotide sequence as compared with EjROP1.1, might be dominant-negative and consequently act as a negative regulator of ROP signal transduction. Increase in expression of EjROP1.1, EjROP2 and EjROP3 was observed during first 4 or 6 d of storage at 20 °C and was positively correlated with the increase in flesh firmness. Expression of EjROP1.2 was constantly low under 20 °C but was quickly, within 6 h, induced under 0 °C, and it increased by about 20 times within 24 h. The expression was induced under 5 °C as well but not so strong as that under 0 °C, and transfer of fruit from 5 °C to 0 °C re-stimulated the expression. The possible roles of EjROPs played during senescence and cold regulated lignification was discussed, and the simultaneous increase in the expression of three functional EjROPs and the negative regulator EjROP1.2 was suggested to be important for maintaining a ROP rheostat to protect cells from excessive lignification. To our knowledge, this is the first study on a dominant-negative ROP resulted from a deletion mutation, and a ROP responded to low temperature.     

Pre- and postharvest treatment with alternatives to synthetic fungicides to control postharvest decay of sweet cherry

The effectiveness of alternatives to synthetic fungicides for the control of pathogens causing postharvest diseases of sweet cherry was tested in vitro and in vivo. When amended to potato dextrose-agar, oligosaccharides, benzothiadiazole, chitosan, calcium plus organic acids, and nettle macerate reduced the growth of Monilinia laxa, Botrytis cinerea and Rhizopus stolonifer. Treatment of sweet cherries three days before harvest or soon after harvest with oligosaccharides, benzothiadiazole, chitosan, calcium plus organic acids, nettle extract, fir extract, laminarin, or potassium bicarbonate reduced brown rot, gray mold, Rhizopus rot, Alternaria rot, blue mold and green rot of cherries kept 10 d at 20 ± 1 °C, or 14 d at 0.5 ± 1 °C and then exposed to 7 d of shelf-life at 20 ± 1 °C. Among these resistance inducers, when applied either preharvest or postharvest, chitosan was one of the most effective in reducing storage decay of sweet cherry, and its antimicrobial activity in vitro and in field trials was comparable to that of the fungicide fenhexamid. Benzothiadiazole was more effective when applied postharvest than with preharvest spraying. These resistance inducers could represent good options for organic growers and food companies, or they can complement the use of synthetic fungicides in an integrated disease management strategy.     

Continuous microwave treatment to control postharvest brown rot in stone fruit

Monilinia spp. are the most important causes of brown rot in stone fruit and no chemical fungicides are allowed in the European Union to be applied to stone fruit after harvest. From preliminary studies, microwave (MW) treatments at 17.5 kW for 50 s and 10 kW for 95 s were selected as effective conditions to control brown rot. Both treatments were investigated to control Monilinia fructicola in fruit with different weights and maturity levels and in naturally infected fruit. Fruit weight only had a significant effect on microwave efficacy in ‘Placido’ peaches treated by MW at 10 kW for 95 s in which better brown rot control was observed in small than large fruit. Maturity level did not have a significant effect on efficacy of MW treatments in any of the varieties evaluated. When both MW treatments were studied in naturally infected peaches and nectarines, brown rot incidence was significantly reduced to less than 14% compared with untreated fruit where brown rot incidence was higher than 45%. The effect of both treatments on fruit quality was also evaluated. Fruit firmness was not negatively affected in the varieties tested and even a delay of fruit softening was observed. However, internal damage around the stone was observed, especially in the smallest fruit in which high temperature is achieved at the end of both MW treatments.     

Vitamin C in broccoli (Brassica oleracea L. var. italica) flower buds as affected by postharvest light,UV-B irradiation and temperature

In this study, the changes in vitamin C, l-ascorbic acid (AA) and l-dehydroascorbic acid (DHA) levels in broccoli flower buds were examined during pre-storage and storage periods, simulating refrigerated transport with wholesale distribution and retail, respectively. Broccoli heads were pre-stored for 4 or 7 days at 0 °C or 4 °C in the dark and then stored for 3 days at 10 °C or 18 °C. During storage the broccoli heads were exposed for 12 h per day to three different levels of visible light (13, 19 or 25 μmol m⁻² s⁻¹) or a combination of visible light (19 μmol m⁻² s⁻¹) and UV-B irradiation (20 kJ m⁻² d⁻¹), or they were stored in the dark. The vitamin C content in broccoli flower buds during storage was significantly affected by pre-storage period and temperature. Higher vitamin C levels in flower buds after storage were observed for broccoli heads pre-stored for 4 days or at 0 °C as compared to those pre-stored for 7 days or at 4 °C. Storage temperature also affected vitamin C in broccoli flower buds, with higher levels observed for broccoli stored at 10 °C than at 18 °C. Hence, vitamin C in broccoli flower buds was demonstrated to decrease together with increasing pre-storage period, pre-storage temperature and storage temperature. AA in broccoli flower buds was influenced mainly by storage temperature and to a minor extent by pre-storage temperature. The DHA level and DHA/AA ratio were stable in flower buds of broccoli pre-stored for 7 days, whereas increasing tendencies for both DHA level and ratio were observed after pre-storage for 4 days. These results indicate a shift in the ascorbate metabolism in broccoli flower buds during storage at low temperatures, with its higher rate observed for broccoli pre-stored for shorter time. There were no effects of the light and UV-B irradiation treatments on vitamin C, AA and DHA levels in broccoli flower buds.     

The effect of CO2 and 1-methylcyclopropene on the regulation of postharvest senescence of mint,Mentha longifolia L.

The regulatory effects of 5 kPa CO₂ and of the ethylene action inhibitor, 1-methylcyclopropene (1-MCP) at 0.5 μmol/l on the senescence of harvested mint, Mentha longifolia L. were assessed. Visual parameters of senescence including yellowing, browning, decay and leaf abscission were recorded and scored on scales linking the onset and progression of senescence to marketability. The effects of plant age on the rate of postharvest senescence and on the efficacy of the CO₂ and 1-MCP treatments were also investigated. All experiments were repeated with and without the presence of exogenous ethylene. Two experimental formats were used, with 6 days storage at room temperature representing local market conditions, and 6 days cold storage at 1.5 °C followed by 4 days at room temperature representing export market conditions. Sprigs from old plants were no longer of marketable quality after 6 days storage at room temperature. Exogenous ethylene accelerated the onset of senescence causing unacceptably high rates of leaf abscission. Raised levels of CO₂ in a controlled atmosphere system were found to be more effective in inhibiting senescence without the presence of exogenous ethylene than pre-treatment with 1-MCP, and no additive effect was found. However in the presence of exogenous ethylene, a combined treatment with 1-MCP together with raised CO₂ levels resulted in a significant additive effect in nullifying the ethylene-induced leaf abscission. Respiration rates as measured by CO₂ production, and ethylene production, were recorded throughout all experiments. While CO₂ levels were not affected by any experimental treatment, ethylene production was elevated in mint sprigs exposed to an initial dose of gaseous 1-MCP, and was further increased under a combined treatment of 1-MCP together with 5 kPa CO₂. However in the presence of exogenous ethylene, CO₂ strongly suppressed the 1-MCP induced ethylene production.