Small heat shock proteins (sHSPs) identified in nodules of tropical woody legumes

Biological nitrogen fixation (BNF) is a primary input of nitrogen to natural and agricultural systems globally. BNF is a temperature-dependant enzymatic process and can be conducted by microbes (including Rhizobia ) hosted symbiotically in root nodules of some plants. Heat shock proteins (Hsps) have been implicated in the process of acquiring thermotolerance or acclimating to elevated temperature, as they play a vital role in maintaining cell integrity and homeostasis during heat stress. Although the BNF response to temperature may crucially impact future ecosystem productivity in the face of global climate change, little is known about Hsp expression in nodules of N-fixing non-agricultural species, such as tropical N-fixing trees in the Fabaceae family. This project aimed to characterize small (15-20 kDa) Hsp (sHsp) expression in nodule tissue to examine the biochemical mechanisms of heat response in these tissues. To first identify Hsps in nodule tissues, Vachellia farnesiana and Acacia confusa nodules were excised, heat shock was induced, and protein content was isolated via chemical treatment before separation of protein species and analysis with SDS-PAGE. Two polyacrylamide gels yielded bands in the 15-20 kDa region that displayed differential Coomassie staining, which were sent for further characterization by HPLC-MS analysis for protein sequencing. Ten rhizobial sHsps were detected in these samples in addition to seven Acacia sHsps when compared independently to reference rhizobial and plant proteome databases. In an attempt quantify relative expression of Hsps in nodule and root tissue, we performed western blot experiments using Anti-Hsp20 antibodies raised against human and mouse Hsp proteins, with anti-beta actin loading control. While nonuniform beta-actin expression across tissue types ( A. confusa nodules versus root control) prevented quantitative analysis, the experiments validated that Hsp20s are expressed in Acacia nodules as well as in root tissue. These experiments provide a foundation for future studies to determine variation in responses to key stressors predicted to increase with global climate change and help determine the implications of warming across the tropics and beyond. Proteomics data are available via ProteomeXchange with identifier PXD055599.