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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (8): 1996-2006.doi: 10.3724/SP.J.1006.2022.13047

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ROS production and ATP synthase subunit mRNAs integrity in artificially aged maize embryos

WANG Tian-Bo(), HE Wen-Xue, ZHANG Jun-Ming, LYU Wei-Zeng, LIANG Yu-Huan, LU Yang, WANG Yu-Lu, GU Feng-Xu, SONG Ci, CHEN Jun-Ying*()   

  1. College of Agronomy, Henan Agricultural University, Zhengzhou 450046, Henan, China
  • Received:2021-07-09 Accepted:2021-11-29 Online:2022-08-12 Published:2021-12-24
  • Contact: CHEN Jun-Ying E-mail:15093455085@163.com;chenjunying3978@126.com
  • Supported by:
    National Natural Science Foundation of China(31971998);National Natural Science Foundation of China(31571761)

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Abstract:

In this study, hybrid maize (Zea mays L.) cultivar ‘Zhengdan 958' seeds were used as materials to reveal the mechanism of seed aging. After artificial aging treatment (45℃, 100% relative humidity), the seed vigor, O2- production, ATP content, and ATP synthase activity of seed embryo were investigated, and the integrity of ATP synthase subunit mRNAs in seed embryo and different organs (scutellum, radicle, and plumule) was analyzed by the reverse transcription blocking and double-primer amplification method. The results showed that the germination rate and radicle growth rate of maize seeds decreased, the viability of embryo decreased and the respiration intensity of seeds decreased after artificial aging treatment. The O2- production rate in embryo reached the peak on the 3rd day of aging treatment, and then decreased, the O2- production rate and content in radicle and plumule increased significantly. The ATP content and ATP synthase activity in embryo decreased. The mRNA integrity of ATP synthase ε-subunit and γ-subunit increased first and then decreased. The mRNA integrity of δ-subunit did not change significantly. The mRNA integrity of α-subunit and β-subunit decreased continuously in artificial aging treatment. The damage degree of mRNA integrity of ATP synthase subunits in radicle and plumule was much greater than that in scutellum. These results revealed that the production and accumulation of ROS in radicle and plumule could lead to different degrees of damage to ATP synthase subunit mRNAs and the activity of ATP synthase and the content of ATP in embryo decreased, which resulted in the lack of energy supply during germination, and this maybe one of the major factors causing maize seed aging.

Key words: maize embryo, artificial aging, ROS production, ATP synthesis, integrity of mRNAs

Fig. 1

Reverse transcription blocking and double primers amplification schematics The dots are oxidative damage sites."

Fig. 2

Germination rate (a), radicle length (b) during imbibition of aged maize seeds, and growth status (c) at 60 hours of imbibition Bar: 1.0 cm."

Fig. 3

Effects of artificial aging treatment on seed viability in maize (a) TTC staining of maize embryo; S: scutellum; R: radicle; P: plumule; bar: 2.0 mm. (b) Effects of aging treatment on dehydrogenase activity of maize embryo. Bars superscripted by different lowercase letters are significantly different at the 0.05 probability level."

Fig. 4

Change of respiration rates of aged maize seeds during imbibition period Different lowercase letters indicate significant difference among different aging treatments at the same imbibition time."

Fig. 5

NBT staining of maize embryos after aging treatment S: scutellum; R: radicle; P: plumule. "

Fig. 6

Effects of artificial aging on O2- production rate of embryos (a) and different organs (b) Different lowercase letters indicate significant difference among different aging treatments of the same organ."

Fig. 7

Change of ATP content in aged maize seeds during imbibition Different lowercase letters indicate significant difference among different aging treatments at the same imbibition time."

Fig. 8

Change of ATP synthase activities in maize embryos (a) and different organs (b) after aging treatment Different lowercase letters indicate significant difference among different aging treatments of the same organ."

Table 1

ATP synthase subunit genes and primer sequences"

基因ID
Gene ID		 引物方向
Primer direction		 引物序列
Primer sequence (5′-3′)		 位置
Position		 退火温度
Tm (℃)		 产物长度
Product length (bp)
Zm00001d040880-5' (ATP-α) F ACAGCCTTCTCTTCTCAT 314 59.0 123
R CTTAGTTCAGAGTTGCCTC 436 59.1
Zm00001d040880-3' (ATP-α) F GCGACTTAGGCATTACTC 1784 59.0 139
R CTAGTGGCATTCGATCAC 1922 59.0
Zm00001d038929-5' (ATP-β) F TGGACGCATCATAAATGTTA 494 59.0 88
R GGCTTCACGATGAATAGG 581 59.0
Zm00001d038929-3' (ATP-β) F GCAGTCATTCTACATGGTT 1589 59.0 88
R GAAGCCTCCTTATGAAGC 1676 58.9
Zm00001d028207-5' (ATP-γ) F CTCAAGTTGTGAGGAACC 305 59.0 188
R AATCACATTCTTCTTGACGT 492 59.1
Zm00001d028207-3' (ATP-γ) F CTCACGCTTACTTACAACA 1054 59.0 99
R TCTCTTCACCTATCCTTCAA 1152 59.1
Zm00001d006439-5' (ATP-δ) F AAGGAGGTTGACATGGTA 384 58.9 87
R CTTGAGCTCTGCAATTGT 470 59.4
Zm00001d006439-3' (ATP-δ) F TGATATTGTGGCTGTTGAG 559 59.0 149
R CACTGTGAACATCGACTC 707 59.2
Zm00001d044441-5' (ATP-ε) F GAAGGTCCATTTCTCCATC 229 59.1 77
R ATTCATCGGATTCTGTGC 305 58.9
Zm00001d044441-3' (ATP-ε) F AGTCATGTGAACATCATGG 479 59.0 84
R TAAGGACCAGACATAGAATCT 562 59.2
Zm00001d010159 (Actin1) F GAACTGCCATGATAAGGTTA — 59.0 194
R GGTGACGAATATACGAGTG 59.0

Fig. 9

Detection of mRNA integrity of ATP synthase subunits during aging Bars superscripted by different lowercase letters are significantly different at the 0.05 probability level."

Fig. 10

Detection of mRNA integrity of ATP synthase subunits in different organs of embryo Different lowercase letters indicate significant difference among different aging treatments of the same organ."

Fig. 11

An assumption of maize seed deterioration model"

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