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    • In plants NO production is

    2022-05-18

    In plants, NO production is one of the early responses to stress (Tossi, Lamattina, & Cassia, 2009). Usually, NO signal transduction requires the involvement of downstream signaling compounds. We further investigated the effects of cGMP on GSK-3 expression. The data showed that LY83583 (a cGMP inhibitor) partly blocked the UV-B stress-enhanced gene (Fig. 2) and protein (Fig. 3) protease inhibitors of GSK-3. 8-Br-cGMP (a cGMP analog) abrogated this inhibition. 8-Br-cGMP used alone also elevated gene (Fig. 2) and protein (Fig. 3) expression of GSK-3. Therefore, the data suggested that cGMP mediated UV-B radiation-induced GSK-3 activation. Both guanine nucleotide-binding protein and cGMP can mediate periodic calcium oscillations (Ewald et al., 1988, Moustafa et al., 2011), and transient elevation in intracellular calcium leads to activation of GSK-3 (Hartigan & Johnson, 1999), which indicates that calcium might be the downstream messenger of cGMP required to activate GSK-3. Subsequently, we further explored the mechanism of the enhancement of cGMP-induced GSK-3 expression. The results showed that KT5823, a PKG inhibitor, significantly reduced the elevation of UV-B-induced gene (Fig. 2) and protein (Fig. 3) expression of GSK-3. 8-Br-cGMP (a cGMP analog) reversed the impact caused by KT5823. This demonstrated that cGMP-induced GSK-3 activation was dependent on PKG. Accordingly, upon cGMP stimulation, overexpressing PKG resulted in a significant increase in phosphorylation of GSK-3 (Das et al., 2008, Sanyal et al., 2013) possibly through regulation of Akt and ERK (Komalavilas et al., 1999). In the light of our previous confirmation that NO stimulates cGMP synthesis (Jiao et al., 2016), we can infer that the cGMP/PKG pathway mediates NO-activated GSK-3 in soybean sprouts under UV-B radiation. In a previous study from our group, we reported that NO, with cGMP, is involved in isoflavone production under UV-B radiation (Jiao et al., 2016). To further explore whether NO-cGMP/PKG pathway-activated GSK-3 mediates UV-B-stimulated isoflavone synthesis, we investigated the effects of bikinin (a GSK-3 inhibitor) on UV-B radiation-triggered isoflavone synthesis in soybean sprouts. The data showed that bikinin treatment resulted in significantly lower isoflavone content (Fig. 4). This effect resulted from a decrease in CHS and IFS activity (Fig. 5A and C) and gene (Fig. 5B and D) and protein (Fig. 6) expression under UV-B radiation. Supplementation with SNP and 8-Br-cGMP reversed the reduction caused by bikinin. Treatments with SNP and 8-Br-cGMP alone also enhanced isoflavone accumulation (Fig. 4), activity (Fig. 5A and C), and gene (Fig. 5B and D) and protein (Fig. 6) expression of CHS and IFS. Therefore, these data demonstrated that isoflavone synthesis up regulated by UV-B-stimulated NO-cGMP was mediated by GSK-3. The regulation of isoflavone synthesis by GSK-3 might be mediated by Ca. The over-expression of AtGSK1 in transgenic plants resulted in the induction of both AtCBL1 and AtCP1, which are two members of the Ca sensor family, AtCBLs. AtCBL1 has been identified to act as a Ca sensor in signaling under stress (Kudla, Xu, Harter, Gruissem, & Luan, 1999), and AtCP1 is a small Ca-binding protein with EF hands (Jang et al., 1998). These proteins interact with protein kinases, called CBL-interacting protein kinases (CIPKs; Shi et al., 1999). Therefore, GSK is an activator of Ca under stress in plants. An elevation in [Ca]i stimulated by UV-B stress could enhance the expression of isoflavone biosynthetic enzymes (Frohnmeyer, Loyall, Blatt, & Grabov, 1999). A previous study also showed that EGTA (a Ca chelator) weakened isoflavone production under UV-B stress (Jiao, Yang, & Gu, 2016). Thus, we speculate that the induction of isoflavone accumulation by GSK-3 may be mediated by Ca. Overall, NO-cGMP-activated GSK-3 up regulated the activity and expression levels of CHS and IFS, and increased isoflavone production.
    Funding sources This project was supported by the National Natural Science Foundation of China (31671862).