Transcription Factor ZmNAC20 Improves Drought Resistance by Promoting Stomatal Closure and Activating Expression of Stress-Responsive Genes in Maize

Abstract

Drought is a major environmental threat that limits crop growth, development, and productivity worldwide. Improving drought resistance with genetic engineering methods is necessary to tackle global climate change. It is well known that NAC (NAM, ATAF and CUC) transcription factors play a critical role in coping with drought stress in plants. In this study, we identified an NAC transcription factor ZmNAC20, which regulates drought stress response in maize. ZmNAC20 expression was rapidly upregulated by drought and abscisic acid (ABA). Under drought conditions, the ZmNAC20-overexpressing plants had higher relative water content and survival rate than the wild-type maize inbred B104, suggesting that overexpression of ZmNAC20 improved drought resistance in maize. The detached leaves of ZmNAC20-overexpressing plants lost less water than those of wild-type B104 after dehydration. Overexpression of ZmNAC20 promoted stomatal closure in response to ABA. ZmNAC20 was localized in the nucleus and regulated the expression of many genes involved in drought stress response using RNA-Seq analysis. The study indicated that ZmNAC20 improved drought resistance by promoting stomatal closure and activating the expression of stress-responsible genes in maize. Our findings provide a valuable gene and new clues on improving crop drought resistance.

Keywords: ABA; ZmNAC20; drought resistance; stomatal closure; transcription factor.

Figure 1

Expression of ZmNAC20 in response to drought stress and ABA. (A) The relative expression level of ZmNAC20 in response to drought stress. (B) The relative expression level of ZmNAC20 after treatment with different concentrations of ABA. (C) The relative expression level of ZmNAC20 after treatment with 1 μM ABA for 0, 1, 2, and 3 h. (D) Relative expression of ZmNAC20 in ZmNAC20-overexpressing transgenic plants. Asterisks indicate significant differences: ** p < 0.01 (Student’s t-test).

Figure 2

Phenotype of ZmNAC20-overexpressing transgenic maize. (A) Drought resistance of transgenic maize overexpressing ZmNAC20. Images were taken before and after drought stress treatment and rewatering. WT represented the wild-type B104 maize and served as the control. ZmNAC20-OE1 and ZmNAC20-OE2 represented the two ZmNAC20-overexpressing maize lines. Bars, 5 cm. (B) Statistical data for the survival rate of WT, ZmNAC20-OE1, and ZmNAC20-OE2. The survival rate was obtained from 30 seedings in three independent tests. (C) The relative water content of maize leaves under drought stress treatment. The second leaves were cut from drought-treated plants. SE values were calculated from three biological replicates and more than 10 plants were examined in each replicate. (D) Fresh weight of maize leaves under drought stress treatment. SD values were calculated from three biological replicates and more than 10 plants were examined in each replicate. Asterisks indicate significant differences: ** p < 0.01 (Student’s t-test).

Figure 3

Stomatal aperture analyses of ZmNAC20-overexpressing maize. (A) Statistics of relative water loss in detached leaves of WT and ZmNAC20-OE1 after dehydration for indicated times. WT represents the wild-type B104 inbred maize. ZmNAC20-OE1 represents ZmNAC20-overexpressing transgenic maize. (B) Photograph of detached leaves after dehydration for 180 min. (C) Photograph of the stomatal aperture of detached leaves from WT and ZmNAC20-OE1 after ABA treatment. Bars, 10 μm (D) Statistics of the stomatal aperture of detached leaves after ABA treatment. Asterisks indicate significant differences, ** p < 0.01 (Student’s t-test).

Figure 4

Subcellular localization of ZmNAC20. 35S:H2B-mCherry is a nuclear localization marker. Either 35S:ZmNAC20-GFP or the empty vector 35S:GFP and 35S:H2B-mCherry were co-transfected into leaves of Nicotiana benthamiana. The fluorescence of leaves was imaged using a confocal microscope. The merged signals of GFP and mCherry showed yellow color, suggesting ZmNAC20 was localized in the nucleus. Bars, 10 μm.

Figure 5

ZmNAC20 regulates multiple signaling under drought stress by RNA-seq analyses. (A) Differential gene expression detected by DESeq2. A twofold change in gene expression levels between ZmNAC20-OE1 and wild-type B104 (WT) with a q-value (adjusted p-value) cutoff of 0.05 was considered as a differential expression. A total of 1361 genes were upregulated and 913 genes were downregulated in ZmNAC20-OE1. (B) GO analyses of upregulated genes in ZmNAC20-OE1. (C) KEGG analyses of upregulated genes in ZmNAC20-OE1.

Figure 6

(A–I) Expression of genes involved in drought stress response in ZmNAC20-overexpressing seedlings under drought stress. The seedlings were treated by dehydration for 3 h. SD values were calculated from three biological replicates Asterisks indicate significant differences: ** p < 0.01 (Student’s t-test), ns indicates no significance.

Figure 7

(A–I) Expression of genes involved in drought stress response by treatment with ABA. The wild-type B104 was treated with different concentrations of ABA for 1 h. SD values were calculated from three biological replicates. Asterisks indicate significant differences: ** p < 0.01 (Student’s t-test).