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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (1): 67-75.doi: 10.3724/SP.J.1006.2024.32012

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

Effects of small peptide Ospep5 on cadmium tolerance in rice

LI Ming-Yue1,3(), ZHANG Wen-Ting1,2, LI Yang1,2, ZHANG Bao-Long1,2, YANG Li-Ming3, WANG Jin-Yan1,2,*()   

  1. 1Institute of Germplasm Resources and Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
    2Zhongshan Biological Breeding Laboratory, Nanjing 210014, Jiangsu, China
    3College of Biology and Environment, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
  • Received:2023-04-01 Accepted:2023-06-29 Online:2024-01-12 Published:2023-07-13
  • Contact: *E-mail: wangjy@jaas.ac.cn
  • Supported by:
    Jiangsu Agricultural Science and Technology Innovation Fund(CX(22)3124)

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Abstract:

Cadmium (Cd) stress is one of the major heavy metal stresses which causes serious impacts on plant growth and development. Although studies have shown that plant small peptides have a mitigating effect on stress, research on their tolerance to cadmium toxicity in rice is limited. In previous studies, a few peptides were identified through translatome, transcriptome, and proteome analysis, among which Ospep5 was found to significantly improve salt tolerance in rice. In this study, the effects of Ospep5 on the growth of rice seedlings under cadmium stress were investigated using the japonica cultivar Nipponbare, Ospep5 overexpressing line Ospep5-OX, and the CRISPR/Cas9 mutant ospep5-3 as the experimental materials. The results showed that 500 μmol L-1 CdCl2 significantly inhibited the morphological growth and chlorophyll content of rice seedlings, while the activities of superoxide dismutase (SOD), contents of proline (Pro), malondialdehyde (MDA), and cadmium ion content were significantly increased. Compared to cadmium stress alone, exogenous application of synthetic Ospep5 effectively alleviated the inhibition of cadmium stress on the morphological growth of rice seedlings. Furthermore, SOD activity was significantly increased, and MDA content, Pro content, and cadmium ion content were significantly reduced. And the relative expression level of cadmium tolerance genes (OsHMA2, OsHMA3, OsCAL1) were upregulated. In conclusion, Ospep5 improves the tolerance of rice seedlings to cadmium stress by regulating various physiological and biochemical reactions and the expression of cadmium tolerance genes.

Key words: rice, cadmium stress, Ospep5, seedling growth

Table 1

Information of primers"

基因名称
Gene ID		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
Ospep5-OE gttacttctgcactaggtaccATGCTTGATCCAACGCACAT tcttagaattcccggggatccTCATGATGGTTTCTCGTAGC
Ospep5-Crispr TGTGCATGTTCGTCCAGCCTCACG AAACCGTGAGGCTGGACGAACATG
Ospep5-RT ATGCTTGATCCAACGCACATG TCATGATGGTTTCTCGTAGCTG
OsHMA2 CGCTGAATCAGGCAAGGTTG AATGCTCGAACAGCGACACG
OsHMA3 GTCCTTCTTCGAGTGGCTCT CGATGAGCATGAGGACGTTG
OsCAL1 GCTTCTGCAAGAAGGTCTGC GCCAAGAACAGAAGCTCGTC
Actin TGGTCGTACCACAGGTATTGTGTT AAGGTCGAGACGAAGGATAGCAT

Fig. 1

Relative expression level of CRISPR/Cas9 mutant and overexpression lines of Ospep5 A: targeted mutagenesis and Sanger sequencing chromatograms of the Ospep5; B: the relative expression level of Ospep5 genes; significant differences are indicated by different lowercase letters (P < 0.05), as determined by ANOVA."

Fig. 2

Phenotypic effects of Ospep5 on rice seedling growth under cadmium stress A: the phenotype of wild-type Nipponbare under different treatments; B: the phenotype of CRISPR/Cas9 mutant ospep5-3 under different treatments; C: overexpression line Ospep5-OX, ospep5-3 under 500 μmol L-1 CdCl2 treatment; the bar is 5 cm. CK: nutrient solution; Cd: nutrient solution containing μmol L-1 CdCl2; Cd+S5: nutrient solution containing 500 μmol L-1 CdCl2 and spraying 300 nmol L-1 Ospep5; R: Nipponbare; -OX: overexpression line ospep5-OX; -3: CRISPR/Cas9 mutant ospep5-3."

Table 2

Effects of Ospep5 on fresh weight, plant height, and root length of rice seedlings under cadmium stress"

材料
Material		 处理
Treatment		 指标Indicator
鲜重 Fresh weight (g) 株高 Plant height (cm) 根长 Root length (cm)
日本晴Nipponbare CK 0.77±0.11 a 36.40±0.60 a 20.18±1.65 a
Cd 0.18±0.04 cd 13.93±1.28 ef 14.08±1.91 c
Cd+S5 0.38±0.04 b 19.90±1.51 d 16.48±0.94 b
Ospep5-3 CK 0.52±0.03 b 29.88±4.05 b 12.71±0.77 cd
Cd 0.16±0.01 d 12.61±0.89 f 9.35±0.64 e
Cd+S5 0.24±0.02 c 15.16±0.62 e 12.01±1.91 d
Ospep5-OX CK 0.50±0.09 b 25.35±0.76 c 16.50±1.00 b
Cd 0.21±0.04 cd 14.35±0.72 ef 14.05±1.28 c

Fig. 3

Effects of Ospep5 on physiological indexes of rice seedlings under cadmium stress A: chlorophyll content; B: malondialdehyde (MDA) content; C: proline (Pro) content; D: superoxide dismutase (SOD) activity. The abscissa is the same as those given in Table 2. Significant differences are indicated by different lowercase letters (P < 0.05), as determined by ANOVA."

Fig. 4

Effects of Ospep5 on cadmium content in rice seedlings under cadmium stress A: cadmium ion content in aboveground parts; B: cadmium ion content in underground parts. CK, Cd and Cd+S5 are the same as in Table 2. Significant differences are indicated by different lowercase letters (P < 0.05), as determined by ANOVA."

Fig. 5

Relative expression level of Ospep5 on rice seedling related genes under cadmium stress A: the relative expression level of Ospep5 genes; B: the relative expression level of OsHMA2 genes; C: the relative expression level of OsHMA3 genes; D: the relative expression level of OsCAL1genes. CK, Cd and Cd+S5 are the same as those given in Table 2. Significant differences are indicated by different lowercase letters (P < 0.05), as determined by ANOVA."

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