Genetius

The authors engineered a double fluorescent reporter line in the tomato cultivar Micro‑Tom, using mTurquoise and mScarlet‑I driven by generative‑cell‑ and sperm‑cell‑specific promoters, respectively. They established a protocol that releases male gametes from pollen, stains them with SYTOX Red, and isolates viable generative cells, sperm cells, and vegetative nuclei via fluorescence‑activated cell sorting (FACS). This tool enables high‑purity, high‑quantity isolation of tomato male gametes for downstream omics studies.

The study characterizes all seven malic enzyme genes in tomato, analyzing their tissue-specific expression, temperature and ethylene responsiveness, and linking specific isoforms to metabolic processes such as starch and lipid biosynthesis during fruit development. Phylogenetic, synteny, recombinant protein biochemical assays, and promoter analyses were used to compare tomato enzymes with Arabidopsis counterparts, revealing complex evolutionary dynamics that decouple phylogeny from functional orthology.

The study investigates the regulation and function of mitochondrial alternative oxidase (AOX) during tomato fruit ripening, revealing that AOX activity peaks at the breaker stage and becomes the dominant pathway for climacteric respiration, driven by increased TCA cycle intermediates rather than gene expression. Using CRISPR‑Cas9 aox1a mutants, the authors show that loss of AOX disrupts ripening‑related metabolite profiles, underscoring AOX’s critical role in supporting ethylene and carotenoid biosynthesis.