Modification of soybean sucrose synthase by S-thiolation with ENOD40 peptide A

Abstract

The gene ENOD40 is expressed at an early stage of root nodule organogenesis and has been postulated to play a central regulatory role in the Rhizobium-legume interaction. In vitro translation of soybean ENOD40 mRNA showed that the gene encodes two peptides of 12 and 24 aa residues (peptides A and B) that bind to sucrose synthase. Here we show that the small Cys-containing peptide A binds to sucrose synthase by disulfide bond formation, which may represent a novel form of posttranslational modification of this important metabolic enzyme. Assays using nanomolar concentrations of peptide A revealed that the monomeric reduced form of this peptide binds to purified sucrose synthase. Using a cysteinyl capture strategy combined with MALDI-TOF MS analysis we identified the Cys residue C264 of soybean sucrose synthase as the binding site of peptide A. Modification of sucrose synthase with ENOD40 peptide A activates sucrose cleavage activity whereas the synthesis activity of the enzyme is unaffected. The results are discussed in relation to the role of sucrose synthase in the control of sucrose utilization in nitrogen-fixing nodules. Ó 2004 Elsevier Inc. All rights reserved. The symbiotic interaction between leguminous plants and rhizobia results in the development of the nodule, a new organ on plant roots. In these nodules the bacteria are hosted intracellularly as bacteroids and find the ideal environment to fix atmospheric nitrogen. Nitrogen fixation is an energy-demanding process which is dependent on the supply of photosynthate. Sucrose, as the major end product of photosynthesis, is synthesized in leaves and is transported in the phloem to various sink tissues such as nodules. This disaccharide is cleaved by the homotetrameric enzyme sucrose synthase (SuSy) catalyzing the reversible, UDP-dependent cleavage of sucrose into UDP-glucose and fructose. This reaction is considered to function in vivo in the sucrose breakdown direction providing substrates for rapidly growing tissues and sink organs. SuSy is one of the most abundant proteins in mature legume nodules [1,2], and breakdown of sucrose is a key step in nitrogen fixation and a necessary prerequisite for normal nodule development and function [3].Due to the important role of SuSy in the control of sucrose utilization in sink tissues, the expression of SuSy genes is highly regulated on the transcriptional and translational levels [4]. Moreover, SuSy is posttranslationally modified by reversible protein phosphorylation [5,6], and the soluble form of SuSy can bind to the actin cytoskeleton in plants [7]. Recently, we reported direct binding of SuSy to peptide A (12 aa residues) and peptide B (24 aa residues) encoded by the early nodulin gene ENOD40 [8]. In addition to the two small peptides, the secondary structure of ENOD40 mRNA has been shown to be a key element in the signaling process underlying nodule organogenesis [9,10].

Peptide Hormones in Plants

“…There has been great controversy over whether peptides encoded by these short ORFs function as hormones (91,92), or whether the mRNA itself possesses biological activity (9,33). Several studies support the former theory, but the available evidence does not exclude the latter theory.…”

Determination of Structural Requirements and Probable Regulatory Effectors for Membrane Association of Maize Sucrose Synthase 1

“…This hydrophobic domain is only one possible contributor to membrane affinity within the N-terminal noncatalytic domain and does not rule out additional regions or modifications that influence m-SUS abundance. In particular, SUS1 is known to have a peptide S-thiolation site (Rohrig et al, 2004) and two sites of phosphorylation (Hardin et al, 2004) within the N-terminal noncatalytic domain, and phosphorylation has been shown to influence the binding of SUS to certain membrane types (Hardin et al, 2004). ) of full-length MBP-SUS1 were analyzed without added microsomes or mixed with equal amounts of carbonatestripped apical leaf microsomes at pH 6.5 and residual (4 mM) Suc (duplicate reactions shown), or after addition of 150 mM Suc (duplicate reactions shown), Glc, or maltose.…”

Soybean miR172c Targets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40 Expression and Regulate Nodule Initiation

“…ENOD40 may function in nodulation as a cellcell signaling molecule; it lacks an open reading frame but does encode two small peptides (Sousa et al, 2001;Röhrig et al, 2002). Notably, ENOD40 peptides can bind to, and enhance the stability of, sucrose synthase (Hardin et al, 2003;Röhrig et al, 2004). Thus, ENOD40 may promote nodule organogenesis by increasing the carbon sink strength of the dividing cells.…”