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A link between magnesium-chelatase H subunit and sucrose nonfermenting 1 (SNF1)-related protein kinase SnRK2.6/OST1 in Arabidopsis guard cell signalling in response to abscisic acid.

Liang S, Lu K, Wu Z, Jiang SC, Yu YT, Bi C, Xin Q, Wang XF, Zhang DP - J. Exp. Bot. (2015)

Bottom Line: Neither mutation nor over-expression of the ABAR gene affects significantly ABA-insensitive phenotypes of stomatal movement in the OST1 knockout mutant allele srk2e.These findings suggest that ABAR shares, at least in part, downstream signalling components with PYR/PYL/RCAR receptors for ABA in guard cells; though cch and rtl1 show strong ABA-insensitive phenotypes in both ABA-induced stomatal closure and inhibition of stomatal opening, while the pyr1 pyl1 pyl2 pyl4 quadruple mutant shows strong ABA insensitivity only in ABA-induced stomatal closure.These data establish a link between ABAR/CHLH and SnRK2.6/OST1 in guard cell signalling in response to ABA.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.

No MeSH data available.


Related in: MedlinePlus

ABAR/CHLH physically interacts with OST1/SnRK2.6/SRK2E. (A) Assays of yeast growth in SD4-drop-out medium (lacking Leu, Trp, His, and Ade) to test for an interaction between ABAR and OST1. BD-ABARc690, truncated ABAR protein [the C-terminal fragment: aa 692–1381 (690 aa)] fused with BD domain; AD-OST1, full-length OST1 fused with AD domain. The yeast co-transformed with the construct pairs AD plus BD-ABARc690 and BD plus AD-OST1, were taken as negative controls. The yeast co-transformed with the construct pair BD-53 plus AD-T was taken as a positive control. Note that the yeast co-transformed with the construct pair AD-OST1 plus BD-ABARc690 was able to grow in the SD4-drop-out medium. (B) Co-IP assays in yeast cells. Myc-ABAR and HA-OST1 were coimmunoprecipitated from yeast total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control. (C) Test of the interaction of three different regions of ABAR with OST1 in the yeast two-hybrid system. ABARc690; ABARn691, N-terminal region of ABAR (aa 1–691); ABARc250, the middle section of ABAR [aa 692–941, (250 aa)]. The yeast were co-transformed with the construct pairs BD-ABARc690/AD-OST1, BD-ABARn691/AD-OST1, and BD-ABARc250/AD-OST1, and only the yeast co-transformed with the construct pair BD-ABARc690/AD-OST1 was able to grow on the SD-4 medium (lacking Leu, Trp, His, and Ade). (D) GST-pull down assay to further test the interaction of the C-terminal half of ABAR with OST1. The GST-tagged C-terminal half of ABAR protein (GST-ABAR) pulled down the His-tagged OST1, which was detected by western blot analysis with anti-His, while GST alone did not pull down His-tagged OST1, which was taken as a negative control. (E) LCI to test the interaction of ABAR with OST1. The N. benthamiana leaves were co-transformed by infiltration using a needleless syringe with construct pairs as indicated in the left panel (Bright field). NLuc and CLuc, N-terminal and C-terminal half of the luciferase (Luc), respectively. ABAR-NLuc, full-length ABAR fused with NLuc; OST1-CLuc, full-length OST1 fused with CLuc. The right panel shows the luciferin fluorescence of the treated leaf. (F) ABAR co-immunoprecipitates with Myc-tagged OST1 protein from transgenic Arabidopsis (expressing Myc-tagged OST1) total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control.
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Figure 1: ABAR/CHLH physically interacts with OST1/SnRK2.6/SRK2E. (A) Assays of yeast growth in SD4-drop-out medium (lacking Leu, Trp, His, and Ade) to test for an interaction between ABAR and OST1. BD-ABARc690, truncated ABAR protein [the C-terminal fragment: aa 692–1381 (690 aa)] fused with BD domain; AD-OST1, full-length OST1 fused with AD domain. The yeast co-transformed with the construct pairs AD plus BD-ABARc690 and BD plus AD-OST1, were taken as negative controls. The yeast co-transformed with the construct pair BD-53 plus AD-T was taken as a positive control. Note that the yeast co-transformed with the construct pair AD-OST1 plus BD-ABARc690 was able to grow in the SD4-drop-out medium. (B) Co-IP assays in yeast cells. Myc-ABAR and HA-OST1 were coimmunoprecipitated from yeast total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control. (C) Test of the interaction of three different regions of ABAR with OST1 in the yeast two-hybrid system. ABARc690; ABARn691, N-terminal region of ABAR (aa 1–691); ABARc250, the middle section of ABAR [aa 692–941, (250 aa)]. The yeast were co-transformed with the construct pairs BD-ABARc690/AD-OST1, BD-ABARn691/AD-OST1, and BD-ABARc250/AD-OST1, and only the yeast co-transformed with the construct pair BD-ABARc690/AD-OST1 was able to grow on the SD-4 medium (lacking Leu, Trp, His, and Ade). (D) GST-pull down assay to further test the interaction of the C-terminal half of ABAR with OST1. The GST-tagged C-terminal half of ABAR protein (GST-ABAR) pulled down the His-tagged OST1, which was detected by western blot analysis with anti-His, while GST alone did not pull down His-tagged OST1, which was taken as a negative control. (E) LCI to test the interaction of ABAR with OST1. The N. benthamiana leaves were co-transformed by infiltration using a needleless syringe with construct pairs as indicated in the left panel (Bright field). NLuc and CLuc, N-terminal and C-terminal half of the luciferase (Luc), respectively. ABAR-NLuc, full-length ABAR fused with NLuc; OST1-CLuc, full-length OST1 fused with CLuc. The right panel shows the luciferin fluorescence of the treated leaf. (F) ABAR co-immunoprecipitates with Myc-tagged OST1 protein from transgenic Arabidopsis (expressing Myc-tagged OST1) total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control.

