Maize leaves salt-responsive genes revealed by comparative transcriptome of salt-tolerant and salt-sensitive cultivars during the seedling stage

Abstract

Maize (Zea mays) is a crop of significant global importance, yet its productivity is considerably hindered by salt stress. In this study, we investigated two maize cultivars, one exhibiting high salt tolerance (ST) and the other showing salt sensitivity (SS) at the seedling stage. The ST cultivar demonstrated superior seedling survival rates, higher relative water content, and lower electrolyte leakage and malondialdehyde levels in its leaves after both 3-day and 7-day salt treatments, when compared to the SS cultivar. To explore the molecular basis of these differences, we performed comparative transcriptome sequencing under varying salt treatment durations. A total of 980 differentially expressed genes (DEGs) were identified. Gene ontology (GO) functional enrichment analysis of DEGs indicated that the oxidation-reduction process, phosphorylation, plasma membrane, transferase activity, metal ion binding, kinase activity, protein kinase activity and oxidoreductase activity process is deeply involved in the response of ST and SS maize varieties to salt stress. Further analysis highlighted differences in the regulatory patterns of transcription factors encoded by the DEGs between the ST and SS cultivars. Notably, transcription factor families such as AP2/ERF, bZIP, MYB, and WRKY were found to play crucial roles in the salt stress regulatory network of maize. These findings provide valuable insights into the molecular mechanisms underlying salt stress tolerance in maize seedlings.

Keywords: Differentially expressed genes; Maize seedling leaves; RNA sequencing; Salt stress; Salt tolerance.

Figure 1. Salt treatment phenotypic characterization of the ST and SS maize cultivars.

(A) Phenotypes of the ST and SS seedings to salt stress. Plants were grown to the four-leaf stage in soil and then irrigated with saline water containing 300 mM NaCl for 0, 3, and 7 days. The scale bars are six cm. (B) DAB and NBT staining of the third leaves of ST and SS seedings treated with 300 mM NaCl for 0, 3 and 7 days. The scale bars are one cm. (C) The RWC, REL, H₂O₂ and MDA content, SOD and POD activities of ST and SS seedings treated with 300 mM NaCl for 0, 3 and 7. Data means ± SDs (n = 3). The symbols * and ** indicate significant differences between ST and SS seedings at P < 0.05 and P < 0.01, respectively.

Figure 2. Sample-sample correlation heatmap and PCA analysis plot of the differentially expressed genes (DEGs) in three comparisons of ST and SS seedings treated with 300 mM NaCl for 0, 3 and 7 days.

(A) Sample-sample correlation heatmap of the 18 samples. (B) PCA analysis plot.

Figure 3. Numbers of DEGs in ST0 vs. SS0, ST3 vs. SS3, and ST7 vs. SS7 comparisons (A) and overlap between DEGs (B).

Figure 4. Gene ontology (GO) enrichment analysis of the DEGs detected in ST0 vs. SS0, ST3 vs. SS3, and ST7 vs. SS7 comparisons.

The topmost enriched GO terms under the three main GO categories are shown.

Figure 5. KEGG enrichment map of DEGs in ST0 vs. SS0 (A), ST3 vs. SS3 (B), and ST7 vs. SS7 (C) comparisons.

Figure 6. Transcription factors (TFs) encoded by DEGs detected in ST0 vs. SS0, ST3 vs. SS3, and ST7 vs. SS7 comparisons.

(A) Classification and statistics of the TF families. (B) Heatmaps of the FPKM+1 values of the 36 TFs.

Figure 7. Quantitative real-time PCR validations of DEGs characterized by RNA-seq.

(A) Average FPKM values acquired by RNA-seq. (B) The relative expression levels of candidate genes by qRT-PCR.