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Natural polymorphism of ZmICE1 contributes to amino acid metabolism that impacts cold tolerance in maize

Nature Plants volume 8, pages 1176–1190 (2022)Cite this article

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Abstract

Cold stress negatively affects maize (Zea mays L.) growth, development and yield. Metabolic adjustments contribute to the adaptation of maize under cold stress. We show here that the transcription factor INDUCER OF CBF EXPRESSION 1 (ZmICE1) plays a prominent role in reprogramming amino acid metabolome and COLD-RESPONSIVE (COR) genes during cold stress in maize. Derivatives of amino acids glutamate/asparagine (Glu/Asn) induce a burst of mitochondrial reactive oxygen species, which suppress the cold-mediated induction of DEHYDRATION RESPONSE ELEMENT-BINDING PROTEIN 1 (ZmDREB1) genes and impair cold tolerance. ZmICE1 blocks this negative regulation of cold tolerance by directly repressing the expression of the key Glu/Asn biosynthesis genes, ASPARAGINE SYNTHETASEs. Moreover, ZmICE1 directly regulates the expression of DREB1s. Natural variation at the ZmICE1 promoter determines the binding affinity of the transcriptional activator ZmMYB39, a positive regulator of cold tolerance in maize, resulting in different degrees of ZmICE1 transcription and cold tolerance across inbred lines. This study thus unravels a mechanism of cold tolerance in maize and provides potential targets for engineering cold-tolerant varieties.

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Fig. 1: ZmICE1 positively regulates cold tolerance in maize.
Fig. 2: Transcriptome and metabolite analyses show the role of ZmICE1 in regulating amino acid metabolism.
Fig. 3: ZmICE1 directly regulates the expression of genes involved in nitrogen metabolism.
Fig. 4: The Glu–Asn metabolic pathway determines cold tolerance by triggering MtROS.
Fig. 5: Variation in ZmICE1 is associated with free amino acid contents and cold tolerance.
Fig. 6: ZmMYB39 positively regulates maize cold tolerance by directly targeting ZmICE1.
Fig. 7: Proposed model of ZmICE1-mediated amino acid metabolism and cold tolerance in maize.

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Data availability

The mass spectrometry raw data have been released in the Metabolights database (https://www.ebi.ac.uk/metabolights/MTBLS4404). Data sets including RNA-seq and ChIP-seq and detailed information can be viewed and downloaded from http://www.ncbi.nlm.nih.gov/sra/ under accession PRJNA779257. Source data are provided with this paper.

Code availability

The custom codes used in this study are deposited in GitHub (https://github.com/fudiyi/code-for-publication).

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Acknowledgements

We thank J Zuo, F Qin and C Jiang for helpful discussion. The transgenic seeds of maize were created by the Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University. This work was supported by grants from the National Key Research and Development Project (2020YFA0509902), the National Natural Science Foundation of China (32022008, 31700214, 31921001) and the National Science Foundation of Tianjin (19JCYBJC29500).

Author information

Author notes

  1. These authors contributed equally: Haifang Jiang, Yiting Shi, Jingyan Liu, Zhen Li.

Authors and Affiliations

  1. State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing, China

    Haifang Jiang, Yiting Shi, Jingyan Liu, Zhen Li, Diyi Fu, Shifeng Wu, Minze Li, Zijia Yang, Yunlu Shi, Jinsheng Lai, Xiaohong Yang, Zhizhong Gong & Shuhua Yang

  2. Fresh Corn Research Center of BTH, Tianjin Agricultural University, Tianjin, China

    Jingyan Liu

  3. National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing, China

    Jinsheng Lai & Xiaohong Yang

  4. Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, USA

    Jian Hua

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  1. Haifang Jiang
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Contributions

S.Y. conceived the project. Y.T.S. and S.Y. designed the experiments. H.J., Y.T.S. and J.Y.L. performed most of the experiments. Z.L. and Z.Y. performed amino acid measurement. Y.L.S. performed the maize transformation. X.Y., D.F., S.W. and M.L. performed GWAS, RNA-seq and ChIP-seq analyses. X.Y. and J.S.L. provided the inbred population. All authors analysed the data. Y.T.S., J.H. and S.Y. wrote the manuscript with comments from all authors.

