How a single transcription factor fine-tunes the tang in citrus fruits
image: Functional characterization of CsAIL6 in transgenic citrus fruits and tomato lines. A) Transient OE and VIGS of CsAIL6 in citrus fruits. EV, empty vector. B, D) Relative expression levels of CsAIL6 in transgenic citrus fruits. C, E) Citric acid contents in citrus fruits. F) Phenotypes of mature tomato fruits of the WT and OE transgenic lines (#3 and #5). G) Analysis of CsAIL6 expression in transgenic and WT tomato fruits by RT-PCR. H) Citric acid contents in transgenic and WT tomato fruits. Asterisks (*) indicate significant differences determined by the t-test (P < 0.05). The error bars indicate the standard deviation of three replicates (±SD). FW, fresh weight. WT, wild-type. Image link: https://academic.oup.com/view-large/figure/507964337/uhaf002f3.tif?login=false
Credit: Horticulture Research
The characteristic sour-sweet balance of citrus fruits is largely defined by citric acid accumulation in the vacuoles of juice sacs. Previous studies identified several proton pumps and transcription factors, such as CsPH8, CsAN1, and CsPH4, that control acid storage and transport. However, little was known about how CsAN11, a WD40 protein within the MBW complex, is regulated at the transcriptional level. Moreover, many citrus cultivars exhibit wide variations in acidity, even among closely related varieties, suggesting the presence of additional genetic regulators. Due to these challenges, it is essential to investigate the upstream molecular mechanisms that modulate citric acid metabolism and storage in citrus fruits.
A research team from the College of Horticulture and Forestry Sciences, Huazhong Agricultural University, has revealed how the AP2 transcription factor CsAIL6 influences fruit acidity by repressing the WD40 protein CsAN11. The study, published (DOI: 10.1093/hr/uhaf002) online in Horticulture Research on April 1, 2025, provides fresh insights into the genetic control of citric acid accumulation in citrus. By integrating molecular, physiological, and biochemical analyses, the authors demonstrated that CsAIL6 acts as a negative regulator of fruit acidity through its interaction with CsAN11, thereby uncovering a new layer of control within the MBW regulatory network.
The researchers first found that CsAIL6 expression was high in low-acid citrus cultivars but low in high-acid varieties, showing a strong negative correlation with titratable acid content. During fruit development, CsAIL6 transcript levels increased as citric acid declined, suggesting a suppressive role in acid buildup. Functional validation confirmed this: transient overexpression of CsAIL6 in citrus and stable transformation in tomato (Micro-Tom) both reduced fruit citric acid content, while silencing CsAIL6 had the opposite effect.
Further transcriptomic analysis of transgenic lines showed that overexpressing CsAIL6 specifically downregulated CsAN11, without significantly affecting CsAN1 or CsPH4. Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed a direct interaction between CsAIL6 and CsAN11, indicating that CsAIL6 modulates acidity via the MBW complex. Overexpression of CsAN11 increased citric acid content, confirming its downstream role. Together, these findings establish that CsAIL6 functions as a key repressor of citric acid accumulation by targeting CsAN11, thus modulating the MBW-mediated proton-pump pathway responsible for vacuolar acidification.
“Our findings reveal an elegant regulatory mechanism where CsAIL6 acts like a molecular brake on fruit acidity,” said corresponding author Prof. Yong-Zhong Liu. “By binding to CsAN11, it prevents excessive citric acid buildup, providing a genetic explanation for why some citrus cultivars taste less sour. This work expands our understanding of acid metabolism beyond previously known regulators like CsPH8 and CsAN1 and introduces a new AP2–WD40 interaction that could be exploited to control fruit flavor through precise breeding or gene-editing approaches.”
The discovery of CsAIL6 as a key repressor of citric acid accumulation offers valuable genetic resources for developing citrus varieties with tailored acidity and improved consumer appeal. Manipulating CsAIL6 expression could help breeders produce sweeter or more balanced citrus fruits without compromising other quality traits. Furthermore, the identified AP2–WD40 regulatory module may serve as a model for understanding organic acid metabolism in other fruit crops such as tomato and apple. This study lays a foundation for molecular breeding and precision agriculture aimed at optimizing fruit flavor, storage stability, and overall market competitiveness.
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References
DOI
Original URL
https://doi.org/10.1093/hr/uhaf002
Funding information
This work is supported by the Earmarked Fund for CARS 26.
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2024. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.