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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (10): 2001-2011.doi: 10.3724/SP.J.1006.2021.04238

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

Effects of exogenous 2,4-Epibrassinolide on nitrogen metabolism and TAs metabolism of Atropa belladonna L. under NaCl stress

XIN Zheng-Qi1(), DAI Huan-Huan2, XIN Yu-Feng1, HE Xiao1, XIE Hai-Yan1, WU Neng-Biao1,*()   

  1. 1School of Life Science, Southwest University / Key Laboratory of Eco-environments in Three Gorges Region, Ministry of Education, Chongqing 400715, China
    2The Fifth Middle School of Qingyang, Qingyang 745000, Gansu, China
  • Received:2020-11-01 Accepted:2021-01-13 Online:2021-10-12 Published:2021-02-25
  • Contact: WU Neng-Biao E-mail:490992699@qq.com;wunb@swu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(30500041)

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Abstract:

To explore the physiological mechanism of exogenous 2,4-Epibrassinolide (EBR) regulating NaCl tolerance of A. belladonna, potted A. belladonna seedlings was used as the experimental materials, and exogenous EBR was applied to A. belladonna seedlings with the different concentrations (0.05, 0.1, 0.2, and 0.4 mg L-1) of exogenous EBR and different treatment times (5, 10, 15, and 20 days) on the nitrogen metabolism, the contents of the secondary metabolites and precursor substances in the synthesis pathway of TAs, and the relative expression levels of key enzyme genes. NaCl stress caused inhibitory effect on nitrogen metabolism in A. belladonna, however the content of nitrate nitrogen increased significantly, the content of ammonium nitrogen decreased, the content of free amino acids, soluble protein, and the activity of key enzymes of nitrogen metabolism increased to some extent under exogenous EBR treatment, which indicating the exogenous EBR could effectively enhance the nitrogen metabolism capacity. NaCl stress was not conducive to the synthesis and accumulation of alkaloids. The synthesis of precursor substances and the relative expression levels of key enzyme genes in TAs pathway significantly were reduced under NaCl stress. The contents of ornithine, arginine, polyamine, and the activities of key enzymes in putrescine synthesis were increased with the exogenous EBR of 0.1 mg L-1. Moreover, exogenous EBR could effectively enhance the contents of hyoscyamine and scopolamine by increasing the relative expression levels of key enzyme genes TR I and H6H in TAs pathway. In conclusion, the appropriate concentration of exogenous EBR could effectively relieve the damage of NaCl stress to the physiological metabolism in A. belladonna, and the exogenous EBR could improve NaCl tolerance of belladonna seedlings by increasing nitrogen metabolism and promoting the production and accumulation of TAs.

Key words: Atropa belladonna L., NaCl stress, nitrogen metabolism, secondary metabolism

Table 1

Different treatments of Atropa belladonna L."

处理组
Group		 NaCl盐浓度
NaCl concentration (mmol L-1)		 EBR浓度
EBR concentration
(mg L-1)
CK 0 0
T0 100 0
B1 100 0.05
B2 100 0.1
B3 100 0.2
B4 100 0.4

Table 2

Primers for qRT-PCR"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
F-qPGK TCGCTCTTGGAGAAGGTTGAC
R-qPGK CTTGTCCGCAATCACTACATCAG
F-AbTR Ⅰ TTCTTTGCTTCCCTGCTGCTTC
R-AbTR Ⅰ CAGGCCAACCTTAGTATCACACAG
F-AbH6H TTCCACTTGAGCAGAAAGCAAAGC
R-AbH6H CCTCATGGTCAACTTCCTCACTTCC
F-AbPMT CCTACTTACCCTACTGGTGTTATC
R-AbPMT GCGAAAGATGGCAAAATAAAAGC
F-AbPPAR GATTGGAGTTGTTGGATTGG
R-AbPPAR CCTTGAAGTGTAAGAGATGGA

Fig. 1

Growth of Atropa belladonna with 0.1 mg L-1 EBR treatment after 20 days of NaCl stress treatment Treatments are the same as those given in Table 1. Bar: 10 cm."