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Correspondence to Shuhua Yang.

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Nature Plants thanks Baoxing Song and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Analysis of metabolic profiling under cold stress in maize young seedlings.

a, Heatmap displaying the log10 value of differentially accumulated metabolites before or after cold treatment (4 °C, 12 h) in maize varieties. b, Metabolic pathway analysis plot created using MetaboAnalyst 4.0. Plots depict several metabolic pathway alterations induced by cold treatment. The x-axis represents the pathway impact value computed from pathway topological analysis, and the y-axis is the -log10 of the P-value obtained from pathway enrichment analysis. The pathways that were most substantially changed are characterized by both a high -log(P) value and high impact value (top right region). c, Distribution of mGWAS signals across the maize genome under warm temperature.

Extended Data Fig. 2 Characterization of ZmICE1-overexpression transgenic plants and zmice1 mutants.

a, Schematics of mutations in the coding region of ZmICE1. CRISPR/Cas9 technology was used to generate two mutant alleles, zmice1-1 (deleting 19 bp) and zmice1-2 (deleting 44 bp). Dots indicate deleted bases. b, The calculation method of relative injury percentage of the maize seedlings after cold treatment. c-d, Cold phenotypes (c) and leaf injury (d) of transgenic lines of zmice1-1×zmice1-2 (F1 plants generated by crossing zmice1-1 with zmice1-2). V2 seedlings were treated with or without at 4 °C for 4 d. Shown in (d) are means ± s.d. from 3 biological replicates (n = 5 plants for each replicate). P-values are from two-sided t-test. e, Growth phenotype of the zmice1-1 mutant. WT and zmice1-1 were grown at green house for 4 weeks. f, Relative expression levels of ZmICE1 in WT and ZmICE1-overexpression seedlings. Total RNAs were extracted from 12-day-old seedlings. g, ZmICE1 gene expression level in various tissues. h, ZmICE1 expression in V2-stage maize seedlings under cold stress. Data are means ± s.d. from 3 biologically independent samples. P-values shown are from two-sided t-test. i, Subcellular localization of ZmICE1-GFP in maize protoplasts and ZmICE1-GFP plants. ZmICE1-GFP was transformed into maize protoplasts, and GFP was used as a control. The roots of ZmICE1-GFP plants were treated with or without 4 °C. GFP signals were visualized by laser confocal-scanning microscope Leica sp5. Scale bars, 10 μm. j, Immunoblotting analysis of ZmICE1-GFP nuclear protein in maize protoplasts with or without 4 °C treatment. ZmICE1 was detected with anti-GFP antibody (1:3000). Anti-H3 antibody (1:10000) was used as a loading control. Relative binding intensity was quantified with ImageJ software. Data in f, g and h are mean values ± s.d. from 3 biologically independent samples; P-values are from two-sided t-test. The experiments in f, g, h, i and j were independently performed three times with similar results.

Source data

Extended Data Fig. 3 Identification of direct targets of ZmICE1 by RNA-seq and Chip-seq.

a, Functional classification of putative ZmICE1 target genes using gene ontology (GO) annotations. The color scale represents the −Log10 of false discovery rate (FDR) values. b, Distributions of ZmICE1 binding sites for COR genes-associated loci, as shown in Integrated Genome Browser. Blue lines (WT) or red lines (ZmICE1-GFP) indicate notable peaks calculated by MACS2. The bottom track indicates the gene’s direction on the chromosome. Blue bars indicate peaks of ZmICE1. c, Transcript abundance of COR genes in wild type and zmice1-1 in FPKM. d, Expression of ZmDREB1s in WT and zmice1 mutants treated at 4 °C for 0 h and 12 h. Data are means ± s.d. from 3 biologically independent samples; P-values shown are from two-sided t-test. The experiments were independently performed three times with similar results.

