JIA-2018-09

1968 WANG Ling-shuang et al. Journal of Integrative Agriculture 2018, 17(9): 1959–1971 A close association has been proposed between 2,4-D and stress response (Cho et al. 2000; Teixeira et al. 2006; Pan et al. 2010). Therefore, this study mainly focused on the regulation of GmBIN2 in response to stress tolerance. GmBIN2 was shown to contain a serine/threonine protein kinase catalytic domain (Fig. 1). The protein structure, amino acid sequence and evolutionary relationship of the GmBIN2 protein indicated that GmBIN2 belonged to GSK3 kinase family. GmBIN2 expression was increased by salt and drought stresses (Fig. 2). When subjected to different NaCl and mannitol concentrations, transgenic Arabidopsis seeds observably increased in germination rate and root length compared to wild-type seeds (Fig. 3). Our results indicated that transgenic Arabidopsis plants showed a high resistance to salt and drought stresses. Further examination of Na + , K + , and Ca 2+ contents revealed that GmBIN2 could reduce cellular Na + content and increase cellular Ca 2+ content in transgenic plants under NaCl treatment (Fig. 4). We know Ca 2+ is an important messenger in plants, and plays an important role in regulating plant resistance to salt stress. It has been shown that increased Ca 2+ content may restrict the Na + influx and K + efflux to maintain cellular K + /Na + homeostasis (Amtmann and Sanders 1999; White and Davenport 2002; Shabala et al. 2006). Additionally, GmBIN2 may trigger a Ca 2+ -dependent pathway to control the ion content to enhance salt tolerance in transgenic Arabidopsis (Liu and Zhu 1998; Pardo et al. 1998). In addition, two NaCl stress-responsive genes, RD29A and AtCBL1 , showed altered expression in transgenic Arabidopsis plants (Fig. 5). RD29A is involved in response to salt, drought, and cold stresses (Yamaguchi-Shinozaki and Shinozaki 1993), whereas AtCBL1 is reported to function as Ca 2+ sensor in osmotic stress signaling and could also be induced by salt and drought stresses (Kudla et al. 1999; Cheong et al. 2003). In the present study, expression levels of RD29A and AtCBL1 genes were higher in transgenic Arabidopsis than in wild-type plants under NaCl stress and overexpression of GmBIN2 up-regulated RD29A expression even in the absence of NaCl stress, suggesting that GmBIN2 may play different roles in inducing these stress- responsive genes under different conditions and is involved in a complicated regulatory stress response network. To date, there are 10 AtSK genes in the GSK3/SHAGGY-like protein kinases family (Dornelas et al. 1998). We know that AtSK22 and AtSK23 are the two closest homologues of AtBIN2 ( AtSK21 ). Like in mammals, Arabidopsis GSK3 functions both redundantly and specifically to regulate plant development. The specificity of GSK3 has been exhibited in several studies. AtBIN2 has been shown to have a primary role in the BR signaling pathway and is not responsive to abiotic stress (Charrier et al. 2002; Yan et al. 2009). In addition, overexpression of GmBIN2 in transgenic Arabidopsis did not cause a differential expression of AtBIN2 compared to wild type (Appendix B). However, AtGSK1 ( AtSK22 ) has been shown to be involved in the NaCl- stressed signal pathway, and overexpression of AtGSK1 enhanced NaCl tolerance in Arabidopsis . When transgenic plants were under NaCl stress, AtGSK1 was seen to activate a Ca 2+ -dependent signaling pathway and several small Ca 2+ -binding proteins, including AtCBL1, that may activate downstream components of NaCl stress signaling and enhance NaCl tolerance (Piao et al. 2001). In our study, we posit that the GmBIN2 gene may encode protein that is functionally homologous to AtGSK1 and may act through similar pathways to regulate the stress signaling. Furthermore, overexpression of GmBIN2 in soybean hairy roots also showed significantly higher relative root growth rate than control roots under stress treatments (Fig. 6). Under salt and drought conditions, transgenic hairy roots showed a lower relative electrical conductivity compared to control roots, meanwhile, transgenic roots accumulated more proline and SOD (Fig. 7). When plant tissues are under stress conditions, the structure of the cell membrane is destroyed, and the membrane permeability is greatly increased, resulting in an increase of the relative electrical conductivity of the tissue exudate (Feng et al. 2005). The lower relative electrical conductivity of transgenic hairy roots meant a lower degree of damage to the cell membrane. SOD is an antioxidant enzyme used to eliminate oxygen free radicals and counteract the toxic effects on cells. For example, overexpression of tomato Cu/Zn superoxide dismutase could enhance oxidative stress defense in transgenic potato (Perl et al. 1993). Our findings showed that SOD activities in GmBIN2 -overexpression hairy roots were higher than in control roots after treatment with salt and drought (Fig. 7-B). This might be the result of reducing the toxic effects of superoxide radicals and enhancing stress tolerance. Plants accumulate a large amount of proline in response to environmental stresses (Saradhi and Mohanty 1997) and high proline content can result from injury to the plants (Liu and Zhu 1997; Wang et al. 2003). Therefore, proline accumulation may be a signal of stress tolerance in plants. The present results inferred that the high proline contents of transgenic soybean hairy roots may be an indicator for salt and drought stresses, and high proline accumulations in soybean hairy roots could protect transgenic lines against injury from salt and drought stresses. Moreover, we investigated the expression of the GmCBL1 gene, a homolog of AtCBL1 in soybean hairy roots. CBL1 is a calcineurin B-like calcium sensor and plays a critical role in plant responses to abiotic stress signaling pathways. Importantly, CBL1 functions as a positive regulator of salt and drought responses (Cheong et al. 2003). It has been