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Functional characterization and reconstitution of ABA signaling components using transient gene expression in rice protoplasts.

Kim N, Moon SJ, Min MK, Choi EH, Kim JA, Koh EY, Yoon I, Byun MO, Yoo SD, Kim BG - Front Plant Sci (2015)

Bottom Line: These might be able to make thousands of combinations through interaction networks resulting in diverse signaling responses.By using TGERP, we could characterize successfully the effects of ABA dependent gene expression signaling components in rice.In conclusion, TGERP represents very useful technology to study systemic functional genomics in rice or other monocots.

View Article: PubMed Central - PubMed

Affiliation: Molecular Breeding Division, National Academy of Agricultural Science, Rural Development Administration Jeonju, South Korea.

ABSTRACT
The core components of ABA-dependent gene expression signaling have been identified in Arabidopsis and rice. This signaling pathway consists of four major components; group A OsbZIPs, SAPKs, subclass A OsPP2Cs and OsPYL/RCARs in rice. These might be able to make thousands of combinations through interaction networks resulting in diverse signaling responses. We tried to characterize those gene functions using transient gene expression for rice protoplasts (TGERP) because it is instantaneous and convenient system. Firstly, in order to monitor the ABA signaling output, we developed reporter system named pRab16A-fLUC which consists of Rab16A promoter of rice and luciferase gene. It responses more rapidly and sensitively to ABA than pABRC3-fLUC that consists of ABRC3 of HVA1 promoter in TGERP. We screened the reporter responses for over-expression of each signaling components from group A OsbZIPs to OsPYL/RCARs with or without ABA in TGERP. OsbZIP46 induced reporter most strongly among OsbZIPs tested in the presence of ABA. SAPKs could activate the OsbZIP46 even in the ABA independence. Subclass A OsPP2C6 and -8 almost completely inhibited the OsbZIP46 activity in the different degree through the SAPK9. Lastly, OsPYL/RCAR2 and -5 rescued the OsbZIP46 activity in the presence of SAPK9 and OsPP2C6 dependent on ABA concentration and expression level. By using TGERP, we could characterize successfully the effects of ABA dependent gene expression signaling components in rice. In conclusion, TGERP represents very useful technology to study systemic functional genomics in rice or other monocots.

No MeSH data available.


Rab16A promoter responds rapidly and significantly to ABA in rice protoplasts. Dual luciferase assay driven by ABA-responsive promoters. (A) pABRC3-fLUC reporter. (B) pRab16A-fLUC reporter. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; analysis of variance (ANOVA) with Tukey’s test, ∗P < 0.05, ∗∗∗P < 0.001.
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Figure 1: Rab16A promoter responds rapidly and significantly to ABA in rice protoplasts. Dual luciferase assay driven by ABA-responsive promoters. (A) pABRC3-fLUC reporter. (B) pRab16A-fLUC reporter. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; analysis of variance (ANOVA) with Tukey’s test, ∗P < 0.05, ∗∗∗P < 0.001.

Mentions: The first step to investigate ABA-dependent gene expression regulation using TGERP is to establish ABA-responsive reporter systems. Accordingly, we constructed a reporter vector consisting of Rab16A promoter fused with fLUC. The reason for using Rab16A promoter is that it has been known as a representative ABA-responsive marker gene in rice (Mundy and Chua, 1988; Mundy et al., 1990; Nakagawa et al., 1996; Miyoshi et al., 1999; Xu et al., 2006; Lu et al., 2009; Kim et al., 2012; Joo et al., 2014). We examined the ABA-responsive induction of fLUC using pRab16A-fLUC and pABRC3-fLUC, which has been used as a control compared to pRab16A. Both pABRC3-fLUC and pRab16A-fLUC were individually transfected with pAtUBQ-rLUC as internal control and transiently over-expressed for 2, 4, and 16 h in the presence of 0, 5, 10, and 20 μM ABA in rice protoplasts. As shown in Figure 1A, pABRC3-fLUC expression was not induced under any concentration of ABA at 2 and 4 h. However, as ABA concentration increased from 5 to 20 μM, pABRC3-fLUC expression at 16 h was induced 36, 50, and 63%, respectively. By contrast, pRab16A-fLUC expression was induced under ABA treatments beginning at 2 h (Figure 1B). Thus, ABA treatments led to rapid and significant induction of pRab16A-fLUC expression compared to the pABRC3-fLUC expression under the same conditions. In particular, the increasing rate of pRab16A-fLUC induction by addition of 5 μM ABA, that is 4.4-, 7.2- and 6.5-fold at 2, 4, and 16 h, respectively, was more efficient compared to that of pRab16A-fLUC induction by addition of 10 and 20 μM ABA suggesting that 5 μM ABA was sufficient to induce pRab16A-fLUC expression (Figure 1B). When we compared the increase of fLUC expression at each time under different ABA concentrations, the relative rate of fLUC induction was very similar at 4 and 16 h. Induction rates were 7.2-, 8.6-, and 10-fold at 4 h, and 6.5-, 8.6-, and 10-fold at 16 h under 5, 10, 20 μM ABA conditions, respectively (Figure 1B). However, at 2 h, fLUC was induced 4.4-, 5-, and 6.9-fold (Figure 1B). These data suggest that the proper time to monitor the ABA-mediated regulation of gene expression using pRab16A-fLUC is 4 h after ABA treatment in TGERP. Overall, these results indicate that Rab16A promoter fused to fLUC can be used as a reporter system for ABA-dependent gene expression due to rapid and significant response to ABA in TGERP.


