1a. Objectives (from AD-416):
Characterize the effect of methionine oxidation on phosphorylation of synthetic peptides. Determine the impact of methionine oxidation on the phosphoproteome in vivo. Develop novel motif antibodies to identify specific phosphoproteins that may be sensitive to methionine oxidation in vivo.
1b. Approach (from AD-416):
We will test the emerging concept that reversible methionine oxidation participates in cellular responses to mild oxidants such as H2O2, by attenuating the phosphorylation of key cellular proteins. To do this, we will employ both in vitro and in vivo approaches in the proposed studies. First, we will build on the observation that methionine oxidation can inhibit peptide phosphorylation by soybean protein kinases in vitro. We will consider canonical and non-canonical motifs targeted by calcium-dependent protein kinases and SNF1-related protein kinases, and also determine the biochemical basis for the inhibition. Second, we will examine the in vivo significance of methionine oxidation using transgenic Arabidopsis with altered expression of methionine sulfoxide repair enzymes (cytosolic PMSRA3 or plastid-targeted MsrB1/2 and PMSRA4), and assess the impact on the leaf and root phosphoproteome. In addition to this broad discovery mode approach to identify phosphoproteins sensitive to methionine oxidation, we will also employ a candidate protein approach. We will specifically focus on selected metabolic enzymes that have a methionine residue within known phosphorylation motifs, such as nitrate reductase and chloroplast elongation factor EF-Tu, which was identified in preliminary studies as an abundant phosphoprotein that is sensitive to methionine oxidation in vivo. In addition, we will also develop targeted-phosphospecific antibodies that may help to identify low abundance proteins that are phosphorylated and are sensitive to oxidative signals in vivo.
3. Progress Report:
Activities primarily involved conducting and analyzing experiments related to the impact of methionine oxidation on critical biological processes and mechanisms of enzyme regulation. Dissemination involved sharing results with the scientific community through publications, and various presentations, including four invited seminars and five symposium presentations. Outcomes primarily involve changes in knowledge as a result of project activities. The overall notion to emerge is that methionine (Met) oxidation may function, in some cases at least, as a regulatory posttranslational modification rather than simply as oxidative damage. Specifically, we tested the postulate that oxidation of Met to Met sulfoxide (MetSO) can couple oxidative signals to changes in protein phosphorylation and used nitrate reductase (NR) as a candidate protein that may be regulated by this mechanism in vivo. We demonstrated that exogenous peroxide selectively inhibited phosphorylation of NR at the Ser-534 site relative to the overall phosphoproteome, suggesting that this mechanism may operate in vivo. We developed an “on-blot phosphorylation assay” using recombinant CDPK to show that changes in the NR protein rather than endogenous protein kinases or protein phosphatases were likely responsible for the effects of exogenous peroxide in vivo. We also developed transgenic plants that may ultimately help determine whether this mechanism occurs in vivo; if so, this may explain the activation of NR that occurs when plants are subjected to ROS-generating stresses such as root flooding. During the course of these studies we also observed that exogenous hydrogen peroxide strongly attenuated gene expression induced by the growth-promoting hormone, brassinolide (BL). We speculated that the effect of peroxide on BL signaling might involve the extracellular leucine-rich repeat domain of the BRI1 receptor kinase. It is known that BL binds to a specific portion of the extracellular domain with a leucine-rich repeat (LRR) that is unusual in that many of the Leu residues are replaced with Met residues that might be oxidized by apoplast ROS. To test this idea, we produced transgenic plants expressing directed mutants of BRI1 where the five Met residues in LRR21 were individually substituted with leucine or glutamine. The transgenes were expressed in the bri1-5 weak allele background to test their ability to function in BR signaling and rescue the strong dwarf phenotype of the bri1-5 mutant. Substitutions at two positions (Met-657 and Met-665) were of considerable interest. At the 657 position, substitution of Leu rescued the dwarf phenotype of the bri1-5 line whereas substitution of Gln did not, suggesting that oxidation of Met-657 in the wild type protein may be responsible for at least a portion of the cross talk between ROS and BL signaling. At the 665 position, substitution of Gln resulted in plants that were larger than those expressing the wild type gene (with Met at the 665 position). These results suggest that oxidation of some residues (such as Met-665) may actually enhance signaling and provides a new target for manipulation in crop plants to enhance growth and productivity. Cross-talk between ROS and BL signaling has not been reported previously and may explain, in part, how stress signals result in inhibition of plant growth. Identifying the mechanisms and specific site(s) at which ROS impacts BL signaling may allow the engineering of plants to react to stress in a less conservative fashion.