Table 3

Effects of exogenous EBR on nitrogen compounds content of leaves under NaCl stress in Atropa belladonna L."

指标
Indicator		 处理组
Group		 处理时间Treatment days
5 d 10 d 15 d 20 d
硝态氮
Nitrate nitrogen
(μg g-1 FW)		 CK 235.270±19.155 a 219.531±17.631 a 228.121±17.588 a 223.681±10.459 a
T0 172.379±10.148 c 165.078±13.045 d 144.347±15.771 d 126.918±15.774 d
B1 196.475±15.896 bc 184.987±15.751 cd 171.979±18.628 cd 155.752±12.174 c
B2 221.515±18.481 ab 212.715±25.806 ab 192.219±10.863 bc 192.379±16.613 b
B3 238.206±17.135 a 206.361±16.391 ab 209.203±15.735 ab 180.566±18.503 bc
B4 209.142±23.651 ab 193.902±15.395 bc 184.267±11.701 bc 177.438±15.189 bc
铵态氮
Ammonium nitrogen
(μg g-1 FW)		 CK 86.897±4.105 a 76.944±2.499 c 67.929±2.828 d 71.087±4.672 d
T0 72.957±2.013 c 91.748±1.776 a 106.025±1.289 a 124.603±5.316 a
B1 81.997±1.921 ab 83.524±3.315 b 90.275±1.066 b 113.194±1.749 ab
B2 78.304±3.566 bc 74.326±2.223 cd 71.299±3.039 d 82.622±7.815 cd
B3 73.251±0.933 c 69.352±5.986 d 81.022±3.893 c 93.441±3.973 bc
B4 77.815±2.184 bc 79.089±3.618 bc 86.931±5.621 bc 98.837±5.552 bc
游离氨基酸
Free amino acids
(μg g-1 FW)		 CK 821.721±30.434 a 784.398±26.959 a 797.848±43.402 a 758.979±36.011 a
T0 691.934±11.070 c 651.923±5.596 d 606.195±35.981 d 586.021±28.179 c
B1 740.688±15.033 bc 685.546±18.517 cd 647.551±15.952 cd 663.018±11.939 b
B2 782.717±20.377 ab 716.479±14.387 bc 732.619±16.309 ab 708.746±20.148 ab
B3 754.810±13.649 b 741.024±11.110 ab 744.723±17.181 ab 680.879±17.181 b
B4 728.920±8.578 bc 704.375±12.221 bc 692.271±6.623 bc 654.612±9.047 bc
可溶性蛋白
Soluble proteins
(mg g-1 FW)		 CK 2.926±0.072 c 2.721±0.085 bc 2.625±0.121 a 2.501±0.058 a
T0 2.839±0.241 bc 2.542±0.109 c 1.816±0.094 c 1.667±0.050 d
B1 3.021±0.094 ab 2.741±0.095 bc 1.979±0.129 bc 1.927±0.104 cd
B2 3.361±0.110 a 3.002±0.051 ab 2.374±0.234 ab 2.308±0.144 ab
B3 3.122±0.071 ab 3.106±0.118 a 2.557±0.146 a 2.149±0.119 bc
B4 2.895±0.104 bc 3.038±0.142 ab 2.056±0.122 bc 2.095±0.126 bc

Table 4

Effects of exogenous EBR on NR, GS, and GDH activity of leaves under NaCl stress in Atropa belladonna L."

