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Acta Agronomica Sinica ›› 2026, Vol. 52 ›› Issue (1): 249-261.doi: 10.3724/SP.J.1006.2026.54013

• TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY • Previous Articles     Next Articles

Cloning of the NtCEP7 gene in tobacco and functional analysis of its encoded peptide in seedling-stage drought resistance

Kong Na1(), Liu Tao1,2, Liu Wen-Ting1,2, Chen Gang1, Wen Li-Chao3, Deng Zhi-Chao1,2, Guo Mei1, Li Wei1,*(), Guo Yong-Feng1,*()   

  1. 1Tobacco Research Institute, Chinese Academy of Agricultural Sciences / Key Laboratory of Tobacco Gene Resources, Qingdao 266101, Shandong, China
    2Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3Chongqing Three Gorges University, Chongqing 404020, China
  • Received:2025-01-24 Accepted:2025-09-10 Online:2026-01-12 Published:2025-10-13
  • Contact: *E-mail: liwei06@caas.cn; E-mail: guoyongfeng@caas.cn
  • Supported by:
    Key Science and Technology Project of Shandong Provincial Bureau of China National Tobacco Corporation(KN307);Agricultural Science and Technology Innovation Program(ASTIP-TRIC-ZD04);National Natural Science Foundation of China(31970204)

Abstract:

Although tobacco exhibits strong drought tolerance at maturity, its seedlings are particularly sensitive to water deficiency during early developmental stages. Drought stress following field transplantation significantly disrupts plant growth, ultimately reducing yield and compromising leaf quality. C-terminally encoded peptides (CEPs), a class of small peptide hormones, are known to play crucial roles in regulating plant responses to abiotic stresses. However, the drought resistance functions of CEP family members in tobacco remain largely unexplored. In this study, we cloned the NtCEP7 gene from Nicotiana tabacum cv. K326. The full-length coding sequence (CDS) of NtCEP7 is 261 bp, encoding a protein of 86 amino acids. The predicted propeptide contains a typical secretory signal peptide and a transmembrane domain. Phylogenetic analysis placed NtCEP7 in Group I, and promoter analysis revealed the presence of cis-acting elements responsive to abscisic acid (ABA) and drought stress. Quantitative real-time PCR showed that NtCEP7 expression was significantly upregulated by both drought stress and ABA treatment. To assess its function, drought stress was simulated by withholding water from potted plants. Compared to the control group (sprayed with water), plants treated with NtCEP7a maintained a better growth status under drought conditions, which was manifested by milder leaf yellowing and wilting, exhibited increased chlorophyll content, net photosynthetic rate, dry weight of shoot and proline accumulation, along with decreased electrolyte leakage and malondialdehyde (MDA) levels. Further physiological analysis revealed that NtCEP7a treatment under drought conditions reduced stomatal aperture, increased relative leaf water content, enhanced antioxidant enzyme activities, and lowered levels of superoxide anions and hydrogen peroxide. Collectively, these results indicate that NtCEP7a alleviates drought-induced damage in tobacco by regulating stomatal dynamics, promoting osmoprotectant accumulation, and activating antioxidant defenses, thereby enhancing drought tolerance.

Key words: tobacco, NtCEP7a peptide, drought stress, stomatal aperture, antioxidant enzyme activity

Table 1

Primer sequences"

引物名称
Primer name
引物序列
Primer sequence (5'-3')
目的
Purpose
NtCEP7-F ATGGCTAGGGTCATTCAATATT 基因克隆
Gene cloning
NtCEP7-R TTACTTGTTCCCCACGC
q-NtCEP7-F CTTTAGTGGTGGTGAATAATTCTGG 基因表达分析
Gene expression analysis
q-NtCEP7-R ATCATCAACTTCAGCATCTTCAGTA
q-NtActin-F CAAGGAAATCACCGCTTTGG
q-NtActin-R AAGGGATGCGAGGATGGA

Fig. 1

Cloning, gene structure, and physicochemical property analysis of the NtCEP7 gene in tobacco A: PCR amplification of the NtCEP7 gene; M: DL500 bp DNA Marker; 1, 2: PCR products. B: gene structure of NtCEP7; C: prediction of the signal peptide of NtCEP7 pro-protein; D: nucleotide and amino acid sequences of NtCEP7 gene; *: translation termination point. E: prediction of transmembrane domains pro-protein."

