Elevated expression of CsCit and CsALMT9-like plays a key role in lowering citrate and increasing malate accumulation in Citrus tamurana × natsudaidai ‘Haruka’ fruit

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.