指标
Indicator		 处理组
Group		 处理时间Treatment days
5 d 10 d 15 d 20 d
硝酸还原酶
NR
(U g-1 FW h-1)		 CK 1.445±0.028 ab 1.396±0.065 ab 1.462±0.027 a 1.429±0.037 a
T0 1.233±0.063 d 1.065±0.074 d 0.932±0.104 d 0.798±0.076 d
B1 1.392±0.041 cd 1.174±0.045 cd 0.974±0.070 cd 0.874±0.053 d
B2 1.394±0.017 bc 1.455±0.073 a 1.251±0.926 b 1.185±0.063 b
B3 1.566±0.043 a 1.396±0.026 ab 1.149±0.060 bc 1.049±0.052 bc
B4 1.511±0.055 ab 1.257±0.028 bc 1.106±0.026 cd 0.939±0.074 cd
谷氨酰胺合成酶
GS
(OD540 mg-1 protein FW h-1)		 CK 3.676±0.126 a 3.482±0.172 a 3.304±0.168 a 3.392±0.067 a
T0 2.992±0.154 c 2.733±0.164 c 2.467±0.143 d 2.303±0.133 d
B1 3.148±0.048 bc 2.867±0.102 bc 2.698±0.081 cd 2.465±0.085 cd
B2 3.570±0.140 a 3.187±0.173 ab 3.225±0.102 ab 3.044±0.036 b
B3 3.512±0.067 ab 3.437±0.042 a 3.025±0.108 bc 2.698±0.124 c
B4 3.429±0.102 ab 3.258±0.080 ab 2.889±0.098 bc 2.527±0.113 cd
谷氨酸脱氢酶
GDH
(U mg-1 FW min-1)		 CK 0.252±0.026 c 0.271±0.016 bc 0.772±0.016 b 0.283±0.033 ab
T0 0.293±0.019 bc 0.202±0.008 d 0.154±0.025 d 0.184±0.027 c
B1 0.271±0.024 bc 0.241±0.027 cd 0.198±0.017 cd 0.230±0.019 bc
B2 0.356±0.025 a 0.305±0.022 ab 0.283±0.026 b 0.305±0.024 ab
B3 0.315±0.022 ab 0.346±0.026 a 0.359±0.034 a 0.362±0.016 a
B4 0.236±0.163 c 0.242±0.031 cd 0.258±0.037 bc 0.248±0.025 bc

Fig. 2

Effects of exogenous EBR on hyoscyamine (A) and scopolamine (B) content of leaves under NaCl stress in Atropa belladonna L. Different lowercase letters represent significantly different at the 0.05 probability level among the treatments. Treatments are the same as those given in Table 1."

Fig. 3

Effects of exogenous EBR with different concentrations on ornithine and arginine content of leaves after 20 days of NaCl stress treatment in Atropa belladonna L. Different lowercase letters represent significantly different at the 0.05 probability level among treatments. Treatments are the same as those given in Table 1."

Fig. 4

Effects of exogenous EBR with different concentrations on polyamine content (A), PAs content (B) content of Atropa belladonna L. leaves after 20 days of NaCl stress treatment Different lowercase letters represent significantly different at the 0.05 probability level among treatments. Treatments are the same as those given in Table 1."

Fig. 5

Effects of exogenous EBR with different concentrations on key enzyme activity for polyamine synthesis of Atropa belladonna L. leaves after 20 days of NaCl stress treatment Different lowercase letters represent significantly different at the 0.05 probability level among treatments. Treatments are the same as those given in Table 1."

Fig. 6

Effects of exogenous EBR on relative gene expression level of TR I (A), PMT (B), H6H (C), and PPAR (D) in roots and leaves of Atropa belladonna L. under NaCl stress Different lowercase letters represent significantly different at the 0.05 probability level among treatments. Treatments are the same as those given in Table 1."

Fig. 7

Nitrogen metabolism and TAs metabolic pathway network changes of Atropa belladonna after 0.1 mg L-1 EBR treatment under NaCl stress at 20 days Red represents a significant increase; green represents a significant decrease; white represents no obvious change; FAA: free amino acids; SP: soluble protein; Orn: ornithine; Arg: arginine; Hyo: hyoscyamine; Sco: scopolamine."

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