Internal browning disorders of 'Rocha' pear during long-term storage

A pera 'Rocha' (Pyrus communis L.) é uma cultivar Portuguesa que pode ser armazenada em atmosfera controlada (AC) durante 10 meses. No entanto, sabe-se que é suscetível ao acastanhamento interno (AI), uma das principais causas de perdas económicas durante o armazenamento prolongado. Embora muitos estudos tenham sido realizados para melhor compreender o AI em peras, os mecanismos subjacentes não permanecem por elucidar. O objetivo desta tese é investigar os mecanismos fisiológicos, bioquímicos e moleculares envolvidos no desenvolvimento do AI em pera 'Rocha', estabelecendo a base científica para o desenvolvimento de modelos preditivos para o AI e recomendações de manuseio pós-colheita, visando reduzir a incidência de AI em um ambiente regulatório em que as ferramentas químicas convencionais já não se encontram disponíveis. O AI em pera pode ter vários sintomas e foi demostrado que a suscetibilidade da pera para desenvolver este acidente fisiológico é afetada pela maturação dos frutos à colheita. Neste trabalho, os AIs em pera 'Rocha' foram classificados em duas categorias: decomposição necrótica húmida e cavidades secas, que podem coexistir no mesmo fruto. A ocorrência de cavidades foi associada a períodos de armazenamento mais prolongados e à exposição dos frutos a alto CO2. Os resultados mostraram claramente que as peras colhidas tardiamente eram mais suscetíveis ao AI do que as colhidas num estado de maturação precoce ou ótimo. Para o estudo da base bioquímica do AI foram conduzidos três ensaios em três anos consecutivos. Na nossa primeira avaliação, realizada numa base de longo prazo, a fermentação desempenhou um papel importante no AI, mas o sistema antioxidante não. No entanto, no segundo ensaio, realizado numa base de curto prazo, os resultados sugeriram que o AI em pera 'Rocha' era desencadeado por stress oxidativo. Finalmente, no terceiro ensaio, a base bioquímica do AI relacionado com o CO2 em pera 'Rocha' foi estabelecida. Os resultados mostraram que o mecanismo subjacente ao AI em pera 'Rocha' envolve a conjugação de ambos os metabolismos, antioxidante e fermentativo. A partir dos resultados obtidos um mecanismo o para o desenvolvimento do AI relacionada com o CO2 foi proposto. Neste estudo também se analisou pela primeira vez a regulação molecular de genes que codificam enzimas antioxidantes e fermentativas. As diferenças encontradas na regulação da transcrição destes genes apoiaram os dados bioquímicos. Os resultados também mostraram que o armazenamento dos frutos em alto CO2 leva a uma disfunção do sistema antioxidante e que, quando o alto CO2 é combinado com níveis muito baixos de O2 a fermentação é altamente induzida, com ambos os fatores a atuar sinergicamente no desenvolvimento de IBD. Também trabalhamos para identificar marcadores de predisposição dos frutos para desenvolver AI, o que poderá ser útil para uma avaliação precoce do risco de desenvolvimento de AI. Entre os marcadores bioquímicos, os resultados evidenciaram o acetaldeído, o etanol e o ácido ascórbico como os mais promissores, ao passo que, entre os minerais, o cobre (Cu) era o melhor candidato. Em geral, modelos de previsão do AI em pera 'Rocha' foram desenvolvidos e validados neste trabalho, representando um importante passo na previsão do AI em pera 'Rocha'. De forma a encontrar estratégias de controlo eficazes para prevenir o AI em pera 'Rocha' foram realizados três ensaios em dois anos consecutivos. Dois dos ensaios foram realizados no primeiro ano e o outro no ano seguinte. No primeiro ensaio, atmosferas controladas dinâmicas monitorizadas por fluorescência da clorofila (ACD-FC) e etanol (ACD-EtOH) foram avaliadas para a prevenção do AI. Pela primeira vez mostramos que o armazenamento dos frutos em ACD-FC conduz a uma incidência reduzida de AI, sendo esta uma metodologia promissora para o armazenamento prolongado de pera 'Rocha'. Pelo contrário, mostramos também que o armazenamento em ACD-EtOH poderá não ser adequado para prevenir o AI. No segundo e terceiro ensaios, a eficácia do tratamento com 1-metilciclopropeno (1-MCP) e do armazenamento em atmosfera controlada diferida na prevenção do AI foi avaliada. Pela primeira vez, mostrámos que a eficácia do 1-MCP depende da maturidade dos frutos à colheita e que, quando aplicado em frutos tardios, induz uma maior incidência de AI. Os resultados também mostraram que o armazenamento de frutas atmosfera controlada diferida, em contraste com o observado para outras cultivares de pera, não foi eficaz na prevenção do AI em pera 'Rocha', podendo mesmo induzir a sua incidência. Em geral, os resultados obtidos no decorrer desta tese proporcionaram novo conhecimento e uma melhor compreensão do AI em pera 'Rocha'. Os mecanismos bioquímicos subjacentes foram estabelecidos e pistas moleculares foram desvendadas; novas perspetivas para a modelação preditiva do AI foram dadas; uma nova e promissora metodologia para a prevenção do AI foi revelada e as limitações das estratégias de controlo atualmente usadas, quando aplicadas à pera 'Rocha', foram evidenciadas.