Diverse peptide hormones affecting root growth identified in the Medicago truncatula secreted peptidome

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.This work was supported by ARC grants to MAD: DP150104050 and LP150100826. NP was partly supported by an Endeavor Fellowship. NAMR was supported by an ANU Ph.D. scholarship supported by DP120101893. AI was supported by an Australian Post-graduate Award and an AW Howard Memorial Award. LC was supported by the Bruce-Veness Chandler and the John A. Lamberton research scholarship

Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula

Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two domains encoded by Medicago truncatula CEP1 (MtCEP1). Novel arabinosylated and hydroxylated peptides were identified in root cultures overexpressing MtCEP1. The five most abundant CEP peptides were hydroxylated and these species were detected also in low amounts in vector control samples. Synthetic peptides with different hydroxylation patterns differentially affected root development. Notably, the domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5mM KNO3 and stimulated the highest increase in nodule number when grown with 0mM KNO3. Inhibition of lateral root emergence by D1:HyP4,11 was not alleviated by removing peptide exposure. In contrast, the domain 2 peptide hydroxylated at Pro11 (D2:HyP11) increased stage III-IV lateral root primordium numbers by 6-fold (P < 0.001) which failed to emerge. Auxin addition at levels which stimulated lateral root formation in wild-type plants had little or no ameliorating effect on CEP peptide-mediated inhibition of lateral root formation or emergence. Both peptides increased and altered the root staining pattern of the auxin-responsive reporter GH3:GUS suggesting CEPs alter auxin sensitivity or distribution. The results showed that CEP primary sequence and post-translational modifications influence peptide activities and the improved isolation procedure effectively and reproducibly identifies and characterises CEPs

Remote Ischemic Preconditioning (RIPC) Modifies the Plasma Proteome in Children Undergoing Repair of Tetralogy of Fallot: A Randomized Controlled Trial

<div><p>Background</p><p>Remote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair.</p><p>Methods and Results</p><p>Children (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation.</p><p>Conclusions</p><p>We provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery.</p><p>Trial Registration</p><p>ACTR.org.au <a href="http://www.anzctr.org.au/trial_view.aspx/ACTRN12610000496011" target="_blank">ACTRN12610000496011</a></p></div

Global Survey of the Bovine Salivary Proteome: Integrating Multidimensional Prefractionation, Targeted, and Glycocapture Strategies

Saliva is easily obtainable from a large number of animals in a noninvasive manner and contains a wide diversity of compounds including hormones, metabolites, and proteins that may be a good source of biomarkers of health and disease. Here we have used a combination of multidimensional prefractionation, targeted, and glycocapture methodologies to profile the bovine salivary proteome. The nontargeted approach used four different separation methodologies consisting of SDS-PAGE, Off-gel fractionation, RP-HPLC, and SCX-HPLC. In the targeted approach, we’ve employed a hypothesis-based methodology by only selecting extracellular proteins from <i>in silico</i> data. Finally, the hydrazide capture methodology not only enabled us to identify formerly N-linked glycoproteins but it also provided a selective enrichment process for the identification of low abundance proteins. Together, the three different approaches identified 402 salivary proteins and 45 N-linked glycoproteins. A large number of these proteins have previously been uncharacterized in bovine saliva. To date, this is the largest global survey of the bovine salivary proteome and expands the potential of the diagnostic utility of this fluid to guide development of experiments seeking biomarkers for health traits (i.e., disease resistance) as well as feed conversion efficiency and productivity traits in dairy and beef cattle

Time points of the sample collection in panel A.

<p>Pooling of the plasma samples at each time point in panel <b>B.</b> Plasma from all 20 patients of one group was pooled into 5 samples (each representing plasma of 4 patients).</p

CONSORT diagram describing flow of the participants though the enrolment, allocation, follow up and analysis phases of the trial.

<p>CONSORT diagram describing flow of the participants though the enrolment, allocation, follow up and analysis phases of the trial.</p

Global Survey of the Bovine Salivary Proteome: Integrating Multidimensional Prefractionation, Targeted, and Glycocapture Strategies

Saliva is easily obtainable from a large number of animals in a noninvasive manner and contains a wide diversity of compounds including hormones, metabolites, and proteins that may be a good source of biomarkers of health and disease. Here we have used a combination of multidimensional prefractionation, targeted, and glycocapture methodologies to profile the bovine salivary proteome. The nontargeted approach used four different separation methodologies consisting of SDS-PAGE, Off-gel fractionation, RP-HPLC, and SCX-HPLC. In the targeted approach, we’ve employed a hypothesis-based methodology by only selecting extracellular proteins from <i>in silico</i> data. Finally, the hydrazide capture methodology not only enabled us to identify formerly N-linked glycoproteins but it also provided a selective enrichment process for the identification of low abundance proteins. Together, the three different approaches identified 402 salivary proteins and 45 N-linked glycoproteins. A large number of these proteins have previously been uncharacterized in bovine saliva. To date, this is the largest global survey of the bovine salivary proteome and expands the potential of the diagnostic utility of this fluid to guide development of experiments seeking biomarkers for health traits (i.e., disease resistance) as well as feed conversion efficiency and productivity traits in dairy and beef cattle

Protein data for the 6 proteins that were up-regulated in RIPC group as compared to the control group at 6 hours after CPB.

<p>Protein data for the 6 proteins that were up-regulated in RIPC group as compared to the control group at 6 hours after CPB.</p