Bruno G. Defilippi

Effect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple fruit

The role of ethylene in regulating sugar, acid, texture and volatile components of fruit quality was investigated in transgenic apple fruit modified in their capacity to syntheize endogenous ethylene. Fruit obtained from plants silenced for either ACS (ACC synthase; ACC – 1-aminocyclopropane-1-carboxylic acid) or ACO (ACC oxidase), key enzymes responsible for ethylene biosynthesis, expectedly showed reduced autocatalytic ethylene production. Ethylene suppressed fruits were significantly firmer than controls and displayed an increased shelf-life. No significant difference was observed in sugar or acid accumulation suggesting that sugar and acid composition and accumulation is not directly under ethylene control. Interestingly, a significant and dramatic suppression of the synthesis of volatile esters was observed in fruit silenced for ethylene. However, no significant suppression was observed for the aldehyde and alcohol precursors of these esters. Our results indicate that ethylene differentially regulates fruit quality components and the availability of these transgenic apple trees provides a unique resource to define the role of ethylene and other factors that regulate fruit development.

Characterization of Sugars and Organic Acids in Commercial Varieties of Table Grapes

La composicion del sabor ha sido definida como un atributo complejo de la calidad de la fruta, en el cual la mezcla de azucares, acidos y volatiles juega un rol determinante. En uva de mesa (Vitis vinifera L.), el dulzor y la acidez son los atributos primordiales para su consumo fresco. Sin embargo, la mayoria de los estudios realizados al respecto se han desarrollado en uva para la produccion de vino, la cual crece, se cultiva y se procesa de manera diferente a la uva de mesa. Por lo tanto, el objetivo del presente trabajo se centro en caracterizar los cambios en azucares y acidos organicos a lo largo de su desarrollo en las variedades ‘Thompson Seedless’, ‘Red Globe’, y ‘Crimson Seedless’, producidas bajo las mismas condiciones agroclimaticas. Para cada variedad se realizaron muestreos semanales desde 2 semanas previas a pinta hasta la cosecha comercial. Los azucares y acidos organicos fueron cuantificados mediante cromatografia liquida de alto rendimiento (HPLC) equipados con detectores evaporativos de dispersion de la luz (ELSD) y ultra violeta, respectivamente. El rango de concentraciones de acidos y azucares encontrados en uva fueron los siguientes: acido tartarico, 1,28-7,45 g L-1; acido malico, 0,38-29,92 g L-1, acido citrico trazas-1,03 g L-1; fructosa, 0,15-8,74 g (azucar) 100 g (uva)-1; glucosa, 0,19-8,71 g (azucar) 100 g (uva)-1 and sacarosa 0.02-0.91 g (azucar) 100 g(uva)-1. Entre los azucares, glucosa fue la mas abundante en etapas tempranas y luego disminuyo hasta el periodo de cosecha, cuando la cantidad de fructosa y glucosa converge en un promedio de 47% para cada azucar. A pesar de que los acidos organicos alcanzaron un nivel constante entre 3 a 4 semanas antes de su cosecha, existen diferencias importantes en el perfil de acidos organicos entre las variedades, con ‘Thompson Seedless’ mostrando la mas baja relacion de acidos tartarico/malico de 1,19. Estas diferencias son un punto importante al considerar el sabor general para cada variedad.

Chapter 1 Aroma Volatiles: Biosynthesis and Mechanisms of Modulation During Fruit Ripening

Abstract Flavor composition has been defined as a complex attribute of quality, in which the mix of sugars, acids, and volatiles play a primary role. In addition to the four basic flavors (sweet, sour, salty, and bitter) that humans can recognize in fruits and vegetables, aroma has an important influence on the final consumer acceptance of the commodity. Fruit aroma is determined by a complex mixture of a large number of volatile compounds including alcohols, aldehydes, and esters. During fruit development, especially at ripening, there are many changes of these metabolites caused by their synthesis, transport or degradation. In terms of volatile biosynthesis, several studies have been performed identifying and characterizing the most important genes and encoded enzymes involved in aroma-related volatiles; however, research in the mechanisms of regulation or modulation is still limited. To have an updated overview about aroma biosynthesis in fruit species, the main objective of this manuscript was to review the recent advances in this topic, mainly in terms of the new insights in volatile characterization, gene identification, and regulation of aroma during fruit ripening.

Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.).

Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adhEU395433; Pa-pdcEU395434; and Pa-loxEU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.

Browning in Annona cherimola fruit: Role of polyphenol oxidase and characterization of a coding sequence of the enzyme

Cherimoya (Annona cherimola Mill.) fruit is an attractive candidate for food processing applications as fresh cut. However, along with its desirable delicate taste, cherimoya shows a marked susceptibility to browning. This condition is mainly attributed to polyphenol oxidase activity (PPO). A general lack of knowledge regarding PPO and its role in the oxidative loss of quality in processed cherimoya fruit requires a better understanding of the mechanisms involved. The work carried out included the cloning of a full-length cDNA, an analysis of its properties in the deduced amino sequence, and linkage of its mRNA levels with enzyme activity in mature and ripe fruits after wounding. The results showed one gene different at the nucleotide level when compared with previously reported genes, but a well-conserved protein, either in functional and in structural terms. Cherimoya PPO gene (Ac-ppo, GenBank DQ990911) showed to be present apparently in one copy of the genome, and its transcripts could be significantly detected in leaves and less abundantly in flowers and fruits. Analysis of wounded matured and ripened fruits revealed an inductive behavior for mRNA levels in the flesh of mature cherimoya after 16 h. Although the highest enzymatic activity was observed on rind, a consistent PPO activity was detected on flesh samples. A lack of correlation between PPO mRNA level and PPO activity was observed, especially in flesh tissue. This is probably due to the presence of monophenolic substrates inducing a lag period, enzyme inhibitors and/or diphenolic substrates causing suicide inactivation, and proenzyme or latent isoforms of PPO. To our knowledge this is the first report of a complete PPO sequence in cherimoya. Furthermore, the gene is highly divergent from known nucleotide sequences but shows a well conserved protein in terms of its function, deduced structure, and physiological role.

Effect of harvest maturity stage on the sensory quality of 'Palsteyn' apricot (Prunus armeniaca L.) after cold storage

Summary Fresh apricots (Prunus armeniaca L.) are in high demand, but are available for only a short period during the Spring and the beginning of the Summer. There is no information on the correct harvest maturity stage that should be chosen to ensure a long post-harvest life and high sensory quality. The objective of this study was to evaluate the effect of maturity stage at harvest on the sensory quality of ‘Palsteyn’ apricots after 14, 28, or 42 d in cold storage. Sourness, flavour, and acceptability showed major changes during cold storage. Ripe fruit (orange-yellow skin colour) showed the highest acceptability, as assessed by a “mini-consumer test” (i.e., an untrained panel; n = 36). Fruit harvested at an intermediate stage of maturity (light yellow skin colour) reached an average acceptability, while unripe fruit (greenish skin colour) were not acceptable. After 28 d of cold storage, fruits were still acceptable; but, after 42 d, fruits reached the “dislike” zone. On a principal component analysis, acceptability was shown to be positively associated with sweetness, flavour, juiciness, and aroma, and negatively associated with sourness. After 28 d and 42 d of cold storage, unripe fruit appeared to have low acceptability and quality attributes. ‘Palsteyn’ apricot harvested with an orange-yellow skin colour, as the least ripe fruits, showed that they could reach high sensory quality standards and were able to withstand long periods of cold storage.

Proteomic analysis of a segregant population reveals candidate proteins linked to mealiness in peach

Peaches are stored at low temperatures to delay ripening and increase postharvest life. However some varieties are susceptible to chilling injury,which leads to fruit mealiness, browning and flesh bleeding. In order to identify potentialmarkers associated with chilling injury,we performed proteomic analyses on a segregating population with contrasting susceptibility to chilling-induced mealiness. Chilling-induced mealiness was assessed by measuring juiciness in fruits that have been stored in cold and then allowed to ripen. Fruitmesocarp and leaf proteome from contrasting segregants were analyzed using 2-DE gels. Comparison of protein abundance between segregants revealed 133 spots from fruit mesocarp and 36 from leaf. Thirty four fruit mesocarp proteins were identified from these spots. Most of these proteins were related to ethylene synthesis, ABA response and stress response. Leaf protein analyses identified 22 proteins, most of which related to energy metabolism. Some of the genes that code for these proteins have been previously correlated with chilling injury through transcript analyses and co-segregation with mealiness QTLs. The results from this study, further deciphers the molecular mechanisms associated with chilling response in peach fruit, and identifies candidate proteins linked to mealiness in peach which may be used as putative markers for this trait.

