Global warming poses severe threats to agricultural production, including soybean. One of the major mechanisms for organisms to combat heat stress is through heat shock proteins (HSPs) that stabilize protein structures at above‐optimum temperatures, by assisting in the folding of nascent, misfolded, or unfolded proteins. The HSP40 subgroups, or the J‐domain proteins, functions as co‐chaperones. They capture proteins that require folding or refolding and pass them on to HSP70 for processing. In this study, we have identified a type‐I HSP40 gene in soybean, GmDNJ1, with high basal expression under normal growth conditions and also highly inducible under abiotic stresses, especially heat. Gmdnj1‐knockout mutants had diminished growth in normal conditions, and when under heat stress, exhibited more severe browning, reduced chlorophyll contents, higher reactive oxygen species (ROS) contents, and higher induction of heat stress‐responsive transcription factors and ROS‐scavenging enzyme‐encoding genes. Under both normal and heat‐stress conditions, the mutant lines accumulated more aggregated proteins involved in protein catabolism, sugar metabolism, and membrane transportation, in both roots and leaves. In summary, GmDNJ1 plays crucial roles in the overall plant growth and heat tolerance in soybean, probably through the surveillance of misfolded proteins for refolding to maintain the full capacity of cellular functions.
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