The purpose of this study is to elucidate the changes of chloroplast metabolites during wheat resistance to stripe rust, and to clarify the role of key chloroplast metabolites in wheat resistance to stripe rust.

Wheat cultivar Suwon11 was used as the experimental material, wheat chloroplasts were extracted 48 and 72 h after spraying sterile water (control group) and Puccinia striiformis f. sp. tritici CYR23 (experimental group), and a widespread non-targeted metabolomics analysis was conducted via ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Principal component analysis (PCA) and correlation analysis were employed to visualize inter-group and intra-group differences and associations. VIP (variable importance in projection) values derived from the orthogonal partial least squares discriminant analysis (OPLS-DA) model were employed to identify differential chloroplast metabolites between the disease-resistant and control groups. Pathway enrichment analysis was performed on differential chloroplast metabolites using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to investigate key metabolic pathways in chloroplasts during the resistance response of wheat to stripe rust.

The metabolomics data exhibited high quality, with good intra-group reproducibility and significant inter-groups variability validated by multivariate statistical analyses. A total of 1 496 metabolites were detected, which predominantly included lipids and lipid-like molecules, organic acids and their derivatives, organic heterocyclic compounds, phenylpropanoids and polyphenolic compounds, organic oxides, and benzoid compounds. The metabolite species were generally similar between different groups, but significant variations in metabolite levels were observed at different infection time points. At 48 hours post infection (hpi) compared to the control group (CK), 121 differential metabolites were identified, including 21 up-regulated and 100 down-regulated metabolites; At 72 hpi compared to the CK, 58 differential metabolites were detected, with 35 up-regulated and 23 down-regulated; Between 72 and 48 hpi groups, 53 differential metabolites were observed, of which 33 were up-regulated and 20 were down-regulated. Four differential metabolites were shared between the 48 hpi vs CK and 72 hpi vs CK comparisons. KEGG analysis revealed that the differential metabolites were enriched in multiple metabolic pathways. Among them, the expression of linoleic acid which in the linoleic acid metabolic pathway exhibited 2.75- and 2.93- fold increases after inoculation 48 and 72 h, respectively, indicating that linoleic acid was continuously synthesized induced by P. striiformis f. sp. tritici.

The chloroplast metabolites associated with wheat resistance to stripe rust primarily include lipids, fatty acids and their derivatives, organic acids and their derivatives, organic oxides, as well as phenylpropanoids and polyphenolic compounds. Notably, linoleic acid may play a crucial role in the sustained resistance of wheat against stripe rust.