ذوالفقار غانم محمد المحنه

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing economic losses; Thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress sensitivity and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing the accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further featured.

Upon pathogen infection, receptors in plants will activate a localized immune response, the effector-triggered immunity (ETI), and a systemic immune response, the systemic acquired response (SAR). Infection also induces oscillations in the redox environment of plant cells, triggering response mechanisms involving sensitive cysteine residues that subsequently alter protein function. Arabidopsis thaliana thimet oligopeptidases TOP1 and TOP2 are required for plant defense against pathogens and the oxidative stress response. Herein, we evaluated the biochemical attributes of TOP isoforms to determine their redox sensitivity using ex vivo Escherichia coli cultures and recombinant proteins. Moreover, we explored the link between their redox regulation and plant immunity in wild-type and mutant Arabidopsis lines. These analyses revealed that redox regulation of TOPs occurs through two mechanisms: (1) oxidative dimerization of full-length TOP1 via intermolecular disulfides engaging cysteines in the N-terminal signal peptide, and (2) oxidative activation of all TOPs via cysteines that are unique and conserved. Further, we detected increased TOP activity in wild-type plants undergoing ETI or SAR following inoculation with Pseudomonas syringae strains. Mutants unable to express the chloroplast NADPH-dependent thioredoxin reductase C (NTRC) showed elevated TOP activity under unstressed conditions and were SAR-incompetent. A top1top2 knockout mutant challenged with P. syringae exhibited misregulation of ROS-induced gene expression in pathogen-inoculated and distal tissues. Furthermore, TOP1 and TOP2 could cleave a peptide derived from the immune component ROC1 with distinct efficiencies at common and specific sites. We propose that Arabidopsis TOPs are thiol-regulated peptidases active in redox-mediated signaling of local and systemic immunity.

The complexity in chemical composition alongside the genomic complexity of crop plants poses significant challenges for the characterization of their proteomes. This chapter provides specific methods that can be used for the extraction and identification of proteins from sweet potato, and a proteogenomic method for the subsequent peptide mapping on the haplotype-derived sweet potato genome assembly. We outline two basic methods for extracting proteins expressed in root and leaf tissues for the label-free quantitative proteomics-one phenol-based procedure and one polyethylene glycol (PEG) 4000-based fractionation method-and discuss strategies for the organ-specific protein extraction and increased recovery of low-abundance proteins. Next, we describe computational methods for improved proteome annotation of sweet potato based on aggregated genomics and transcriptomics resources available in our and public databases. Lastly, we describe an easily customizable proteogenomics approach for mapping sweet potato peptides back to their genome location and exemplify its use in improving genome annotations using a mass spectrometry data set.

Two complementary protein extraction methodologies coupled with an automated proteomic platform were employed to analyze tissue-specific proteomes and characterize biological and metabolic processes in sweetpotato. A total of 74 255 peptides corresponding to 4321 nonredundant proteins were successfully identified. Data were compared to predicted protein accessions for Ipomoea species and mapped on the sweetpotato transcriptome and haplotype-resolved genome. The two methodologies exhibited differences in the number and class of the unique proteins extracted. Overall, 39 916 peptides mapped to 3143 unique proteins in leaves, and 34 339 peptides mapped to 2928 unique proteins in roots. Primary metabolism and protein translation processes were enriched in leaves, whereas genetic pathways associated with protein folding, transport, sorting, as well as pathways in the primary carbohydrate metabolism were enriched in storage roots. A proteogenomics analysis successfully mapped 90.4% of the total uniquely identified peptides against the sweetpotato transcriptome and genome, predicted 741 new protein-coding genes, and specified 2056 loci where gene annotations can be further improved. The proteogenomics results provide evidence for the translation of new open reading frames (ORFs), alternative ORFs, exon extensions, and intronic ORF sequences. Data are available via ProteomeXchange with identifier PXD012999.