The plant cell wall is a dynamic cellular compartment consisting of a complex matrix of components that can change dramatically in response to environmental stresses. During pathogen attack, for instance, a wide spectrum of proteins that participate in various sequential processes involved in plant defense is secreted into the cell wall. In this study, a mass spectrometry, data-independent acquisition approach known as LC/MS (E) was used to assess temporal changes in the cell wall proteome in response to different levels of an endogenous inducer of plant disease defense responses, salicylic acid (SA). LC/MS (E) was used as a label-free method that enabled simultaneous protein identification and absolute femtomole quantification of each protein secreted into the extracellular matrix. A total of 74 secreted proteins were identified, 63 of which showed increased specific secretion in response to SA. A majority of this induced secretion occurred within 2 h of treatment, indicating that many proteins are involved in the early stages of plant defenses. We also identified a number of apparently nonclassically secreted proteins, suggesting that, as in many nonplant systems, Golgi/ER-independent mechanisms exist for plant protein secretion. These results provide new insight into plant apoplastic defense mechanisms and demonstrate that LC/MS (E) is a powerful tool for obtaining both relative and absolute proteome-scale quantification that can be applied to complex, time- and dose-dependent experimental designs.

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Leukoreduction in blood units could prevent patients undergoing transfusions from transfusion-associated adverse reactions (TAARs) such as febrile nonhemolytic transfusion reactions (FNHTRs). However, the effect of prestorage and poststorage leukoreduction on TAARs and its underlying mechanisms in stored blood components remains to be determined. Therefore, we investigated the impact of prestorage leukocyte-reduced (pre-LR) and poststorage leukocyte-reduced (post-LR) blood products, including red blood cells (RBCs) and apheresis platelets (PHs), on the incidence of FNHTRs and other TAARs in patients who received transfusions from 2009 to 2014 in a tertiary care center. We also investigated the difference of leukocyte-related bioactive mediators between pre- and post-LR blood components. The results indicated that prevalence of TAARs was significantly reduced in the transfusions of pre-LR blood components. Particularly, the prevalence of FNHTRs was significantly reduced in the pre-LR RBC transfusions and the prevalence of allergy reactions was markedly reduced in the pre-LR PH transfusions. Furthermore, in vitro evaluation of cytokines in the pre- and post-LR blood components revealed that IL-1β, IL-8 and RANTES levels were significantly elevated in the post-LR RBCs during the storage. In contrast, IL-1β, IL-6 and IL-8 levels were significantly elevated in the post-LR PHs during the storage. These findings suggested that prestorage leukoreduction had a diminishing effect on the development of TAARs, which could be associated with less accumulation of cytokines in the stored blood components.

The sugar alcohol mannitol is an important carbohydrate with well-documented roles in both metabolism and osmoprotection in many plants and fungi. In addition to these traditionally recognized roles, mannitol is reported to be an antioxidant and as such may play a role in host-pathogen interactions. Current research suggests that pathogenic fungi can secrete mannitol into the apoplast to suppress reactive oxygen-mediated host defenses. Immunoelectron microscopy, immunoblot, and biochemical data reported here show that the normally symplastic plant enzyme, mannitol dehydrogenase (MTD), is secreted into the apoplast after treatment with the endogenous inducer of plant defense responses salicylic acid (SA). In contrast, a cytoplasmic marker protein, hexokinase, remained cytoplasmic after SA-treatment. Secreted MTD retained activity after export to the apoplast. Given that MTD converts mannitol to the sugar mannose, MTD secretion may be an important component of plant defense against mannitol-secreting fungal pathogens such as Alternaria. After SA treatment, MTD was not detected in the Golgi apparatus, and its SA-induced secretion was resistant to brefeldin A, an inhibitor of Golgi-mediated protein transport. Together with the absence of a known extracellular targeting sequence on the MTD protein, these data suggest that a plant's response to pathogen challenge may include secretion of selected defensive proteins by as yet uncharacterized, non-Golgi mechanisms.

Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, mannitol dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted mannitol. In the current study, LC/MS(E) was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MS(E) can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis.