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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.


Related in: MedlinePlus

Over-expression of SAPKs cannot increase trans-activation activity of OsbZIP46 significantly in wild-type rice protoplasts in the presence of ABA. (A,B) Expression analysis of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 in rice protoplasts. After transfection, protoplasts were incubated for (A) 2 h and (B) 4 h. GFP signal of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 was detected after 2 h incubation and gradually increased. GFP:SAPKs were used at 10 μg per transfection. Exposure time of GFP fluorescence was 600 ms. Chlorophyll autofluorescence is in red to distinguish it from GFP (green) fluorescence. (C,D) Dual luciferase assay after 2 h (C) and 4 h (D) incubations. Flag-tagged SAPK2, -6, and -9 were transfected with HA-tagged OsbZIP46, pRab16A-fLUC reporter plasmid and pArUBQ-rLUC plasmid as an internal control. After transfection, protoplasts were incubated for 2 and 4 h in the presence of 0 and 5 μM ABA under light. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; ANOVA with Tukey’s test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
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Figure 3: Over-expression of SAPKs cannot increase trans-activation activity of OsbZIP46 significantly in wild-type rice protoplasts in the presence of ABA. (A,B) Expression analysis of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 in rice protoplasts. After transfection, protoplasts were incubated for (A) 2 h and (B) 4 h. GFP signal of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 was detected after 2 h incubation and gradually increased. GFP:SAPKs were used at 10 μg per transfection. Exposure time of GFP fluorescence was 600 ms. Chlorophyll autofluorescence is in red to distinguish it from GFP (green) fluorescence. (C,D) Dual luciferase assay after 2 h (C) and 4 h (D) incubations. Flag-tagged SAPK2, -6, and -9 were transfected with HA-tagged OsbZIP46, pRab16A-fLUC reporter plasmid and pArUBQ-rLUC plasmid as an internal control. After transfection, protoplasts were incubated for 2 and 4 h in the presence of 0 and 5 μM ABA under light. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; ANOVA with Tukey’s test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Mentions: SAPKs can be classified into three different subclasses in terms of ABA-dependent kinase activity (Kobayashi et al., 2004; Kulik et al., 2011). SAPK2, -6, and -9 belonging to each subclass (I, II, and III, respectively) has been shown to bind OsbZIP46 directly and SAPK2 and -6 can phosphorylate OsbZIP46 without ABA in in vitro phosphorylation assay (Tang et al., 2012). However, transcriptional activity of OsbZIP46 enhanced directly by these SAPKs has not been confirmed yet. Thus we examined whether SAPK2, -6, and -9 could activate the OsbZIP46 in TGERP. Firstly, we confirmed whether the protein synthesis of SAPK2, -6, and -9 is enough at 2 and 4 h. GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 started to show much weaker GFP fluorescence after 2 h incubation than OsbZIP (Figure 3A) and GFP signal was significantly enhanced at 4 h for all SAPKs (Figure 3B). It seems that the expression of SAPKs required more induction time as compared to OsbZIPs. To examine the effects of SAPKs through the OsbZIP46 in the presence or absence of ABA, SAPK2, -6, and -9 were co-transfected with OsbZIP46, respectively. After 2 h incubations, fLUC expression was up to 48% greater with over-expression of SAPK2, whereas the over-expression of SAPK6 and -9 decreased fLUC expression without ABA (Figure 3C). After 4 h incubations, over-expression of SAPK2, -6, and -9 enhanced the fLUC expression by 3.1-, 1.7-, and 1.5-fold without ABA, respectively (Figure 3D). With 5 μM ABA at 2 h, over-expression of SAPK2 and -9 increased fLUC expression about 1.3-fold (Figure 3C). With 5 μM ABA at 4 h, over-expression of SAPK2, -6, and -9 increase fLUC expression about 1.3-, 1-, and 1.2-fold, respectively, but one-way ANOVA showed no significant effects (Figure 3D). Taken together, in the absence of ABA, SAPK2 can activate OsbZIP46 most significantly among three different subfamilies of SAPKs.


