Identification of Indicator Proteins Associated with Flooding Injury in Soybean Seedlings Using Label-free Quantitative Proteomics

Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean

Identification of Indicator Proteins Associated with Flooding Injury in Soybean Seedlings Using Label-free Quantitative Proteomics

Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean

Predicting arbuscular mycorrhizal fungal colonization of soybean in farmers’ fields by using infection unit density

Estimating arbuscular mycorrhizal (AM) fungal activity to colonize crop root before cultivation is prerequisite for effective utilization of their functions which enhance growth and yield of the plant especially under low fertilizer input. We have hypothesized that the infection unit (IU) density formed on test plant roots grown for short period (12 days) with soil sampled from soybean production fields would be an effective indicator to predict AM fungal colonization intensity to the plant. In order to test this hypothesis, three-year farmland survey was conducted, in which soil samples before sowing soybean and the plant root samples at third trifoliate (V3) and full bloom (R2) stage were collected from farmers’ fields in two regions in Hokkaido, Iwamizawa and Tokachi. For each sampling spot, IU density was determined by using test plants, and intensity of AM fungal colonization of soybean root was measured. Before pursuing field survey, laboratory experiments were conducted to find out proper soil storage condition that keeps IU density unchanged while handling many soil samples. Our results indicated that IU density was almost comparable to the original value after six-month storage if soil samples were kept in a refrigerator, although storing at ambient temperature significantly decreased the measurement. Air drying also had negative impact on IU density. According to the field survey, IU densities determined using field soil were positively and significantly correlated with AM fungal colonization of soybean roots at both V3 and R2 stages. Differences in climate, soil type, and style of agriculture between Iwamizawa and Tokachi seemed to have little effect on IU density-AM fungal colonization relationship. Other than IU density, soil pH and soil penetration resistance at 10 cm depth were selected as significant explanatory variables for predicting AM fungal colonization by multiple regression analysis. However, IU density was the most influential factor among three. Therefore, IU density is recognized as an effective measure to evaluate AM fungal colonizing activity in field soil.</p

(A) Expression of mRNA in salivary gland epithelial cells.

<p>Salivary gland epithelial cells were isolated from SS patients and cultured. Total RNA was isolated from these cells, and EGF-R, α amylase-1, and CD3δ mRNAs were assayed by RT-PCR, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045689#s2" target="_blank">Materials and Methods</a> section. Lane 1: salivary gland epithelial cells, Lane 2: labial salivary gland of the same patient, Lane 3: normal lymph node as a control for CD3δ. <b>(B and C) Effects of IFNγ on human SGE cells.</b> SGE cells were incubated with various concentration of IFNγ for 48 hours (B) or with 1000 U/ml of IFNγ for the indicated times (C), and the surface expression of CD40 was examined by FACS analysis.</p

Profile of patients included in the study.

<p>Nine patients (all women; mean age, 48±14 years) met both the 2002 American-European consensus group (AECG) criteria and the SICCA criteria for Sjögren's syndrome (SS), whereas the other six (all women; mean age, 57±8 years) did not (No).</p>*<p>Titers of anti-nuclear antibody (ANA).</p

Quantification of cytokines secreted into the culture supernatants of SGE cells.

<p>Confluent SGE cells obtained from 9 SS patients and 6 normal controls (non-SS) were incubated in the presence of IFNγ (1000 U/ml), and the culture supernatants were collected on days 0, 2, 4, and 6. Median concentrations (pg/ml) of IL-6 (A) and TGFβ (B) in the supernatants were determined by ELISA and compared by non-parametric Mann-Whitney tests (*, p<0.05).</p

Effects of IFNγ on the proliferation and apoptosis of SGE cells.

<p>(A) SGE cells, human airway epithelial cells (HBTEC) and human umbilical vein endothelial cells (HUVEC)were incubated with the indicated concentration of IFNγ, and proliferative responses were assessed at 48 h. Each bar shows mean + SD. IFNγ did not significantly affect the proliferation of any of these cells. The results shown are representative of three independent experiments. (B) SGE cells were incubated with the indicated concentration of IFNγ, and apoptosis was determined at 12 h by flow cytometry. Numbers in R1 and R2 indicate early and late apoptosis, respectively.</p

Decreased Expression of Innate Immunity-Related Genes in Peripheral Blood Mononuclear Cells from Patients with IgG4-Related Disease

<div><p>Background</p><p>IgG4-related disease (IgG4-RD) is a new clinical entity of unknown etiology characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. Although aberrancies in acquired immune system functions, including increases in Th2 and Treg cytokines observed in patients with IgG4-RD, its true etiology remains unclear. To investigate the pathogenesis of IgG4-RD, this study compared the expression of genes related to innate immunity in patients with IgG4-RD and healthy controls.</p><p>Materials and Methods</p><p>Peripheral blood mononuclear cells (PBMCs) were obtained from patients with IgG4-RD before and after steroid therapy and from healthy controls. Total RNA was extracted and DNA microarray analysis was performed in two IgG4-RD patients to screen for genes showing changes in expression. Candidate genes were validated by real-time RT-PCR in 27 patients with IgG4-RD and 13 healthy controls.</p><p>Results</p><p>DNA microarray analysis identified 21 genes that showed a greater than 3-fold difference in expression between IgG4-RD patients and healthy controls and 30 genes that showed a greater than 3-fold change in IgG4-RD patients following steroid therapy. Candidate genes related to innate immunity, including those encoding Charcot–Leyden crystal protein (CLC), membrane-spanning 4-domain subfamily A member 3 (MS4A3), defensin alpha (DEFA) 3 and 4, and interleukin-8 receptors (IL8R), were validated by real-time RT-PCR. Expression of all genes was significantly lower in IgG4-RD patients than in healthy controls. Steroid therapy significantly increased the expression of DEFA3, DEFA4 and MS4A3, but had no effect on the expression of CLC, IL8RA and IL8RB.</p><p>Conclusions</p><p>The expression of genes related to allergy or innate immunity, including CLC, MS4A3, DEFA3, DEFA4, IL8RA and IL8RB, was lower in PBMCs from patients with IgG4-RD than from healthy controls. Although there is the limitation in the number of patients applied in DNA microarray, impaired expression of genes related to innate immunity may be involved in the pathogenesis of IgG4-RD as well as in abnormalities of acquired immunity.</p></div

Comparison of gene expression between patients with IgG4-RD and healthy controls.

<p>Relative expression of genes in PBMCs from 27 patients with IgG4-RD and 20 healthy controls. (A) CLC; Charcot—Leyden crystal protein. (B) IL8RA; interleukin 8 receptor alpha. (C) IL8RB; interleukin 8 receptor beta. (D) MS4A3; membrane-spanning 4-domain subfamily A member 3. (E) DEFA3; defensin alpha 3. (F) DEFA4; defensin alpha 4. Expression of all genes was significantly lower in PBMCs from untreated IgG4-RD patients than from healthy controls (p< 0.01).</p

Gene expression in PBMCs from 20 patients with IgG4-RD, before and after steroid treatment.

<p>(A) CLC; Charcot—Leyden crystal protein. (B) IL8RA; interleukin 8 receptor alpha. (C) IL8RB; interleukin 8 receptor beta. (D) MS4A3; membrane-spanning 4-domain subfamily A member 3. (E) DEFA3; defensin alpha 3. (F) DEFA4; defensin alpha 4. Levels of CLC, IL8RA and IL8RB mRNA were not altered in IgG4-RD patients by steroid therapy (A-C), whereas those of MS4A3, DEFA3 and DEFA4 were significantly increased following steroid therapy (D-F, p<0.01).</p