Mentions: To identify the interaction partners of ABAR, a fragment encoding the C-terminus of ABAR (aa 692–1381, ABARc690) was used as a bait to screen the Arabidopsis cDNA library in a yeast two-hybrid system, and OST1 was identified as a candidate. Further assays were performed to confirm the interaction between ABAR and OST1. Full-length OST1 was cloned to pGADT7 fused with the AD, and the ABAR fragment encoding ABARc690 was cloned into pGBKT7 fused with the BD. The yeast cells co-transformed with the construct pair AD-OST1 plus BD-ABARc690 or BD-53 plus AD-T (a positive control) were able to grow in the SD4-drop-out medium (lacking Leu, Trp, His, and Ade) and turned blue in the presence of α-Gal (Fig. 1A), while the yeast cells co-expressing the construct pairs AD plus BD-ABARc690 and BD plus AD-OST1, taken as negative controls, were not able to grow in the SD4-drop-out medium (Fig. 1A), indicating that ABAR interacts with OST1 and that the interaction detected in this yeast system is specific and reliable. Co-IP assays in the yeast cells confirmed the interaction of ABAR with OST1 in the yeast system (Fig. 1B). The further experiments showed that, whereas ABARc690—the C-terminal half of ABAR—is an interaction domain, neither the N-terminal region of ABAR (aa 1–691, ABARn691) nor the middle section of ABAR (aa 692–941, ABARc250) interacts with OST1 (Fig. 1C). The interaction of the C-terminal half of ABAR with OST1 was further confirmed in a pull down assay with the recombinant C-terminal half of ABAR and OST1 proteins (Fig. 1D), consistent with the idea that ABAR interacts with OST1 in the cytosolic space through its C-terminal half.


A link between magnesium-chelatase H subunit and sucrose nonfermenting 1 (SNF1)-related protein kinase SnRK2.6/OST1 in Arabidopsis guard cell signalling in response to abscisic acid.

Liang S, Lu K, Wu Z, Jiang SC, Yu YT, Bi C, Xin Q, Wang XF, Zhang DP - J. Exp. Bot. (2015)

