César Luis Girardi

Identification of a novel α-L-arabinofuranosidase gene associated with mealiness in apple

In order to investigate the genetic bases of the physiological syndrome mealiness that causes abnormal fruit softening and juice loss in apples, an integrative approach was devised, consisting of sensory, instrumental, biochemical, genetic, and genomic methods. High levels of activity of α-L-arabinofuranosidase (α-AFase), a hydrolase acting on the pectic component of the cell walls, were found in individuals exhibiting the mealiness phenotype in a segregating population. The expression levels of the previously uncharacterized apple AF gene MdAF3 are higher in fruits from plants consistently showing mealiness symptons and high α-AFase activity. The transcription of MdAF3 is differentially regulated in distinct genomic contexts and appears to be independent of ethylene. Thus, it is likely to be controlled by endogenous developmental mechanisms associated with fruit ripening. The use of integrative approaches has allowed the identification of a novel contributor to the mealiness phenotype in apple and it has been possible to overcome the problems posed by the unavailability of near-isogenic lines to dissect the genetic bases of a complex physiological trait in woody perennial species.

CONSERVATION OF PERSIMMONS FRUITS (Diospyros kaki, L.), cv. FUYU WITH APLICATION OF 1- METHYLYCLOPROPENE

The present work evaluated the effects of the 1-MCP (1-methylcyclopropene) on persimmons fruits (Diospyrus kaki L.) of cv. Fuyu stored at 0oC for up to 90 days. Fruits were picked from a commercial orchard in Farroupilha-RS, when skin color was yellow-orange. Three concentrations of 1-MCP (312, 625 and 1250 nL.L-1) were applied for 24 hours in an ambient temperature (±25oC). Control fruit were kept under identical conditions without 1-MCP treatment. Immediately after treatment application, the fruits were transferred to a cold storage at 0oC and approximately 90% of relative humidity. Persimmons were analysed on the day of harvest, after 30, 60 and 90 days of refrigerated storage plus 3 more days at ambient temperature to simulate the commercialization period. Total soluble solids, pH and titratable acidity were not influenced by 1-MCP treatments. Ethylene production rates did not reach detectable levels. Skin color showed a higher development of red color in fruits treated with 1-MCP. Flesh firmness was significantly higher in treated fruits in comparison to control fruits, indicating a positive effect of 1-MCP in the postharvest life of persimmons fruits. There was no significant difference among the different concentrations of 1-MCP.

Efeito da aplicação de ácido giberélico e cloreto de cálcio no retardamento da colheita e na conservabilidade de caqui, Fuyu

The present work studied the effects of the gibeberellic acid(GA3) and of the CaCl2, in the harvest sprays on ripening control and on the postharvest quality of persimmons. For the field treatments, groups of 4 plants were selected that were sprayed with: a)30 ppm GA3; b)1%(m/v) CaCl2; c)1% CaCl2 + 30ppm GA3 and d) water as control. Fruits were harvested with green-yellowish epidermal color. They were stored at ambient conditions and in plastic boxes. At the harvest and every 4 days during 20 days, persimmons were evaluated for weight loss, flesh firmness, titratable acidity, ethylene production, total soluble solids, chlorophylls and carotenoids. GA3 treatments delayed harvest for 15 days and increased the storage potential of the fruits. CaCl2 sprays did not contribute delaying the maturation and reduced the beneficial effects of the GA3 after harvest, when combined.

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

Effects of pre-harvest gibberellic acid spraying on gene transcript accumulation during peach fruit development

In order to understand early molecular events associated with increase in fruit size and woolliness prevention induced by pre-harvest gibberellic acid (GA3) spraying, differential transcript accumulation of genes encoding proteins putatively involved in protein folding and protection, cell wall metabolism, and endomembrane transport was studied during fruit development of ‘Chiripá’ peach. Woolliness occurrence reached 100% in untreated peach, was reduced by 15% with GA3 spraying at the end of the pit hardening stage and was significantly reduced (by 78%) in peaches treated at the beginning of the pit hardening stage despite a significantly increased fruit size. Low incidence of woolliness after cold storage and fruit size increase in early GA3 treated peach was correlated with high transcript accumulation of genes encoding proteins putatively involved in protein folding, and protection of the endoplasmic reticulum (heat shock proteins—HSP40 er) and chloroplast (HSP17.8 ch), endomembrane transport (GTPase), as well as genes involved in cell wall loosening (expansins—Exp1, Exp2, Exp3, Exp4).

