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.
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.
Affiliation: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
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Figure 7: Phosphorylation assessment of the ABAR and KAT1 proteins by Phos-tag SDS-PAGE test. (A) ABAR phosphorylation in the srk2e mutant, detected with ABAR-immunoblot assay using Phos-tag SDS-PAGE test. Three-week-old plants of wild-type Col or srk2e mutant were treated with ABA-free (−ABA) or ABA-containing solution (+ABA) [50 μM (±)ABA] for 90min, then ABAR phosphorylation status was detected with anti-ABAR serum (+Phos-tag, top panel). Black and open arrows indicate the phosphorylated and dephosphorylated forms of ABAR, respectively. -Phos-tag SDS-PAGE (bottom panel) shows a loading control and indicates the ABAR protein with the expected molecular mass of ABAR (about 150kDa). The experiment was replicated five times with similar results. (B) Phosphorylation level of the His-tagged C-terminal domain of KAT1. The recombinant truncated KAT1 containing C-terminal region His301–Asn677 (KAT1301–677) was treated with alkaline phosphatase-containing (+AP) or AP-free buffer (−AP) for 6h. The phosphorylation of KAT1301–677 was assayed by a slower mobility in a Phos-tag SDS-PAGE that selectively retards phospho- KAT1301–677 (black arrow), where the His-tagged KAT1301–677 was detected by anti-His-Tag serum. Note that the AP treatment increases the amount of the dephosphorylation form of KAT1301–677 (open arrow). The bottom panel (indicated by -Phos-tag) shows a loading control. (C) ABA-activated phosphorylation of the KAT1301–677 protein in wild-type Col, quadruple, and cch mutant plants. Three-week-old plants were treated with ABA-free (−ABA) or 50 μM (±)ABA-containing medium (+ABA) for 90min. The phosphorylation was assayed in the extracted total protein, using KAT1301–677 as a substrate, and the His-tagged KAT1301–677 was detected by anti-His-Tag serum (top panel). Black and open arrows indicate phosphorylated and dephosphorylated KAT1301–677, respectively. Note that the amounts of phosphorylated KAT1301–677 protein are apparently comparable among the three genotypes Col, quadruple, and cch with ABA-free treatment (−ABA). The amounts of the phosphorylated KAT1301–677 protein with ABA treatment were evaluated by scanning the protein bands, and relative band intensities, normalized relative to the intensity of the ABA-free treatment sample (as 100%), are indicated by numbers above the bands. Note that, in comparison with Col, the levels of the ABA-activated phosphorylated KAT1301–677 protein in quadruple and cch decrease. -Phos-tag SDS-PAGE (bottom) shows a loading control and indicates the expected molecular mass of His-tagged KAT1301–677 (about 60kDa). The experiment was repeated five times with similar results.
Upon activation by ABA, OST1 modulates the activities of downstream effectors to regulate stomatal movement by phosphorylation (Sato et al., 2009; Sirichandra et al., 2009; Geiger et al., 2009, 2010; Lee et al., 2009, 2013; Brandt et al., 2012; Acharya et al., 2013; Imes et al., 2013; Osakabe et al., 2013; Liang and Zhang, 2014). A recent report suggests that ABAR may be phosphorylated (Wang et al., 2013a). It was tested whether ABAR is a substrate of OST1. In the Phos-tag SDS-PAGE assay, in which the phosphorylated proteins with the phosphate group bound to the divalent metal ions decreases the migration speed, separated ABAR bands were observed on the gels (Fig.7A), indicating that ABAR was phosphorylated by the protein kinase (Fig. 7A), which is consistent with a previous observation (Wang et al., 2013a). However, the amount of phosphorylated ABAR in wild-type Col plants was comparable with that in the srk2e mutant, and ABA treatment did not change the amount of phosphorylated ABAR in wild-type Col plants or in the srk2e mutant (Fig. 7A), suggesting that phosphorylation of ABAR is independent of OST1 and ABA.