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Genetic variation in ZmVPP1 contributes to drought tolerance in maize seedlings

Abstract

Maize production is threatened by drought stress worldwide. Identification of the genetic components underlying drought tolerance in maize is of great importance. Here we report a genome-wide association study (GWAS) of maize drought tolerance at the seedling stage that identified 83 genetic variants, which were resolved to 42 candidate genes. The peak GWAS signal showed that the natural variation in ZmVPP1, encoding a vacuolar-type H+ pyrophosphatase, contributes most significantly to the trait. Further analysis showed that a 366-bp insertion in the promoter, containing three MYB cis elements, confers drought-inducible expression of ZmVPP1 in drought-tolerant genotypes. Transgenic maize with enhanced ZmVPP1 expression exhibits improved drought tolerance that is most likely due to enhanced photosynthetic efficiency and root development. Taken together, this information provides important genetic insights into the natural variation of maize drought tolerance. The identified loci or genes can serve as direct targets for both genetic engineering and selection for maize trait improvement.

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Figure 1: GWAS for drought tolerance in maize seedlings.
Figure 2: Natural variations in ZmVPP1 were significantly associated with maize drought tolerance.
Figure 3: Functional characterization of ZmVPP1 as a H+-PPase and comparison of ZmVPP1B73 and ZmVPP1CIMBL55 protein function in Arabidopsis.
Figure 4: The 366-bp insertion (indel −379) in the ZmVPP1CIMBL55 allele confers stress-inducible expression of ZmVPP1.
Figure 5: The ZmVPP1 tolerant allele improves drought tolerance in maize seedlings.
Figure 6: Drought tolerance of ZmVPP1 transgenic maize.
Figure 7: Photosynthetic capacity and yield performance of transgenic maize.

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Acknowledgements

The authors would like to thank T. Komori (Japan Tobacco, Inc.) for kindly providing us with the maize transformation plasmids pSBI and pSBII. Y. Ma helped in maize transformation vector construction. We would also like to thank S. Li and Z. Li for their excellent technical support in maize transformation and seed propagation. This research was supported by grants from the National High-Tech Research and Development Program of China (2012AA10A306-4), the National Basic Research Program of China (2012CB114302-4), Chinese Academy of Sciences grant (XDA08010206), and the National Natural Science Foundation of China (31471505) to F.Q.

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Authors

Contributions

X.W. resequenced ZmVPP1, analyzed gene and protein expression levels, and performed Arabidopsis transformation and transgenic analysis and the yield test of transgenic maize in fields. H.W. carried out the GWAS of maize drought tolerance and identified the ZmVPP1 gene, and analyzed the phenotype of transgenic maize in the lab. S.L. helped with the phenotypic analysis of maize drought tolerance. X.Y., J.Y., and J.L. provided the maize materials and the SNP information, and X.Y. advised on the experiments. A.F. provided the fugu5 mutant seeds and advised on the experiments. F.Q. designed and advised on the experiments and wrote the manuscript.

Corresponding authors

Correspondence to Xiaohong Yang or Feng Qin.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Phenotypic variation of maize drought tolerance at the seedling stage in the natural variation population.

(a) Distribution of plant survival rate in a drought test (SR) of genotypes from different origins. Temperate origins contain non-stiff stalk (NSS) and stiff stalk (SS). (b) Box-plot of the SR of genotypes from different origins. The solid lines in the boxes denotes the median value; dashed lines indicate variability outside the upper and lower quartiles; and dots denote outliers.

Supplementary Figure 2 Analysis of conditional GWAS and two sub-population GWAS.

Manhattan plot of the conditional GWAS (a), GWAS of TST (c) and NSS (e) subpopulations. The dashed horizontal line depicts the significance threshold (P = 1.0 × 10-5). The SNPs locating within the candidate genes, identified by the GWAS, are labeled in red dots. The X-axis indicates the SNP location along the 10 chromosomes, with chromosomes separated by different colors; Y-axis is the -log10(Pobserved) for each analysis. Quantile-quantile plot of conditional GWAS (b), GWAS of TST (d) and NSS (f) subpopulations under GLM (black dots) and MLM (red dots).

Supplementary Figure 3 The BAC sequence of ZmVPP1CIMBL55 and its synteny with the B73 genomic sequence.

Red and blue arrows represent the generic region of ZmVPP1 and GRMZM2G105167 (the gene upstream from ZmVPP1), respectively. Homologous regions are indicated by numbered boxes. Transposons are indicated by empty arrows. Sequence synteny is indicated by light-blue shading.

Supplementary Figure 4 Comparison of the 3'-UTR of ZmVPP1B73 and ZmVPP1CIMBL55 on GFP expression.

The GFP induction rate under ABA and mannitol treatments. “CK” indicates that the transfected protoplasts were cultured under normal conditions. The GFP expression levels in the samples transfected by the empty vector “GFP-NosT” were defined as 1. pUbi:Luciferase is co-transfected and acts as a reference gene. The means and errors (s.d.) were calculated from at least three biological replicates.

Supplementary Figure 5 Investigation of the performance of ZmVPP1 transgenics under field conditions.

(a) Comparison of plant height, leaf number, leaf length, leaf width, leaf numbers above the ear (LAE), days to anthesis (DTA), days to silking (DTS), tassel length, tassel branch number (TBN), ear length, row number, kernel per row between WT and ZmVPP1 transgenic maize under watered conditions. Leaf length and width were measured on the ear leaf and data were obtained from at least 30 plants of each kind. (b) Representative photo of ears for each kind of plant. (c) Photosynthesis parameters (PS, SC, TR and WUE) were measured and calculated under the drought-2 conditions. Data were obtained from 8 seedlings. Error bars represent the s.d. and significance was derived from a two-sided t-test, * P < 0.05, ** P < 0.01.

Supplementary Figure 6 Phylogenetic tree of ZmVPP1 from different plant species and gene expression profile of the paralogous ZmVPP1 gene.

(a) The abbreviation in the gene code or name of “Zm” stands for Zea mays, “Os” for Oryza sativa; “Sb” for Sorghum bicolor; “At” for Arabidopsis thaliana; “Vr” for Vigna radiate; and “Th” for Thellungiella halophila. Bootstrap values from 1,000 replicates were indicated at each node and the scale represents branch length. (b) A heat map illustrating levels of type-I ZmVPP gene expression in fifteen different tissues from various developmental stages. Normalized gene expression values (Plant J. 66, 553–563, 2011) are shown in different colors that represent the levels of expression as indicated by the scale bar.

Supplementary Figure 7 The original photo of western-blot analysis of the transgenic maize with increased ZmVPP1 protein levels as detected by ZmVPP1 anti-serum.

WT denotes the transgenic-negative siblings; “others” denotes a failed sample; “OE” denotes the independent transgenic lines with enhanced ZmVPP1 expression. CBB staining indicates equivalent loading of samples.

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Wang, X., Wang, H., Liu, S. et al. Genetic variation in ZmVPP1 contributes to drought tolerance in maize seedlings. Nat Genet 48, 1233–1241 (2016). https://doi.org/10.1038/ng.3636

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