Fig. 2

Phylogenetic analysis of CEP proteins and cis-acting elements in the NtCEP7 promoter in tobacco A: phylogenetic tree of the proteins of Arabidopsis thaliana (At), Solanum lycopersicum (Sl), and Nicotiana tabacum (Nt) CEP; B: analysis of cis-acting elements in promoters. ABRE: cis-acting element involved in the abscisic acid responsiveness; as-1: auxin and salicylic acid responsive element; CGTCA-motif: MeJA-responsive; TGACG-motif: MeJA-responsive; TCA-element: cis-acting element involved in salicylic acid responsiveness; TGA-element: auxin-responsive element; MYC: MYC binding site element; LTR: cis-acting element involved in low-temperature responsiveness; MYB: MYB binding site element; ARE: cis-acting regulatory element essential for the anaerobic induction; W box: the inducer, injury, and pathogen responses bind to WRKY class transcription factors; Box 4: part of the conserved DNA module of cis-acting regulatory elements involved in light response; G-Box: cis-acting regulatory element involved in light responsiveness; A-box: cis-acting regulatory element; MRE: metal response element; ACE: cis-acting element involved in light responsiveness."

Fig. 3

Expression analysis of the NtCEP7 gene in leaves and roots under drought stress T0: control; T1: dehydration at room temperature; T2: ABA treatment. *, **, and *** denote significant difference at P < 0.05, P < 0.01, and P < 0.001, respectively."

Fig. 4

Effect of the NtCEP7a peptide on drought resistance in tobacco A: analysis of tobacco plant growth status; B: analysis of leaf water content of tobacco plants; C: analysis of shoot dry weight of tobacco plants; D: analysis of root dry weight of tobacco plants; E: analysis of MDA content of tobacco plants; F: analysis of Pro content of tobacco plants; G: analysis of electrical conductivity of tobacco plants. CK: normal conditions+foliar spraying of equal volume of clear water; CK+CEP: normal conditions+foliar spraying of 1 μmol L-1 NtCEP7a peptide; D: drought conditions+foliar spraying of equal volume of clear water; D+CEP: drought conditions+foliar spraying of 1 μmol L-1 NtCEP7a peptide. Lowercase letters indicate significant differences between treatments (P < 0.05); Fig. A scale bar is 7 cm. Physiological and biochemical data were obtained from the leaves of tobacco seedlings after seven days of treatment, as shown in Fig. 4-A."

Fig. 5

Effects of NtCEP7a peptide on stomatal movement and in vitro water loss rate of leaves in tobacco A: phenotypic map of tobacco stomata; B: stomatal width to length ratio; C: leaf water loss rate in vitro. Different lowercase letters indicate statistically significant differences between treatments (P < 0.05). Scale bar = 30 μm. All physiological and biochemical data were obtained from the leaves of tobacco seedlings after seven days of treatment, as shown in Fig. 4-A. Treatments are the same as those given in Fig. 4."

Table 2

Effects of foliar application of NtCEP7a peptide on photosynthetic characteristics of tobacco under drought stress"

处理
Treatment
叶绿素含量
Chlorophyll content (mg g-1)
胞间CO2浓度
Ci (μmol mol-1)
净光合速率
Pn (μmol m-2 s-1)
气孔导度
Gs (mmol m-2 s-1)
蒸腾速率
Tr (μmol m-2 s-1)
CK 2.29±0.18 a 212.3±17.4 c 0.340±0.018 a 3.28±0.16 a 0.100±0.005 a
CK+CEP 2.33±0.19 a 210.8±13.3 c 0.350±0.019 a 3.23±0.08 a 0.110±0.003 a
D 1.42±0.14 c 368.1±23.5 a 0.030±0.014 b 1.67±0.20 b 0.043±0.009 b
D+CEP 1.75±0.09 b 318.0±24.8 b 0.051±0.020 c 1.00±0.18 c 0.028±0.005 c

Fig. 6

Effects of the NtCEP7a peptide on the activities of SOD, POD, and CAT in tobacco leaves A: NBT staining; B: $\mathrm{O}_{2}^{\bar{.}}$content; C: H2O2 content; D: SOD activity; E: POD activity; F: CAT activity. Different lowercase letters indicate significant differences between treatments at P < 0.05. All physiological and biochemical data were obtained from the leaves of tobacco seedlings after seven days of treatment, as shown in Fig. 4A. Treatments are the same as those given in Fig. 4."

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