Short communication
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

https://doi.org/10.1016/j.jplph.2020.153312Get rights and content

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.

Introduction

Conjugation of plant hormones (phytohormones) is widely spread in the plant kingdom as a molecular mechanism that maintains a proper hormone concentration during plant physiological processes and responses to abiotic and biotic stimuli (Ludwig-Müller, 2011).

It is widely known that most of the total auxin pool (about 99 %) in plant tissues does not exist as a free indole-3-acetic acid (IAA) but is converted to its storage forms (conjugates) (Bandurski and Schulze, 1977; Bandurski et al., 1995). There are two types of these low molecular weight conjugates. Ester-linked forms of bound auxin, such as IAA-glucose and IAA-myo-inositol, are found in monocotyledonous plants (Hall, 1980; Nowacki and Bandurski, 1980), however, UDP-glucosyltransferases that conjugate IAA to glucose were also identified in some dicots (Arabidopsis thaliana, Phaseolus vulgaris L., Pisumsativum L.) (Jackson et al., 2001; Jakubowska and Kowalczyk, 2004). On the other hand, amide conjugates of IAA (IAA-aspartate/IAA-Asp, IAA-glutamate, IAA-alanine, etc.) predominate in dicots, even though genes that encode IAA-amino acid synthetases are also found in tissues of some monocotyledonous plants (Bandurski and Schulze, 1977; Feng et al., 2015).

Gretchen Hagen 3 (GH3) belong to one of the three families of early auxin-responsive genes and encode anacyl acid amidosynthetases that conjugate various acyl substrates to l-amino acids in ATP- and Mg2+- dependent manner (Hagen and Guilfoyle, 2002; Westfall et al., 2012). GH3 amidosynthetases are cytosolic, acidic, 67−72 kDa enzymes that act according to the Bi UniUni Bi ping pong mechanism of the catalysis. These synthetases convert various acyl substrates such as jasmonate (Staswick et al., 2002), salicylic acid (Westfall et al., 2016; Mackelprang et al., 2017), 4-substituted benzoates (Chen et al., 2013) and auxins: IAA (Staswick et al., 2005), IBA (Sherp et al., 2018a) into their amino acid conjugates. Recently, Sherp et al. (2018b) reported that synthetic auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) are substrates of AtGH3.15 for conjugation with amino acids. Interestingly, a synthesis of isochorismate-glutamate conjugate by AtGH3.12/PBS3 is a part of a novel salicylate biosynthesis pathway (Rekhter et al., 2019). Holland et al. (2019) have found that AtGH3.12/PBS3 and AtGH3.7 are able to conjugate chorismate to form chorismic acid-glutamate and chorismic acid-cysteine, respectively. In addition, Wang et al. (2012) indicated that AtGH3.5 is able to synthesize indole-3-acyl-cysteinate (ICA-Cys) that acts as a precursor of phytoalexin.

