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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (10): 1527-1538.doi: 10.3724/SP.J.1006.2018.01527

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

Effect of Exogenous Ca 2+ on Physiological Characteristics and Secondary Metabolites accumulation of Atropa belladonna L. Seedlings under UV-B Stress

Ke-Huan LU1,Xing LIU1,Yi YANG1,Zhi-Hua LIAO2,Neng-Biao WU1,*()   

  1. 1 Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education / School of Life Science, Southwest University, Chongqing 400715, China
    2 School of Life Science, Southwest University, Chongqing 400715, China
  • Received:2018-01-15 Accepted:2018-07-20 Online:2018-10-10 Published:2018-08-01
  • Contact: Neng-Biao WU E-mail:wunb@swu.edu.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(30500041);the Science Technology Breakthrough Plan Project of Chongqing(cstc2012gg-yyjs80013)

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Abstract:

Ultraviolet B (UV-B) radiation does harm to plant, for which growth and development. Ca 2+is one of the most necessary elements. Exogenous Ca 2+ can enhance plant abilities to resist stress and regulate secondary metabolism. In this research, we used Atropa belladonna L. seedlings to investigate the effects of different concentrations of exogenous Ca 2+ and different treatment days on the physiological characteristics, nitrogen metabolism, secondary metabolites content and relative genes expression levels of three key enzymes in secondary metabolism under the background of UV-B radiation. The UV-B radiation not only caused inhibitory effect on the Atropa belladonna L. photosynthesis, nitrogen metabolism, alkaloid content, but also increased peroxidation of membrane lipid. The Fo, MDA content gradually decreased, but the Fv/Fm, photosynthetic pigment content, antioxidant enzyme activity gradually increased under Ca 2+ treatment, showing that Ca 2+ is propitious to relieve inhibitory effect on photosynthesis and enhance resistance to UV-B stress. Moreover, Ca 2+ reduced the content of nitrate nitrogen significantly, promoted the contents of free amino acids, soluble proteins, atropine alkaloids, and raised the activities of key enzymes in nitrogen metabolism. Using real-time fluorescence quantitative PCR, we found that exogenous Ca 2+ increased the relative gene expression levels of three key enzymes in secondary metabolism in different degrees. This research could provide a theoretical references for plantation in the field.

Key words: UV-B stress, Atropa belladonna L., physiological characteristics, nitrogen metabolism, tropane alkaloids

Table 1

Different treatments of Atropa belladonna L."

处理组
Group		 CaCl2 浓度
CaCl2 concentration (mmol L-1)		 UV-B辐射强度
UV-B radiation intensity (μW cm-2)		 处理时间
Treatment time (d)
T0 0 0 4, 8, 12
T1 0 10 4, 8, 12
T2 2 10 4, 8, 12
T3 4 10 4, 8, 12
T4 6 10 4, 8, 12
T5 8 10 4, 8, 12

Table 2

Primer pairs for quantitative PCR"

基因
Gene		 登录号
Accession number		 上游引物
Forward primer (5'-3')		 下游引物
Reverse primer (5'-3')		 退火温度
Annealing temperature (°C)
PGK JX154676 TCGCTCTTGGAGAAGGTTGAC CTTGTCCGCAATCACTACATCAG 59.5
PMT AB018570 CCTACTTACCCTACTGGTGTTATC GCGAAAGATGGCAAAATAAAAGC 56.4
H6H JN542537 TTCCACTTGAGCAGAAAGCAAAGC CCTCATGGTCAACTTCCTCACTTCC 56.4
TR I JX155757 TTCTTTGCTTCCCTGCTGCTTC CAGGCCAACCTTAGTATCACACAG 61.4

Fig. 1

Effects of Ca2+ on Fo (A) and Fv/Fm (B) of Atropa belladonna L. under UV-B stress Bars superscripted by different letters are significantly different among different treatments of Ca2+concentration in the same treating days at the 0.05 level. T1 to T5 indicate different treatments of Ca2+ concentration (0, 2, 4, 6, and 8 mmol L-1) under UV-B stress. T0 indicates untreated blank control groups."

Fig. 2

Effects of Ca2+ on the content of total chlorophyll (A) and carotenoid (B) of Atropa belladonna L. leaves under UV-B stress Bars superscripted by different letters are significantly different among different treatments of Ca2+concentration in the same treating days at the 0.05 probability level. T1 to T5 indicate different treatments of Ca2+ concentration (0, 2, 4, 6, and 8 mmol L-1) under UV-B stress. T0 indicates untreated blank control groups."

Fig. 3

Effects of Ca2+ on the activities of antioxidant enzymes (A, B, C) and the contents of MDA (D) of Atropa belladonna L. under UV-B stress Bars superscripted by different letters are significantly different among different treatments of Ca2+concentration in the same treating days at the 0.05 probability level. T1 to T5 indicate different treatments of Ca2+ concentration (0, 2, 4, 6, and 8 mmol L-1) under UV-B stress. T0 indicates untreated blank control groups."

