Journal: Plant Physiology

Article Title: SYNTAXIN OF PLANTS 132 underpins secretion of cargoes associated with salicylic acid signaling and pathogen defense

doi: 10.1093/plphys/kiae541

Figure Lengend Snippet: Analysis of leaf apoplast secreted proteins associated with trafficking SNARE SYP132 using wild type and syp121/syp122 mutant Arabidopsis (c.f. , and ). A) Images (representative) of 4-wk-old soil-grown A. thaliana wild-type and syp121/syp122 mutant plants used in the experiments. Scale bar = 1 cm (for both panels) (c.f. for images from corresponding experiments with plate-grown plants for flood inoculation with bacterial pathogens Pst ). Images were digitally extracted for comparison. B) SYP132 transcript levels in wild type and syp121/syp122 mutant Arabidopsis leaf tissue relative to 18S using RT-qPCR with gene-specific primers (c.f. ). Data are mean ± Se ( n = 3). Statistical significance using 2-tailed Mann–Whitney t test is indicated. C) Immunoblots (representative) of purified PM proteins derived from wild-type and syp121/syp122 mutant Arabidopsis leaves. Proteins were resolved using SDS-PAGE (c.f. for corresponding immunoblots detecting lumen-binding protein BiP [∼74 kDa], a marker for IMs, and Coomassie-stained membranes for total protein). Purity of the PM fractions using BiP bands as reference was estimated as >99%. Native SYP132 proteins (∼35 kDa) were detected using anti-SYP132 antibodies. A low molecular weight nonspecific band detected is marked n.s. Black lines (left) indicate position of molecular mass markers, and black arrows (right) point to expected band positions. D) Mean ± Se ( n = 6) SYP132 protein density in PM fractions of wild type and syp121/syp122 mutant Arabidopsis leaf tissue. Values are obtained from densitometric analysis of immunoblots and normalized to total protein in each lane detected using Coomassie stain. Statistical significance using 2-tailed Mann–Whitney t test is indicated. E) Workflow for TMT-MS analysis of Arabidopsis secretory proteome. Apoplast flush was collected from wild-type and syp121/syp122 mutant Arabidopsis leaves following infiltration with 10 m m MgCl 2 buffer using centrifugation ( ; ; ). Samples from 3 independent experiments were trypsin digested, labeled with reagents from TMT-11plex isobaric mass tagging kit (Thermo Scientific), and detected using electrospray ionization MS. Acquired MS/MS spectra were analyzed for protein identification and quantification using Proteome Discoverer software 3.0 (Thermo Scientific) using the MSPepSearch node. For quantitative changes, protein ratios for cargoes detected in wild-type vs syp121/syp122 mutant plants were computed (c.f. ). F) Venn diagrams depicting distribution of secreted cargo (+)/(−) signal peptides identified in wild type and syp121/syp122 mutant plants (top) and the overlap of cargoes with signal peptides detected in wild type and syp121/syp122 mutant Arabidopsis (bottom). Cargo proteins with signal peptides are considered as secretory vesicle cargoes. G) Bar graphs showing the assignment of GO terms to cargoes that undergo secretory vesicle trafficking, based on GO annotation categories, including molecular and biological functions for cargoes that show upregulation (left) and downregulation (right) in abundance in the apoplast flush. Analysis was performed using the PANTHER database program ( www.pantherdb.org ). H) Heatmap depicting hierarchical clustering of secretory cargoes detected in syp121/syp122 mutant compared against wild-type Arabidopsis using TMT-MS analysis. Cargoes with statistically significant differences in TMT ratio analyzed by ANOVA are shown ( P ≤ 0.05). Cargoes associated with hormones SA, JA, and auxin (Aux) signals are indicated (gray bars). Data are from 3 independent experiments.

Article Snippet: Isolation of microsomal total membranes and purification of the PM and IM fractions were performed using Minute Plant Plasma Membrane Protein Isolation Kit (Invent Biotech, Plymouth, USA) as per the manufacturer's instruction.

Techniques: Mutagenesis, Comparison, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Purification, Derivative Assay, SDS Page, Binding Assay, Marker, Staining, Molecular Weight, Centrifugation, Labeling, Tandem Mass Spectroscopy, Software