ABAR/CHLH physically interacts with OST1/SnRK2.6/SRK2E. (A) Assays of yeast growth in SD4-drop-out medium (lacking Leu, Trp, His, and Ade) to test for an interaction between ABAR and OST1. BD-ABARc690, truncated ABAR protein [the C-terminal fragment: aa 692–1381 (690 aa)] fused with BD domain; AD-OST1, full-length OST1 fused with AD domain. The yeast co-transformed with the construct pairs AD plus BD-ABARc690 and BD plus AD-OST1, were taken as negative controls. The yeast co-transformed with the construct pair BD-53 plus AD-T was taken as a positive control. Note that the yeast co-transformed with the construct pair AD-OST1 plus BD-ABARc690 was able to grow in the SD4-drop-out medium. (B) Co-IP assays in yeast cells. Myc-ABAR and HA-OST1 were coimmunoprecipitated from yeast total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control. (C) Test of the interaction of three different regions of ABAR with OST1 in the yeast two-hybrid system. ABARc690; ABARn691, N-terminal region of ABAR (aa 1–691); ABARc250, the middle section of ABAR [aa 692–941, (250 aa)]. The yeast were co-transformed with the construct pairs BD-ABARc690/AD-OST1, BD-ABARn691/AD-OST1, and BD-ABARc250/AD-OST1, and only the yeast co-transformed with the construct pair BD-ABARc690/AD-OST1 was able to grow on the SD-4 medium (lacking Leu, Trp, His, and Ade). (D) GST-pull down assay to further test the interaction of the C-terminal half of ABAR with OST1. The GST-tagged C-terminal half of ABAR protein (GST-ABAR) pulled down the His-tagged OST1, which was detected by western blot analysis with anti-His, while GST alone did not pull down His-tagged OST1, which was taken as a negative control. (E) LCI to test the interaction of ABAR with OST1. The N. benthamiana leaves were co-transformed by infiltration using a needleless syringe with construct pairs as indicated in the left panel (Bright field). NLuc and CLuc, N-terminal and C-terminal half of the luciferase (Luc), respectively. ABAR-NLuc, full-length ABAR fused with NLuc; OST1-CLuc, full-length OST1 fused with CLuc. The right panel shows the luciferin fluorescence of the treated leaf. (F) ABAR co-immunoprecipitates with Myc-tagged OST1 protein from transgenic Arabidopsis (expressing Myc-tagged OST1) total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control.
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Figure 1: ABAR/CHLH physically interacts with OST1/SnRK2.6/SRK2E. (A) Assays of yeast growth in SD4-drop-out medium (lacking Leu, Trp, His, and Ade) to test for an interaction between ABAR and OST1. BD-ABARc690, truncated ABAR protein [the C-terminal fragment: aa 692–1381 (690 aa)] fused with BD domain; AD-OST1, full-length OST1 fused with AD domain. The yeast co-transformed with the construct pairs AD plus BD-ABARc690 and BD plus AD-OST1, were taken as negative controls. The yeast co-transformed with the construct pair BD-53 plus AD-T was taken as a positive control. Note that the yeast co-transformed with the construct pair AD-OST1 plus BD-ABARc690 was able to grow in the SD4-drop-out medium. (B) Co-IP assays in yeast cells. Myc-ABAR and HA-OST1 were coimmunoprecipitated from yeast total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control. (C) Test of the interaction of three different regions of ABAR with OST1 in the yeast two-hybrid system. ABARc690; ABARn691, N-terminal region of ABAR (aa 1–691); ABARc250, the middle section of ABAR [aa 692–941, (250 aa)]. The yeast were co-transformed with the construct pairs BD-ABARc690/AD-OST1, BD-ABARn691/AD-OST1, and BD-ABARc250/AD-OST1, and only the yeast co-transformed with the construct pair BD-ABARc690/AD-OST1 was able to grow on the SD-4 medium (lacking Leu, Trp, His, and Ade). (D) GST-pull down assay to further test the interaction of the C-terminal half of ABAR with OST1. The GST-tagged C-terminal half of ABAR protein (GST-ABAR) pulled down the His-tagged OST1, which was detected by western blot analysis with anti-His, while GST alone did not pull down His-tagged OST1, which was taken as a negative control. (E) LCI to test the interaction of ABAR with OST1. The N. benthamiana leaves were co-transformed by infiltration using a needleless syringe with construct pairs as indicated in the left panel (Bright field). NLuc and CLuc, N-terminal and C-terminal half of the luciferase (Luc), respectively. ABAR-NLuc, full-length ABAR fused with NLuc; OST1-CLuc, full-length OST1 fused with CLuc. The right panel shows the luciferin fluorescence of the treated leaf. (F) ABAR co-immunoprecipitates with Myc-tagged OST1 protein from transgenic Arabidopsis (expressing Myc-tagged OST1) total proteins. Immunoprecipitation with pre-immune serum was taken as a negative control.
Mentions: To identify the interaction partners of ABAR, a fragment encoding the C-terminus of ABAR (aa 692–1381, ABARc690) was used as a bait to screen the Arabidopsis cDNA library in a yeast two-hybrid system, and OST1 was identified as a candidate. Further assays were performed to confirm the interaction between ABAR and OST1. Full-length OST1 was cloned to pGADT7 fused with the AD, and the ABAR fragment encoding ABARc690 was cloned into pGBKT7 fused with the BD. The yeast cells co-transformed with the construct pair AD-OST1 plus BD-ABARc690 or BD-53 plus AD-T (a positive control) were able to grow in the SD4-drop-out medium (lacking Leu, Trp, His, and Ade) and turned blue in the presence of α-Gal (Fig. 1A), while the yeast cells co-expressing the construct pairs AD plus BD-ABARc690 and BD plus AD-OST1, taken as negative controls, were not able to grow in the SD4-drop-out medium (Fig. 1A), indicating that ABAR interacts with OST1 and that the interaction detected in this yeast system is specific and reliable. Co-IP assays in the yeast cells confirmed the interaction of ABAR with OST1 in the yeast system (Fig. 1B). The further experiments showed that, whereas ABARc690—the C-terminal half of ABAR—is an interaction domain, neither the N-terminal region of ABAR (aa 1–691, ABARn691) nor the middle section of ABAR (aa 692–941, ABARc250) interacts with OST1 (Fig. 1C). The interaction of the C-terminal half of ABAR with OST1 was further confirmed in a pull down assay with the recombinant C-terminal half of ABAR and OST1 proteins (Fig. 1D), consistent with the idea that ABAR interacts with OST1 in the cytosolic space through its C-terminal half.

Bottom Line: Neither mutation nor over-expression of the ABAR gene affects significantly ABA-insensitive phenotypes of stomatal movement in the OST1 knockout mutant allele srk2e.These findings suggest that ABAR shares, at least in part, downstream signalling components with PYR/PYL/RCAR receptors for ABA in guard cells; though cch and rtl1 show strong ABA-insensitive phenotypes in both ABA-induced stomatal closure and inhibition of stomatal opening, while the pyr1 pyl1 pyl2 pyl4 quadruple mutant shows strong ABA insensitivity only in ABA-induced stomatal closure.These data establish a link between ABAR/CHLH and SnRK2.6/OST1 in guard cell signalling in response to ABA.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.

No MeSH data available.


Related in: MedlinePlus