JIA-2018-09

1967 WANG Ling-shuang et al. Journal of Integrative Agriculture 2018, 17(9): 1959–1971 roots. Consistent with the finding of higher proline contents in transgenic hairy roots, the SOD activity in transgenic roots was dramatically higher than that of in the control roots under drought and salt stress conditions (Fig. 7-C). In addition, we investigated GmCBL1 gene expression, a homolog of AtCBL1 in soybean hairy roots. As a result, transcript abundance of GmCBL1 in transgenic roots was higher than that in wild-type roots under salt and drought stresses (Fig. 7-D). The analysis above implied that transgenic roots overexpressing GmBIN2 suffered less damage to cell membranes and accumulated more proline and SOD to resist the damage caused by stresses. It was also demonstrated that the overexpression of GmBIN2 in transgenic soybean hairy roots may activate a stress- signaling pathway through the induction of the GmCBL1 gene. 4. Discussion In the present study, we found that the GmBIN2 gene, a member of GSK3 protein kinase family in soybean, enhanced the resistance to salt and drought stresses in Arabidopsis and soybean hairy roots when overexpressed. It has been shown that plants have more divergent GSK3 isoforms compared with mammalian GSK3s, with 10 identified in Arabidopsis and nine identified in rice (Yoo et al. 2006). Youn and Kim (2015) investigated the phylogenetic relationships, gene structure, and the diverse cellular signal transduction pathways of the GSK3 kinase in Arabidopsis and rice to understand the versatile functions of plant GSK3 kinases. It has been demonstrated that plant GSK3 kinases play a prominent part in multiple signaling pathways in response to diverse environmental stimuli, however, the specific functions of GSK3 kinase in soybean remain largely unknown. According to the data analyzed in the Phytozome database, 22 soybean GSK3 kinases existed in the soybean genome and five of these GSK3 members exhibited a close relationship with GmBIN2 protein. In the previous work of our laboratory, GmBIN2 was screened under different 2,4-D treatment concentrations in the soybean Dongnong 50 cultivar to determine the differentially expressed genes. 0 10 20 30 40 50 60 70 80 A B C D Control 150 mmol L –1 NaCl 200 mmol L –1 mannitol Relative electrical conductivity (%) K599 GmBIN2 -oe ** ** 0 10 20 30 40 50 60 70 Control 150 mmol L –1 NaCl 200 mmol L –1 mannitol Proline content (μg g –1 ) 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Control 150 mmol L –1 NaCl 200 mmol L –1 mannitol SOD activity (U mg –1 protein) 0 2 4 6 8 10 12 14 Control 150 mmol L –1 NaCl 200 mmol L –1 mannitol Relative expression GmCBL1 ** ** ** ** ** ** Fig. 7 Altered physiological indicators and gene expression in soybean hairy roots. A, relative electrical conductivity between K599 and GmBIN2 -oe (transgenic roots) with and without 150 mmol L –1 NaCl or 200 mmol L –1 mannitol treatments for 7 d. B, proline contents between K599 and GmBIN2 -oe with and without 150 mmol L –1 NaCl or 200 mmol L –1 mannitol treatments for 7 d. C, superoxide dismutase (SOD) activities between K599 and GmBIN2 -oe with and without 150 mmol L –1 NaCl or 200 mmol L –1 mannitol treatments for 7 d. D, expression of GmCBL1 in K599 and GmBIN2 -oe with and without 150 mmol L –1 NaCl or 200 mmol L –1 mannitol treatments for 2 d. Transcript abundances were normalized against the soybean reference gene Actin4 . Values are mean±SD ( n =5). Each experiment was performed three times with similar results. Asterisks indicate significant differences ( ** , P <0.01).