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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (11): 2826-2839.doi: 10.3724/SP.J.1006.2022.14172

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

Effects of shading at grain filling stages on anthocyanin accumulation and related gene expression characteristics in seed coat of black mung bean

MA Chao(), FENG Ya-Lan, WU Shan-Wei, ZHANG Jun, GUO Bin-Bin, XIONG Ying, LI Chun-Xia*(), LI You-Jun*()   

  1. Agronomy College, Henan University of Science and Technology / Dry-land Agricultural Engineering Technology Research Center in Henan, Luoyang 471000, Henan, China
  • Received:2021-09-24 Accepted:2022-03-25 Online:2022-11-12 Published:2022-04-20
  • Contact: LI Chun-Xia,LI You-Jun E-mail:machao840508@163.com;lichx@haust.edu.cn;kdnxylyj@126.com
  • Supported by:
    The National Natural Science Foundation of China(31401323);The Natural Science Foundation of Henan Province(222300420430);The Training Program for University Young Key Teachers in Henan Province(2021GGJS050)

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Abstract:

To investigate the accumulation of anthocyanin in the seed coat of mung bean and the effects of shading on it, two cultivars with different seed colors (green and black) were both shaded after flowering 5 days under the conditions of field cultivation. The accumulation of various anthocyanin components in seed coat were assessed using spectrophotometry combined with liquid chromatography tandem mass spectrometer. The relative expressions of structural genes and the activity of three anthocyanin synthase enzymes were both analyzed. The results showed that the seed coat of black mung bean began to accumulate anthocyanins at 16 days after flowering (S3 period), and the detectable anthocyanins at the near-mature period (S4 period) were delphinidin (88.67%, main pigment), cyanidin (8.68%), procyanidin (1.05%), petunidin (0.94%), pelargonidin (0.34%), and peonidin (0.32%) according to the proportion of each component. In the black mung bean seed coat at S4 period, the main pigment components of the three anthocyanins were delphinidin-3-O-glucoside (76.61%), cyanidin-3-O-glucoside (89.17%), and procyanidin B3 (28.64%). In general, the relative expression levels of the anthocyanin synthesis structural genes (PAL, C4H, 4CL, CHS, CHI, F3’H, F3’5’H, DFR, LDOX, and UFGT) in the seed coat of black mung bean were significantly higher than green mung bean, among which DFR (37185%), LDOX (4409%), and F3’5’H (3299%) had the largest differential expression multiples. The activities of PAL, CHI, and UFGT in black mung bean seed coat were also greater than those of green mung bean. The relative expression levels of nine anthocyanin synthesis structural genes and the activity of two anthocyanin synthesis-related enzymes were dramatically positively correlated with the contents and the total contents of most anthocyanin components. The relative expression levels of eleven anthocyanin synthesis structural genes and the activities of three anthocyanin synthesis-related enzymes were remarkably reduced under shading conditions, which ultimately led to a noticeable decrease in the content and total amount of anthocyanin components. The results provide a theoretical foundation for the regulation mechanism of black mung bean seed coat coloration, and also contribute to the auxiliary selection of black mung bean breeding.

Key words: black mung bean, shading, anthocyanin, seed coat, the relative expression levels of genes

Fig. 1

Phenotypes of mature seeds of green mung bean and black mung bean"

Table 1

Primers in this study"

基因名称
Gene name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
VrPAL TCTTCATCCTATCACCCCACA TCATGCTATCAATCACCCAATC
VrC4H ATACTAAAGCATTCCACCGTT AGATACATCACCACCTACCCA
Vr4CL GCTGGAAGAAGAACGCTTGTT GTTGTTGGATTGAATGGTGGG
VrCHS GTCCATTCCCAAACCACCTCA TCGGCACTTCCACAACCACTA
VrCHI AAGCTAATAAGAGGATCGAAG AGAGAAACTAAGCCCCAAG
VrF3H CAAATCAAAGGAACCACAAG TTATCACAATCATACACAACAACT
VrF3'H GGTGGTGGAGGTGATGGTG ACTTGGGTCAGGATTCTTGGA
VrF3'5'H CAAAGTGATAGGTAGGGAGCG GGTTGAGAGGAGTTGAAGGGT
VrDFR AATGAAGTGATAAAGCCGACA GCATAAGAAAGGGACCAACG
VrLDOX AGGAGTTGGAGAACATAGGAA AGGATAAACAAGGTGGAAGAA
VrUFGT GGTGTGAGGATAAGATGGTTGT TGCGAGGTAGGGAATGAAGT
VrACTIN GTTCTGTTCCAGCCATCCAT GTGGTGCGACAACCTTGATT

Fig. 2

Phenotypes of seed coat of green mung bean and black mung bean under shaded treatment at different stages S1: 10 days post anthesis; S2: 13 days post anthesis; S3: 16 days post anthesis; S4: 10 days post anthesis."