‘Rocha’ pear (Pyrus communis L.) is a Portuguese native cultivar which can be stored under controlled atmosphere (CA) for up to ten months. However, it is known to be susceptible to internal browning disorders (IBD), one of the major causes of economic losses during long-term storage. Although many studies have been conducted to better understand IBDs in pears, the underlying mechanisms of this disorder remains to be elucidated. The aim of this thesis is to investigate the physiological, biochemical and molecular mechanisms involved on IBD development in ‘Rocha’ pear, establishing the scientific basis for the development of IBD predictive models and postharvest handling recommendations aiming at reducing the incidence of IBD in a regulatory environment where conventional chemical tools are no longer available. IBDs in pear can have various symptoms and pear susceptibility to develop this disorder has been shown to be affected by fruit maturity at harvest. In the current work IBDs in ‘Rocha’ pear were classified into two categories: wet necrotic breakdown and dry cavities, which may coexist in the same fruit. The occurrence of cavities was associated to longer storage durations and fruit exposure to high CO2. The results clearly showed that late harvested pears were more susceptible to IBD than early and optimally harvested. For the study of the biochemical basis of IBD three trials were conducted in three consecutive years. In our first assessment, conducted on a long-term basis, fermentation played a major role on IBD whereas the antioxidant system did not. However, in the second trial, conducted on a short-term basis, the results suggested that IBD in ‘Rocha’ pear was triggered by oxidative stress. Finally, in the third trial, the biochemical basis of CO2-related IBD in ‘Rocha’ pear was established. The results showed that the underlying mechanism of IBD in ‘Rocha’ pear involves the conjugation of both, the antioxidant and fermentative metabolisms. From the results obtained a mechanism of CO2-related IBD development was proposed. In this study we also looked for the first time to the molecular regulation of genes codifying antioxidant and fermentative enzymes. The differences found in the transcriptional regulation of these genes supported the biochemical findings. The results also showed that fruit storage under high CO2 leads to an impairment of the antioxidant system and that, when high CO2 is combined with very low O2 levels, there is a highly induction of fermentation with both factors acting synergistically on IBD development. We also worked to identify markers of fruit predisposition to develop IBD, which could be useful for an early assessment of the risk of IBD development. Among the biochemical markers, the results evidenced acetaldehyde (AcDH), ethanol (EtOH) and ascorbic acid (AA) as the most promising ones, whereas, among the minerals, copper (Cu) was the best candidate. Overall, IBD prediction models for ‘Rocha’ pear have been developed and validated in this work, representing a major step forward in the prediction of IBD in ‘Rocha’ pear. In order to find effective control strategies to prevent IBD in ‘Rocha’ pear three trials were conducted in two consecutive years. Two trials were conducted in the first year and the other in the following year. In the first trial, dynamic controlled atmospheres monitored by chlorophyll fluorescence (DCA-CF) and ethanol (DCA-EtOH) were evaluated for IBD prevention. For the first time, we showed that fruit storage under DCA-CF leads to a reduced IBD incidence, making it a promising methodology for ‘Rocha’ pear long-term storage. On the contrary, we also showed that storage under DCA-EtOH may not be suitable to prevent IBD. In the second and third trials, the efficacy of 1-MCP treatment and storage under delayed CA, in preventing IBD was assessed. For the first time, we showed that 1-MCP efficacy is dependent on fruit maturity at harvest and that, when applied to late harvested fruits, induces higher IBD incidence. The results also showed that fruit storage under delayed CA, in contrast to the observed for other pear cultivars, was not effective in preventing IBD in ‘Rocha’ pear, and may even induce its incidence. Overall the results obtained in the course of this thesis provided novel knowledge and a better understanding of IBDs in ‘Rocha’ pear. The underlying biochemical mechanisms were established and molecular clues were unraveled; new perspectives for IBD predictive modelling were provided; a new promising methodology for IBD prevention was revealed and the limitations of currently used control strategies, when applied to ‘Rocha’ pears, were evidenced

