An <i>in vivo</i> GA‐ and ABA‐responsive dual‐luciferase reporter system for simultaneous detection of GA and ABA responses, hormone crosstalk and heat stress response in rice

Abstract

Gibberellic acid (GA) and abscisic acid (ABA) are the primary phytohormones that play antagonistic roles in the control of the transition from embryogenesis to germination in seeds (Finch-Savage and Leubner-Metzger, 2006). Given the low concentrations of GA and ABA in seeds and plants, biosynthesis inhibitors (Toh et al., 2008) or deficient mutants (Debeaujon and Koornneef, 2000) have been used to investigate the relative importance of their physiological functions. However, biosynthetic inhibitors may have pleiotropic effects in plants, and the use of GA- or ABA-deficient mutants does not permit simultaneous monitoring of dynamic changes in GA and ABA during seed development or germination (Dejonghe et al., 2018). Analytical determination provides accurate quantification of GA and ABA contents, but the extraction procedure is time-consuming, and the antagonism and dynamic changes between GA and ABA may not be detected. Reporter genes directed by an ABA- or a GA-responsive promoter have been used to study the concentration or signalling of GA or ABA. The ABA-responsive complex (ABRC) has been manipulated in the control of several stress-tolerance genes expressed in transgenic plants (Chen et al., 2015; Garg et al., 2002; Shen and Ho, 1995). In addition, the α-amylase32b (Amy32b) promoter has been known to be highly induced by GA during seed germination in barley (Gomez-Cadenas et al., 2001; Lanahan et al., 1992). An individual ABRC or Amy32b promoter has been used to direct the expression of reporter genes. However, a simple and versatile system for rapid and simultaneous determination of antagonistic and dynamic changes in GA and ABA remains elusive. In this study, an in vivo GA/ABA dual-luciferase reporter system was established (Figure 1a). Transgenic rice harbouring a firefly luciferase (firefly LUC) directed by two copies of the ABRC together with a Renilla luciferase (Renilla LUC) directed by the Amy32b promoter was generated. In developing seeds, firefly LUC activity was notably enhanced 323-fold at 28 days after flowering (DAF), which indicates that the ABA response was strongly and positively correlated with seed development. In contrast, Renilla LUC activity showed 30%–40% reduction at 7–21 DAF and increased slightly at 28 DAF (Figure 1b). During seed germination, a 30%–40% decrease in firefly LUC activity was observed at 12–48 hours after imbibition (HAI). In contrast, Renilla LUC activity significantly increased at 12–48 HAI (Figure 1c). In the embryo, firefly LUC activity declined significantly at 24 and 48 HAI. However, the decline in firefly LUC activity at 24 and 48 HAI in the endosperm was less pronounced than that in the embryo (Figure 1d). In contrast, Renilla LUC activity increased greatly in the embryo and endosperm at 12-48 HAI (Figure 1e). These data indicate that the activation of the GA response preceded that of the ABA response after imbibition. Therefore, the dual-luciferase reporter system faithfully reflects the effects of ABA and GA in transgenic rice during seed germination. To assess the sensitivity of the ABA-responsive promoter to ABA, luciferase activities were measured in roots of rice seedlings due to high ABA concentrations in mature seeds. Measurement of firefly LUC activity revealed that 0.5, 1 and 5 μm ABA significantly increased promoter activity with treatment for 12–24 h. However, ABA treatment for 48-h attenuated firefly LUC activity (Figure 1f). The sensitivity of the Amy32b promoter in transgenic rice to GA was determined using embryoless half-seeds. Measurement of Renilla LUC activity revealed that GA concentrations higher than 10−3 μm significantly activated Amy32b promoter activity with treatment for 12 h. The GA response was increased strongly by GA treatment for 48 h (Figure 1g). The effects of methyl jasmonate (MJ), kinetin (KT) and salicylic acid (SA) on LUC activity in the roots were measured to examine the potential crosstalk of phytohormones in rice seedlings. After testing a range of phytohormone concentrations, rice seedlings were treated with or without 100 μm MJ, 100 μm KT or 1000 μm SA for 24 and 48 h. The firefly LUC activity was increased significantly by MJ at 24 h, but was strongly suppressed by MJ at 48 h. In contrast, Renilla LUC activity was increased by MJ at 24 and 48 h. Kinetin treatment markedly reduced firefly LUC activity at 24 and 48 h. However, Renilla LUC activity was notably enhanced at 24 and 48 h. The activation of Renilla LUC activity by kinetin is possibly caused by two putative cytokinin responsive elements in the Amy32b promoter. Treatment with SA significantly increased firefly LUC activity at 24 h but suppressed activity at 48 h. Renilla LUC activity was increased by SA at 24 h (Figure 1h and i). These results provide evidence for crosstalk between exogenous and endogenous phytohormones. Nevertheless, a significant difference in luminescence signal was observed within the control group without exogenous hormone at the different time points. This indicates that this system is quantitative but time-dependent in root tissues. Few studies have focused on the correlation between ABA and heat stress. Incubation of rice seeds at 27–37 °C results in 90%–97% germination. However, above this temperature range the germination percentage declines markedly (Fujino et al., 2004). The dual-luciferase reporter system was applied to investigate the changes in GA and ABA responses during seed germination under a suboptimal temperature (27 °C), supraoptimal temperature (37 °C) and heat stress (40°C). The expression of OsRab16A and OsAmy7, which are known to be highly induced by ABA and GA, respectively, was determined by qRT-PCR to compare with luciferase signal. After imbibition for 24 h, seed germination was observed only at 37 °C. At 36 h, 98%, 64% and 6% seed germination was observed at 37 °C, 27 °C and 40 °C, respectively (Figure 1j). Simultaneous detection of the changes in GA/ABA responses revealed that firefly LUC activity notably declined with imbibition for 8 and 2 h at 37 °C and 40 °C, respectively, compared with that observed at 27 °C. Almost no firefly LUC activity was detectable at 8 and 48 h of imbibition at 37 °C and 40 °C, respectively (Figure 1k). In contrast, firefly LUC activity declined slowly after seed germination at 27 °C (Figure 1l). These data indicate that the sensitivity of these plants to ABA is hampered or the degradation of ABA was strongly correlated with the increasing temperature during seed germination in rice. Renilla LUC activity increased significantly during the first 4 h of heat treatment (37°C and 40°C) compared with that observed at 27°C. With imbibition for 12–48 h, Renilla LUC activity gradually increased at 27°C and markedly increased at 37°C, whereas the GA response was suppressed under heat stress (40°C) (Figure 1l). Therefore, the GA response gradually increased followed by gradual degradation of ABA under suboptimal and supraoptimal temperatures. In contrast, both the ABA and GA responses were suppressed under heat stress. The GA/ABA dual-luciferase reporter system provides a simple, sensitive and reliable method to elucidate the role of genes crucial for signalling and antagonism of GA and/or ABA by stable transformation or transient expression in protoplasts. This system is also useful for studying the effect of exogenous environmental factors and endogenous factors on the dynamic changes in GA and ABA responses simultaneously. Our system measures transcriptional responses to GA and ABA, in contrast to the FRET reporters which directly measure GA and ABA concentration changes. These tools are complementary to address different biological questions. This work was supported by the Ministry of Science and Technology of Taiwan (MOST 108-2313-B-002-055-MY3). The authors declare no competing financial interests. CYW and CYH designed the experiments and wrote the manuscript. CYW performed the experiments and analysed the data. Both authors participated in data interpretation.

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