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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (7): 1747-1757.doi: 10.3724/SP.J.1006.2023.23054

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Heat-inducible transcription factor ZmNF-YC13 regulates heat stress response genes to improve heat tolerance in maize

MEI Xiu-Peng1,2(), ZHAO Zi-Kun1,2(), JIA Xin-Yao1,2, BAI Yang1,2, LI Mei3, GAN Yu-Ling1,2, YANG Qiu-Yue1,2, CAI Yi-Lin1,2,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
    3Department of Agriculture and Horticulture, Guangxi Agricultural Vocational University, Nanning 530007, Guangxi, China
  • Received:2022-08-03 Accepted:2022-11-25 Online:2023-07-12 Published:2022-12-05
  • Contact: *E-mail: caiyilin1789@163.com E-mail:swumxp2009@163.com;404683331@qq.com;caiyilin1789@163.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    The National Natural Science Foundation of China(31901555);The China Postdoctoral Science Foundation(2019M663879XB);The Innovation and Entrepreneurship Training Program for College Students of Southwest University(S202210635315)

Abstract:

Heat stress is an essential factor affecting maize growth and yield formation. The exploration and mechanism analysis of related tolerance genes is an important field for breeding maize heat stress tolerant varieties. However, there is few study in this aspect. In this study, we identified a nuclear factor ZmNF-YC13 associated with heat stress response, and its encoding gene expression was rapidly induced by high temperature and osmotic stress. The promoter of Arabidopsis thaliana heat stress-inducible expression gene AtHSP70 was used to drive ZmNF-YC13 and the heat-inducible expression maize material of ZmNF-YC13 (HSP21Pro:ZmNF-YC13-myc) was successfully screened. Phenotypic analysis after high temperature treatment demonstrated that leaf length, leaf width, shoot thickness, fresh and dry weight of shoot and root were significantly higher than wild type. The relative expression level showed that ZmNF-YC13 could enhance the inducible level of downstream heat stress response genes in response to heat stress. Luciferase reporter assay and ChIP-qPCR assay also revealed that ZmNF-YC13 could regulate the expression of heat stress transcription factor ZmHsfA2c. These results confirmed preliminarily that ZmNF-YC13 could improve the heat tolerance of maize by regulating downstream heat stress response genes, which could provide a theoretical basis for marker-assisted selection and germplasm identification using the polymorphism of this locus.

Key words: maize, heat stress, heat stress transcription factor, NF-Y

Table S1

Primers used in this study"

引物名称 Primer 序列Sequence (5′-3′)
ZmNF-YC13RT-F TAGGGCTGGTCTGCCACCCAT
ZmNF-YC13RT-R GGCTCCTGCCACAAATAAGTCACT
ZmDnaJ2RT-1F TCAAGTTCTGCGTTTGGGGTGTTA
ZmDnaJ2RT-1R GAAGCTAGCAAACGTATTCGAGCG
ZmDnaJ1RT-1F TACACCGCGCACGTGACGACGC
ZmDnaJ1RT-1R ATCGGCATGCCCTCCCCTCGCA
ZmHSP70RT-1F GTCGACTAAATGAGGAAATTCTGATA
ZmHSP70RT-1R AATAAGATCCACTTTGTAATTGACGC
AtHSP21Pro-F CAGCTATGACATGATTACGAATTCCTTACCAAGCTTCTGAGCATCTCC
AtHSP21Pro-R CTGAGGGGATGGTTCCATGGATCCTTGTTTCGAGTATGAGCCAAAAAT
ZmHsfA2cPro-1F AGCCTACCTTTTATGTGATACTCC
ZmHsfA2cPro-1R CAGTAGCCAAGTGTGAATCATTGT
ZmHsfA2cPro-2F TCTTGTCTCTCCTCTCCAGAACCT
ZmHsfA2cPro-2R GTCGAGACACGTCGCGAGGCTT
ZmHsfA2cPro-3F ACAGAAATATCCTAAGCGCTGAC
ZmHsfA2cPro-3R AACACGGGGCAAGGACTCGATT
ZmHsfA2cPro-4F TTCTCTTGGTCAGGGCTTGCTAAT
ZmHsfA2cPro-4R TATTGCAGAGGATTCGGATGCTCT
ZmHsfA2cPro-F GAGGTCGACGGTATCGATAAGCTTTAGTGAAGATCCAAAAGATAATA
ZmHsfA2cPro-R GGCCGCTCTAGAACTAGTGGATCCGGCCTCCGCCCAGGCCCAAGAACC
18S rRNA-F ACCTTACCAGCCCTTGACATATG
18S rRNA-R GACTTGACCAAACATCTCACGAC

Fig. 1

Relative expression level of ZmNF-YC13 under different conditions and inbred lines in maize A: relative expression level of ZmNF-YC13 genes in maize leaves under heat stress and osmotic conditions. B: ZmNF-YC13 gene expression in heat-tolerant and heat-sensitive maize inbred plants leaves under normal condition. C: ZmNF-YC13 gene expression in heat-tolerant and heat-sensitive maize inbred plants leaves after heat stress. Data are means of three biological replicates. Differences among data were tested using two-tailed Student’s t-test. *: P < 0.05, **: P < 0.01. D: ZmNF-YC13-myc fused protein analysis of the HSP21Pro:ZmNF-YC13-myc plants leaves under heat stress by Western blot."

Fig. 2

Phenotypic analysis of wild-type (WT) and HSP21Pro:ZmNF-YC13-myc plants under heat stress A-D: Plant morphologies of wild-type (WT) and HSP21Pro:ZmNF-YC13-myc plants before and after high temperature treatment. The arrow in Fig. 2-D indicates the fifth leaf, Bar: 5 cm. E: leaf length, leaf width, shoot thickness, and biomass of the wild-type (WT) and HSP21Pro: ZmNF-YC13-myc plants after high temperature treatment. Data are means of three biological replicates. Differences among data were tested using two-tailed Student’s t-test. *: P < 0.05, **: P < 0.01."

Fig. 3

Relative expression patterns of heat stress responsive genes in wild-type and HSP21Pro:ZmNF-YC13-myc plants"

Fig. 4

Relative expression levels of ZmHsfA2c genes regulated by ZmNF-YC13 A: the transactivation of ZmHsfA2c promoter and ZmNF-YC13. For the transactivation,-ZmNF-YC13 means empty effector plasmid and +ZmNF-YC13 means ZmNF-YC13-fused effector plasmid. Data are means ± SDs of three independent experiments. **: P < 0.01. B: the promoter binding site analysis of ZmNF-YC13. The experiment was performed by three independent experiments. C: the schematic diagram of the ZmHsfA2c promoter fragments using for ChIP-qPCR analysis."

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