Regulatory control of the symbiotic enhanced soybean bHLH transcription factor, GmSAT1.

Abstract

GmSAT1 is a basic Helix-Loop-Helix (bHLH) DNA binding transcription factor expressed in soybean root nodules. GmSAT1 is a unique protein, in that it is localised on cellular membranes including the symbiosome membrane, which encircles nitrogen-fixing bacteroids in soybean nodules. Its role in the regulation of gene transcription in nodules or in other plant tissues is poorly understood. In this study, GmSAT1’s functional activity was investigated through a series of studies that investigated the link between gene activities to functional phenotypes. This analysis included the influence of symbiotic partnerships with rhizobia and AM fungi and non-symbiotic root tissues. In this context, an evaluation of changes in gene transcription with or without GmSAT1 expression (RNAi-based silencing of GmSAT1) was explored at the individual and global gene levels. The data indicates that GmSAT1;1 and a close relative GmSAT1;2, are both expressed in roots and nodules but GmSAT1;1 displayed an overall enhancement in the symbiotic root nodule. Expression of both genes was reduced with external nitrogen supply to the nodule and inoculated root. Both genes were up-regulated in root and nodule tissues when plants were supplied low levels of phosphate. Using an improved method for transgenic hairy roots, developed as part of this thesis project, GmSAT1 was silenced using a RNAi construct. Tissues (roots and nodules) were analysed for changes in global gene expression using microarray analysis, the impact on symbiotic relationships (rhizobia and AM fungi) and genetic and biochemical responses to phosphorus supply. Transcriptome analysis identified networks that GmSAT1;1 may be associated with, including a suite of putatively active circadian clock regulators operating in nodules, phosphorus responsive genes in roots, cell wall maintenance and or stress defence signaling pathways, nitrogen transport and metabolism and genes linked to auxin and gibberellin regulatory pathways. The influence of phosphorus and the AM fungal symbiosis was investigated in more detail. Loss of GmSAT1 activity altered AM colonisation, causing a reduction in root colonisation when grown at reduced external P. At higher P levels, colonisation remained unchanged. Shoot P content was significantly increased at both low and high external P supply in the GmSAT1 silenced plants, indicating a potential role of GmSAT1 in mediating P homeostasis. The impact of gibberellins (GA₃) on GmSAT1 expression and activity was also investigated. Using both qPCR and native promoter:GUS fusion constructs in transformed soybean hairy roots and nodules the expression of GmSAT1;1 in roots and nodules decreased with external supply of GA₃. In parallel experiments, RNAi SAT1-silenced plants showed similar responses with GA₃ treated plants, where nodule number and weight decreased while plant height significantly increased. Furthermore, microarray analysis indicated GmSAT1 negatively interacts with known gibberellin-responsive genes, including GASA6, GAMA-TIP, CLE2, MTO3, GIP1, TPS11, and GBF1. The overall findings of this study have shown that GmSAT1 is an important TF to soybean with a broad transcriptional imprint which influences both root nodule symbiosis and AM fungal symbioses. Its activity appears to be linked to multiple genetic signaling networks that involve phosphorus and nitrogen metabolism, hormone activity and regulation of the circadian clock.