Spatial Mapping of Plant N-Glycosylation Cellular Heterogeneity Inside Soybean Root Nodules Provided Insights Into Legume-Rhizobia Symbiosis

Abstract

Although ubiquitously present, information on the function of complex N-glycan posttranslational modification in plants is very limited and is often neglected. In this work, we adopted an enzyme-assisted matrix-assisted laser desorption/ionization mass spectrometry imaging strategy to visualize the distribution and identity of N-glycans in soybean root nodules at a cellular resolution. We additionally performed proteomics analysis to probe the potential correlation to proteome changes during symbiotic rhizobia-legume interactions. Our ion images reveal that intense N-glycosylation occurs in the sclerenchyma layer, and inside the infected cells within the infection zone, while morphological structures such as the cortex, uninfected cells, and cells that form the attachment with the root are fewer N-glycosylated. Notably, we observed different N-glycan profiles between soybean root nodules infected with wild-type rhizobia and those infected with mutant rhizobia incapable of efficiently fixing atmospheric nitrogen. The majority of complex N-glycan structures, particularly those with characteristic Lewis-a epitopes, are more abundant in the mutant nodules. Our proteomic results revealed that these glycans likely originated from proteins that maintain the redox balance crucial for proper nitrogen fixation, but also from enzymes involved in N-glycan and phenylpropanoid biosynthesis. These findings indicate the possible involvement of Lewis-a glycans in these critical pathways during legume-rhizobia symbiosis.

Discovery top-down proteomics in symbiotic soybean root nodules

“…Only two of the sixteen fragments showed low signal in mutant samples in LFQ after alignment and match-between-runs (MBR), which can likely be attributed to LC carry-over or false positive matches during MBR. Compared to the 3,653 soybean and 2,863 bacterial proteins from previous shotgun BUP data (Veličković et al, 2022) on the same sample, the global proteome coverage is lower in TDP. This is as expected given the known high protein molecular weight detection limitations in TDP.…”

“…Plant growth and soybean nodule collection was performed at the University of Missouri as previously described. (Agtuca et al, 2020;Veličković et al, 2022) Briefly, the plants were inoculated at 3 days post-germination, and soybean nodules were harvested at day 21, plunge frozen into liquid nitrogen, and stored in −80 °C. Five WT and five nifH-nodules were used for protein extraction and bulk LCMS analysis.…”

“…Five biological replicates of nifH − and WT soybean nodules were prepared for top-down proteomics. Proteins were extracted from homogenized frozen soybean nodules with the same methanol/choloroform protocol as described in detail previously (Veličković et al, 2022). The extracted proteins in lysis buffer (8 M urea, 50 mM ammonium bicarbonate, pH 8.0) were split for TDP and BUP with matching sample names in the raw data files.…”

PNGase H + variant from Rudaea cellulosilytica with improved deglycosylation efficiency for rapid analysis of eukaryotic N‐glycans and hydrogen deuterium exchange mass spectrometry analysis of glycoproteins

“…36 Furthermore, the higher yields and homogeneity after protein purification allowed the immobilization of PNGase Dj on microfluidic chips for studying the glycosylated sema-domain of the tyrosine-protein kinase MET, 36 or the use as an in-situ deglycosylation agent for matrix-assisted laser desorption-mass spectrometry (MALDI)-imaging of N-glycans in soybean root nodules. 41 Herein, we describe and characterize a previously unstudied recombinant acidic PNGase variant from Rudaea cellulosilytica with improved enzymatic properties and expression yields compared to previously described superacidic PNGases. Our results indicate that PNGase Rc should be a highly suitable enzyme for use in LC/LC-MS workflows for the analysis of released N-linked glycans from a broad array of complex glycoprotein samples as well as for the HDX-MS technique that requires highly efficient deglycosylation at low pH to allow comprehensive analysis of glycoproteins.…”

“…The most active variant originating from Dyella japonica (in short, PNGase Dj) showed a ~ fourfold increase in deglycosylation activity of horseradish peroxidase (HRP) when compared to PNGase Tr, and outperformed PNGase A in deglycosylating Trastuzumab glycopeptides 36 . Furthermore, the higher yields and homogeneity after protein purification allowed the immobilization of PNGase Dj on microfluidic chips for studying the glycosylated sema‐domain of the tyrosine‐protein kinase MET, 36 or the use as an in‐situ deglycosylation agent for matrix‐assisted laser desorption‐mass spectrometry (MALDI)‐imaging of N ‐glycans in soybean root nodules 41 …”