Source data

Extended Data Fig. 4 ZmICE1 participates in the regulation of amino acid metabolism pathway.

a, The GO terms and KEGG pathway enrichment analysis of ZmICE1-regulated metabolism under normal condition. Cutoff value is FDR < 0.05. b, Analysis of free amino acid contents in the leaves of V2-stage young WT and zmice1-2 seedlings with or without cold treatment at 4 °C for 12 h. Data are means ± s.d. from 4 biologically independent samples. Different letters represent differences (P < 0.05) determined by one-way ANOVA with Tukey’s multiple comparisons test. The experiment were independently performed twice with similar results.

Source data

Extended Data Fig. 5 The effect of cold stress on the content of endogenous amino acids in maize.

a, The -log10 (peak area) value of free amino acids evaluated by LC-MS/MS before or after cold treatment (4 °C, 12 h) in maize association panel. The plots shown are means ± s.d. (n is shown in figures; two-sided t-test). b, Relative expression of ZmAS3 and ZmGS1-3 in WT, ZmAS3- and ZmGS1-3-overexpression seedlings. Data are means ± s.d. from 3 biologically independent samples. P-values shown are from two-sided t-test. The experiments were independently performed three times with similar results. c, Relative leaf injury of cold-treated seedlings without or with application of Leu or Val. Maize seedlings at V2 stage were treated with 20 mM Leu or Val for 12 h, followed by 4 °C treatment for 2 d. Data are means ± s.d. from 3 biological replicates (n = 5 plants for each replicate). P-values are from two-sided t-test. d, Relative amount of endogenous amino acids in maize seedlings with exogenously applied Asn and Glu at 20 mM. Data are means ± s.d. from 4 biologically independent samples. P-values shown are from two-sided t-test.

Source data

Extended Data Fig. 6 SNP sites associated with amino acid content and evolution of haplotypes of maize inbred lines.

a, Quantile-quantile (Q–Q) plots with -log10 P-values of Thr, Asn, Lys and Ser, 95% confidence interval (blue shading) for linear regression. b, Association plot for Ser content is shown for the region at 156.0–158.0 Mb on chromosome 3 (x axis). Negative log10-transformed P values from the compressed mixed linear model are plotted on the y axis. The horizontal dashed line indicates the genome-wide significance threshold. The locations of the predicted open reading frame in B73 genome are indicated by color boxes. The 9 annotated genes associated with Ser content are listed in the left panel. c, Conditional association analysis. Top is the non-conditioned plot and the bottom is the association plot after conditioning on the lead SNP. d, Phylogenetic tree of inbred lines. All SNPS in the ZmICE1 gene were used to construct the maximum-likelihood tree by MEGA5.0. Bootstrap value is indicated on the tree. The HapA inbred lines (12 total) were highlighted in red. Others (177 lines) are HapB. e, Violin plot of expression levels of ZmDREB1s in maize inbred lines of HapA and HapB. Shown are the log2 of FPKM values from RNA-seq data in developing kernels. P-values are from two-sided student t-test.

Source data

Extended Data Fig. 7 ROS burst in zmice1 mutant, ZmAS3-OE and ZmGS1-3-OE with and without cold treatment.

a-c, Histochemical staining by NBT (nitroblue tetrazolium) and DAB (3′,3′-diaminobenzidine) of leaves of WT, zmice1-1 (a), ZmAS3-OE (b), and ZmGS1-3-OE (c) seedlings with or without cold treatment for 12 h. Representative images of leaf histochemical staining are shown from three independent experiments. Scale bars, 1 mm.

