The miR164-dependent regulatory pathway in developing maize seed

  • Lanjie Zheng
  • Xiangge Zhang
  • Haojun Zhang
  • Yong Gu
  • Xinrong Huang
  • Huanhuan Huang
  • Hanmei Liu
  • Junjie Zhang
  • Yufeng Hu
  • Yangping Li
  • Guowu Yu
  • Yinghong Liu
  • Shaneka S. Lawson
  • Yubi HuangEmail author
Original Article


MicroRNA164 (miR164) plays a key role in leaf and flower development, lateral root initiation, and stress responses. However, little is known about the regulatory roles of miR164 during seed development, particularly in maize. The aim of this study was to discover the developmental function of miR164 in maize seed. Small RNA sequencing (sRNA-seq) was performed at two key stages. The results indicated that miR164 was down-regulated during maize seed development. In addition, degradome library sequencing and transient expression assays identified the target genes for miR164. Two microRNA (miRNA) pairs, miR164-NAM, ATAF, and CUC32 (NAC32) and miR164-NAC40, were isolated. The developmental function of miR164 was determined by analyzing the differentially expressed genes (DEGs) between the wild-type and miR164 transgenic lines using RNA sequencing (RNA-seq) and by screening the DEGs related to NAC32 and NAC40 via co-expression and transient expression analysis. These results identified two beta-expansin genes, EXPB14 and EXPB15, which were located downstream of the NAC32 and NAC40 genes. This study revealed, for the first time, a miR164-dependent regulatory pathway, miR164-NAC32/NAC40-EXPB14/EXPB15, which participates in maize seed expansion. These findings highlight the significance of miR164 in maize seed development, and can be used to explore the role of miRNA in seed development.


Maize Seed development MiR64 NAC transcription factor Regulatory pathway 



We are grateful to Biomarker NovelBio, Shanghai, China for providing technical support.


This work was financially supported by the National Natural Science Foundation of China (no. 31571684).

Compliance with ethical standards

Conflict of interest

All authors declare no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

438_2018_1524_MOESM1_ESM.tif (15.3 mb)
Fig. S1. Diagram of modified binary vector pCambia1301 (TIF 15659 KB)
438_2018_1524_MOESM2_ESM.tif (4.7 mb)
Fig. S2. Total reads of 18-30 nt small RNAs (TIF 4762 KB)
438_2018_1524_MOESM3_ESM.tif (15.8 mb)
Fig. S3. Identification of miR164 target site by degradome sequencing. Red rhombus indicates target signature of NAC32 (a), NAC40 (b), NAC108 (c), and NAC113 (d) produced by miR164-directed cleavage (TIF 16133 KB)
438_2018_1524_MOESM4_ESM.xlsx (13 kb)
Table S1. Primers used in this work (XLSX 13 KB)
438_2018_1524_MOESM5_ESM.xlsx (22 kb)
Table S2. Known miRNA expression changes during early development stage (S1) and middle development stage (S2) (XLSX 21 KB)
438_2018_1524_MOESM6_ESM.xlsx (16 kb)
Table S3. Identified targets of known miRNAs in early development stage (S1) and middle development stage (S2) of maize seeds (XLSX 16 KB)
438_2018_1524_MOESM7_ESM.xls (195 kb)
Table S4. Unigenes expression changes in wild-type and MIR164-transgenic seeds of 6 DAP (XLS 195 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Lanjie Zheng
    • 1
  • Xiangge Zhang
    • 1
  • Haojun Zhang
    • 1
  • Yong Gu
    • 4
  • Xinrong Huang
    • 1
  • Huanhuan Huang
    • 1
  • Hanmei Liu
    • 2
  • Junjie Zhang
    • 2
  • Yufeng Hu
    • 1
  • Yangping Li
    • 1
  • Guowu Yu
    • 1
  • Yinghong Liu
    • 3
  • Shaneka S. Lawson
    • 5
  • Yubi Huang
    • 1
    Email author
  1. 1.College of AgronomySichuan Agricultural UniversityChengduChina
  2. 2.College of Life ScienceSichuan Agricultural UniversityYa’anChina
  3. 3.Maize Research InstituteSichuan Agricultural UniversityChengduChina
  4. 4.Sichuan Yuliang Biotechnological Co. LtdChengduChina
  5. 5.USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center (HTIRC)Purdue UniversityWest LafayetteUSA

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