Expression Characteristics and Functions of CaPIF4 in Capsicum annuum Under Salt Stress

doi:10.13560/j.cnki.biotech.bull.1985.2023-1045

Abstract

Abstract:

【Objective】Studying the molecular mechanisms of the pepper photoreceptor pigment phytochrome-interacting factor 4（PIF4）in response to salt stress plays an important role in understanding the molecular mechanisms of pepper's response to salt stress and breeding salt-tolerant varieties. 【Method】Using ‘Zunla-1’ cDNA as a template, CaPIF4 was cloned. Then the physical and chemical properties of CaPIF4 were analyzed using bioinformatics software. And the expression pattern and salt stress-induced expression was studied by real-time fluorescence quantitative PCR（RT-qPCR）and virus-induced gene silencing technology（VIGS）. 【Result】CaPIF4 was expressed in various tissues of pepper, including roots, stems, leaves, flowers, and fruits, with the highest expression in the leaves. And the expression of CaPIF4 increased with the extension of 300 mmol/L NaCl treatment, and reached the highest at 2 h after treatment. CaPIF4 gene was silenced using VIGS technology. The control group（CK）and gene transient silencing group（pTRV-CaPIF4）were treated in 300 mmol/L NaCl. And the phenotype showed that pTRV-CaPIF4 plants after salt stress presented more obvious salty damage compared with CK. Using VIGS technology to silence CaPIF4, the CK and the pTRV-CaPIF4 were treated with 300 mmol/L NaCl. It was found that the plants in the pTRV-CaPIF4 group were more severely wilted compared to CK after gene silencing, indicating a decrease in salt tolerance in the pTRV-CaPIF4 group. Subcellular localization results showed that CaPIF4 was located in the nucleus. Yeast transcriptional activity analysis revealed that CaPIF4 had transcriptional activation activity. Under high salt stress, the hydrogen peroxide content and antioxidant enzyme activity in the pepper leaves increased. Staining results showed that silencing the CaPIF4 gene resulted in a significant increase in H₂O₂ and O^2- levels. 【Conclusion】CaPIF4, as a transcription factor, may be involved in regulating salt tolerance in peppers.

Key words: Capsicum annuum, salt stress, CaPIF4, transcription factor, ROS

LI Hui, WEN Yu-fang, WANG Yue, JI Chao, SHI Guo-you, LUO Ying, ZHOU Yong, LI Zhi-min, WU Xiao-yu, YANG You-xin, LIU Jian-ping. Expression Characteristics and Functions of CaPIF4 in Capsicum annuum Under Salt Stress[J]. Biotechnology Bulletin, 2024, 40(4): 148-158.

Figures/Tables 12

Table1 List of primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
CaPIF4-F	ATGAATCCTTGTCTTCCTG
CaPIF4-R	TCAAACCTGAGAAGCACC
CaPIF4-qPCR-F	TGGTTGATCCATCAATCCCTT
CaPIF4-qPCR-R	CAAACCTGAGAAGCACCCTGA
CaPIF4-VIGS-F	TGAGTAAGGTTACCGAATTCATGAATCCTTGTC- TTCCTGAAT
CaPIF4-VIGS-R	GTGAGCTCGGTACCGGATCCACAAAATACTTTG- TCAAATGAAT
pGBKT7-CaPIF4-F	ATGGCCATGGAGGCCGAATTCATGAATCCTTGT- CTTCCTGAATGG
pGBKT7-CaPIF4-R	CCGCTGCAGGTCGACGGATCCAACCTGAGAAGCACCCTG
p3301-CaPIF4-F	ACGGGGGACTCTTGACCATGGATGAATCCTTG- TCTTCCTGAATGG
p3301-CaPIF4-F	GCCCTTGCTCACCATCCATGGAACCTGAGAAGCACCCTG
CaACTIN-F	AGGGATGGGTCAAAAGGATGC
CaACTIN-F	GAGACAACACCGCCTGAATAGC

Fig. 1 PCR amplification of CaPIF4 M: BM2000 DNA marker. 1-2: Two repeats

Fig. 2 Sequence analysis of CaPIF4 A: Hydrophilicity analysis of CaPIF4 protein. B: Conserved domain analysis of CaPIF4. C: Secondary structure of CaPIF4 protein. D: Tertiary structure of CaPIF4 protein

Fig. 3 Expression pattern analysis of CaPIF4 A: Expression analysis of CaPIF4 in different tissues. B: Expression analysis of CaPIF4 in the leaves under salt stress. Mean and SD values were obtained from three independent experiments, with 3 plants per experiments. The different letters indicate significant differences at P ≤0.05

Fig. 4 Construction of pTRV2-CaPIF4 vector A: Enzyme digesting verification of pTRV2-CaPIF4 vector; B: pTRV2-CaPIF4 vector sequence alignment; M: BM2000 DNA marker; 1-3: three repeats

Fig. 5 Phenotypic observation of pepper plants silenced by VIGS

Fig. 6 Detection of CaPIF4 gene silencing efficiency The error line in the figure refers to the standard deviation. * indicate statistical significance （*P<0.05，**P<0.01，***P<0.001 ） compared to control. The same below

Fig. 7 Subcellular localization of CaPIF4-GFP fusion protein

Fig. 8 Transcription self-activation activity analyses of CaPIF4

Fig. 9 Phenotype analysis of CaPIF4 gene silencing plants under salt stress

Fig. 10 ROS accumulation in the control group and VIGS silenced plants in the 3rd and 4th leaves before and after salt stress A: 3,3'-Diaminobenzidine (DAB) staining. B: Nitro-blue tetrazolium (NBT) staining

Fig. 11 Determination of physiological and biochemical indexes of pepper plants under salt stress

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