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在动态受控气氛储存期间调节苹果的呼吸代谢

来源: Boeckx,J  发布日期: 2019-07-04  访问量: 315


收获后,苹果(Malus x domestica Borkh。)果实仍保持代谢活性并继续成熟。采后贮藏的主要目标是减缓这种代谢活动及其相关的果实成熟。因此,大量的苹果果实通常在受控气氛(CA)条件下(低温,低氧和高二氧化碳分压)储存,最近的发展趋向于动态CA(DCA)储存。在DCA储存期间,冷藏库中的氧分压朝向无氧补偿点降低,直到果实的生理反应表明其代谢已经从有氧呼吸转变为发酵。通过这种方式,可以比常规CA储存更有效地延迟果实成熟。然而,改变正常的大气条件在果实生理水平上并非没有任何风险:它可能诱导低氧胁迫。为了进一步改善DCA储存技术的发展,充分了解低氧胁迫对苹果果实生理的影响非常重要。如今,关于低氧浓度对苹果果实代谢过程及其调节的精确作用方式知之甚少。因此,本论文的主要目的是揭示'Jonagold'苹果果实中低碳胁迫引起的中心碳代谢的代谢变化及其调控...
标签: 动态受控气氛、苹果、呼吸代谢、SafePod
 

Regulation of the respiratory metabolism of apple during (dynamic) controlled atmosphere storage

在(动态)受控气氛储存期间调节苹果的呼吸代谢 

 

Author: Boeckx, J

 

Abstract:

After harvest, apple (Malus x domestica Borkh.) fruit remains metabolically active and continues to ripen. The main goal of postharvest storage is to slow down this metabolic activity and its associated fruit ripening. Therefore, large volumes of apple fruit are routinely stored under controlled atmosphere (CA) conditions (low temperature, low oxygen and high carbon dioxide partial pressures) with recent developments moving towards dynamic CA (DCA) storage. During DCA storage, the oxygen partial pressure in the cool store is reduced towards the anaerobic compensation point until the physiological response of the fruit indicates that its metabolism has switched from aerobic respiration to fermentation. In this way, fruit ripening can be delayed more effectively than under regular CA storage. However, altering the normal atmospheric conditions is not without any risk at the fruit physiology level: it may induce low-oxygen stress. To be able to further improve the development of the DCA storage technology, it is important to fully understand the impact of low-oxygen stress on the physiology of apple fruit. Nowadays, little is known about the precise mode of action of low-oxygen concentrations on the metabolic processes and their regulation in apple fruit. Therefore, the main objective of this dissertation was to unravel the metabolic changes of the central carbon metabolism and its regulation introduced by low-oxygen stress in 'Jonagold' apple fruit. Due to oxygen consumption and slow diffusion present in the apple fruit, the oxygen concentration declines towards the centre of the fruit. In order to avoid oxygen gradients experiments were all conducted on thin slices of the apple cortex tissue. To diminish the influence of wound ethylene on the metabolism, the apple slices were flushed with humidified air before the start of the experiments. If the oxygen concentration in the fruit tissue is too low, fermentation will be induced, which can lead to alcoholic off-flavours and internal storage disorders. However, as of today, little information is available on the in vivo regulatory mechanism of the fermentative metabolism under low-oxygen stress. Based on in vitro experiments, it was proposed that fermentation could be regulated via a molecular and a metabolic control system, but experimental conditions in vitro are not necessarily a correct representation of the situation in vivo. Therefore, the first focus of this study was to get a better insight into the regulation of the fermentative metabolism in vivo under different oxygen concentrations and temperature levels, and this immediately after harvest as well as after storage. A combination of experimental techniques and a kinetic modelling approach was used to study the regulation of the fermentative metabolism. To correctly measure the in vitro activity of the fermentation enzymes, pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), enzyme assays were optimized to increase the signal to noise ratio. Furthermore, an elaborated kinetic model based method was developed to accurately estimate the in vitro specific enzyme activity of PDC and ADH from spectrophotometric assay data. The models are based on Michaelis-Menten and first-order kinetics, which describe the reaction mechanism catalysed by the enzymes. In contrast to the linear regression approach, the models can be used to estimate the enzyme activity regardless of whether linearity is achieved since they take into account the complete progress curve. The advantage of using a kinetic modelling approach is that the maximum activity of the enzyme can be estimated through extrapolation, even if saturating substrate conditions are not fully realized. The regulation of the fermentative metabolism appeared to be highly depending on the conditions applied. Temperature changed the overall metabolic reaction rate, which appeared to have a large influence on the induction and regulation of the fermentative metabolism. At 1 °C, the metabolic rate was low and fermentation was only slightly induced and largely metabolically controlled. However, at 18 °C, the overall metabolic rate was faster and a ten-fold increase in ethanol levels was observed. In this case, the ethanol pathway was regulated by a combination of molecular and metabolic control systems. Also, the model indicated that under anoxic conditions at harvest the in vivo regulation was located at the fast induction of PDC activity, whether after six months of storage an extra regulation on ADH activity seemed to be present. Furthermore, a different regulation of the ethanol pathway was observed between hypoxic and anoxic conditions. Even though fermentation was clearly induced under all experimental conditions applied, the flux through the fermentative metabolism started to decrease again after prolonged low-oxygen stress. To investigate whether 'Jonagold' apple fruit reduces the negative effects of fermentation by shuttling pyruvate into alternative pathways, a detailed pathway analysis, based on RNA-sequencing data, was performed to study the transcriptional regulation of the central carbon metabolism during low-oxygen stress in more detail. This revealed that apple fruit, exposed to 0.5 kPa O2, diminishes the energy crisis by upregulating the glycolytic flux and the PPi-dependent reactions, saves carbon atoms by shuttling pyruvate into the alanine metabolism and limits cytosolic acidification by activating the alanine metabolism and the GABA shunt. Furthermore, evidence was provided supporting the presence of an oxygen sensing mechanism in 'Jonagold' apple fruit, indicating that besides the substrate inhibition of the electron transport chain by limiting oxygen levels, an active regulatory mechanism of the central carbon metabolism is present. Future research is necessary to characterize the influence of different (dynamically changing) low-oxygen partial pressures on the central carbon metabolism of multiple cultivars and to better understand the operation of the oxygen sensors in apple fruit. Furthermore, a system biology model should be developed, implementing data of all the organizational levels, to help us better understand the influence of low-oxygen stress on the physiology of apple fruit.

