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Breeding crops with high zinc (Zn) density is an effective way to alleviate human dietary Zn deficiencies. We characterized a mutant Lilizhi (LLZ) accumulating at least 35% higher Zn concentration in grain than the wild type (WT) in hydroponic experiments. The mutant stored less Zn content in the root and transported more Zn to the grain. Metabolite profiling demonstrated that, with high Zn treatment, the contents of proline, asparagine, citric acid, and malic acid were enhanced in both LLZ and the WT, which were thought to be involved in Zn transport in rice. Furthermore, the contents of cysteine, allothreonine, alanine, tyrosine, homoserine, β-alanine, and nicotianamine required for the production of many metal-binding proteins were specifically increased in LLZ. LLZ had higher capability of amino acid biosynthesis and metal cation transportation. The current research extends our understanding on the physiological mechanisms of Zn uploading into grain and provides references for further Zn biofortification breeding in rice.

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