Combined effect of elevated CO2 and Fe deficiency on common bean metabolism and mineral profile

Aims: Elevated atmospheric CO2 (eCO2) and restricted iron (Fe) supply are known to impact plant growth and nutritional quality of food crops. However, studies aimed at understanding how eCO2 will interact with Fe deficiency are scarce. Changes in the nutritional status of the common bean (Phaseolus vulgaris L.) may significantly impact the nutritional status of populations that rely heavily on this crop. Methods: To understand the combined effects of eCO2 and Fe deficiency on mechanisms relevant to plant nutrient uptake and accumulation, common bean plants were grown under Fe sufficiency (Fe+, 20 mM Fe-EDDHA) and Fe deficiency (Fe-, 0 mM Fe-EDDHA) combined with eCO2 (800 ppm) or ambient CO2 (aCO2, 400 ppm) in hydroponics until maturity. Results: Elevated CO2, besides stimulating photosynthesis and stomatal closure, highly affected plant Fe metabolism: stimulated root ferric chelate reductase (FCR) activity by 6-fold and downregulated the expression of root FRO1 and IRT1 expressions by about 4-fold. In leaves, citrate and oxalate increased, but ferritin expression decreased by 9-fold. Such changes may have determined the differences on mineral accumulation patterns particularly the lower levels of Fe in roots (62%), leaves (38%) and seeds (50%). The combination of Fe deficiency and eCO2 doubled the effect of a single factor on FCR up-regulation, balanced the internal pH of Fe deficient plants, and resulted in the lowest Fe accumulation in all plant parts. Conclusions: These results suggest that eCO2 directly affects the Fe uptake mechanism of common bean plants, decreasing plant Fe content.info:eu-repo/semantics/publishedVersio

Intraspecific variation in the response of bean (Phaseolus vulgaris L.) and soybean (Glycine max L.) genotypes to elevated CO2 in an indoor farming environment

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The impact of metabolic engineering of yeast, designed to produce valuable biomolecules through fermentation, on resulting spent yeast composition

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Impact of elevated CO2 and restricted iron supply on soybean (Glycine max) and common bean (Phaseolus vulgaris) growth and nutrition

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Antimicrobial potential of formulations, incorporating spent yeast derived from synthetic biotechnology, against Pseudomonas spp.

