Purcellmorsing8220

Jasmonoyl-L-isoleucine (JA-Ile) is a powerful oxylipin responsible for the genome-wide transcriptional reprogramming in plants that results in major physiological shifts from growth to defense. The double T-DNA insertion Arabidopsis mutant, cyp94b1cyp94b3 (b1b3), defective in cytochrome p450s, CYP94B1 and CYP94B3, which are responsible for oxidizing JA-Ile, accumulates several fold higher levels of JA-Ile yet displays dampened JA-Ile-dependent wound responses-the opposite of what is expected. Transcriptomic and proteomic analyses showed that while the transcriptional response to wounding was largely unchanged in b1b3 compared to wild type (WT), many proteins were found to be significantly reduced in the mutant, which was verified by immunoblot analyses of marker proteins. To understand this protein phenotype and their hypothesized contribution to the b1b3 phenotypes, wounded rosette leaf samples from both WT and b1b3 were subject to a translating ribosome affinity purification RNA sequencing analysis. More than 1,600 genes whose transcripts do not change in abundance by wounding changed their association with the ribosomes after wounding in WT leaves. Consistent with previous observations, the total pool of mRNA transcripts was similar between WT and b1b3; however, the ribosome-associated pool of transcripts was changed significantly. Most notably, fewer transcripts were associated with the ribosome pool in b1b3 than in WT, potentially explaining the reduction of many proteins in the mutant. Among those genes with fewer ribosome-associated transcripts in b1b3 were genes relating to stress response, specialized metabolism, protein metabolism, ribosomal subunits, and transcription factors, consistent with the biochemical phenotypes of the mutant. These results show previously unrecognized regulations at the translational level that are affected by misregulation of JA homeostasis during the wound response in plants.Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.The length of the day (photoperiod) is a robust seasonal signal originated by earth orbital and translational movements, a resilient external cue to the global climate change, and a predictable hint to initiate or complete different developmental programs. In eukaryotic algae, the gene expression network that controls the cellular response to photoperiod also regulates other basic physiological functions such as starch synthesis or redox homeostasis. Land plants, evolving in a novel and demanding environment, imbued these external signals within the regulatory networks controlling organogenesis and developmental programs. Unlike algae that largely have to deal with cellular physical cues, within the course of evolution land plants had to transfer this external information from the receiving organs to the target tissues, and mobile signals such as hormones were recruited and incorporated in the regulomes. Control of senescence by photoperiod, as suggested in this perspective, would be an accurate way to feed seasonal information into a newly developed function (senescence) using an ancient route (photoperiodic signaling). This way, the plant would assure that two coordinated aspects of development such as flowering and organ senescence were sequentially controlled. As in the case of senescence, there is growing evidence to support the idea that harnessing the reliability of photoperiod regulation over other, more labile signaling pathways could be used as a robust breeding tool to enhance plants against the harmful effects of climate change.N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. Selleckchem Chroman 1 thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants.