Overwhelming glycyrrhizin production in Glycyrrhiza glabra induced by rihizobial symbiosis

Abstract

We reported that Glycyrrhiza uralensis inoculated with rhizobium tended to increase biomass production and glycyrrhizic acid (GL) production, in this study we have also achieved drastically increase in biomass and GL production in Glycyrrhiza glabra. At thirty days after inoculation (DAI), a significant increase in SPAD values was observed, and the expression of GL synthesis marker genes was also significantly increased. At 150 DAI, a significant increase in biomass was observed. Characteristically, it was also found that thick roots were enlarged by rhizobial inoculation. In addition, the expression of GL synthesis marker genes was also significantly increased. Moreover, GL content per unit root dry weight reached 4%, and GL production per plant increased six times compared to uninoculated plants. Moreover, we tried to reveal the mechanism of induction of GL production by rhizobial inoculation. Since it has been reported that the expression of jasmonic acid (JA) synthesis marker genes is increased by rhizobium in soybean, we investigated the expression of those genes in G. glabra, and found that GgMYC2 and GgJAR1 were up-regulated at Thirty DAI. Furthermore, methyl jasmonate treatment increased the expression of GL synthesis marker genes, suggesting that JA signaling is involved in the increased GL production due to rhizobial inoculation. These results aid in understanding the mechanism of increased GL production through the introduction of rhizobial symbiosis, and show the potential for providing a technology to significantly shorten the cultivation period for the production of Glycyrrhiza that meets the criteria for herbal medicines.

Keywords: Glycyrrhiza; Glycyrrhizic acid; Nodulation; Rhizobium; Symbiosis.

Fig. 1

Phenotype of root nodule formed on the G. glabra prepared from cuttings and grown at 30 days after rhizobial inoculation. Arrows indicate nodules formed on the roots

Fig. 2

Growth, nodulation and nitrogen fixation ability of G. glabra prepared from cuttings at 30 days after rhizobial inoculation. J8(−) indicates uninoculated plants and J8(+) indicates plants inoculated with strain J8. Shoot length (A), shoot weight (B), root length (C), root weight (D) were measured. Leaf SPAD values (E) were measured on three leaves of each plant and the average value was calculated. Number of nodules (F), nodule weight (G), acetylene reduction activity (H) were measured. Shoot length and weight; J8(−), J8(+): n = 11, n = 10, respectively. Root length and weight; n = 5. Leaf SPAD value; n = 10. Nodule number and acetylene reduction activity; J8(−), J8(+):n = 7, n = 5, respectively. Nodule weight; J8(−), J8(+):n = 7, n = 4, respectively. Error bars indicate standard errors. *, ** indicate significant differences. (Student’s t-test: *p < 0.05, **p < 0.01)

Fig. 3

Expression of genes involved in the GL biosynthetic pathway in the root of G. glabra at 30 days after rhizobium inoculation. (n = 4 in uninoculated lateral root or J8-inoculated adventitious root, n = 5 in uninoculated adventitious root or J8-inoculated lateral root) Expression levels of CYP88D6 gene (A), CYP72A154 gene (B), CSyGT gene (C). 18S rRNA gene was used as the internal control. For the respective gene, the expression level of J8(−) in the adventitious root was set to 1. Error bars indicate standard errors. *, ** indicate significant differences. (Student’s t-test: *p < 0.05, **p < 0.01)

Fig. 4

Growth, nodulation and nitrogen fixation activity of G. glabra prepared from cuttings grown at 150 days after rhizobium inoculation. Shoot length (A), shoot weight (B), root length (C), root weight (D), leaf SPAD value (E), number of nodules (F), nodule weight (G), acetylene reduction activity (H) was measured. Shoot length, shoot weight, root weight, SPAD value, nodule number, and nodule weight; J8(−), J8(+), n = 8, n = 21, respectively. Root length; J8(−), J8(+): n = 8, n = 16, respectively. Acetylene reduction activity; J8(−), J8(+): n = 7, n = 17, respectively. Error bars indicate standard errors. *, ** indicate significant differences. (Student’s t-test: *p < 0.05, **p < 0.01)

Fig. 5

Root phenotype of G. glabra prepared from cuttings and grown for 150 days after rhizobial inoculation. Phenotypes of shoot of uninoculated plants (A), shoot of plants inoculated with rhizobia (B), nodules formed on roots (C), roots of uninoculated plants (D), thick roots with thin roots removed from uninoculated roots (E), roots of plants inoculated with rhizobia (F), thick roots with fine roots removed from roots of plants inoculated with rhizobia (G)

Fig. 6

Shoot and root diameters of G. glabra prepared from cuttings grown for 150 days after rhizobial inoculation. Shoot and thick root diameters were measured at 1 cm above and below the base. J8(−), J8(+): n = 8, n = 19, respectively. Shoot diameter (A), root diameter (B) was measured. Error bars indicate standard errors. *, ** indicate significant differences. (Student’s t-test: *p < 0.05, **p < 0.01)

Fig. 7

Expression of genes involved in the GL biosynthetic pathway in the root of G. glabra at 150 days after rhizobial inoculation. Expression levels of CYP88D6 gene (A), CYP72 A154 gene (B), CSyGT gene (C). J8(−), J8(+): n = 5, n = 5. 18S rRNA gene was used as the internal control. For the respective gene, the expression level of J8(−) was set to 1. Error bars indicate standard errors. *, ** indicate significant differences. (Student’s t-test: *p < 0.05, **p < 0.01)

Fig. 8

GL content of G. glabra prepared from cuttings and grown for 150 days after rhizobium inoculation. Total GL content per unit of roots weight (A), GL content per root dry weight (B) was measured. J8(−), J8(+); n = 5. Error bars indicate standard errors. *, ** indicate significant differences. (Student’s t-test: *p < 0.05, **p < 0.01)

Fig. 9

Expression of JA synthesis marker genes in the root of G. glabra at 30 days after rhizobial inoculation (n = 4 in uninoculated lateral root or J8-inoculated adventitious root, n = 5 in uninoculated adventitious root or J8-inoculated lateral root) Expression levels of JAR1 gene (A), MYC2 gene (B). 18S rRNA gene was used as the internal control. For the respective gene, the expression level of J8(−) in the adventitious root was set to 1. Error bars indicate standard errors. *, ** indicate significant differences. (Student’s t-test: *p < 0.05, **p < 0.01)

Fig. 10

Expression of genes involved in the GL biosynthetic pathway in the root of G. glabra on MeJA-treated. Expression levels of CYP88D6 gene (A), CYP72A154 gene (B), and CSyGT gene (C). (0 µM: n = 5, 10 µM: n = 5, 100 µM: n = 5) 18S rRNA gene was used as the internal control. For the respective gene, the expression level of J8(−) in the adventitious root was set to 1. Error bars indicate standard errors. Different letters indicate significant differences (p < 0.05). Statistical methods were performed using analysis of variance (ANOVA) and the Turkey method