Yeasts are currently used as cell factories for the sustainable production of high value biomolecules for applications in the pharmaceutical and cosmetic sectors. However, the production of such molecules through fermentation, conducted in bioreactors and making use of genetically engineered yeast strains, generates a number of waste-streams, with spent yeast as the second main by-product of fermentation processes, representing more than 20% of the total waste produced. Since this by-product is rich in several molecules including proteins, vitamins and several minerals, being also a natural source of glucans and mannoproteins with known bioactivities, it is of interest to develop valorization strategies for these residues. In this work, spent yeast was autolysed and a set of formulations, incorporating the resulting supernatant and pellet at different concentrations, were tested as potential antimicrobial solutions to prevent diseases in plants and fungi caused by Pseudomonas spp. The formulations were initially tested against P. aeruginosa and then against strains affecting cultivated mushrooms (Agaricus bisporus), P. tolaasi, and P. agarici; and a number of P. syringae strains responsible for plant diseases including the pathovars actinidifoliorium, tomato, pisi, syringae and atrofaciens. It has been recently shown that spent yeast hydrolysates possess antimicrobial activity against Salmonella enterica, Aeromonas salmonicida, Bacillus cereus and Bacillus subtilis (Martin et al. 2021), suggesting that spent yeast extracts may have potential antimicrobial effect against a range of microorganisms. To date, there are no studies showing the potential of spent yeast extracts against Pseudomonas strains. The results showed that the supernatant of the autolyzed yeast at 0.1% inhibited the growth of P. aeruginosa by about 20% and P. tolaasii by 10% but increased the growth of P. agarici by up to 34%. The combination of supernatant with lactose also slightly increased P. tolaasii growth inhibition (12%) but it reduced the inhibitory effect of supernatant against P. aeruginosa. With regard to plant pathogens, the formulations containing 0.1% and 0.3% of pellet inhibited by 25% the growth P. syringae pv. actinidifoliorium, the bacterium responsible for kiwi canker, but for all the other pathovars, the inclusion of spent yeast extracts in the formulation induced bacterial growth. Among all the tested formulations, the ones including supernatant and pellet at low concentrations were the most promising leading to slight growth inhibition of some environmental Pseudomonas spp.info:eu-repo/semantics/publishedVersio

Growth and nutritional responses of bean and soybean genotypes to elevated CO2 in a controlled environment

In the current situation of a constant increase in the atmospheric CO2 concentration, there is a potential risk of decreased nutritional value and food crop quality. Therefore, selecting strong-responsive varieties to elevated CO2 (eCO2) conditions in terms of yield and nutritional quality is an important decision for improving crop productivity under future CO2 conditions. Using bean and soybean varieties of contrasting responses to eCO2 and different origins, we assessed the effects of eCO2 (800 ppm) in a controlled environment on the yield performance and the concentration of protein, fat, and mineral elements in seeds. The range of seed yield responses to eCO2 was −11.0 to 32.7% (average change of 5%) in beans and −23.8 to 39.6% (average change of 7.1%) in soybeans. There was a significant correlation between seed yield enhancement and aboveground biomass, seed number, and pod number per plant. At maturity, eCO2 increased seed protein concentration in beans, while it did not affect soybean. Lipid concentration was not affected by eCO2 in either legume species. Compared with ambient CO2 (aCO2), the concentrations of manganese (Mn), iron (Fe), and potassium (K) decreased significantly, magnesium (Mg) increased, while zinc (Zn), phosphorus (P), and calcium (Ca) were not changed under eCO2 in bean seeds. However, in soybean, Mn and K concentrations decreased significantly, Ca increased, and Zn, Fe, P, and Mg concentrations were not significantly affected by eCO2 conditions. Our results suggest that intraspecific variation in seed yield improvement and reduced sensitivity to mineral losses might be suitable parameters for breeders to begin selecting lines that maximize yield and nutrition under eCO2.info:eu-repo/semantics/publishedVersio

Valorization of biotechnology derived spent yeast as potential ruminant feed additive

The production of high quantities of spent yeast is a transverse problem to most industries based on fermentation processes. Traditional fermentation industries, such as breweries, and the ones that use engineered yeast to produce high commercial interest molecules, have been looking for strategies to minimize this problem. Spent yeast represents a valuable nutrient source due to its abundant levels of several high value bioactive compounds. Through an optimized autolysis process, high levels of these bioactive compounds, such as β-glucans, mannans, peptides, and other nutrients can be more easily available, targeting several commercial applications. Some of these compounds have prebiotic applicability in animal feed industries. The benefits of supplementing yeast in diets of ruminants trusts on the increase of cellulolytic bacteria in the rumen, increasing the energy extracted from the diets, and consequently the animal’s performance (Bortoluzzi et al., 2018). Yeast derivatives have shown the potential to modulate ruminal fermentation by decreasing the starch degradation rate or by stimulating the growth of lactate-utilizers in the rumen. As the effect of it may be increased by low pH or by high-concentrate diets, supplementing the ruminant's diet with autolyzed yeast may help to counteract the negative effects of high-concentrate diets (Kröger et al., 2017; Humer et al., 2018; Neubauer et al., 2018). This research aimed to characterize a synthetic biotechnology fermentation derived spent yeast as potential additive to ruminant feed. Autolyzed spent yeast was spray dried and characterized in terms of composition and prebiotic potential. The challenge in the utilization of traditional fermentation derived spent yeast, for ruminant consumption, is to guarantee the level of protein and essential amino acids, their ratio and stability along storage. Results demonstrate that synthetic biology associated fermentation derived spent yeast presented both nutritional value and prebiotic activity showing high potential to be incorporated as an additive for ruminants feed, with an interesting amino acids profile.info:eu-repo/semantics/publishedVersio

