GmGLU1 and GmRR4 contribute to iron deficiency tolerance in soybean

Authors: KOHLHASE, DANIEL - Iowa State University; O`Rourke, Jamie; Graham, Michelle

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/2/2024
Publication Date: 2/27/2024
Citation: Kohlhase, D.R., O'Rourke, J.A., Graham, M.A. 2024. GmGLU1 and GmRR4 contribute to iron deficiency tolerance in soybean. Frontiers in Plant Science. 15. https://doi.org/10.3389/fpls.2024.1295952.
DOI: https://doi.org/10.3389/fpls.2024.1295952

Interpretive Summary: Iron deficiency chlorosis (IDC) is an abiotic stress that can cause significant decreases to soybean yield. In a previous study, we demonstrated that a region on soybean chromosome 3, originally thought to contain a single iron stress tolerance gene, likely contains multiple genes helping to confer iron stress tolerance. In this study, we take advantage of virus induced gene silencing (VIGS) to validate three different candidate genes: GmGLU1 (GLUTAMATE SYNTHASE 1, Glyma.03G128300), GmRR4 (RESPONSE REGULATOR 4, Glyma.03G130000), and GmbHLH38 (beta Helix Loop Helix 38, Glyma.03G130400 and Glyma.03G130600). We developed VIGS constructs to targeted each of these genes alone and in combination. We found significant decreases in chlorophyll content using the GmGLU1 and the double construct GmRR4+GmGLU1. Whole genome expression analyses revealed a wide range of genes effected by gene silencing including those with involved with iron stress responses, defense and hormone signaling, photosynthesis and cell wall structure. These findings highlight the importance of GmGLU1 and GmRR4 in soybean iron stress responses and provide evidence that iron stress tolerance is controlled by multiple genes.

Technical Abstract: Iron deficiency chlorosis (IDC) is an abiotic stress that can cause significant decreases to soybean yield. In a previous study, we demonstrated the historical IDC QTL on soybean chromosome Gm03 was composed of four distinct linkage blocks, each containing candidate genes for IDC tolerance. Here, we take advantage of virus induced gene silencing (VIGS) to validate three high priority candidate genes, corresponding to three different linkage blocks in the historical IDC QTL. In multiple previous studies, the three genes were differentially expressed in response to iron stress conditions. We built three single gene constructs to target GmGLU1 (GLUTAMATE SYNTHASE 1, Glyma.03G128300), GmRR4 (RESPONSE REGULATOR 4, Glyma.03G130000), and GmbHLH38 (beta Helix Loop Helix 38, Glyma.03G130400 and Glyma.03G130600). Given the polygenic nature of the iron stress tolerance trait, we also silenced the genes in combination. We built two constructs targeting GmRR4+GmGLU1 and GmbHLH38+GmGLU1. All constructs were tested on the iron efficient soybean genotype Clark grown in iron sufficient conditions. We found significant decreases in SPAD measurements using the GmGLU1 and both double constructs, with potential additive effects in the GmRR4+GmGLU1 construct. Whole genome expression analyses (RNA-seq) revealed a wide range of effected processes including known iron stress responses, defense and hormone signaling, photosynthesis and cell wall structure. These findings highlight the importance of GmGLU1 in soybean iron stress responses and provide evidence that IDC is truly a polygenic trait, with multiple genes within the historic QTL contributing to IDC tolerance.