A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

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.

Genome-Wide Identification, Molecular Evolution, and Expression Divergence of Aluminum-Activated Malate Transporters in Apples.

Aluminum-activated malate transporters (ALMTs) play an important role in aluminum tolerance, stomatal opening, and fruit acidity in plants. However, the evolutionary pattern of the ALMT gene family in apples remains relatively unknown. In this study, a total of 25 MdALMT genes were identified from the apple reference genome of the "Golden Delicious" doubled-haploid tree (GDDH13). The physiological and biochemical properties, gene structure, and conserved motifs of MdALMT genes were examined. Chromosome location and gene-duplication analysis indicated that whole-genome duplication/segmental duplication played an important role in the expansion of the MdALMT gene family. The Ka/Ks ratio of duplicated MdALMT genes showed that members of this family have undergone strong purifying selection. Through exploration of the phylogenetic relationships, seven subgroups were classified, and higher old gene duplication frequency and significantly different evolutionary rates of the ALMT gene families were detected. In addition, the functional divergence of ALMT genes occurred during the evolutionary process of Rosaceae species. Furthermore, the functional divergence of MdALMT genes was confirmed by expression discrepancy and different subcellular localizations. This study provides the foundation to better understand the molecular evolution of MdALMT genes and further facilitate functional analysis to unravel their exact role in apples.