Ioannis S. Minas

Physiological and proteomic approaches to address the active role of ozone in kiwifruit post-harvest ripening

Post-harvest ozone application has recently been shown to inhibit the onset of senescence symptoms on fleshy fruit and vegetables; however, the exact mechanism of action is yet unknown. To characterize the impact of ozone on the post-harvest performance of kiwifruit (Actinidia deliciosa cv. ‘Hayward’), fruits were cold stored (0 °C, 95% relative humidity) in a commercial ethylene-free room for 1, 3, or 5 months in the absence (control) or presence of ozone (0.3 μl l−1) and subsequently were allowed to ripen at a higher temperature (20 °C), herein defined as the shelf-life period, for up to 12 days. Ozone blocked ethylene production, delayed ripening, and stimulated antioxidant and anti-radical activities of fruits. Proteomic analysis using 1D-SDS-PAGE and mass spectrometry identified 102 kiwifruit proteins during ripening, which are mainly involved in energy, protein metabolism, defence, and cell structure. Ripening induced protein carbonylation in kiwifruit but this effect was depressed by ozone. A set of candidate kiwifruit proteins that are sensitive to carbonylation was also discovered. Overall, the present data indicate that ozone improved kiwifruit post-harvest behaviour, thus providing a first step towards understanding the active role of this molecule in fruit ripening.

Postharvest Fruit Rots of Apple in Greece: Pathogen Incidence and Relationships Between Fruit Quality Parameters, Cultivar Susceptibility, and Patulin Production

The incidence of pathogens associated with postharvest fruit rots on the four most extensively cultivated apple cultivars (Red Delicious, Golden Delicious, Granny Smith, and Fuji) in Greece was surveyed during two consecutive storage periods (2008-09 and 2009-10) in five packinghouses located in northern Greece. The fungi isolated were identified based on their morphological characteristics and internal transcribed spacer gene sequencing. In the four cultivars sampled, Penicillium expansum and Botrytis cinerea were the predominant pathogens, accounting for averages of 44.2 and 23.6%, respectively, of the pathogens isolated from the sampled fruit. Two other important rot pathogens were Alternaria tenuissima and Mucor pyriformis, accounting for 16.1 and 6.6%, respectively, of the diseased apple fruit. Other pathogens such as Monilinia laxa, M. fructigena, Botryosphaeria obtusa, Geotrichum candidum, Fusarium avenaceum, and F. proliferatum were isolated at low frequencies and are considered of minor importance. Measurements of the resistance level of the four apple cultivars to fruit rot caused by P. expansum and Botrytis cinerea revealed that Golden Delicious was the most susceptible to blue mold while Fuji was the most susceptible to gray mold infections. Susceptibility to gray mold was negatively correlated with flavonoid and phenol concentration as well to fruit antioxidant activity, while susceptibility to blue mold was negatively correlated with fruit firmness and phenol concentration. Patulin production was significantly higher in Red Delicious and Golden Delicious fruit than in Granny Smith and Fuji fruit and was negatively correlated with the acidity of the fruit. The high incidence of P. expansum and A. tenuissima along with the presence of F. avenaceum and F. proliferatum, all of which are potentially mycotoxin producers, emphasize the risk for mycotoxin contamination of apple fruit juices and by-products. Furthermore, information on the distribution of the pathogens on the main cultivars may be useful for the implementation of strategies to control the diseases and minimize the threat of mycotoxin contamination on each cultivar.

The impact of sodium nitroprusside and ozone in kiwifruit ripening physiology: a combined gene and protein expression profiling approach

