The role of GmHSP23.9 in regulating soybean nodulation under elevated CO₂ condition

Legume-rhizobia symbiosis offers a unique approach to increase leguminous crop yields. Previous studies have indicated that the number of soybean nodules are increased under elevated CO₂ concentration. However, the underlying mechanism behind this phenomenon remains elusive. In this study, transcriptome analysis was applied to identify candidate genes involved in regulating soybean nodulation mediated by elevated CO₂ concentration. Among the different expression genes (DEGs), we identified a gene encoding small heat shock protein (sHSP) called GmHSP23.9, which mainly expressed in soybean roots and nodules, and its expression was significantly induced by rhizobium USDA110 infection at 14 days after inoculation (DAI) under elevated CO₂ conditions. We further investigated the role of GmHSP23.9 by generating transgenic composite plants carrying GmHSP23.9 overexpression (GmHSP23.9-OE), RNA interference (GmHSP23.9-RNAi), and CRISPR-Cas9 (GmHSP23.9-KO), and these modifications resulted in notable changes in nodule number and the root hairs deformation and suggesting that GmHSP23.9 function as an important positive regulator in soybean. Moreover, we found that altering the expression of GmHSP23.9 influenced the expression of genes involved in the Nod factor signaling pathway and AON signaling pathway to modulate soybean nodulation. Interestingly, we found that knocking down of GmHSP23.9 prevented the increase in the nodule number of soybean in response to elevated CO₂ concentration. This research has successfully identified a crucial regulator that influences soybean nodulation under elevated CO₂ level and shedding new light on the role of sHSPs in legume nodulation.