Fig. 3

Effects of shading on anthocyanin contents in seed coat of green mung bean and black mung bean A: HCl-Methanol method; B: LC-MC/MC method; C: the correlation analysis of result between HCl-Methanol method and LC-MC/MC method. Values are means of three biological replicates with error bars indicating standard derivations (SD). The same treatment with different letters is significantly different at the 0.05 probability level in different stage. GS1: 10 days post anthesis of green mung bean; GS2: 13 days post anthesis of green mung bean; GS3: 16 days post anthesis of green mung bean; GS4: 19 days post anthesis of green mung bean; BS1: 10 days post anthesis of black mung bean; BS2: 13 days post anthesis of black mung bean; BS3: 16 days post anthesis of black mung bean; BS4: 19 days post anthesis of black mung bean."

Fig. 4

Effects of shading on the proportion of anthocyanin components in seed coat of green mung bean and black mung bean A: illuminated; B: shaded. Abbreviations are the same as those given in Fig. 3."

Table 2

Efects of shading on the rhaire contet of depinidin in sed coat of gre mg bean and black mung bean"

Table 3

Effects of shading on relative content of cyanidins in seed coat of cyanidins in sed coat of green mung bean and black mung bean"

Table 4

Effect of shading on relative content of procyanidins in seed coat of green mung bean and black mung bea n."

Fig. 5

Effects of shading on the relative expression of biosynthesis genes of anthocyanin in seed coat of green mung bean and black mung bean Abbreviations are the same as those given in Fig. 3. Values are means of three biological replicates with error bars (SDs). The same treatment with different letters is significantly different at the 0.05 probability level at different stages."

Fig. 6

Effects of shading on the activity of biosynthesis related enzyme of anthocyanin in seed coat of green mung bean and black mung bean Abbreviations are the same as those given in Fig. 3. Values are means of three biological replicates with error bars (SDs). The same treatment with different letters is significantly different at the 0.05 probability level at different stages."

Table 5

Correlation coefficients between anthocyanin content and related gene expression and enzymatic activity"

指标
Index		 总花青素
Total
anthocyanin		 飞燕草色素
Delphinidin		 矢车菊色素
Cyanidin		 矮牵牛色素
Petunidin		 原花青素
Procyanidine		 天竺葵色素
Pelargonidin		 芍药花色素
Peonidin
基因相对表达量
Relative expression
level of genes		 PAL 0.97** 0.99** 0.98** 0.99** 0.33 0.63 0.82**
C4H 0.89** 0.96** 0.92** 0.95** 0.46 0.73* 0.90**
4CL 0.93** 0.98** 0.95** 0.98** 0.37 0.66 0.86**
CHS 0.74* 0.84** 0.78** 0.84** 0.61 0.80** 0.91**
CHI 0.37 0.50 0.42 0.50 0.85** 0.86** 0.83**
F3H 0.01 0.02 -0.01 0.02 0.73* 0.62 0.45
F3'H 0.99** 0.98** 0.99** 0.99** 0.33 0.63 0.80**
F3'5'H 0.92** 0.93** 0.91** 0.94** 0.43 0.69 0.86**
DFR 0.94** 0.94** 0.93** 0.97** 0.42 0.69 0.85**
LDOX 0.93** 0.94** 0.93** 0.96** 0.44 0.70* 0.86**
UFGT 0.84** 0.82** 0.83** 0.83** 0.49 0.72* 0.81**
酶活性
Activity
of enzymes		 PAL 0.87** 0.93** 0.88** 0.91** 0.42 0.69 0.87**
CHI 0.43 0.08 0.01 0.08 0.65 0.54 0.44
UFGT 0.77** 0.89** 0.83** 0.87** 0.44 0.69 0.87**
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