BACKGROUND AND AIMS Despite their importance in many aspects of plant physiology, information about the function of oxidative and, particularly, of nitrosative signalling in fruit biology is limited. This study examined the possible implications of O3 and sodium nitroprusside (SNP) in kiwifruit ripening, and their interacting effects. It also aimed to investigate changes in the kiwifruit proteome in response to SNP and O3 treatments, together with selected transcript analysis, as a way to enhance our understanding of the fruit ripening syndrome. METHODS Kiwifruits following harvest were pre-treated with 100 μm SNP, then cold-stored (0 °C, relative humidity 95 %) for either 2 or 6 months in the absence or in the presence of O3 (0·3 μL L(-1)), and subsequently were allowed to ripen at 20 °C. The ripening behaviour of fruit was characterized using several approaches: together with ethylene production, several genes, enzymes and metabolites involved in ethylene biosynthesis were analysed. Kiwifruit proteins were identified using 2-D electrophoresis coupled with nanoliquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Expression patterns of kiwifruit ripening-related genes were also analysed using real-time quantitative reverse transcription-PCR (RT-qPCR). KEY RESULTS O3 treatment markedly delayed fruit softening and depressed the ethylene biosynthetic mechanism. Although SNP alone was relatively ineffective in regulating ripening, SNP treatment prior to O3 exposure attenuated the O3-induced ripening inhibition. Proteomic analysis revealed a considerable overlap between proteins affected by both SNP and O3. Consistent with this, the temporal dynamics in the expression of selected kiwifruit ripening-related genes were noticeably different between individual O3 and combined SNP and O3 treatments. CONCLUSIONS This study demonstrates that O3-induced ripening inhibition could be reversed by SNP and provides insights into the interaction between oxidative and nitrosative signalling in climacteric fruit ripening.

1H NMR Metabolic Fingerprinting to Probe Temporal Postharvest Changes on Qualitative Attributes and Phytochemical Profile of Sweet Cherry Fruit

Sweet cherry fruits (Prunus avium cvs. ‘Canada Giant’, ‘Ferrovia’) were harvested at commercial maturity stage and analyzed at harvest and after maintenance at room temperature (storage at ∼20°C, shelf life) for 1, 2, 4, 6, and 8 days, respectively. Fruit were initially analyzed for respiration rate, qualitative attributes and textural properties: ‘Canada Giant’ fruit were characterized by higher weight losses and stem browning index, being more intense over the late stages of shelf life period; meanwhile ‘Ferrovia’ possessed appreciably better performance even after extended shelf life period. A gradual decrease of respiration rate was monitored in both cultivars, culminated after 8 days at 20°C. The sweet cherry fruit nutraceutical profile was monitored using an array of instrumental techniques (spectrophotometric assays, HPLC, 1H-NMR). Fruit antioxidant capacity was enhanced with the progress of shelf life period, concomitant with the increased levels of total anthocyanin and of phenolic compounds. ‘Ferrovia’ fruit presented higher contents of neochlorogenic acid and p-coumaroylquinic acid throughout the shelf life period. We further developed an 1H-NMR method that allows the study of primary and secondary metabolites in a single running, without previous separation and isolation procedures. Diagnostic peaks were located in the aliphatic region for sugars and organic acids, in the aromatic region for phenolic compounds and at 8.2–8.6 ppm for anthocyanins. This NMR-based methodology provides a unifying tool for quantitative and qualitative characterization of metabolite changes of sweet cherry fruits; it is also expected to be further exploited for monitoring temporal changes in other fleshy fruits.

Discovery of non-climacteric and suppressed climacteric bud sport mutations originating from a climacteric Japanese plum cultivar (Prunus salicina Lindl.)

Japanese plums are classified as climacteric; however, some economically important cultivars selected in California produce very little ethylene and require long ripening both “on” and “off” the tree to reach eating-ripe firmness. To unravel the ripening behavior of different Japanese plum cultivars, ripening was examined in the absence (air) or in the presence of ethylene or propylene (an ethylene analog) following a treatment or not with 1-methylcyclopropene (1-MCP, an ethylene action inhibitor). Detailed physiological studies revealed for the first time three distinct ripening types in plum fruit: climacteric, suppressed-climacteric, and non-climacteric. Responding to exogenous ethylene or propylene, the slow-softening supressed-climacteric cultivars produced detectable amounts of ethylene, in contrast to the novel non-climacteric cultivar that produced no ethylene and softened extremely slowly. Genetic analysis using microsatellite markers produced identical DNA profiles for the climacteric cultivars “Santa Rosa” and “July Santa Rosa,” the suppressed-climacteric cultivars “Late Santa Rosa,” “Casselman,” and “Roysum” and the novel non-climacteric “Sweet Miriam,” as expected since historic records present most of these cultivars as bud-sport mutations derived initially from “Santa Rosa.” This present study provides a novel fruit system to address the molecular basis of ripening and to develop markers that assist breeders in providing high-quality stone fruit cultivars that can remain “on-tree,” increasing fruit flavor, saving harvesting costs, and potentially reducing the need for low-temperature storage during postharvest handling.