Abstract

Cadmium (Cd) contamination has long been a concern because this heavy metal is a major threat to plant growth and human health. Citrate synthase (CS) plays a key role in the tricarboxylic acid cycle, with the reaction it catalyzes producing citric acid (CA), which serves as a major chelating agent contributing to heavy metal detoxification. In sheepgrass ( Leymus chinensis ), the expression levels of three CS-encoding genes ( LcCS2 , LcCS3 , and LcCS4 ) are substantially upregulated under Cd stress conditions. In this study, the heterologous expression of these genes in yeast and rice significantly enhanced Cd tolerance. Subcellular localization analyses showed that LcCS2 and LcCS3 are localized in the cytoplasmic matrix, while LcCS4 is localized in mitochondria. LcCS2 and LcCS3 overexpression in rice promoted Cd efflux from roots and decreased Cd accumulation in shoots and grains. However, LcCS4 overexpression increased Cd influx into roots, leading to Cd accumulation in rice. In all examined LcCS2/3/4 -overexpressing rice lines, CS activities and CA contents increased following a Cd treatment. Accordingly, Cd detoxification mechanisms mediated by LcCS2 and LcCS3 may involve increased CA synthesis and chelation of Cd ions, followed by the transport of CA–Cd complexes out of root cells via plasma membrane proteins. By contrast, LcCS4 facilitates the sequestration of CA–Cd complexes in vacuoles via vacuolar membrane transporters. Our study results demonstrate that LcCS2/3/4 enhance plant Cd tolerance through unique, but complementary, mechanisms, thereby providing valuable molecular insights applicable to improving crop safety as well as the phytoremediation of Cd-contaminated soil.