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)

Over-expression of SAPKs cannot increase trans-activation activity of OsbZIP46 significantly in wild-type rice protoplasts in the presence of ABA. (A,B) Expression analysis of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 in rice protoplasts. After transfection, protoplasts were incubated for (A) 2 h and (B) 4 h. GFP signal of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 was detected after 2 h incubation and gradually increased. GFP:SAPKs were used at 10 μg per transfection. Exposure time of GFP fluorescence was 600 ms. Chlorophyll autofluorescence is in red to distinguish it from GFP (green) fluorescence. (C,D) Dual luciferase assay after 2 h (C) and 4 h (D) incubations. Flag-tagged SAPK2, -6, and -9 were transfected with HA-tagged OsbZIP46, pRab16A-fLUC reporter plasmid and pArUBQ-rLUC plasmid as an internal control. After transfection, protoplasts were incubated for 2 and 4 h in the presence of 0 and 5 μM ABA under light. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; ANOVA with Tukey’s test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
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Figure 3: Over-expression of SAPKs cannot increase trans-activation activity of OsbZIP46 significantly in wild-type rice protoplasts in the presence of ABA. (A,B) Expression analysis of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 in rice protoplasts. After transfection, protoplasts were incubated for (A) 2 h and (B) 4 h. GFP signal of GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 was detected after 2 h incubation and gradually increased. GFP:SAPKs were used at 10 μg per transfection. Exposure time of GFP fluorescence was 600 ms. Chlorophyll autofluorescence is in red to distinguish it from GFP (green) fluorescence. (C,D) Dual luciferase assay after 2 h (C) and 4 h (D) incubations. Flag-tagged SAPK2, -6, and -9 were transfected with HA-tagged OsbZIP46, pRab16A-fLUC reporter plasmid and pArUBQ-rLUC plasmid as an internal control. After transfection, protoplasts were incubated for 2 and 4 h in the presence of 0 and 5 μM ABA under light. The mean value of relative luciferase activity for three independent experiments is shown, and error bars indicate SD; ANOVA with Tukey’s test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
Mentions: SAPKs can be classified into three different subclasses in terms of ABA-dependent kinase activity (Kobayashi et al., 2004; Kulik et al., 2011). SAPK2, -6, and -9 belonging to each subclass (I, II, and III, respectively) has been shown to bind OsbZIP46 directly and SAPK2 and -6 can phosphorylate OsbZIP46 without ABA in in vitro phosphorylation assay (Tang et al., 2012). However, transcriptional activity of OsbZIP46 enhanced directly by these SAPKs has not been confirmed yet. Thus we examined whether SAPK2, -6, and -9 could activate the OsbZIP46 in TGERP. Firstly, we confirmed whether the protein synthesis of SAPK2, -6, and -9 is enough at 2 and 4 h. GFP:SAPK2, GFP:SAPK6, and GFP:SAPK9 started to show much weaker GFP fluorescence after 2 h incubation than OsbZIP (Figure 3A) and GFP signal was significantly enhanced at 4 h for all SAPKs (Figure 3B). It seems that the expression of SAPKs required more induction time as compared to OsbZIPs. To examine the effects of SAPKs through the OsbZIP46 in the presence or absence of ABA, SAPK2, -6, and -9 were co-transfected with OsbZIP46, respectively. After 2 h incubations, fLUC expression was up to 48% greater with over-expression of SAPK2, whereas the over-expression of SAPK6 and -9 decreased fLUC expression without ABA (Figure 3C). After 4 h incubations, over-expression of SAPK2, -6, and -9 enhanced the fLUC expression by 3.1-, 1.7-, and 1.5-fold without ABA, respectively (Figure 3D). With 5 μM ABA at 2 h, over-expression of SAPK2 and -9 increased fLUC expression about 1.3-fold (Figure 3C). With 5 μM ABA at 4 h, over-expression of SAPK2, -6, and -9 increase fLUC expression about 1.3-, 1-, and 1.2-fold, respectively, but one-way ANOVA showed no significant effects (Figure 3D). Taken together, in the absence of ABA, SAPK2 can activate OsbZIP46 most significantly among three different subfamilies of SAPKs.

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.


Related in: MedlinePlus