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Pea GH3 acyl acid amidosynthetase conjugates IAA to proteins in immature seeds of Pisum sativum L. - A new perspective on formation of high-molecular weight conjugates of auxin.
J Plant Physiol. 2021 Jan; 256:153312.JP

Abstract

Gretchen Hagen 3 (GH3) acyl acid amidosynthetases are encoded by early auxin-responsive genes and catalyze an ATP-dependent biosynthesis of IAA-amino acid conjugates. An amide conjugate of IAA, indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp), is a predominant form of bound auxin in immature seeds of pea. However, there is some evidence that IAA is also able to form high molecular weight amide conjugates with proteins in pea and other plant species. In this short study we report that recombinant PsGH3 IAA-amino acid synthetase, which exhibits a preference for the formation of IAA-Asp, can also conjugate IAA with the protein fraction from immature seeds of pea (S-10 fraction). We studied [14C]IAA incorporation to the S-10 protein fraction by two assays: TLC method and protein precipitation by trichloroacetic acid (TCA). In both cases, radioactivity of [14C]IAA in the protein fraction increases in comparison to the control (without PsGH3), about 9.3- and 3.17-fold, respectively. l-Asp, as a preferred substrate in the IAA conjugation catalyzed by PsGH3, down-regulates [14C]IAA conjugation to the proteins as shown by the TLC assay (∼2.8-fold decrease) and the TCA precipitation variant (∼2-fold decrease). Moreover, l-Trp that competes with Asp for the catalytic site of PsGH3 and inhibits activity of the enzyme, diminished radioactivity of [14C]IAA-proteins about 1.2- and 2.8-fold, respectively. Taking into account that amino group of an amino acid or a protein acts as an acceptor of the indole-3-acetyl moiety from IAA-AMP intermediate during GH3-dependent conjugation, we masked amine groups (α- and ε-NH2) of the S-10 protein fraction from pea seeds by reductive alkylation. The alkylated proteins revealed about 3- and 2.8-fold lower radioactivity of [14C]IAA than non-alkylated fraction for TLC and TCA precipitation variant, respectively. This is a first study demonstrating that formation of high molecular weight IAA conjugates with proteins is catalyzed by a GH3 acyl acid amidosynthetase.

Authors+Show Affiliations

Department of Biochemistry, Nicolaus Copernicus University, Lwowska 1, 87-100, Torun, Poland. Electronic address: maciejost@umk.pl.Department of Biochemistry, Nicolaus Copernicus University, Lwowska 1, 87-100, Torun, Poland. Electronic address: annaciar@doktorant.umk.pl.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

33161181

Citation

Ostrowski, Maciej, and Anna Ciarkowska. "Pea GH3 Acyl Acid Amidosynthetase Conjugates IAA to Proteins in Immature Seeds of Pisum Sativum L. - a New Perspective On Formation of High-molecular Weight Conjugates of Auxin." Journal of Plant Physiology, vol. 256, 2021, p. 153312.
Ostrowski M, Ciarkowska A. Pea GH3 acyl acid amidosynthetase conjugates IAA to proteins in immature seeds of Pisum sativum L. - A new perspective on formation of high-molecular weight conjugates of auxin. J Plant Physiol. 2021;256:153312.
Ostrowski, M., & Ciarkowska, A. (2021). Pea GH3 acyl acid amidosynthetase conjugates IAA to proteins in immature seeds of Pisum sativum L. - A new perspective on formation of high-molecular weight conjugates of auxin. Journal of Plant Physiology, 256, 153312. https://doi.org/10.1016/j.jplph.2020.153312
Ostrowski M, Ciarkowska A. Pea GH3 Acyl Acid Amidosynthetase Conjugates IAA to Proteins in Immature Seeds of Pisum Sativum L. - a New Perspective On Formation of High-molecular Weight Conjugates of Auxin. J Plant Physiol. 2021;256:153312. PubMed PMID: 33161181.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Pea GH3 acyl acid amidosynthetase conjugates IAA to proteins in immature seeds of Pisum sativum L. - A new perspective on formation of high-molecular weight conjugates of auxin. AU - Ostrowski,Maciej, AU - Ciarkowska,Anna, Y1 - 2020/11/04/ PY - 2020/06/03/received PY - 2020/10/12/revised PY - 2020/10/27/accepted PY - 2020/11/9/pubmed PY - 2021/5/4/medline PY - 2020/11/8/entrez KW - Auxin KW - Auxin-amide conjugates KW - Auxin-protein KW - GH3 KW - Pisumsativum KW - indole-3-acetic acid SP - 153312 EP - 153312 JF - Journal of plant physiology JO - J Plant Physiol VL - 256 N2 - Gretchen Hagen 3 (GH3) acyl acid amidosynthetases are encoded by early auxin-responsive genes and catalyze an ATP-dependent biosynthesis of IAA-amino acid conjugates. An amide conjugate of IAA, indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp), is a predominant form of bound auxin in immature seeds of pea. However, there is some evidence that IAA is also able to form high molecular weight amide conjugates with proteins in pea and other plant species. In this short study we report that recombinant PsGH3 IAA-amino acid synthetase, which exhibits a preference for the formation of IAA-Asp, can also conjugate IAA with the protein fraction from immature seeds of pea (S-10 fraction). We studied [14C]IAA incorporation to the S-10 protein fraction by two assays: TLC method and protein precipitation by trichloroacetic acid (TCA). In both cases, radioactivity of [14C]IAA in the protein fraction increases in comparison to the control (without PsGH3), about 9.3- and 3.17-fold, respectively. l-Asp, as a preferred substrate in the IAA conjugation catalyzed by PsGH3, down-regulates [14C]IAA conjugation to the proteins as shown by the TLC assay (∼2.8-fold decrease) and the TCA precipitation variant (∼2-fold decrease). Moreover, l-Trp that competes with Asp for the catalytic site of PsGH3 and inhibits activity of the enzyme, diminished radioactivity of [14C]IAA-proteins about 1.2- and 2.8-fold, respectively. Taking into account that amino group of an amino acid or a protein acts as an acceptor of the indole-3-acetyl moiety from IAA-AMP intermediate during GH3-dependent conjugation, we masked amine groups (α- and ε-NH2) of the S-10 protein fraction from pea seeds by reductive alkylation. The alkylated proteins revealed about 3- and 2.8-fold lower radioactivity of [14C]IAA than non-alkylated fraction for TLC and TCA precipitation variant, respectively. This is a first study demonstrating that formation of high molecular weight IAA conjugates with proteins is catalyzed by a GH3 acyl acid amidosynthetase. SN - 1618-1328 UR - https://www.unboundmedicine.com/medline/citation/33161181/Pea_GH3_acyl_acid_amidosynthetase_conjugates_IAA_to_proteins_in_immature_seeds_of_Pisum_sativum_L____A_new_perspective_on_formation_of_high_molecular_weight_conjugates_of_auxin_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0176-1617(20)30202-9 DB - PRIME DP - Unbound Medicine ER -