Despite the occurrence of low molecular weight conjugates of auxin, plants have high molecular weight compounds that contain covalently attached IAA. For example, maize seeds have high amounts of IAA-β-1,4-glucan as a high molecular weight ester conjugate of auxin (Bandurski et al., 1995). Bialek and Cohen (1986) have isolated from bean seeds a high molecular weight fraction that released IAA after strong alkaline hydrolysis. The bean seeds contain 3.6-kDa peptide that binds two IAA molecules per one molecule of the peptide. Moreover, antibodies anti-3.6-kDa recognized several proteins of bean seeds (Walz et al., 2002), among which an IAA containing protein, Phaseolus vulgaris IAP1 protein (42 kDa), has been identified. IAP1 is encoded by a single copy of a gene and its expression is tightly associated with maturation of bean seeds. Different iap1 homologues have been found in various plants, such as Arabidopsis, Glycine, Triticum, Oryza (Seidel et al., 2006). The high amount of the IAP1 fraction was accumulated in the endomembrane fraction. Taking into account that the endoplasmic reticulum compartment contains IAA-amino acid hydrolases, the co-localization of IAP1 and these hydrolytic enzymes might be involved in hydrolysis of IAA-proteins (Seidel et al., 2006). Interestingly, Park et al. (2006) have used anti-IAA-Gly-BSA antibodies which recognized a 76-kDa-polypeptide in strawberry fruit that has been identified as an ATP synthase. This finding may suggest that formation of IAA conjugates with proteins is a part of a covalent post-translational modification that regulates biological activity of some proteins (Seidel et al., 2006). In addition, pea fruit and seeds also synthesize both IAA and 4-chloro-indole-3-acetic acid (4-Cl-IAA) conjugates with proteins (Park et al., 2010). Surprisingly, the amount of high molecular weight conjugates of IAA in young pericarp of pea is higher than low-molecular (solvent-soluble) weight conjugates. This is an interesting finding, because pea seeds accumulate an amide conjugate, IAA-Asp, as a pivotal bound auxin that is synthesized by two amidosynthetases, IAA-Asp synthetase and PsGH3 (Ostrowski and Jakubowska, 2011; Ostrowski et al., 2016). Our previous study indicated that both pea enzymes belong to the GH3 family and prefer IAA and l-Asp as substrates for ATP-dependent conjugating activity (Ostrowski and Jakubowska, 2011; Ostrowski et al., 2016). Moreover, PsGH3 is inhibited by l-Trp that competes with l-Asp for the catalytic site of the enzyme. Apart from l-Asp, PsGH3 synthetase is able to conjugate IAA to aromatic amino acids (L-Phe and l-Tyr) and l-Met, but these activities are much weaker than IAA-Asp synthesis.

Despite the fact that various plant species produce conjugates of IAA with proteins, biochemical mechanism of this synthesis and its regulation remain unknown. In this study, we used recombinant PsGH3 synthetase to analyze formation of high molecular weight linkages of IAA in immature seeds of pea. This is a first report that shows enzyme-dependent synthesis of IAA-protein conjugates.

Section snippets

Plant material

Pea (Pisumsativum L.) used for experiments was grown under field conditions during the summer of 2019. The young pods of pea were harvested, and seeds with a diameter of more than 5 mm were selected, immediately frozen, and stored in freezer bags at −20 °C.

Isolation of protein fraction from pea seeds

3 g of immature pea seeds was homogenized at 0−4 °C with 2 mL of 50 mMTris−HCl buffer pH 7.6 using mortar and pestle and an UltraTurax T25 homogenizer (IKA, Germany). The homogenate was filtered through a nylon cloth and centrifuged at 16 000

Purification of PsGH3 by ÄKTA start chromatography system

Recombinant PsGH3 was synthesized in E. coli cells as described by Ostrowski et al. (2016). N-terminally His-tagged PsGH3 was purified from the cell lysate by Ni2+ affinity chromatography using ÄKTA start chromatography system. PsGH3 was eluted as two peaks with 77 mM and 170.5 mM imidazole in the buffer A, respectively. SDS-PAGE analysis of the protein fractions revealed a single protein band which corresponds to His-tagged PsGH3 molecular mass (Fig. 1A). In the previous work (Ostrowski et

Conclusion

This short communication is the first study that reports the involvement of the GH3 enzymatic activity in synthesis of high molecular weight conjugates of IAA with proteins. In contrast to IAA-amino acid conjugates biosynthesis, where mechanism of catalysis has been well documented, formation of IAA-protein linkages remains still elusive. In this study, we show that uncharacterized protein substrate can compete with Asp and Trp for catalytic site of GH3 synthetase during IAA conjugation. The

CRediT authorship contribution statement

Maciej Ostrowski: Conceptualization, Methodology, Investigation, Writing - review & editing. Anna Ciarkowska: Investigation, Writing - original draft.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgments

We are grateful to Doctor Anna Kozakiewicz from Department of Biomedical Chemistry and Polymers, Faculty of Chemistry of Nicolaus Copernicus University in Torun for providing ÄKTA start chromatography system.

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