Functional characterisation of GmSALT3: a candidate gene for conferring salt tolerance in soybean [Glycine max (L.) Merr.]

Soybean (Glycine max (L.) Merrill) is native to East Asia, which includes China that has a cultivation history stretching back at least 5,000 years. Now soybean is widely cultivated around the world as an important crop. It is an annual plant and its seeds are processed to produce two major products, oil and meal. Many biotic and abiotic stresses threaten soybean production in different areas of the world, such as fungal, bacterial and viral diseases; aluminium, drought, and salinity. In this thesis, the focus is on investigating the salinity stress responses in soybean and how GmSALT3 (salt tolerance-associated gene on chromosome 3), a dominant gene that is associated with limiting the accumulation of sodium ions in shoots, contributes to soybean’s salinity tolerance. GmSALT3 was identified through fine-mapping; it encodes a protein from the cation/H⁺ exchanger (CHX) family that I localized to the endoplasmic reticulum (ER) and which is preferentially expressed in the salt-tolerant parent Tiefeng 8 within root cells associated with phloem and xylem. In the salt-sensitive parent, 85-140, a 3.78-kb copia retrotransposon insertion in exon 3 of Gmsalt3 was identified that truncates the transcript. In addition, nine haplotypes including two salt-tolerant haplotypes and seven salt-sensitive haplotypes were identified by sequencing 31 soybean landraces and 22 wild soybean (Glycine soja) cultivars in China. By analysing the distribution of haplotypes, it was found that haplotype 1 (H1, found in Tiefeng 8) was strongly associated with salt tolerance and is likely to be the ancestral allele. H1, unlike other alleles, has wide geographical range including saline areas, which indicates it is maintained when required but its potent stress tolerance can be lost during natural selection and domestication. Then, I evaluated the impact of GmSALT3 on soybean performance under saline or non-saline treatments, with both field and controlled conditions experiments being performed. Three sets of near isogenic lines (NILs), with genetic similarity of 95.6–99.3% between each pair of NIL-T (salt-tolerant) and NIL-S (salt-sensitive), were generated from a cross between 85–140 and Tiefeng 8 by using marker-assisted selection. It was shown that GmSALT3 does not contribute to an improvement in seedling emergence rate or early vigor under salt stress. However, when 12-day-old seedlings were exposed to NaCl stress, I found that the NIL-T lines accumulated significantly less leaf Na⁺ and Cl⁻ compared with their corresponding NIL-S, while no significant difference of K⁺ concentration was observed between NIL-T and NIL-S. In addition, I found that the NIL-T lines accumulated less Cl⁻ in the leaf and more in the root prior to any difference in Na⁺; in the field, NIL-T accumulated less pod wall Cl⁻ than the corresponding NIL-S lines. Under non-saline field conditions, no significant differences were observed for yield related traits within each pair of NIL-T and NIL-S lines, indicating there was no observable yield penalty for having the GmSALT3 gene. In contrast, under saline field conditions the NIL-T lines had significantly greater plant seed weight and 100-seed weight than the corresponding NIL-S lines, meaning GmSALT3 conferred a yield advantage to soybean plants in salinized fields. In addition to confirming that Cl⁻ exclusion occurs prior to Na⁺ exclusion using a time course analysis, I found that stem secretion of Na⁺ contributes to its exclusion from leaves; NIL-T also accumulated less K⁺ in the leaf compared to NIL-S. I observed that Cl⁻ concentration is significantly higher in both the stem xylem and phloem sap of NIL-T. This likely means that whilst more Cl⁻ is transported from root-to-shoot more Cl⁻ is recirculated back to roots, and this contributes to a greater accumulation of Cl⁻ in NIL-T roots. Na⁺ is significantly greater in concentration in NIL-S xylem sap but no differences were detected in phloem sap and roots between NILs, which indicates Na⁺ is most likely regulated by exclusion at the root xylem, so in a different way in NIL-T compared to Cl⁻. Plants with full-length GmSALT3 maintain a significantly higher photosynthetic rate than NIL-S plants before and after salt treatment. In heterologous expression systems, GmSALT3 could restore bacterial growth of E. coli strain LB2003 (trkAΔ, kup1Δ, kdpABCDEΔ) that is defective in K⁺ uptake systems; when expressed in Xenopus laevis oocytes, GmSALT3 contributes to higher accumulation of Na⁺, K⁺, and Cl⁻ and higher net influx of Na⁺, K⁺, and Cl⁻ (measured by MIFE, Microelectrode Ion Flux Estimation) compared to water-injected oocytes. In an attempt to reveal new insights to the potential underlying mechanisms I used RNAseq analysis of roots from soybean NIL (Near Isogenic Lines); NIL-S (salt-sensitive, Gmsalt3) and NIL-T (salt-tolerant, GmSALT3). Thirty RNA-seq libraries were constructed and sequenced, including NIL-T and -S roots from three time points of 14 day old plants, 0 hours, 6h, and 3d following salt-treatment (200mM NaCl) and their corresponding non-treatment controls. Gene ontology (GO) analysis showed that unique DEGs under salt treatment in NIL-T are clustered into GO terms such as response to biotic stimulus, oxidation reduction and oxidoredutase activity, while in NIL-S GO terms are more diverse including cell communication, signalling, and biological regulation. Accordingly, reactive oxygen species (ROS) generation and detoxification was measured and differed in NIL consistent with the RNA-seq data. As such, I propose that GmSALT3 affects the ROS status of roots, which improves the ability of NIL-T to cope with stress. Overall, the collective findings of this thesis provide new insights into the transport activity of GmSALT3 and how GmSALT3 contributes to salinity tolerance in soybean.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2018