A comparative study of differences in condensed tannin synthesis between Medicago sativa and Onobrychis viciifolia at different growth stages

doi:10.11686/cyxb2025115

Abstract

Abstract:

In this study， we explored the differences in condensed tannin （CT） synthesis and accumulation between alfalfa （Medicago sativa） and sainfoin （Onobrychis viciifolia）. Stems and leaves of the alfalfa cultivars ‘Gannong No. 3’， ‘Gannong No. 7’， and ‘Elite’， and the sainfoin ‘Gansu’ were sampled across six growth stages from seedling to maturity. The contents of total phenolics， total flavonoids， CTs， and secondary metabolites （rutin， gallic acid， epicatechin gallate， epicatechin， catechin） were determined. In addition， to decipher the regulatory mechanism of condensed tannin synthesis， we measured the activities of key enzymes in the phenylpropanoid metabolic pathway （phenylalanine ammonia-lyase， PAL； 4-coumarate-CoA ligase， 4CL； cinnamate 4-hydroxylase， C4H） and the flavonoid pathway （anthocyanidin reductase， ANR and leucoanthocyanidin reductase， LAR）. The results show that： 1） The condensed tannin content varied significantly among growth stages， peaking at the bud stage in O. viciifolia ‘Gansu’ and the pod-setting stage in M. sativa， while the condensed tannin content was generally higher in leaves than in stems. 2） The trends in the contents of total flavonoids and rutin were analogous to those of condensed tannins. 3） The activities of 4CL， ANR， PAL， LAR， and C4H significantly increased at the bud stage of O. viciifolia ‘Gansu’ and the pod-setting stage of M. sativa， indicative of enhanced metabolic flow of the phenylpropanoid pathway towards condensed tannin synthesis. Among the alfalfa varieties， M. sativa ‘Gannong No. 3’ exhibited significantly higher condensed tannin content at the pod-setting stage （P<0.05）， suggesting stronger tannin synthesis potential. The results of this study clarify the dynamic correlations between key CT synthesis enzymes and metabolites， providing a theoretical basis for metabolic regulation of CT in forage crops and for breeding high-tannin varieties.

Key words: Medicago sativa, Onobrychis viciifolia, condensed tannins, phenolic acids

Li-juan CHEN, Rong GAO, Jian-xi WANG, Hui-ling MA. A comparative study of differences in condensed tannin synthesis between Medicago sativa and Onobrychis viciifolia at different growth stages[J]. Acta Prataculturae Sinica, 2026, 35(2): 221-236.

Figures/Tables 15

Table 1 Gradient elution program of the mobile phase

时间 Time （min）	流速 Flow rate （mL·min^-1）	流动相 Mobile phase （A，%）	流动相 Mobile phase （B，%）
0.0	1.0	5	95
15.0	1.0	20	80
23.0	1.0	25	75
25.0	1.0	30	70
26.0	1.0	40	60
26.5	1.0	5	95
31.0	1.0	5	95

Table 2 Calibration curve analysis of flavonoid compounds by ultra performance liquid chromatography （UPLC）

化合物 Compound	保留时间 Retention time （min）	回归方程 Calibration curve equation	决定系数 Determination coefficient （R2）
芦丁 Rutin	20.911	y=6072.9x+3.537	1.0000
没食子酸 Gallic acid	7.043	y=30842.0x-157055.000	0.9999
表儿茶素没食子酸酯 Epicatechin gallate	21.957	y=16244.0x-60958.000	0.9994
表儿茶素 Epicatechin	16.745	y=7278.5x-4627.600	1.0000
儿茶素 Eatechin	13.979	y=7794.4x-25134.000	0.9993

Fig.1 Rutin content in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.2 Gallic acid content in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.3 Temporal variation in epicatechin gallate content of alfalfa cultivars and O. viciifolia ‘Gansu’ during distinct growth stages

Fig.4 Epicatechin content in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.5 Catechin content in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.6 Total phenolic content in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.7 Total flavonoid content in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.8 Condensed tannins content in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.9 Dynamic analysis of 4CL， ANR， C4H， LAR and PAL enzyme activities in M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.10 Analysis of phenolic and flavonoid compound contents in the stems of M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.11 Analysis of phenolic and flavonoid compound contents in the leaves of M. sativa and O. viciifolia ‘Gansu’ at different growth stages

Fig.12 Analysis of phenolic， flavonoid compounds and enzyme activities in M. sativa and O. viciifolia ‘Gansu’

Fig.13 Analysis of the correlation among phenolic and flavonoid compounds and enzyme activities in M. sativa ‘Gannong No.3’ and O. viciifolia ‘Gansu’

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