Transcriptional regulatory networks controlling woolliness in peach in response to preharvest gibberellin application and cold storage

BackgroundPostharvest fruit conservation relies on low temperatures and manipulations of hormone metabolism to maintain sensory properties. Peaches are susceptible to chilling injuries, such as ‘woolliness’ that is caused by juice loss leading to a ‘wooly’ fruit texture. Application of gibberellic acid at the initial stages of pit hardening impairs woolliness incidence, however the mechanisms controlling the response remain unknown. We have employed genome wide transcriptional profiling to investigate the effects of gibberellic acid application and cold storage on harvested peaches.ResultsApproximately half of the investigated genes exhibited significant differential expression in response to the treatments. Cellular and developmental process gene ontologies were overrepresented among the differentially regulated genes, whereas sequences in cell death and immune response categories were underrepresented. Gene set enrichment demonstrated a predominant role of cold storage in repressing the transcription of genes associated to cell wall metabolism. In contrast, genes involved in hormone responses exhibited a more complex transcriptional response, indicating an extensive network of crosstalk between hormone signaling and low temperatures. Time course transcriptional analyses demonstrate the large contribution of gene expression regulation on the biochemical changes leading to woolliness in peach.ConclusionOverall, our results provide insights on the mechanisms controlling the complex phenotypes associated to postharvest textural changes in peach and suggest that hormone mediated reprogramming previous to pit hardening affects the onset of chilling injuries.

Ethylene-dependent regulation of an α-L-arabinofuranosidase is associated to firmness loss in 'Gala' apples under long term cold storage.

Fruit texture changes impair the quality of apples submitted to long term storage, especially under cold. The changes are due to cell wall modifications during ripening and senescence and are associated to ethylene. We have investigated the activity of α-l-arabinofuranosidase, a glycosyl hydrolase acting on the side chains of pectin in the cell wall and middle lamella. The transcription of arabinofuranosidase coding sequences 1 and 3 was investigated in plant organs and in response to ethylene, employing hormone application and 1-methylcyclopropene. The transcription of arabinofuranosidase genes is not restricted to fruits, although upregulated by ripening and ethylene. Transcripts of the genes were detected under cold storage up to 180 days. Similarly, arabinofuranosidase activity increased with rising levels of ethylene and under cold storage. Levels of arabinofuranosidase3 transcripts were higher than those of arabinofuranosidase1, suggesting that the first is an important contributor to enzyme activity and texture changes during cold storage.

Contrasting Transcriptional Programs Control Postharvest Development of Apples (Malus x domestica Borkh.) Submitted to Cold Storage and Ethylene Blockage

Apple is commercially important worldwide. Favorable genomic contexts and postharvest technologies allow year-round availability. Although ripening is considered a unidirectional developmental process toward senescence, storage at low temperatures, alone or in combination with ethylene blockage, is effective in preserving apple properties. Quality traits and genome wide expression were integrated to investigate the mechanisms underlying postharvest changes. Development and conservation techniques were responsible for transcriptional reprogramming and distinct programs associated with quality traits. A large portion of the differentially regulated genes constitutes a program involved in ripening and senescence, whereas a smaller module consists of genes associated with reestablishment and maintenance of juvenile traits after harvest. Ethylene inhibition was associated with a reversal of ripening by transcriptional induction of anabolic pathways. Our results demonstrate that the blockage of ethylene perception and signaling leads to upregulation of genes in anabolic pathways. We also associated complex phenotypes to subsets of differentially regulated genes.

Atmosfera controlada associada ao 1-metilciclopropeno na preservação da qualidade de kiwi ‘tewi’

The aim of this study was to evaluate the effect of different storage technologies on the physical and chemical qualities of ‘Tewi’ kiwifruits. After harvesting, part of the fruits was treated with 1-methylcyclopropene (1-MCP), and then both the treated and non-treated fruits were stored under different conditions, as follows: storage at room temperature (20 °C ± 0.5 °C) for twelve days; refrigerated storage (0°C ± 0.5°C), with or without a controlled atmosphere (3% O2 and 5% CO2); and storage with or without ethylene adsorption for two and four months. The firmness of the flesh, soluble solids content and titratable acidity were evaluated during storage. It was noted that regardless of storage technology there was a reduction in firmness after two months. The controlled atmosphere conditions with 1-MCP were the most effective in retaining firmness. Fruits kept at room temperature showed a direct relationship between the reduction in flesh firmness and the increase in soluble solids content, although this relationship was not always observed after storage. The highest titratable acidity was observed in the fruits treated with 1-MCP. ‘Tewi’ kiwis showed good conservation potential, and could be stored for up to four months in a controlled atmosphere with ethylene management using 1-MCP or ethylene absorption.

Manejo do resfriamento e da classificação de pêssegos cv granada na ocorrência de podridões e qualidade de consumo

The loss of quality in peach after harvest is associated with metabolic changes, wounding, loss of pulp firmness, physiological disorders and decay. In the Granada cultivar, decay is the most important determinant of storage-life. Aiming to prevent this problem, different procedures of postharvest handling were tested in two experiments: 1) harvest, pre-cooling, cooled transportation and storage at 23±2°C and 75%±5% relative humidity (RH), 2) harvest, pre-cooling, cold storage for 15 days, classification in equipment with or without water to transport the fruit, with or without application of wax coating on fruit, cooled transportation, and storage at 23±2°C and 60-75% RH. In both experiments, we evaluated: fruit decay, colour, pulp firmness and general acceptability. Decay was lower in pre-cooled peach than in not pre-cooled peach before cooled transportation. The classification, in equipment with water, favoured decay occurrence, in comparison to the classification in equipment without water, if the shelf life exceeds three days. The wax treatment in equipment without water improved the appearance of the fruit and contributed to prevent decay.