Extended Data Fig. 8 Co-expression analysis identifies ZmMYB39 as a positive regulator of ZmICE1.

a, Transient dual-LUC expression assay in maize protoplasts. The LUC reporter was driven by 1.5-kb ZmICE1 promoter fragments from inbred lines SK (HapA) and two mutated forms (substitution of the nucleotide at position −576 from T to A or C. Data are means ± s.d. from 4 biological replicates. P-values shown are from two-sided t-test. b, Alignment of ZmICE1 promoter sequences from Zheng58 and SK. The positions of SNP were labelled with blue arrows. MRE indicates MYB Recognition Element (AACCTAA). c, Interaction network of ZmICE1-related genes in maize. d, The maximum-likelihood tree based on protein sequences of ZmMYB39 homologues in plant species. e, Expression of ZmMYB39, ZmMYB41 and ZmICE1 in WT under cold stress. f-g, Expression of ZmMYB39 and ZmMYB41 in WT and overexpression seedlings. h, Cumulative germination rates of ZmMYB39-OE seeds when subjected to cold stress. Data are means ± s.d. from 3 biological replicates (n = 32/34/35 seeds in in each independent experiment; * P < 0.05, two-sided t-test). i, Leaf injury of ZmMYB41-OE plants. Data are means ± s.d. from 3 biological replicates (n = 5 plants for each replicate). The P-values are from two-sided t-test. j, Expression of ZmICE1 in WT and ZmMYB41-OE lines. k, Schematics of mutation in the coding region of ZmMYB39 generated by CRISPR/Cas9. (l-m) Cold tolerance phenotype (l) and leaf injury percentage (m) of zmmyb39. Shown in (m) are means ± s.d. from 3 biological replicates (n = 5 plants for each replicate). P-values are from two-sided t-test. (n) EMSA showing the association of ZmMYB39 with the ZmICE1 promoter. The intensity of the shifted band was quantified by ImageJ software, and the relative intensity is shown below the quantified band. Three independent experiments were done with the similar results. In e, f, g and j, data are means ± s.d. from 3 biologically independent samples. The P-values shown are from two-sided t-test. These experiments were independently performed three times with similar results.

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Extended Data Fig. 9 Kernel phenotypes of ZmICE1-overexpression lines.

Maize plants were grown under optimal nutrition conditions and harvested at maturity in three locations. 5 biological replicates were performed with similar results. n is shown in figures, The P-values shown are from two-sided t-test.

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Extended Data Fig. 10 Effect of Glu and Arg on cold tolerance differ in plant species.

a, Freezing phenotypes of 12-day-old Arabidopsis seedlings treated with amino acids. The seedlings were grown under a 16-h-white light/8-h-dark photoperiod at 22 °C and then sprayed with 20 mM amino acids for 12 h before being exposed to freezing treatment. At least three plates were treated in each treatment. Representative photographs were taken after a 3-day recovery period. b, Cold tolerance phenotypes in different species with exogenous application of 20 mM Glu. c, Expression of DREB1B and DREB1A genes in Arabidopsis treated at 4 °C for 3 h. 2-week-old plants grown on 1/2 MS plates at 22 °C were sprayed with 20 mM Glu and Arg for 6 h followed by a 4 °C treatment of 3 h. The qPCR data were normalized to the mock. Data are means ± s.d. from 3 biologically independent samples. P-values shown are from two-sided t-test. ACTIN gene was used as internal reference. The experiments were independently performed three times with similar results. d, Relative amount of endogenous amino acids in Arabidopsis seedlings after sprayed with 20 mM Glu. The 12-day-old plants grown on 1/2 MS plates at 22 °C were sprayed with 20 mM Glu. Materials were collected at 12 h after spray to detected free amino acid content. Data are means ± s.d. from 3 biologically independent samples. P-values shown are from two-sided t-test. Experiments were independently performed twice with similar results. (e) Difference of Glu metabolic pathway in Arabidopsis and maize.

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Supplementary information

Reporting Summary

Supplementary Table

List of primer sequences used in this study.

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Jiang, H., Shi, Y., Liu, J. et al. Natural polymorphism of ZmICE1 contributes to amino acid metabolism that impacts cold tolerance in maize. Nat. Plants 8, 1176–1190 (2022). https://doi.org/10.1038/s41477-022-01254-3

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