<scp>PAM18</scp> ‐3, a J‐domain protein, maintains mitochondrial integrity and plant growth and development in <i>Arabidopsis thaliana</i>

Mitochondrial protein import is indispensable for organelle biogenesis and function, and is powered by the evolutionarily conserved presequence translocase-associated motor (PAM) complex. In Arabidopsis thaliana, three paralogs: PAM18-1, PAM18-2, and PAM18-3 encode J-domain proteins homologous to yeast PAM18, which stimulates the ATPase activity of mitochondrial HSP70 (mtHSP70) during protein translocation. Here, we identify PAM18-3 as the most highly and ubiquitously expressed paralog and demonstrate its critical role in mitochondrial function and plant development. Genetic disruption of PAM18-3 caused severe vegetative and reproductive defects, including reduced root length, smaller rosette size with fewer leaves, decreased plant height, shorter siliques, reduced seed set, and increased seed abortion. These phenotypes were fully rescued in complemented lines expressing PAM18-3. Ultrastructural analyses revealed profound mitochondrial abnormalities in mutants, whereas chloroplast architecture remained unaffected. Functional assays showed reduced mitochondrial membrane potential and altered respiratory flux with a compensatory induction of the alternative oxidase (AOX) pathway. Transcript profiling revealed upregulation of AOX genes and multiple components of the mitochondrial TIM23 import apparatus and associated chaperones. Import assays demonstrated reduced mitochondrial accumulation of canonical TIM23 substrates, including IDH, ATPβ, and SHMT1, confirming a defect in matrix protein translocation. Consistently, pam18-3 mutants accumulated elevated reactive oxygen species (ROS) and exhibited strong induction of mitochondrial dysfunction stimulon (MDS) genes, including key transcription factors mediating retrograde signaling. Together, our findings establish PAM18-3 as a central component of the mitochondrial protein import machinery, supporting plant growth and development in A. thaliana.