Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

中文翻译：

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NAC 转录因子 AcNAC1 诱变导致水果柠檬酸急剧下降

柠檬酸盐是一种常见的初级代谢物，通常具有水果风味的特征。人们对水果和蔬菜中柠檬酸盐积累的关键调节剂知之甚少。我们系统地分析了猕猴桃（ Actinidia spp.）发育过程中有机酸成分的动态特征。柠檬酸盐不断积累，成为收获时总酸度的主要贡献者。基于使用不同猕猴桃品种的共表达网络分析，Al-ACTIVATED MALATE TRANSPORTER基因（AcALMT1）被确定为负责柠檬酸盐积累的候选基因。使用该基因在非洲爪蟾卵母细胞中表达的电生理学测定表明，AcALMT1 具有柠檬酸转运蛋白的功能。此外，猕猴桃中AcALMT1的瞬时过表达显着增加了柠檬酸盐含量，而AcALMT1表达较高的组织则积累了更多柠檬酸盐。AcALMT1的表达与 17 个候选转录因子高度相关。然而，双荧光素酶和 EMSA 测定表明，只有 NAC 转录因子 AcNAC1通过直接结合其启动子来激活AcALMT1表达。猕猴桃中AcNAC1的靶向 CRISPR-Cas9 诱导诱变导致柠檬酸水平急剧下降，而苹果酸和奎宁酸水平并未受到实质性影响。我们的研究结果表明，NAC 转录因子对主要柠檬酸转运蛋白的转录调节负责猕猴桃中柠檬酸的积累，这对其他水果和蔬菜具有广泛的影响。