Maize WRKY Transcription Factor ZmWRKY106 Confers Drought and Heat Tolerance in Transgenic Plants

Abstract

WRKY transcription factors constitute one of the largest transcription factor families in plants, and play crucial roles in plant growth and development, defense regulation and stress responses. However, knowledge about this family in maize is limited. In the present study, we identified a drought-induced WRKY gene, ZmWRKY106, based on the maize drought de novo transcriptome sequencing data. ZmWRKY106 was identified as part of the WRKYII group, and a phylogenetic tree analysis showed that ZmWRKY106 was closer to OsWRKY13. The subcellular localization of ZmWRKY106 was only observed in the nucleus. The promoter region of ZmWRKY106 included the C-repeat/dehydration responsive element (DRE), low-temperature responsive element (LTR), MBS, and TCA-elements, which possibly participate in drought, cold, and salicylic acid (SA) stress responses. The expression of ZmWRKY106 was induced significantly by drought, high temperature, and exogenous abscisic acid (ABA), but was weakly induced by salt. Overexpression of ZmWRKY106 improved the tolerance to drought and heat in transgenic Arabidopsis by regulating stress-related genes through the ABA-signaling pathway, and the reactive oxygen species (ROS) content in transgenic lines was reduced by enhancing the activities of superoxide dismutase (SOD), peroxide dismutase (POD), and catalase (CAT) under drought stress. This suggested that ZmWRKY106 was involved in multiple abiotic stress response pathways and acted as a positive factor under drought and heat stress.

Keywords: WRKY; ZmWRKY106; drought tolerance; maize; thermotolerance.

Figure 1

De novo transcriptome sequencing analysis of maize under drought stress. (A) Cluster analysis of the differentially expressed genes (DEGs) under drought treatment. (B) Transcription levels of the 14 differentially expressed ZmWRKYs under drought treatment. Error bar represent standard deviations (SD). The data represent means ± SD of three biological replications.

Figure 2

Multiple alignment and phylogenetic relationships of ZmWRKY106 with other orthologs in rice, Arabidopsis, and barley. The phylogenetic tree was produced using the aligned file with 1000 bootstraps in the MEGA 5.0 program. (A) Multiple alignment of ZmWRKY106 homologous proteins in rice, Arabidopsis, and barley. The different background colors represent the similar degree of amino acid sequences. (B) Phylogenetic relationship of ZmWRKY106 and other orthologs in different species. The first red box indicates the WRKYGQK motif, and the second indicates the conserved C2H2 zinc-finger motif.

Figure 2

Multiple alignment and phylogenetic relationships of ZmWRKY106 with other orthologs in rice, Arabidopsis, and barley. The phylogenetic tree was produced using the aligned file with 1000 bootstraps in the MEGA 5.0 program. (A) Multiple alignment of ZmWRKY106 homologous proteins in rice, Arabidopsis, and barley. The different background colors represent the similar degree of amino acid sequences. (B) Phylogenetic relationship of ZmWRKY106 and other orthologs in different species. The first red box indicates the WRKYGQK motif, and the second indicates the conserved C2H2 zinc-finger motif.

Figure 3

Subcellular localization of ZmWRKY106. The p16318hGFP and p16318hGFP-ZmWRKY106 constructs were transiently expressed in maize protoplasts. The green indicates green fluorescent, and the red indicates chloroplast autofluorescence. Results were observed after transformation for 18 h with confocal microscopy. Scale bars = 10 μm.

Figure 4

Expression patterns of ZmWRKY106 under (A) drought, (B) high-salt, (C) high-temperature, and (D) exogenous abscisic acid (ABA) stresses. The ordinates are the relative expression level (fold) of ZmWRKY106 compared to the non-stressed control. The horizontal ordinate is treatment time for 0, 0.5, 1, 2, 4, 6, 12 and 24 h. All experiments were repeated three times. Error bars represent standard deviations (SDs). All the data represent the means ± SDs of three independent biological replicates. The different letters in the bar graphs indicate significant differences at p < 0.05.

Figure 5

Phenotypes of ZmWRKY106 transgenic Arabidopsis under drought treatment. (A) Seed germinations of wild-type (WT) and ZmWRKY106-overexpressing lines. (B) Root lengths of WT and ZmWRKY106 transgenic plants. Five-day-old seedlings were transferred to MS medium supplemented with or without PEG6000 for seven days, and then root lengths were measured. All the data represent the means ± SDs of three independent biological replicates and asterisks (**) represent the significant differences at p < 0.01 (Student’s t-test).

Figure 6

Survival rates of WT and ZmWRKY106 transgenic lines under heat stress. Five-day-old seedlings were placed at 45 °C for 5 h and then resumed growth at 22 °C. The data represent the means ± SDs of three independent biological replicates. Asterisks (**) represent the significant differences (p < 0.01) compared with the control (Student’s t-test).

Figure 7

The relative expression of stress-related genes. (A) RD29A, (B) HSP90, (C) DREB2A, (D) CuZnSOD, (E) NCED3, and (F) NCED6 were examined under control and drought conditions for various time points (4, 12, 24 and 36 h). Values are means ± SDs of three replicates, and asterisks (* or **) represent the significant differences at p < 0.05 or p < 0.01, respectively (Student’s t-test).

Figure 8

(A) The reactive oxygen species (ROS) content and the activities of (B) superoxide dismutase (SOD), (C) peroxide dismutase (POD), and (D) catalase (CAT) under different conditions at different time points (0, 4, 12, and 24 h). Values are means ± SDs of three replicates, and asterisks (* or **) represent the significant differences at p < 0.05 or p < 0.01, respectively (Student’s t-test).