A small heat shock protein, GmHSP17.9, from nodule confers symbiotic nitrogen fixation and seed yield in soybean

Abstract

Legume-rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture. Small heat shock proteins (sHSPs) are involved in multiple environmental stresses and plant development processes. However, the role of sHSPs in nodule development in soybean remains largely unknown. In the present study, we identified a nodule-localized sHSP, called GmHSP17.9, in soybean, which was markedly up-regulated during nodule development. GmHSP17.9 was specifically expressed in the infected regions of the nodules. GmHSP17.9 overexpression and RNAi in transgenic composite plants and loss of function in CRISPR-Cas9 gene-editing mutant plants in soybean resulted in remarkable alterations in nodule number, nodule fresh weight, nitrogenase activity, contents of poly b-hydroxybutyrate bodies (PHBs), ureide and total nitrogen content, which caused significant changes in plant growth and seed yield. GmHSP17.9 was also found to act as a chaperone for its interacting partner, GmNOD100, a sucrose synthase in soybean nodules which was also preferentially expressed in the infected zone of nodules, similar to GmHSP17.9. Functional analysis of GmNOD100 in composite transgenic plants revealed that GmNOD100 played an essential role in soybean nodulation. The hsp17.9 lines showed markedly more reduced sucrose synthase activity, lower contents of UDPglucose and acetyl coenzyme A (acetyl-CoA), and decreased activity of succinic dehydrogenase (SDH) in the tricarboxylic acid (TCA) cycle in nodules due to the missing interaction with GmNOD100. Our findings reveal an important role and an unprecedented molecular mechanism of sHSPs in nodule development and nitrogen fixation in soybean.

A Nodule-Localized Small Heat Shock Protein GmHSP17.1 Confers Nodule Development and Nitrogen Fixation in Soybean

“…Directly interaction between GmHSP17.1 and GmRIP1 was confirmed through Y2H, BiFC and pull-down assay ( Figure 6 ). Recently, our team has reported a role of a sHSP, GmHSP17.9, in nodule development and nitrogen fixation through interacting with GmNOD100, a sucrose synthase specifically induced in the nodules of soybean ( Yang et al, 2021 ). GmHSP17.1 and GmHSP17.9 were both cytosolic proteins, while belonged to different subfamily of sHSPs with GmHSP17.1 in CI subfamily and GmHSP17.9 in CII subfamily ( Figure 2D ; Yang et al, 2021 ).…”

“…Recently, our team has reported a role of a sHSP, GmHSP17.9, in nodule development and nitrogen fixation through interacting with GmNOD100, a sucrose synthase specifically induced in the nodules of soybean ( Yang et al, 2021 ). GmHSP17.1 and GmHSP17.9 were both cytosolic proteins, while belonged to different subfamily of sHSPs with GmHSP17.1 in CI subfamily and GmHSP17.9 in CII subfamily ( Figure 2D ; Yang et al, 2021 ). Previously, in Arabidopsis , the expression of either cytosolic class I or II sHSPs were suppressed using RNAi approach and they showed distinct biochemical behavior in vitro and in vivo ( McLoughlin et al, 2016 ).…”

“…Green fluorescent proteins (GFPs) were purified in the same way as GmHSP17.1 and used as negative controls in this experiment. The chaperone activity of GmHSP17.1 was performed by measuring the chemically induced aggregation of insulin (from bovine pancreas, Sigma-Aldrich, St. Louis, MO, United States) and thermal aggregation of malate dehydrogenase (MDH; from porcine heart, Sigma-Aldrich) according to previous protocols ( Yang et al, 2021 ).…”

“…Recently, our research team for the first time reported that sHSP, GmHSP17.9, identified in the proteome of nodules confers nodule development and symbiotic nitrogen fixation via interacting with sucrose synthase GmNOD100 in soybean ( Yang et al, 2021 ). In the meantime, another sHSP, named GmHSP17.1 ( Glyma.06g157800 ), was found to be preferentially expressed in nodules.…”

Improvement of heat stress tolerance in soybean (Glycine max L), by using conventional and molecular tools

“…The mutants would be valuable genetic materials to investigate the molecular mechanism governing soybean response to multiple abiotic stresses ( Xiao et al, 2021 ) and hopefully for heat tolerance. Yang et al (2022) also applied CRISPR/Cas9 to edit the heat shock protein, GmHSP17.9 The resulting mutant plants showed a remarkable variation in nodule number, nodule fresh weight, and nitrogenase activity. These findings reveal an unprecedented molecular mechanism of heat shock protein in nodule development and counter heat stress in soybean.…”