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, 9 (9), e1003790

Genome-wide Analysis of ZmDREB Genes and Their Association With Natural Variation in Drought Tolerance at Seedling Stage of Zea Mays L


Genome-wide Analysis of ZmDREB Genes and Their Association With Natural Variation in Drought Tolerance at Seedling Stage of Zea Mays L

Shengxue Liu et al. PLoS Genet.


The worldwide production of maize (Zea mays L.) is frequently impacted by water scarcity and as a result, increased drought tolerance is a priority target in maize breeding programs. While DREB transcription factors have been demonstrated to play a central role in desiccation tolerance, whether or not natural sequence variations in these genes are associated with the phenotypic variability of this trait is largely unknown. In the present study, eighteen ZmDREB genes present in the maize B73 genome were cloned and systematically analyzed to determine their phylogenetic relationship, synteny with rice, maize and sorghum genomes; pattern of drought-responsive gene expression, and protein transactivation activity. Importantly, the association between the nucleic acid variation of each ZmDREB gene with drought tolerance was evaluated using a diverse population of maize consisting of 368 varieties from tropical and temperate regions. A significant association between the genetic variation of ZmDREB2.7 and drought tolerance at seedling stage was identified. Further analysis found that the DNA polymorphisms in the promoter region of ZmDREB2.7, but not the protein coding region itself, was associated with different levels of drought tolerance among maize varieties, likely due to distinct patterns of gene expression in response to drought stress. In vitro, protein-DNA binding assay demonstrated that ZmDREB2.7 protein could specifically interact with the target DNA sequences. The transgenic Arabidopsis overexpressing ZmDREB2.7 displayed enhanced tolerance to drought stress. Moreover, a favorable allele of ZmDREB2.7, identified in the drought-tolerant maize varieties, was effective in imparting plant tolerance to drought stress. Based upon these findings, we conclude that natural variation in the promoter of ZmDREB2.7 contributes to maize drought tolerance, and that the gene and its favorable allele may be an important genetic resource for the genetic improvement of drought tolerance in maize.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Phylogenetic tree of canonical DREB1 and DREB2 genes in maize, rice, sorghum and Arabidopsis.
The phylogenetic tree was constructed based on the sequence alignments of sixty-six, full-length DREB genes from four species. The gene codes and names are illustrated in red for maize; black for rice; blue for sorghum; and green for Arabidopsis. The gene names used for AtDREBs, OsDREB1s and OsDREB2s were according to Sakuma et al., 2002 , Mao et al., 2012 , and Matsukura et al., 2010 . Genes contained within a blue-box were considered to be direct orthologous genes across species. Bootstrap values from 1,000 replicates were indicated at each node and the scale represents branch lengths.
Figure 2
Figure 2. Synteny of chromosomal segments containing ZmDREB genes among rice, sorghum and maize genomes.
The maize, sorghum, and rice genomes are abbreviated as Zm, Sb, Os, respectively. Homologous chromosome segments between the different genomes are linked by black dotted lines. Each DREB orthologous gene pair is connected by a red line. Yellow boxes indicate homologous regions between the maize and sorghum genomes while gray boxes identify homologous regions in the maize and rice genomes. The blue shaded regions indicate two segmental duplications in the maize genome, corresponding to one rice and/or sorghum segment.
Figure 3
Figure 3. Expression profiles of eighteen ZmDREB genes.
(A) A heat map illustrating levels of gene expression level of the 18 ZmDREB genes in fifteen different tissues from various developmental stages. Normalized gene expression values are shown in different colors that represent the levels of expression indicated by the scale bar. The gray color represents unavailable data. (B) Relative levels of gene expression of 18 ZmDREBs in maize B73 leaf and root tissue grown under normal and drought conditions. The ZmUbi-2 gene, which is constitutively expressed under a wide array of conditions, was used as an internal control. For leaf tissue, the collection time points, 5, 10, and 24 hours, reflected relative leaf water content (RLWC) of 70%, 60% and 58%, respectively. For the sampled root tissues, dehydration stress was applied to hydroponically cultured seedlings for 5, 10, and 24 hours, and RLWC was determined to be approximately 70%, 60% and 58% at the corresponding time points, respectively. Seedlings for the leaf studies were grown and drought stressed in soil (see Materials and Methods). Data represent the mean ± SD of three biological replicates. (T-test, *p<0.05, **p<0.01).
Figure 4
Figure 4. Transactivation activity assay and motif analysis of 18 ZmDREB proteins.
(A) Cultures of the AH109 yeast, transformed with a plasmid containing different ZmDREB genes, were diluted and inoculated on to a synthetic dropout (SD) media without tryptophan (SD/-T), without tryptophan and histidine (SD/-T-H), or without tryptophan, histidine, and adenine (SD/-T-H-A). The culture plates were amended with different concentrations of 3-aminotriazole (3-AT). Genes were classified into one of three groups (I, II, or III) representing high, medium, or low transactivation activity, respectively. Photos were taken 2 days after inoculation for the plates without 3-AT, and 5 days after inoculation for the plates with various concentrations of 3-AT. (B) Motif analysis of ZmDREB proteins. Conserved protein motifs were identified using the SALAD database ( Different motifs were numbered from 1 to 26 and genes labeled with the same number(s) indicate that the same motif(s) was present in the different ZmDREB proteins. The conserved AP2/ERF domain is highlighted in red. Motifs labeled in purple were specific to ZmDREB1 group proteins, except that Motif 3 was also identified in the ZmDREB2.1/2A protein.