Effect of Harvest Time and L-Cysteine as an Antioxidant on Flesh Browning of Fresh-Cut Cherimoya (Annona cherimola Mill.)

A B S T R A C T Browning development is the most important factor limiting the quality of fresh-cut cherimoya (Annona cherimola Mill.). However, there is little information available about its causes and methods of control. The effectiveness of L-cysteine (0.125, 0.25 and 0.5%) in fresh-cut cherimoya harvested on two occasions (October and November) and stored for 6 and 12 days at 0 °C was studied. In order to understand the biological basis of browning, polyphenol oxidase (PPO) enzyme activity and total phenolic content in fresh-cut pieces were measured. Quality measurements and sensory analysis indicated that 0.5% L-cysteine was somewhat effective in reducing browning development, without affecting other quality attributes. In terms of physiological parameters, PPO activity did not show differences between mature (at harvest) and ripe fruit (at processing) in both harvest times, but cherimoya fruits picked in November presented lower PPO activity than fruit from October. In general, PPO activity and total phenolic content of L-cysteine treated fruits did not show consistent differences with untreated fruit at 6 or 12 days at 0 °C. PPO activity analyses demonstrated that PPO activity was higher in the outer part of cherimoya flesh compared to the middle or inner sector. These results would support the possibility of using L-cysteine as a postharvest treatment to reduce browning development in fresh-cut cherimoya.

Identification and characterization of genes differentially expressed in cherimoya (Annona cherimola Mill) after exposure to chilling injury conditions.

Cherimoyas (Annona cherimola), like other subtropical/tropical fruits, are susceptible to damage from exposure to temperatures between 0 and 5 °C (chilling injury, CI), which may affect fruit quality. To increase our understanding of the molecular mechanisms involved in the CI response, a forward suppression subtractive hybridization (SSH) cDNA library was constructed. In this work, we obtained 75 genes that could potentially be involved in the CI response. The CI induced activation of genes that are involved in a range of metabolic pathways, such as primary metabolism, transport, and endomembrane traffic, among others. We also characterized the expression of 12 selected genes in different A. cherimola tissues by polymerase chain reaction (PCR), and we confirmed the differential expression of a subset in CI fruits by real-time quantitative PCR (qPCR). The expression of six A. cherimola genes: annexin (AcAnex), UDP-glucose pyrophosphorylase (AcUGP), syntaxin of plants 71 (AcSyp71), 1-aminocyclopropane-1-carboxylic-acid synthase (AcACS), ubiquitin carrier-like protein (AcUCP), and enolase (AcEnol), was up-regulated after cold storage for 12 days at 0 °C. These results imply that selected genes could be related to the development of internal browning observed in cherimoyas after exposure to CI conditions. The information generated in this study provides new clues that may aid in understanding the cherimoya ripening process.

Comparative Study of Two Table Grape Varieties with Contrasting Texture during Cold Storage

Postharvest softening of grape berries is one of the main problems affecting grape quality during export. Cell wall disassembly, especially of pectin polysaccharides, has been commonly related to fruit softening, but its influence has been poorly studied in grapes during postharvest life. In order to better understand this process, the Thompson seedless (TS) variety, which has significantly decreased berry texture after prolonged cold storage, was compared to NN107, a new table grape variety with higher berry firmness. Biochemical analysis revealed a greater amount of calcium in the cell wall of the NN107 variety and less reduction of uronic acids than TS during cold storage. In addition, the activity of polygalacturonase was higher in TS than NN107 berries; meanwhile pectin methylesterase activity was similar in both varieties. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) suggests a differential pectin metabolism during prolonged cold storage. Results revealed lower pectin fragments in TS after 60 days of cold storage and shelf life (SL) compared to 30 days of cold storage and 30 + SL, while NN107 maintained the same fragment profile across all time points evaluated. Our results suggest that these important differences in cell wall metabolism during cold storage could be related to the differential berry firmness observed between these contrasting table grape varieties.