Organic acid composition has a major influence on fruit taste and quality. In general, the fruits of common citrus varieties accumulate more citrate than malate. Here, we report that a citrus variety, ‘Haruka’ (Citrus tamurana × natsudaidai), accumulates more malate than citrate at the ripening stage. However, the underlying mechanism is unclear. Organic acid profiles were compared between ‘Haruka’ fruits and a common Ponkan cultivar, ‘Huagan 2’ (Citrus reticulata), during fruit development and ripening. We found that ‘Haruka’ fruit accumulated 75% less citrate than ‘Huagan 2’ fruit and that the malate content was nearly 3-fold greater in ‘Haruka’ fruit than in ‘Huagan 2’ fruit. In addition, 12 aluminum-activated malate transporter (ALMT) genes were identified in the citrus genome, with CsALMT9-like being predominantly expressed in ‘Haruka’ fruit juice sacs. An analysis of genes associated with the accumulation of citrate/malate revealed that the expression levels of genes encoding a P-type proton pump (CsPH8), a citrate/H⁺ symporter (CsCit), and CsALMT9-like were significantly greater in ‘Haruka’ fruit than in ‘Huagan 2’ fruit. Moreover, CsCit overexpression significantly decreased the citrate content, whereas overexpressing and silencing CsALMT9-like significantly increased and decreased the malate content. In addition, shading significantly increased the expression of CsPH8 but reduced the expression of CsCit and CsALMT9-like and significantly increased the citrate content but decreased the malate content, further confirming that CsCit and CsALMT9-like regulate the accumulation of citrate and malate in ‘Haruka’ fruit, respectively. Taken together, these data indicate that the relatively low citrate content of ‘Haruka’ is mainly due to increases in CsCit expression and that the relatively high malate content of ‘Haruka’ is mainly due to increase in CsALMT9-like expression. This study provides insight into the mechanisms that influence malate content in citrus fruit.

Leaf morphology is an important agronomic trait that affects photosynthetic efficiency and plant architecture. Some KNOXI (Class Ⅰ KNOTTED1-LIKE HOMEOBOX) or CIN-TCP (CINCINNATA-LIKE TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL FACTORS) transcription factors control leaf development by influencing GA (gibberellin) content. However, the knowledge about their interaction in regulating leaf morphogenesis is still scarce, especially in woody fruit plants such as citrus. In this study, we found that the expression of CsTCP13 (a citrus CIN-TCP gene) dramatically decreased with a decreasing leaf length-width ratio in citrus. Transiently silencing CsTCP13 in citrus produced wider leaves and significantly decreased CsGA20ox1 (a key citrus GA20-oxidase gene for GA biosynthesis) expression and GAs (GA₃, GA₄, and GA₇) contents. Conversely, stable overexpression of CsTCP13 in tobacco generated narrower leaves and significantly increased GA20ox1 expression and GA₁ content; it also significantly shortened the distance between veins and reduced the number of epidermal cells per unit area of leaf. Moreover, transactivation assays showed that CsTCP13 had transcriptional activation, and yeast two-hybrid, split-luciferase complementation, and bimolecular fluorescence complementation assays confirmed that CsTCP13 interacted with CsKNAT1 (a citrus KNOXI protein). Furthermore, yeast one-hybrid and dual-luciferase assays validated that CsTCP13 promoted while CsKNAT1 and its interacting protein CsKNAT6 inhibited CsGA20ox1 expression by binding to its promoter. Interestingly, CsKNAT1 or the CsKNAT1-CsKNAT6 complex interacted with CsTCP13 to attenuate its promotion effect on CsGA20ox1 expression. Taken together, our findings revealed a novel regulatory mechanism that CsTCP13 regulates citrus leaf width through directly influencing CsGA20ox1 expression and then GA content, which can be negatively affected by the interaction with CsKNAT1 or the CsKNAT1-CsKNAT6 complex.