HSCA2 G87D point mutation enhances Arabidopsis proline tolerance via boosting mitochondrial Fe-S cluster assembly

As a mitochondrial HSP70 chaperone, HSCA2 orchestrates iron-sulfur (Fe-S) cluster assembly through dynamic interactions with scaffold protein ISU1, facilitating Fe-S cluster transfer to recipient proteins critical for electron transport chain (ETC) function. However, its regulatory roles in plant development and stress adaptation remain elusive. This study investigated the potential stress resistance function and molecular mechanisms of a novel G87D mutation in Arabidopsis HSCA2 (HSCA2m). We found that HSCA2m mutant exhibited increased resistance to high proline levels without altering proline uptake capacity. Under proline treatment, HSCA2m seedlings displayed lower malondialdehyde (MDA) and reactive oxygen species (ROS) levels, and higher superoxide dismutase (SOD) activity, indicating reduced stress damage. Molecular characterization revealed the induction of mitochondrial stress-related marker genes AOX1a and AT12CYS-2 in HSCA2m was suppressed. Strikingly, the G87D substitution enhanced intrinsic ATPase activity without disrupting ISU1 binding, while promoting HSCA2 transcript up-regulation under proline stress. Additionally, HSCA2m demonstrated increased tolerance to higher Fe²⁺ concentrations. These findings suggested that this mutation might enhance the supply of Fe-S clusters to Fe-S proteins, thereby mitigating proline-induced mitochondrial stress. Transgenic Arabidopsis overexpressing HSCA2m, but not HSCA2, showed enhanced proline resistance, highlighting the potential of HSCA2m as an elite allele for improving plant stress tolerance.