Figure 5
Figure 5. Association analysis of genetic variation ZmDREB2.7 with maize drought tolerance.
(A) Association analysis of genetic variation ZmDREB2.7 with maize drought tolerance and the pattern of pairwise LD of DNA polymorphisms in the ZmDREB2.7 gene. A schematic diagram of the 2.1 kb genomic fragment, including 600-bp 5′-, 406-bp 3′-UTR, and the protein coding region are presented as the x-axis. The location of the initiation codon (ATG) is marked as “+1”. The P value is shown on a −log10 scale. The five significant polymorphisms in the 5′-UTR and four nonsynonymous variations in the coding region are connected to their locations on the gene diagram by solid lines. “*” indicates a strong LD (r2≥0.8) with these five polymorphisms. (B) Correlation analysis of survival rate with the relative expression level of ZmDREB2.7. Drought stress was applied to the maize seedlings after the RLWC was decreased from 98% (unstressed) to 70% (moderate drought) or 58% (severe drought).
Figure 6
Figure 6. DNA binding analysis of ZmDREB2.7 and drought stress tolerance of 35S:ZmDREB2.7 transgenic Arabidopsis plants.
(A) (a) Dosage-dependent binding of GST-ZmDREB2.7 to DRE1 and DRE2 elements. GST-ZmDREB2.1/2A was used as a positive control. (b) Competitive DNA binding assay of ZmDREB2.7 with DRE1 and DRE2 sequences. 10-, 100-, 500-fold excess amounts of the cold probe were used to compete for the binding of ZmDREB2.7 protein to the labeled probe. (c) The binding assay of GST-ZmDREB2.7 with a GCC-box. The sequences of the three kinds of DNA sequences are listed. Red letters indicate the core sequences. DNA-protein complexes are indicated by arrows; “*” indicates nonspecific bands; “FP” indicates unbound free probes. (B) (a) Drought tolerance of transgenic 35S:ZmDREB2.7 Arabidopsis plants. Photographs were taken both before and after the drought treatment followed by 6 days rewatering. Vector-transformed plants and ZmDREB2.7-OE9, ZmDREB2.7-OE17 and ZmDREB2.7-OE19 transgenic plants were compared. (b) RT-PCR analysis of transcript levels in the three lines of the 35S:ZmDREB2.7 transgenic plants. (c) Statistical analysis of survival rates after the drought-stress treatment. The average survival rates and standard errors were calculated from three independent experiments. Bars with asterisks indicate lines that had significantly higher survival rates than the vector-transformed plants (t-test, *p<0.05, **p<0.01).
Figure 7
Figure 7. The favorable allele of ZmDREB2.7 improves maize drought tolerance.
(A) Haplotypes of ZmDREB2.7 in CIMBL70, 91, 92, CML118, Shen5003 and B73 (as reference genome) maize genotypes. The site of the start codon (ATG) was designated as “+1”. SNP-503, SNP-260, SNP-150, InDel-185 and InDel-154 are the five DNA polymorphisms significantly associated with maize drought tolerance and are located in the 5′-UTR of ZmDREB2.7. The 20-bp InDel upstream of the ATG is in complete LD with the five polymorphisms in the four drought tolerant varieties. These polymorphisms are shaded in red. The location of PCR primers used for genotyping the InDel polymorphism of ZmDREB2.7 in drought tolerant (CIMBL70, 91, 92, and CML118) and drought sensitive (Shen5003) inbred lines are indicated by arrows. The four significant nonsynonymous polymorphisms in the coding region of InDel141, SNP142, SNP436 and SNP661 are shaded in blue. (B) Phenotypic response of CIMBL70, 91, 92, CML118 and Shen5003 to drought stress. The upper panel is a photo of plants growing under favorable water conditions while the lower panel plants re-watered for 6 days after the drought stress treatment was terminated. (C) The survival rate of CIMBL70, 91, 92, CML118 and Shen5003 plants exposed to moderate and severe drought stress. Data represent the mean of triplicates (t-test, *p<0.05, **p<0.01). (D) Relative level of ZmDREB2.7 expression in CIMBL70, 91, 92, CML118 and Shen5003 grown under normal and drought stress conditions. The drought-stress treatment reflected a decrease in RLWC from 98% (unstressed) to 70% (moderate drought), and 58% (severe drought). Data represent the mean of three biological replicates (t-test, **p<0.01). (E) The effect of the ZmDREB2.7 favorable allele on drought tolerance in four F2 segregating populations of maize. In each population, three distinct genotypes for ZmDREB2.7 were identified by DNA amplification: homozygous for the favorable allele, homozygous for the sensitive allele, and heterozygous for both alleles. The survival rate of the different genotypes was assessed and compared in the four populations. N indicates the number of F2 individuals tested in each population (t-test, *p<0.05, **p<0.01).

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Grant support

This research is supported by grants from the National Hi-Tech Research and Development Program of China (2012AA10A306-4), the National Basic Research Program of China (2012CB114302-4), the National Natural Science Foundation of China (31171163) and the Chinese Academy of Sciences (CAS) project KSCX2-YW-N-097. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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