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ZmICE1a regulates the defence–storage trade-off in maize endosperm

Nature Plants volume 10, pages 1999–2013 (2024)Cite this article

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Abstract

The endosperm of cereal grains feeds the entire world as a major food supply; however, little is known about its defence response during endosperm development. The Inducer of CBF Expression 1 (ICE1) is a well-known regulator of cold tolerance in plants. ICE1 has a monocot-specific homologue that is preferentially expressed in cereal endosperms but with an unclear regulatory function. Here we characterized the function of monocot-specific ZmICE1a, which is expressed in the entire endosperm, with a predominant expression in its peripheral regions, including the aleurone layer, subaleurone layer and basal endosperm transfer layer in maize (Zea mays). Loss of function of ZmICE1a reduced starch content and kernel weight. RNA sequencing and CUT&Tag-seq analyses revealed that ZmICE1a positively regulates genes in starch synthesis while negatively regulating genes in aleurone layer-specific defence and the synthesis of indole-3-acetic acid and jasmonic acid (JA). Exogenous indole-3-acetic acid and JA both induce the expression of numerous defence genes, which show distinct spatial-specific expression in the basal endosperm transfer layer and subaleurone layer, respectively. Moreover, we dissected a JA–ZmJAZ9–ZmICE1a–MPI signalling axis involved in JA-mediated defence regulation. Overall, our study revealed ZmICE1a as a key regulator of endosperm defence response and a coordinator of the defence–storage trade-off in endosperm development.

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Fig. 1: Loss of function of monocot-specific ZmICE1a reduces kernel weight.
Fig. 2: zmice1a down-regulates starch synthesis genes while up-regulating defence genes in the peripheral endosperm and phytohormone synthesis genes.
Fig. 3: ZmICE1a positively regulates starch synthesis while negatively regulating defence response and phytohormone synthesis.
Fig. 4: ZmICE1a indirectly regulates defence genes in BETL through IAA.
Fig. 5: ZmICE1a indirectly regulates defence genes in SAL through JA.
Fig. 6: The JA–ZmJAZ9–ZmICE1a–MPI signalling axis regulates defence response in AL and SAL.

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Data availability

RNA-seq and CUT&Tag-seq data are available from the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the series entries GSE254177 and GSE254066, respectively. In addition, the internal control gene UBIQUITIN for gene expression normalization is available under GenBank accession number BT018032. Source data are provided with this paper.

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Acknowledgements

We thank L. Jin and T. Zhu (Shanghai University) for their technical support. We thank Q. Xu and Y. Lu (Shandong Agricultural University) for the measurement of endogenous JA. We thank J. Chu and J. Yan (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for the measurement of endogenous IAA. This study was financially supported by the National Natural Science Foundation of China (32301846 to Q.W.), the Science and Technology Innovation 2030-Major Project (2023ZD0403005 to R.S.) and the National Key Research and Development Program of China (2023YFF1000400 to Z.M.).

Author information

Authors and Affiliations

  1. State Key Laboratory of Maize Bio-breeding, Frontiers Science Center for Molecular Design Breeding, Joint International Research Laboratory of Crop Molecular Breeding, National Maize Improvement Center, College of Agronomy and Biotechnology, China Agricultural University, Beijing, China

    Qun Wang, Kechun Zhang, Yonghui He, Zeyang Ma & Rentao Song

  2. Sanya Institute of China Agricultural University, Sanya, China

    Qun Wang, Zeyang Ma & Rentao Song

  3. Hainan Yazhou Bay Seed Laboratory, Sanya, China

    Qun Wang, Zeyang Ma & Rentao Song

  4. Shanghai Key Laboratory of Bio-Energy Crops, Plant Science Center, School of Life Sciences, Shanghai University, Shanghai, China

    Fan Feng & Weiwei Qi

  5. Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Jiangsu Key Laboratory of Crop Genetics and Physiology, Agricultural College of Yangzhou University, Yangzhou, China

    Yonghui He

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  1. Qun Wang
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  2. Fan Feng
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  3. Kechun Zhang
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  4. Yonghui He
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  5. Weiwei Qi
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  6. Zeyang Ma
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  7. Rentao Song
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Contributions

R.S. conceived the project. R.S., Q.W. and F.F. designed the experiments. Q.W., F.F., K.Z. and Y.H. performed the experiments. All authors analysed the data. Q.W., Z.M. and R.S. wrote the paper.

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Correspondence to Zeyang Ma or Rentao Song.

