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Sulphur and nitrogen fertilization strategy for wheat grain quality improvement and the underlying mechanism

Yu, Zitong (2017) Sulphur and nitrogen fertilization strategy for wheat grain quality improvement and the underlying mechanism. PhD thesis, Murdoch University.

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Australia is the fourth largest wheat (Triticum aestivum) exporter around the world,exporting about18 million tonnes annually. To achieve a desirable profit, the focus of the Australian wheat industry is to concurrently increase grain yield (GY) and grain protein content(GPC). However, due to the negative correlation between wheat GY and GPC,this is a challenging task. Apart from complex genetic approaches, modifyingnitrogen and sulphur fertilizer regime is a strategic method to achieve this goal. It is known that without adequate sulphur application, wheat cannot reach its full yield potential and make efficient use of nitrogen for protein biosynthesis. During grain filling, changes in nitrogen availability mainly affect GPC, while variable sulphur concentration exerts major impact on grain protein compositions.

In this study, the impacts of a range of nitrogen and sulphur treatments on a series of nitrogen use efficiency (NUE) related agronomic traits, morphological traits and protein parameters were investigated using multi-year & site field trials and glasshouse experiments, and the accumulation patterns of individual storage protein composition. Six varying quality Australian bread wheat varieties,including Spitfire, Mace, Wyalkatchem, Westonia, Bonnie Rock,and Livingston,were selected for this study. Grain protein yield is used as a parameter to represent protein efficiency which is obtained by GY multiplying GPC. The results indicate that nitrogen application increases grain protein yield and optimizes grain protein parameters, but the NUE in relation to GY and grain protein yield (NUE-GY & NUE-PY) are both decreased. The correlation of neck diameter with GY and NUE-GY under different nitrogen treatments indicates that the improvement of neck diameter can be achieved through optimizing nitrogen treatments, which ultimately increases GY and NUE-GY. Wheat GY and GPC react to the nitrogen availability at the similar level with the GY being slightly more sensitive, and genotype is a vital determinant of grain protein yield. In another hand, high sulphur application can increase GY and NUE-GY simultaneously. All peduncle traits are positively correlated with GY and NUE-GY under different sulphur treatments, indicating that peduncle trait enhancement is a route for GY and NUE-GY appreciation through high sulphur availability.

To understand the impact of nitrogen availability on grain protein biosynthesis during grain filling, the GS activity and SDS-unextractable polymeric proteins (UPP)accumulation in high and low nitrogen treatments were studied, followed by a comparative proteomics study to identify major functional proteins in response to nitrogen availability using developing grains at7, 14, 21,and 28 days post-anthesis (DPA). The results illustrate that glutamine synthetase (GS) activity of developing grains is flag leaf dependent, and high nitrogen application improves protein polymerization to form UPP. Its underlying mechanism was revealed by comparative proteomics study and validated by yeast two-hybrid assay, which shows that PPIase can be SUMOylated with the assistance of SUMO1, and high nitrogen availability facilitates this connection for subsequent protein polymerization.

Since fertilizer management exerts major impacts on gene expression, a comparative transcriptomic study was carried out to explore the role of sulphur in regulating gene expression for nitrogen metabolism and grain protein biosynthesis. Eighteen developing grains at7, 14,and 21 DPA from high and low sulphur treatments were collected for RNA-seq assay. The results indicated that high sulphur supply led to one upregulated differentially expressed gene (DEG)and 63downregulated DEGs in 7 DPA. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that three of 63downregulated DEGs were significantly enriched in nitrogen metabolic pathway, and all annotated as GS, which is the pivotal enzyme in GOGAT cycle for nitrogen assimilation, revealingthat GS is a bridge of sulphur and nitrogen metabolism. Accordingly, the dynamics of GS activity were traced and compared between high and low sulphur treatments.Results indicate that high sulphur availability results in an increased GS activity of developing grains. Meanwhile, the transcriptomics assay of the developing grains from high and low sulphur treatments identifies a total of 20,997 DEGs in four libraries, and there are 1,004 DEGs enriched in significant GO items and KEGG pathway. After aligning their promoter regions with an in-house developed cis-regulatory elements database, 40 cis-regulatory elements in response to phytohormone were identified and most of them show response to ABA. Subsequently, with the use of on-line database WheatNet, a sulphur-dependent epigenetics regulatory mechanism for wheat seed storage proteins biosynthesis was proposed, which suggests that high sulphur availability induces HMT-1 expression for methionine (Met) biosynthesis for an increased amount of Met for grain storage protein (20% Met) and secondary metabolites biosynthesis (80% Met). It also maintains redox homeostasis for protein polymerization. The proposed network was validated by the comparison of free amino acid dynamics between high and low sulphur treatments during grain filling, revealing that high sulphur availability can take advantage of more free amino acids to participate in biological processes for grain growth than low sulphur availability. Specifically, high sulphur application results in reduced asparagine residuals of mature grain (42 DPA), which will potentially decrease the acrylamide formation during breadmaking and thus reducethe chance of cancer development in human.

In conclusion, this study demonstrates that although an incremental nitrogen application can increase GY, GPC,and grain protein yield, the NUE-GY and NUE-PY are decreased.High sulphur availability can increase GY and NUE-GY simultaneously. Finally,a nitrogen regulatory mechanism and a sulphur dependent mechanism of wheat grain protein polymerization were respectively revealed byproteomics study and transcriptomics study.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Ma, Wujun
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