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SUMMARY The establishment of the nitrogen‐fixing symbiosis between soybean andBradyrhizobium japonicumis a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization‐mass spectrometry (LAESI‐MS) forin situmetabolic profiling of wild‐type nodules, nodules infected with aB. japonicum nifHmutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in thestearoyl‐acyl carrier protein desaturase(sacpd‐c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. ThenifHmutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+) and ineffective (nifHmutant, fix−) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lackingsacpd‐cdisplayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI‐MS for high‐throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.
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GGCX mutants that impair hemostasis reveal the importance of processivity and full carboxylation to VKD protein function
The gamma-glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout the multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M, S300F) whose combined heterozygosity causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation: complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX (fIX)) or calcification (Matrix Gla Protein (MGP)) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of VKD protein in the complex. The VKD protein in the complex with wild type carboxylase was carboxylated before challenge protein carboxylation occurred, and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time, and did not completely carboxylate fIX in the complex. S300F carboxylation was poor with both fIX and MGP. Additional studies analyzed fIX and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The V255M mutant generated more carboxylated peptide than wild type GGCX, however the peptides were partially carboxylated. Analysis of the V255M mutant in fIX-HEK293 cells lacking endogenous GGCX revealed poor fIX clotting activity. The studies show that disrupted processivity causes disease, and explain the defect in the patient. The kinetic analyses also suggest that disrupted processivity may occur in wild type carboxylase under some conditions, e.g. warfarin therapy or vitamin K deficiency.
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- Award ID(s):
- 1908875
- PAR ID:
- 10345129
- Date Published:
- Journal Name:
- Blood
- ISSN:
- 0006-4971
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1182/blood.2021014275
