Phenotype analysis of WT and LMM 5368 lines
A barley LMM 5386 line was generated through the application of ethyl methanesulfonate (EMS) to the ‘Tamalpais’ wild type (WT) cultivar. We found that several brown spots were spontaneously produced in the leaves of LMM 5368 lines under field conditions (Fig. 1a–b). The brown spot area per leaf was quantified in WT and LMM 5368 lines, and that in LMM 5368 lines was significantly higher than in the WT (Fig. 1c).
We also compared the phenotypes of WT and LMM 5368 individuals (Fig. 2a). The tiller number of LMM 5368 was significantly lower (approximately 16.7%) than that of the WT (Fig. 2b), but there were no obvious differences in the plant height (Fig. 2c). Further, the number of leaf brown spots per plant was significantly higher in LMM 5368 plants than that in WT plants (Fig. 1d).
RNA-seq analysis of WT and LMM 5368 lines
To better understand the mechanism of LM formation in LMM lines, we performed an RNA-seq analysis using the flag leaves of WT and LMM 5368 lines. The RNA-seq analysis provided 65.80 Gb of clean bases. The percentage of Q30 in each sample was not less than 93.89%, 91.62–92.09% of reads could be accurately mapped to the reference genome, and 2.38–3.69% of reads could be mapped to multiple genome sequences (Table 1). The Pearson correlation coefficients among biological replicates were found to be higher than 0.95 (Fig. S1).
Compared with WT lines, 1453 differentially expressed genes (DEGs) were found in LMM 5368 lines, of which 1260 were upregulated and 193 were downregulated (Fig. 3). DEGs with the same or similar expression patterns were placed into 16 groups via hierarchical clustering analysis (Fig. S2). For example, the nine DEGs in group 1 were enriched in the glutathione (GSH) metabolic process, GSH transferase activity, anchored component of the plasma membrane, aleurone grain membrane, and cytokinin biosynthetic process (Fig. S3).
The functions of the 1453 DEGs were verified using the gene ontology (GO) database (http://www.geneontology.org), which provides annotations of biological processes, molecular functions, and cellular components. Specifically, we compared the biological process between the WT and LMM 5368 lines. Among the upregulated DEGs, those encoding the protein phosphorylation, oxidation-reduction process, and defense response were clearly over-represented (Fig. 4a). Among the downregulated DEGs, we found that many were significantly enriched in oxidation-reduction and redox homeostasis processes (Fig. 4b).
To compare the metabolic pathways of DEGs in WT and LMM 5368 lines, we analyzed the obtained DEGs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/pathway.html). Among the upregulated DEGs, many were significantly enriched in GSH metabolism, plant-pathogen interactivity, and amino acid biosynthesis (Fig. 5a). Among the downregulated DEGs, many were significantly enriched in photosynthesis antenna proteins, glyoxylate and dicarboxylate metabolism, and carbon fixation in photosynthetic organisms (Fig. 5b).
ROS analysis of WT and LMM 5368 lines
GO analysis showed that many DEGs were enriched in the oxidation-reduction process (Fig. 4). Therefore, we compared the H2O2 content and O2•− production rate between WT and LMM 5368 lines (Fig. 6). The accumulation of O2•− was detected via nitroblue tetrazolium (NBT) staining, and O2•− accumulation in the brown spots of LMM 5368 lines was significantly higher than that in WT plants (Fig. 6a). Similarly, diaminobenzidine (DAB) staining suggested that H2O2 content in brown spots of LMM 5368 lines was significantly higher than that in the WT (Fig. 6c). Finally, we quantified the ROS accumulation. As shown in Fig. 6b and d, LMM 5368 lines showed a relatively higher O2•− production rate and H2O2 content than seen in the WT lines.
GSH and glycine (Gly) analysis of WT and LMM 5368 lines
KEGG analysis showed that many upregulated DEGs were significantly enriched in GSH metabolism (Fig. 5a). Therefore, the GSH and Gly content was compared between WT and LMM 5368 lines (Fig. 7a and b, respectively); both were significantly lower in LMM 5368 lines than in the WT.
Antioxidant competence analysis of WT and LMM 5368 lines
Antioxidant enzyme activities were also compared between WT and LMM 5368 lines (Fig. 8). We measured SOD (Fig. 8a), catalase (CAT) (Fig. 8b), ascorbate peroxidase (APX) (Fig. 8c), and peroxidase (POD) (Fig. 8d) activity levels, which were significant in LMM 5368 lines than in the WT. The activities of glutathione reductase (GR; Fig. 8e) and GST (Fig. 8f) were consistent with those of SOD, CAT, APX, and POD. Nevertheless, the downregulation of GR and GST was significantly greater than that of SOD, CAT, APX, and POD in LMM 5368 lines. Further, the antioxidant enzyme-encoding genes of WT and LMM 5368 were compared (Fig. 9). The expression of Cu/Zn-SOD (Fig. 9a), HvCAT1 (Fig. 9b), HvAPX1 (Fig. 9c), and HvGST6 (Fig. 9d) genes in LMM 5368 lines were significantly lower than those in the WT.
Resistance to F. graminearum analysis of WT and LMM 5368 lines
RNA-seq analysis indicated that a large number of genes associated with disease resistance were altered between WT and LMM 5368 lines. Therefore, we determined the expression of six disease resistance-related genes, namely, isochorismate synthase (HvICS) (Fig. 10a), ethylene response factor 1 (HvERF1) (Fig. 10b), HvWRKY38 (Fig. 10c), pathogenesis related protein-1a (HvPR1a) (Fig. 10d), ethylene-responsive transcription factor 3 (HvERFC3) (Fig. 10e), and flavonoid O-methyltransferase protein (HvFme) (Fig. 10f). Their expressions in the LMM 5368 lines were up-regulated compared to those in the WT (Fig. 10). We also compared the F. graminearum lesions on infected leaves between WT and LMM 5368 lines (Fig. 11). There were no differences in F. graminearum lesions at 3DAI between WT and LMM 5368 lines (Fig. 11a, b). After infected with F. graminearum at 7d, the length of lesions was significantly larger in all lines. But the lesions in the LMM 5368 were significantly smaller than those in WT (Fig. 11a, b).