Functional characterization and reconstitution of ABA signaling components using transient gene expression in rice protoplasts.

Kim N, Moon SJ, Min MK, Choi EH, Kim JA, Koh EY, Yoon I, Byun MO, Yoo SD, Kim BG - Front Plant Sci (2015)

Rab16A promoter responds rapidly and significantly to ABA in rice protoplasts. Dual luciferase assay driven by ABA-responsive promoters. (A) pABRC3-fLUC reporter. (B) pRab16A-fLUC reporter. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; analysis of variance (ANOVA) with Tukey’s test, ∗P < 0.05, ∗∗∗P < 0.001.
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Figure 1: Rab16A promoter responds rapidly and significantly to ABA in rice protoplasts. Dual luciferase assay driven by ABA-responsive promoters. (A) pABRC3-fLUC reporter. (B) pRab16A-fLUC reporter. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; analysis of variance (ANOVA) with Tukey’s test, ∗P < 0.05, ∗∗∗P < 0.001.
Mentions: The first step to investigate ABA-dependent gene expression regulation using TGERP is to establish ABA-responsive reporter systems. Accordingly, we constructed a reporter vector consisting of Rab16A promoter fused with fLUC. The reason for using Rab16A promoter is that it has been known as a representative ABA-responsive marker gene in rice (Mundy and Chua, 1988; Mundy et al., 1990; Nakagawa et al., 1996; Miyoshi et al., 1999; Xu et al., 2006; Lu et al., 2009; Kim et al., 2012; Joo et al., 2014). We examined the ABA-responsive induction of fLUC using pRab16A-fLUC and pABRC3-fLUC, which has been used as a control compared to pRab16A. Both pABRC3-fLUC and pRab16A-fLUC were individually transfected with pAtUBQ-rLUC as internal control and transiently over-expressed for 2, 4, and 16 h in the presence of 0, 5, 10, and 20 μM ABA in rice protoplasts. As shown in Figure 1A, pABRC3-fLUC expression was not induced under any concentration of ABA at 2 and 4 h. However, as ABA concentration increased from 5 to 20 μM, pABRC3-fLUC expression at 16 h was induced 36, 50, and 63%, respectively. By contrast, pRab16A-fLUC expression was induced under ABA treatments beginning at 2 h (Figure 1B). Thus, ABA treatments led to rapid and significant induction of pRab16A-fLUC expression compared to the pABRC3-fLUC expression under the same conditions. In particular, the increasing rate of pRab16A-fLUC induction by addition of 5 μM ABA, that is 4.4-, 7.2- and 6.5-fold at 2, 4, and 16 h, respectively, was more efficient compared to that of pRab16A-fLUC induction by addition of 10 and 20 μM ABA suggesting that 5 μM ABA was sufficient to induce pRab16A-fLUC expression (Figure 1B). When we compared the increase of fLUC expression at each time under different ABA concentrations, the relative rate of fLUC induction was very similar at 4 and 16 h. Induction rates were 7.2-, 8.6-, and 10-fold at 4 h, and 6.5-, 8.6-, and 10-fold at 16 h under 5, 10, 20 μM ABA conditions, respectively (Figure 1B). However, at 2 h, fLUC was induced 4.4-, 5-, and 6.9-fold (Figure 1B). These data suggest that the proper time to monitor the ABA-mediated regulation of gene expression using pRab16A-fLUC is 4 h after ABA treatment in TGERP. Overall, these results indicate that Rab16A promoter fused to fLUC can be used as a reporter system for ABA-dependent gene expression due to rapid and significant response to ABA in TGERP.

Bottom Line: These might be able to make thousands of combinations through interaction networks resulting in diverse signaling responses.By using TGERP, we could characterize successfully the effects of ABA dependent gene expression signaling components in rice.In conclusion, TGERP represents very useful technology to study systemic functional genomics in rice or other monocots.

View Article: PubMed Central - PubMed

Affiliation: Molecular Breeding Division, National Academy of Agricultural Science, Rural Development Administration Jeonju, South Korea.

ABSTRACT
The core components of ABA-dependent gene expression signaling have been identified in Arabidopsis and rice. This signaling pathway consists of four major components; group A OsbZIPs, SAPKs, subclass A OsPP2Cs and OsPYL/RCARs in rice. These might be able to make thousands of combinations through interaction networks resulting in diverse signaling responses. We tried to characterize those gene functions using transient gene expression for rice protoplasts (TGERP) because it is instantaneous and convenient system. Firstly, in order to monitor the ABA signaling output, we developed reporter system named pRab16A-fLUC which consists of Rab16A promoter of rice and luciferase gene. It responses more rapidly and sensitively to ABA than pABRC3-fLUC that consists of ABRC3 of HVA1 promoter in TGERP. We screened the reporter responses for over-expression of each signaling components from group A OsbZIPs to OsPYL/RCARs with or without ABA in TGERP. OsbZIP46 induced reporter most strongly among OsbZIPs tested in the presence of ABA. SAPKs could activate the OsbZIP46 even in the ABA independence. Subclass A OsPP2C6 and -8 almost completely inhibited the OsbZIP46 activity in the different degree through the SAPK9. Lastly, OsPYL/RCAR2 and -5 rescued the OsbZIP46 activity in the presence of SAPK9 and OsPP2C6 dependent on ABA concentration and expression level. By using TGERP, we could characterize successfully the effects of ABA dependent gene expression signaling components in rice. In conclusion, TGERP represents very useful technology to study systemic functional genomics in rice or other monocots.

No MeSH data available.