收获后,苹果(Malus x domestica Borkh。)果实仍保持代谢活性并继续成熟。采后贮藏的主要目标是减缓这种代谢活动及其相关的果实成熟。因此,大量的苹果果实通常在受控气氛(CA)条件下(低温,低氧和高二氧化碳分压)储存,最近的发展趋向于动态CA(DCA)储存。在DCA储存期间,冷藏库中的氧分压朝向无氧补偿点降低,直到果实的生理反应表明其代谢已经从有氧呼吸转变为发酵。通过这种方式,可以比常规CA储存更有效地延迟果实成熟。然而,改变正常的大气条件在果实生理水平上并非没有任何风险:它可能诱导低氧胁迫。为了进一步改善DCA储存技术的发展,充分了解低氧胁迫对苹果果实生理的影响非常重要。如今,关于低氧浓度对苹果果实代谢过程及其调节的精确作用方式知之甚少。因此,本论文的主要目的是揭示'Jonagold'苹果果实中低碳胁迫引起的中心碳代谢的代谢变化及其调控。由于苹果果实中的氧消耗和缓慢扩散,氧浓度朝向果实的中心下降。为了避免氧气梯度,实验全部在苹果皮层组织的薄片上进行。为了减少伤口乙烯对代谢的影响,在实验开始之前用加湿的空气冲洗苹果切片。如果水果组织中的氧浓度太低,将诱发发酵,这可能导致酒精异味和内部储存障碍。然而,截至目前,关于低氧胁迫下发酵代谢的体内调节机制的信息很少。基于体外实验,提出可以通过分子和代谢控制系统调节发酵,但体外实验条件不一定是体内情况的正确表示。因此,本研究的第一个重点是更好地了解不同氧浓度和温度水平下体内发酵代谢的调节,以及收获后和储存后的情况。实验技术和动力学建模方法的组合用于研究发酵代谢的调节。为了正确测量发酵酶,丙酮酸脱羧酶(PDC)和醇脱氢酶(ADH)的体外活性,优化酶测定以增加信噪比。此外,开发了一种精心设计的基于动力学模型的方法,用于从分光光度测定数据准确估计PDC和ADH的体外特异性酶活性。这些模型基于Michaelis-Menten和一阶动力学,它描述了酶催化的反应机理。与线性回归方法相比,该模型可用于估计酶活性,无论是否达到线性​​,因为它们考虑了完整的进展曲线。使用动力学建模方法的优点是,即使没有完全实现饱和的底物条件,也可以通过外推估计酶的最大活性。发酵代谢的调节似乎高度取决于所应用的条件。温度改变了整体代谢反应速率,这似乎对发酵代谢的诱导和调节具有很大影响。在1°C时,代谢率很低,发酵只是轻微诱导并且在很大程度上代谢控制。然而,在18℃时,总体代谢速率更快,并且观察到乙醇水平增加10倍。在这种情况下,乙醇途径由分子和代谢控制系统的组合调节。此外,该模型表明,在收获时的缺氧条件下,体内调节定位于PDC活性的快速诱导,无论是在储存六个月后,似乎存在对ADH活性的额外调节。此外,在缺氧和缺氧条件下观察到乙醇途径的不同调节。即使在所应用的所有实验条件下明显诱导发酵,通过发酵代谢的通量在延长的低氧应力后也开始再次降低。调查是否'Jonagold' 苹果果实通过将丙酮酸穿梭到替代途径来减少发酵的负面影响,基于RNA测序数据进行详细的途径分析,以更详细地研究低氧应激期间中心碳代谢的转录调节。这表明暴露于0.5 kPa O2的苹果果实通过上调糖酵解通量和PPi依赖性反应来减少能量危机,通过将丙酮酸穿梭到丙氨酸代谢中来节省碳原子并通过激活丙氨酸代谢和GABA来限制细胞溶质酸化。分流。此外,提供的证据支持'Jonagold'苹果果实中存在氧传感机制,表明除了底物通过限制氧水平抑制电子传递链,存在中枢碳代谢的主动调节机制。未来的研究有必要表征不同(动态变化)低氧分压对多品种中心碳代谢的影响,并更好地了解苹果果实中氧传感器的运行。此外,应开发系统生物学模型,实施所有组织水平的数据,以帮助我们更好地了解低氧胁迫对苹果果实生理的影响。未来的研究有必要表征不同(动态变化)低氧分压对多品种中心碳代谢的影响,并更好地了解苹果果实中氧传感器的运行。此外,应开发系统生物学模型,实施所有组织水平的数据,以帮助我们更好地了解低氧胁迫对苹果果实生理的影响。未来的研究有必要表征不同(动态变化)低氧分压对多品种中心碳代谢的影响,并更好地了解苹果果实中氧传感器的运行。此外,应开发系统生物学模型,实施所有组织水平的数据,以帮助我们更好地了解低氧胁迫对苹果果实生理的影响。


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