Lailiang Cheng

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY–GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.

Vacuolar malic acid accumulation largely determines fruit acidity, a key trait for the taste and flavor of apple and other fleshy fruits. Aluminum‐activated malate transporter 9 ( ALMT9 / Ma1 ) underlies a major genetic locus, Ma , for fruit acidity in apple, but how the protein transports malate across the tonoplast is unclear. Here, it is shown that overexpression of the coding sequence of Ma1 ( Ma1α ) drastically decreases fruit acidity in “Royal Gala” apple, leading to uncovering alternative splicing underpins Ma1's function. Alternative splicing generates two isoforms: Ma1β is 68 amino acids shorter with much lower expression than the full‐length protein Ma1α. Ma1β does not transport malate itself but interacts with the functional Ma1α to form heterodimers, creating synergy with Ma1α for malate transport in a threshold manner (When Ma1β/Ma1α ≥ 1/8). Overexpression of Ma1α triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1β level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1α and Ma1β or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in ALMT9‐mediated malate transport underlying apple fruit acidity.