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Extended data

Extended Data Fig. 1 Transcription factor features and expression analysis of ZmICE1a.

a, Subcellular localization of ZmICE1a. Fluorescent signals from the expression of ZmICE1a-GFP and GFP alone (negative control) in the protoplasts of maize leaves. Scale bars, 100 μm. Experiments were repeated three times yielding similar results. b, Yeast transactivation assay of ZmICE1a. The β-galactosidase activity resulting from TF transactivation was measured in the yeast. The pGBK-T7 vector alone (BD) was used as a negative control. BD-O2 was used as a positive control. Data are mean ± s.e.m. (n = 3 biologically independent samples). ****, P < 0.0001; two-tailed Student’s t-test; for exact P values, see Source Data. c, d, RNA expression pattern of ZmICE1a in various tissues and developing kernels (c), and the three-components of kernels (d). Data are mean ± s.e.m. (n = 3 biologically independent samples). Per, pericarp; En, endosperm; Em, embryo.

Source data

Extended Data Fig. 2 Biochemical analysis in mutants with loss-of-function of ZmICE1a.

a, Kernel weight of mature WT and zmice1a-2# kernels from a segregated F2 ear. n = 55 kernels in WT, n = 15 kernels in zmice1a-2#. The middle line of the box represents the median, while the lower and upper bounds represent the 25th and 75th percentiles, respectively. The whiskers extend from the minimum to the maximum values. *, P < 0.05; two-sided Wilcoxon Rank Sum test (P = 0.0347). b, Protein content of mature WT and zmice1a kernels. Data are mean ± s.e.m. (n = 3 biologically independent samples). ns, not significant; two-tailed Student’s t-test (P = 0.199). c, The ratio of zein to non-zein in mature WT and zmice1a kernels. Data are mean ± s.e.m. (n = 4 biologically independent samples). ns, not significant; two-tailed Student’s t-test (P = 0.817).

Source data

Extended Data Fig. 3 Phenotypic analysis of zmice1a/1b kernels.

a, Expression analysis of ZmICE1 genes in developing endosperms. Data are mean ± s.e.m. (n = 3 biologically independent samples). For ZmICE1c, one Ct value was not detected in the 12 DAP and 21 DAP endosperm samples. b, Diagram of the CRISPR/Cas9 editing target sites in exon 1 of ZmICE1b. Red arrowheads indicate two editing target sites. The 20-bp targets are highlighted in red. PAM represents the protospacer adjacent motif. Deleted nucleotides are depicted as dashes, and inserted nucleotides are highlighted in blue. c, Mature kernels of WT, zmice1a, zmice1b, and zmice1a /1b from a segregated F2 ear. Scale bars, 1 cm. d, Kernel weight of WT, zmice1a, zmice1b, and zmice1a/1b mature kernels from two segregated F2 ears. The middle line of the box represents the median, while the lower and upper bounds represent the 25th and 75th percentiles, respectively. The whiskers extend from the minimum to the maximum values. Different letters denote significant differences (P < 0.05, one-way ANOVA, Tukey’s HSD test); for exact P values, see Source Data.

Source data

Extended Data Fig. 4 RNA-seq analysis of zmice1a endosperms.

a, RT-qPCR analysis of several representative DEGs in the WT and zmice1a endosperms. Data are mean ± s.e.m. (n = 3 biologically independent samples). **, P < 0.01; *, P < 0.05; two-tailed Student’s t-test; for exact P values, see Source Data. b, Heatmap depicting the log2 (fold change) of representative BETL and AL marker genes involved in defence response in the zmice1a endosperms. c, Expression levels of BETL and AL marker genes (SWEET4c and VPP7) in the WT and zmice1a endosperms. Data are mean ± s.e.m. (n = 3 biologically independent samples). ns, not significant; two-tailed Student’s t-test; for exact P values, see Source Data. d, BETL and AL marker genes used in (b) and (c) were functionally annotated. The genes with changed expression levels in the zmice1a endosperms were highlighted in red. e, f, Pathways of IAA synthesis (e) and JA synthesis (f). The expression of enzyme-encoding genes affected by the zmice1a mutation is highlighted in the red boxes.

