Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

Abstract

Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes.

Crystal Structure of an Indole-3-Acetic Acid Amido Synthetase from Grapevine Involved in Auxin Homeostasis

“…GH3 proteins display differing preferences for both amino acid and acyl substrates (Staswick et al, 2005;Ludwig-Müller et al, 2009;Okrent et al, 2009;Böttcher et al, 2010Böttcher et al, , 2011aBöttcher et al, , 2012Chen et al, 2010). Members of the IAA-amido synthetase family can join IAA to different amino acids, and the particular amino acid used determines the fate of the conjugated auxin: IAA-Asp and IAA-Glu conjugates tend to be converted into oxidized metabolites (Tsurumi and Wada, 1986;Plüss et al, 1989;Östin et al, 1998), whereas, for example, IAA-Ala and IAA-Leu conjugates can be hydrolyzed to release free IAA (LeClere et al, 2002;Savi c et al, 2009).…”

“…In grapevine, there are six auxin-conjugating GH3 proteins (Böttcher et al, 2011a). All six are expressed during grape berry development and may be responsible for regulating the initiation of ripening by reducing IAA levels (Böttcher et al, 2010(Böttcher et al, , 2011a.…”

“…All six are expressed during grape berry development and may be responsible for regulating the initiation of ripening by reducing IAA levels (Böttcher et al, 2010(Böttcher et al, , 2011a. This reduction in IAA concentration appears to be required to allow ripening to progress, and the application of auxins delays ripening (Böttcher et al, 2010(Böttcher et al, , 2011a(Böttcher et al, , 2011b.…”

“…In field-based experiments, different auxins delayed ripening to different extents when applied to berries preripening. The effectiveness of these auxins in delaying ripening was inversely correlated with their ability to act as substrates for GH3 proteins (Böttcher et al, 2011a). The reaction kinetics of three of the six grapevine family members (GH3-1, GH3-2, and GH3-6) have been analyzed in detail (Böttcher et al, 2011a(Böttcher et al, , 2012.…”

“…The effectiveness of these auxins in delaying ripening was inversely correlated with their ability to act as substrates for GH3 proteins (Böttcher et al, 2011a). The reaction kinetics of three of the six grapevine family members (GH3-1, GH3-2, and GH3-6) have been analyzed in detail (Böttcher et al, 2011a(Böttcher et al, , 2012. This article describes the crystallization of the GH3-1 enzyme that, in terms of acyl substrate, has a preference for IAA over the synthetic auxin 1-naphthaleneacetic acid (NAA) and does not conjugate another synthetic auxin-like compound, benzothiazole-2-oxyacetic acid (BTOA).…”