Table 3

Effects of Ca2+ on nitrate nitrogen, free amino acids, and soluble proteins content of Atropa belladonna L. under UV-B stress"

指标
Indicator		 处理组
Group		 处理时间 Treatment time
4 d 8 d 12 d
硝态氮
Nitrate nitrogen
(μg g-1 FW)		 T0 539.543±19.299 b 545.976±29.480 a 526.677±29.480 a
T1 693.935±38.598 d 874.059±58.959 c 1112.080±40.174 d
T2 610.306±40.174 c 803.296±29.480 c 944.822±57.897 c
T3 462.347±19.299 a 726.100±48.568 b 874.059±62.038 c
T4 507.378±48.568 ab 597.440±38.598 a 783.997±29.480 b
T5 494.512±29.480 ab 681.069±44.569 b 861.193±48.568 bc
游离氨基酸
Free amino acids
(mg g-1 FW)		 T0 352.269±32.459 e 329.930±15.145 d 332.723±14.323 d
T1 225.763±38.646 b 177.929±25.124 a 134.465±19.857 a
T2 287.559±33.889 cd 185.213±25.860 a 161.661±18.992 ab
T3 313.176±25.550 de 255.872±30.828 bc 241.789±24.322 c
T4 251.137±33.889 bc 286.224±22.980 cd 264.856±17.015 c
T5 166.153±29.940 a 221.635±35.763 ab 194.076±16.096 b
指标
Indicator		 处理组
Group		 处理时间 Treatment time
4 d 8 d 12 d
可溶性蛋白
Soluble proteins
(mg g-1 FW)		 T0 5.879±0.309 a 5.681±0.364 ab 5.570±0.398 c
T1 6.458±0.401 a 4.953±0.548 a 3.725±0.430 a
T2 7.901±0.653 bc 5.736±0.432 ab 5.264±0.649 bc
T3 9.087±0.399 c 7.415±0.572 c 6.953±0.391 d
T4 6.859±0.491 ab 7.496±0.749 c 4.358±1.058 ab
T5 5.861±1.526 a 6.535±1.007 bc 3.887±0.456 a

Table 4

Effects of Ca2+ on NR, GS, and GDH activities of Atropa belladonna L. under UV-B stress"

指标
Indicator		 处理组
Group		 处理时间 Treatment time
4 d 8 d 12 d
硝酸还原酶 NR
(U g-1 FW h-1)		 T0 1.290±0.048 c 1.282±0.117 c 1.333±0.052 d
T1 1.037±0.022 a 0.813±0.053 a 0.557±0.039 a
T2 1.035±0.061 a 0.939±0.108 a 0.664±0.040 b
T3 1.121±0.009 b 1.083±0.030 b 0.724±0.047 b
T4 1.290±0.018 c 1.213±0.035 bc 0.902±0.043 c
T5 1.135±0.013 b 1.121±0.048 b 0.753±0.080 b
谷氨酰胺合成酶 GS
(OD540 mg-1 FW h-1)		 T0 8.349±0.197 c 7.885±0.259 d 7.696±0.670 d
T1 6.555±0.235 a 4.989±0.767 a 3.952±0.323 a
T2 6.812±0.297 a 5.666±0.155 b 4.323±0.214 ab
T3 7.494±0.285 b 6.442±0.138 c 4.883±0.226 bc
T4 8.037±0.231 c 7.336±0.189 d 5.454±0.201 c
T5 6.870±0.268 a 6.104±0.105 bc 4.592±0.219 ab
谷氨酸脱氢酶 GDH
(U mg-1 FW min-1)		 T0 5.659±0.402 a 5.756±0.658 b 4.968±0.482 b
T1 6.109±0.589 ab 4.244±0.579 a 3.344±0.486 a
T2 6.367±0.841 ab 5.338±0.486 ab 4.887±0.402 b
T3 7.138±0.880 b 5.916±0.990 b 6.881±0.620 c
T4 6.045±0.780 ab 8.039±0.678 c 6.431±0.403 c
T5 5.016±0.696 a 7.395±0.486 c 5.338±0.589 b

Table 5

Effects of Ca2+ on the contents of hyoscyamine and scopolamine of Atropa belladonna L. leaves under UV-B stress"

指标
Indicator		 处理组
Group		 处理时间 Treatment time
4 d 8 d 12 d
莨菪碱
Hyoscyamine
(μg g-1 DW)		 T0 1058.072±37.583 a 1083.528±31.056 d 1052.901±35.189 d
T1 1100.436±16.571 ab 915.777±20.900 a 717.403±22.549 a
T2 1108.081±25.848 ab 923.651±17.580 ab 731.083±22.291 ab
T3 1160.567±33.114 bc 961.774±16.661 b 769.983±19.220 b
T4 1194.663±54.229 c 1005.077±26.956 c 830.383±16.479 c
T5 1160.712±22.361 bc 936.849±19.690 ab 764.317±27.984 b
东莨菪碱
Scopolamine
(μg g-1 DW)		 T0 705.793±9.434 c 712.340±4.758 c 706.746±6.777 e
T1 608.653±18.251 a 436.066±42.860 a 322.495±13.838 a
T2 639.789±29.007 ab 443.938±22.239 a 342.123±13.148 ab
T3 703.185±23.661 c 491.396±19.175 ab 363.689±24.281 bc
T4 691.481±28.849 c 533.542±44.175 b 425.387±36.488 d
T5 665.574±21.782 bc 482.438±38.057 ab 384.401±18.008 c

Fig. 4

Effects of Ca2+ on relative expression level of PMT, H6H, TR I gene in roots (A) and leaves (B) of the Atropa belladonna L. under UV-B stress Bars superscripted by different letters are significantly different among different treatments in the same gene at the 0.05 probability level. T0 group indicates untreated blank control groups; T1 group indicates UV-B radiation treatment for 12 days; T4 group indicates exogenous 6 mmol L-1 Ca2+ treatment for 12 days under UV-B stress."

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