Source data

Extended Data Fig. 5 CUT&Tag-seq analysis of ZmICE1a.

a, Peak number in two replicates of CUT&Tag-seq data. b, Distribution of ZmICE1a binding region peaks (peak count frequency) corresponding to ± 3000-bp regions flanking the TSS. c, ZmICE1a binding motif identified by MEME-ChIP in the 1000 bp flanking sequences around the genic peak summits. d to f, Peak distribution of ZmICE1a-binding sites for CBF genes (d), COR genes (e), and SBT5.3 (f), visualized using IGV. Rep1 and rep2 indicate two replicates of CUT&Tag-seq data. g, Expression levels of SBT5.3 in maize kernel. Data were collected from the Fu et al. gene expression dataset75. AL, aleurone; BETL, basal endosperm transfer layer; CZ, conducting zone; EAS, endosperm adjacent to scutellum; ESR, embryo-surrounding region; PC, placento-chalazal; PE, pericarp; EM, embryo; SCU, scutellum; SE, starchy endosperm; VE, vitreous endosperm.

Source data

Extended Data Fig. 6 ZmICE1a regulates starch synthesis of maize endosperms.

a, Schematic diagram shows constructs used in the dual-LUC transient transcriptional activity assays. b, Transcriptional activities of ZmICE1a on the promoters of two key regulator of starch synthesis. Data are mean ± s.e.m. (n = 3 biologically independent samples). **, P < 0.01; two-tailed Student’s t-test; for exact P values, see Source Data. c, d, Resin section (c) and TEM (d) observations of starch granules (SGs) in 15 DAP WT and zmice1a endosperms. AL, aleurone layer. Scale bars, 100 μm (c) and 10 μm (d). Experiments were repeated three times yielding similar results.

Source data

Extended Data Fig. 7 Def1 and Def2 effectively inhibit the germination and growth of F. verticillioides.

a, Western blot analysis of purified negative control protein (empty vector), Def1, and Def2. Experiments were repeated three times yielding similar results. b, Microscopic observations of F. verticillioides treated with Def1 and Def2. Fv, F. verticillioides. Scale bars, 200 μm. Experiments were repeated three times yielding similar results.

Source data

Extended Data Fig. 8 Exogenous phytohormone treatment represses the expression of starch synthesis genes in maize endosperms.

a, Heatmap depicting the log2 (fold change) of starch synthesis genes in the IAA-treated endosperms. b, Expression of GBSSI in the JA-treated endosperms. Data from RNA-seq of JA-treated endosperms are presented as mean ± s.e.m. (n = 3 biologically independent samples). **, P < 0.01; two-tailed Student’s t-test (P = 0.0021).

Source data

Extended Data Fig. 9 LCI assay confirms the interactions between ZmICE1a and candidate proteins identified by CoIP-MS.

Interaction assays of ZmICE1a and candidate proteins such as ZmICE1b (a), ZmbHLH99 (b), ZmWRI1 (c), and ZmCKB4 (d). The lower-right section shows the interaction signal between ZmICE1a and candidate proteins, while the other three sections show the signals of the negative controls. The fluorescent signal intensity represents their interaction activities.

Extended Data Fig. 10 RNA expression patterns of rice homologs of ZmICE1a and its key targets in the various rice tissues.

Expression of rice homologues of ZmICE1a (a), ZmDef2 (b), ZmSSI (c), ZmSSIIa (d), ZmACX3 (e), and ZmTAR1 (f). Data are from the Yu et al. gene expression dataset76, and the corresponding bar graphs were obtained from the website (http://ipf.sustech.edu.cn/pub/ricerna/). F. reprod. tissue, female reproductive tissue; M. reprod. tissue, male reproductive tissue.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

41477_2024_1845_MOESM3_ESM.xlsx

Supplementary Table 1 DEGs of WT and zmice1a endosperms at 15 DAP. Supplementary Table 2 ZmICE1a binding genes identified by CUT&Tag-seq generated from two biological replicates. Supplementary Table 3 High-confidence targets of ZmICE1a. Supplementary Table 4 DEGs of IAA-untreated and IAA-treated WT endosperms. Supplementary Table 5 DEGs of JA-untreated and JA-treated WT endosperms. Supplementary Table 6 Potential interacting proteins of ZmICE1a. Supplementary Table 7 ZmICE1a-regulated JA-responsive genes. Supplementary Table 8 Primers used in this study.

Source data

Source Data Figs. 1, 3 and 6 and Source Data Extended Data Figs. 1–6 and 8

Statistical source data.

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 7

Unprocessed western blots.

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Wang, Q., Feng, F., Zhang, K. et al. ZmICE1a regulates the defence–storage trade-off in maize endosperm. Nat. Plants 10, 1999–2013 (2024). https://doi.org/10.1038/s41477-024-01845-2

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