基于GABA途径的柑橘柠檬酸降解机制研究

Organic acid, one of the most important traits of fruit quality, was principally governed by the level of citric acid in citrus. Citric acid catabolism, as the primary metabolism in fruit, was also involved in biosynthesis of secondary metabolites, including amino acids, aroma volatiles and bioactive substances, which then further influences fruit flavor and quality. Gamma-aminobutyrate (GABA) pathway has recently been found that may play an important role in the degradation of citric acid in citrus, the results of our preliminary studies also showed that hot air treatment drove citric acid degradation via the GABA shunt pathway in postharvest ponkan fruits, but the regulation mechanism is is relatively unknown. In the project, Gannan navel orange (Citrus sinensis) and Jing'an ponkan (Citrus reticulata) fruits were used to investigate citric acid metabolism (mostly focus on GABA pathway) in citrus during fruit development, storage and postharvest treatments. The internal relations between citric acid degradation and amino acids biosynthesis will be discussed based on quantitative measurement of citric acid, amino acids and medial metabolites. Meanwhile, the activities and gene expression of aconitase (Aco), isocitrate dehydrogenase (IDH), glutamate decarboxylase (GAD), glutamine synthetase (GS), gamma-aminobutyrate aminotrasferase (GABA-AT) and succinate semialdehyde dehydrogenase (SSADH) involved in GABA pathway will be measured to illustrate their function in the degradation of citric acid in citrus. The results will provide the basis for understanding the regulatory mechanism of citrus fruit quality.

柑橘果实以积累柠檬酸为主，柠檬酸代谢不仅决定了柑橘果实有机酸的水平，还为氨基酸、香气物质、生物活性物质等合成提供能量和中间产物，从而影响果实的风味和品质，GABA（γ-氨基丁酸）途径近来被发现可能在柠檬酸降解中起重要作用，我们的前期研究结果也表明，采后热处理也主要通过GABA途径促进椪柑果实柠檬酸降解，但其具体机制尚不明确。本项目拟以赣南脐橙和靖安椪柑为材料，从果实发育、贮藏及采后调控等方面研究柑橘果实柠檬酸的降解代谢，重点围绕GABA途径，通过对柠檬酸、氨基酸及GABA途径中间代谢产物进行定量分析，探讨柠檬酸降解与氨基酸合成的内在联系，从酶活性及基因表达等方面阐明顺乌头酸酶（Aco）、异柠檬酸脱氢酶（IDH）、谷氨酸脱羧酶（GAD）、谷氨酰胺合成酶（GS）、转氨酶（GABA-AT）和琥珀酸半醛脱氢酶（SSADH）在柠檬酸降解过程中的作用，从而为柑橘果实品质形成及调控提供理论依据。

有机酸是影响果实品质的重要指标之一。柑橘果实有机酸积累以柠檬酸为主，其代谢网络极其复杂，涉及到柠檬酸合成、转运和降解的各个方面。大量研究表明，柑橘果实柠檬酸积累与其合成无直接相关，而受转运和降解环节所调控，尤其是发现柠檬酸降解与γ-氨基丁酸（GABA）途径、谷氨酰胺合成酶（GS）及ATP-柠檬酸裂解酶（ACL）等多条代谢路径密切相关。我们的前期研究结果也表明，采后热处理也主要通过GABA途径促进椪柑果实柠檬酸降解，说明GABA途径可能在柠檬酸降解中起重要作用。本项目以赣南‘纽荷尔’脐橙和靖安椪柑为材料，研究了果实发育过程有机酸积累规律、不同采后处理对柑橘果实贮藏期间有机酸含量的影响，并对柠檬酸合成及降解过程相关基因表达进行Q-PCR定量分析，系统探讨GABA途径在柑橘果实柠檬酸降解中的作用。研究结果表明：靖安椪柑果实发育阶段柠檬酸积累主要由CitAco2/3和CitIDH1/2基因共同调节；采后热空气处理能诱导CitAco2/3、CitIDH2/3和CitGAD4等基因的表达，从而激活GABA途径促进果实柠檬酸降解；而壳聚糖处理可以抑制GABA途径CitAco3 、CitGAD4基因表达减缓柠檬酸降解速率；脐橙果实发育阶段柠檬酸水平受GABA和ACL途径调控；热空气处理脐橙幼果可激活GABA、GS和ACL三条途径促进柠檬酸降解，而成熟果主要通过GABA和ACL途径；外源柠檬酸处理提高了果实内柠檬酸的含量，并诱导了GABA途径相关降解基因的表达。这些结果验证了GABA途径在柑橘柠檬酸降解过程的重要作用，也为柑橘果实品质形成及调控提供理论依据。