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A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions.

Planes MD, Niñoles R, Rubio L, Bissoli G, Bueso E, García-Sánchez MJ, Alejandro S, Gonzalez-Guzmán M, Hedrich R, Rodriguez PL, Fernández JA, Serrano R - J. Exp. Bot. (2014)

Bottom Line: Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H(+) efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type.The mechanism of inhibition of the H(+)-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells.ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H(+)-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H(+)-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Camino de Vera, 46022 Valencia, Spain.

No MeSH data available.


Related in: MedlinePlus

SnRK2.2 specifically phosphorylates the C-terminal regulatory domain (Ct) of AHA2 PM H+-ATPase in vitro. (A) 3HA–SnRK2.2, MBP–CtAHA2, and MBP were purified and added to the kinase assay as indicated. Autophosphorylation of SnRK2.2 and phosphorylation of CtAHA2 was observed by autoradiography. MBP was not phosphorylated. The mutated versions of the C-terminal fragment (S899P, S904L, or S931F) were phosphorylated as efficiently as the wild type. The dashed line in the Coomassie staining panel indicates the migration of the immunoprecipitated 3HA–SnRK2.2, which was estimated by overlapping with the anti-HA western blot. (B) His–OST1/SnRK2.6, ΔCABF2, MBP, and MBP–CtAHA2 were purified and added to the kinase assay as indicated. Autophosphorylation of His-OST1/SnRK2.6 and phosphorylation of ΔCABF2 was observed by autoradiography. Neither MBP nor MBP–CtAHA2 were phosphorylated. The experiments were repeated twice with similar results.
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Figure 4: SnRK2.2 specifically phosphorylates the C-terminal regulatory domain (Ct) of AHA2 PM H+-ATPase in vitro. (A) 3HA–SnRK2.2, MBP–CtAHA2, and MBP were purified and added to the kinase assay as indicated. Autophosphorylation of SnRK2.2 and phosphorylation of CtAHA2 was observed by autoradiography. MBP was not phosphorylated. The mutated versions of the C-terminal fragment (S899P, S904L, or S931F) were phosphorylated as efficiently as the wild type. The dashed line in the Coomassie staining panel indicates the migration of the immunoprecipitated 3HA–SnRK2.2, which was estimated by overlapping with the anti-HA western blot. (B) His–OST1/SnRK2.6, ΔCABF2, MBP, and MBP–CtAHA2 were purified and added to the kinase assay as indicated. Autophosphorylation of His-OST1/SnRK2.6 and phosphorylation of ΔCABF2 was observed by autoradiography. Neither MBP nor MBP–CtAHA2 were phosphorylated. The experiments were repeated twice with similar results.

Mentions: An alternative mechanism of regulation could be ABA-induced phosphorylation of inhibitory sites within the regulatory C-terminal domain of the enzyme, such as Ser931 (Fuglsang et al., 2007) and Ser899 (Haruta et al., 2014). As indicated in Fig. 4A (lane 3), the ABA-activated protein kinase SnRK2.2 phosphorylated the C-terminal regulatory domain of AHA2 in vitro. This could not result from non-specific phosphorylation because this kinase could not phosphorylate either MBP (Fig. 4A, lane 2) or the enzyme ABA2 (Supplementary Fig. S3, lane 3 at JXB online) but it did phosphorylate itself (autophosphorylation) and its physiological substrate ABF2 (Supplementary Fig. S3, lane 1). Another piece of evidence against non-specific phosphorylation in our assay is provided in Fig. 4B: the guard-cell protein kinase OST1/SnRK2.6 could phosphorylate ABF2 (substrate of all ABA-activated kinases) but not the C-terminal regulatory domain of AHA2.


A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions.

Planes MD, Niñoles R, Rubio L, Bissoli G, Bueso E, García-Sánchez MJ, Alejandro S, Gonzalez-Guzmán M, Hedrich R, Rodriguez PL, Fernández JA, Serrano R - J. Exp. Bot. (2014)

SnRK2.2 specifically phosphorylates the C-terminal regulatory domain (Ct) of AHA2 PM H+-ATPase in vitro. (A) 3HA–SnRK2.2, MBP–CtAHA2, and MBP were purified and added to the kinase assay as indicated. Autophosphorylation of SnRK2.2 and phosphorylation of CtAHA2 was observed by autoradiography. MBP was not phosphorylated. The mutated versions of the C-terminal fragment (S899P, S904L, or S931F) were phosphorylated as efficiently as the wild type. The dashed line in the Coomassie staining panel indicates the migration of the immunoprecipitated 3HA–SnRK2.2, which was estimated by overlapping with the anti-HA western blot. (B) His–OST1/SnRK2.6, ΔCABF2, MBP, and MBP–CtAHA2 were purified and added to the kinase assay as indicated. Autophosphorylation of His-OST1/SnRK2.6 and phosphorylation of ΔCABF2 was observed by autoradiography. Neither MBP nor MBP–CtAHA2 were phosphorylated. The experiments were repeated twice with similar results.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: SnRK2.2 specifically phosphorylates the C-terminal regulatory domain (Ct) of AHA2 PM H+-ATPase in vitro. (A) 3HA–SnRK2.2, MBP–CtAHA2, and MBP were purified and added to the kinase assay as indicated. Autophosphorylation of SnRK2.2 and phosphorylation of CtAHA2 was observed by autoradiography. MBP was not phosphorylated. The mutated versions of the C-terminal fragment (S899P, S904L, or S931F) were phosphorylated as efficiently as the wild type. The dashed line in the Coomassie staining panel indicates the migration of the immunoprecipitated 3HA–SnRK2.2, which was estimated by overlapping with the anti-HA western blot. (B) His–OST1/SnRK2.6, ΔCABF2, MBP, and MBP–CtAHA2 were purified and added to the kinase assay as indicated. Autophosphorylation of His-OST1/SnRK2.6 and phosphorylation of ΔCABF2 was observed by autoradiography. Neither MBP nor MBP–CtAHA2 were phosphorylated. The experiments were repeated twice with similar results.
Mentions: An alternative mechanism of regulation could be ABA-induced phosphorylation of inhibitory sites within the regulatory C-terminal domain of the enzyme, such as Ser931 (Fuglsang et al., 2007) and Ser899 (Haruta et al., 2014). As indicated in Fig. 4A (lane 3), the ABA-activated protein kinase SnRK2.2 phosphorylated the C-terminal regulatory domain of AHA2 in vitro. This could not result from non-specific phosphorylation because this kinase could not phosphorylate either MBP (Fig. 4A, lane 2) or the enzyme ABA2 (Supplementary Fig. S3, lane 3 at JXB online) but it did phosphorylate itself (autophosphorylation) and its physiological substrate ABF2 (Supplementary Fig. S3, lane 1). Another piece of evidence against non-specific phosphorylation in our assay is provided in Fig. 4B: the guard-cell protein kinase OST1/SnRK2.6 could phosphorylate ABF2 (substrate of all ABA-activated kinases) but not the C-terminal regulatory domain of AHA2.

Bottom Line: Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H(+) efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type.The mechanism of inhibition of the H(+)-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells.ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H(+)-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H(+)-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Camino de Vera, 46022 Valencia, Spain.

No MeSH data available.


Related in: MedlinePlus