Induced autolysis of engineered yeast residue as a means to simplify downstream processing for valorization: a case study

The objective of this work was to study the efficiency of different autolysis processes, combining different temperatures and pH conditions, when applied to a genetically engineered yeast residue. The determination of the supernatants’ dry weight showed that the autolysis time could be reduced to half, from 4 to 2 h, if the residue pH was increased from 5 to 8 at 50 °C (18.20% for 4 h and 18.70% for 2 h with a higher pH). This result allowed us to select a short autolysis time to proceed with the second part of the experiments. The application of this faster induced autolysis process enabled us to obtain supernatants with higher concentrations of relevant compounds, such as some amino acids and minerals. An increase in leucine (of around 7%), aspartic acid, valine, phenylalanine, isoleucine and serine (approximately 2%) was observed in the autolyzed samples, when compared to the untreated ones. Also, regarding minerals, the autolysis process allowed us to obtain significantly higher amounts of potassium in the treated samples’ supernatants. This work allowed the selection of a fast and low-cost induced autolysis process for synthetic biotechnology-derived spent yeast residue to attain a product rich in high-value compounds, which can be used in commercial applications, for example, as an animal feed additive.info:eu-repo/semantics/publishedVersio

Short Term Elevated CO2 Interacts with Iron Deficiency, Further Repressing Growth, Photosynthesis and Mineral Accumulation in Soybean (Glycine max L.) and Common Bean (Phaseolus vulgaris L.)

Elevated CO2 (eCO2) has been reported to cause mineral losses in several important food crops such as soybean (Glycine max L.) and common bean (Phaseolus vulgaris L.). In addition, more than 30% of the world’s arable land is calcareous, leading to iron (Fe) deficiency chlorosis and lower Fe levels in plant tissues. We hypothesize that there will be combinatorial effects of eCO2 and Fe deficiency on the mineral dynamics of these crops at a morphological, biochemical and physiological level. To test this hypothesis, plants were grown hydroponically under Fe sufficiency (20 μM Fe-EDDHA) or deficiency (0 μM Fe-EDDHA) at ambient CO2 (aCO2, 400 ppm) or eCO2 (800 ppm). Plants of both species exposed to eCO2 and Fe deficiency showed the lowest biomass accumulation and the lowest root: shoot ratio. Soybean at eCO2 had significantly higher chlorophyll levels (81%, p < 0.0001) and common bean had significantly higher photosynthetic rates (60%, p < 0.05) but only under Fe sufficiency. In addition, eCO2 increased ferric chelate reductase acivity (FCR) in Fe-sufficient soybean by 4-fold (p < 0.1) and in Fe-deficient common bean plants by 10-fold (p < 0.0001). In common bean, an interactive effect of both environmental factors was observed, resulting in the lowest root Fe levels. The lowering of Fe accumulation in both crops under eCO2 may be linked to the low root citrate accumulation in these plants when grown with unrestricted Fe supply. No changes were observed for malate in soybean, but in common bean, shoot levels were significantly lower under Fe deficiency (77%, p < 0.05) and Fe sufficiency (98%, p < 0.001). These results suggest that the mechanisms involved in reduced Fe accumulation caused by eCO2 and Fe deficiency may not be independent, and an interaction of these factors may lead to further reduced Fe levels