Search Results (8)

Seed germination is a critical phase in rice production and is highly sensitive to environmental and chemical stresses. Chloramphenicol (CAM), a known phytotoxic antibiotic, has been reported to suppress rice seedling establishment, yet its underlying molecular mechanisms remain poorly understood. In this study, we investigated the effects of varying CAM concentrations on rice germination and early seedling establishment. While CAM significantly retarded germination speed and seedling growth, the final germination rates remained largely unaffected, even at high concentrations. To uncover the molecular basis of CAM phytotoxicity, we conducted time-resolved phosphoproteomic profiling during both the germination and early seedling stages. Our analyses revealed dynamic, stage-specific phosphorylation changes: moderate alterations affecting metabolic and cytokinesis-related processes during germination, and extensive disruptions in metabolic pathways, stress response mechanisms, DNA replication, and hormone signaling during early seedling establishment. Collectively, these findings demonstrate that CAM disrupts rice development by remodeling phosphorylation networks and modulating key physiological and signaling pathways. This study provides novel insights into the molecular mechanisms underlying antibiotic-induced growth inhibition and advances our understanding of plant stress responses during early development. Full article

Seed germination is critical for early plantlet development and is tightly controlled by environmental factors. Nevertheless, the signaling networks underlying germination control remain elusive. In this study, the remodeling of Arabidopsis seed phosphoproteome during imbibition was investigated using stable isotope dimethyl labeling and nanoLC-MS/MS analysis. Freshly harvested seeds were imbibed under dark or constant light to restrict or promote germination, respectively. For each light regime, phosphoproteins were extracted and identified from dry and imbibed (6 h, 16 h, and 24 h) seeds. A large repertoire of 10,244 phosphopeptides from 2546 phosphoproteins, including 110 protein kinases and key regulators of seed germination such as Delay Of Germination 1 (DOG1), was established. Most phosphoproteins were only identified in dry seeds. Early imbibition led to a similar massive downregulation in dormant and non-dormant seeds. After 24 h, 411 phosphoproteins were specifically identified in non-dormant seeds. Gene ontology analyses revealed their involvement in RNA and protein metabolism, transport, and signaling. In addition, 489 phosphopeptides were quantified, and 234 exhibited up or downregulation during imbibition. Interaction networks and motif analyses revealed their association with potential signaling modules involved in germination control. Our study provides evidence of a major role of phosphosignaling in the regulation of Arabidopsis seed germination. Full article

High-temperature stress severely affects rice grain quality. While extensive research has been conducted at the physiological, transcriptional, and protein levels, it is still unknown how protein phosphorylation regulates seed development in high-temperature environments. Here, we explore the impact of high-temperature stress on the phosphoproteome of developing grains from two indica rice varieties, 9311 and Guangluai4 (GLA4), with different starch qualities. A total of 9994 phosphosites from 3216 phosphoproteins were identified in all endosperm samples. We identified several consensus phosphorylation motifs ([sP], [LxRxxs], [Rxxs], [tP]) induced by high-temperature treatment and revealed a core set of protein kinases, splicing factors, and regulatory factors in response to high-temperature stress, especially those involved in starch metabolism. A detailed phosphorylation scenario in the regulation of starch biosynthesis (AGPase, GBSSI, SSIIa, SSIIIa, BEI, BEIIb, ISA1, PUL, PHO1, PTST) in rice endosperm was proposed. Furthermore, the dynamic changes in phosphorylated enzymes related to starch synthesis (SSIIIa-Ser94, BEI-Ser562, BEI-Ser620, BEI-Ser821, BEIIb-Ser685, BEIIb-Ser715) were confirmed by Western blot analysis, which revealed that phosphorylation might play specific roles in amylopectin biosynthesis in response to high-temperature stress. The link between phosphorylation-mediated regulation and starch metabolism will provide new insights into the mechanism underlying grain quality development in response to high-temperature stress. Full article

Via inhalation we are continuously exposed to environmental and occupational irritants which can induce adverse health effects, such as irritant-induced asthma (IIA). The airway epithelium forms the first barrier encountered by these agents. We investigated the effect of environmental and occupational irritants on the airway epithelial barrier in vitro. The airway epithelial barrier was mimicked using a coculture model, consisting of bronchial epithelial cells (16HBE) and monocytes (THP-1) seeded on the apical side of a permeable support, and human lung microvascular endothelial cells (HLMVEC) grown on the basal side. Upon exposure to graphene (G) and graphene oxide (GO) in a suspension with fetal calf serum (FCS), ammonium persulfate (AP), sodium persulfate (SP) and hypochlorite (ClO⁻), the transepithelial electrical resistance (TEER) and flux of fluorescent labelled dextran (FD4-flux), was determined. Exposure to graphene nanoparticles (GNPs) induced an immediate negative effect on the epithelial barrier, whereas ClO⁻ only had a negative impact after 24 h of exposure. AP and SP did not affect the barrier properties. The tight junctions (TJ) network showed less connected zonula occludens 1 (ZO-1) and occludin staining in GNP-exposed cocultures. Functional analysis of the phosphoproteomic data indicated that proteins in the adherens junction (AJ) and TJ pathways showed an altered phosphorylation due to GNP exposure. To conclude, the negative effect of GNPs on the epithelial barrier can be explained by the slightly altered the TJ organization which could be caused by alterations in the phosphorylation level of proteins in the AJ and TJ pathway. Full article

The membrane phosphoproteome in plant seed changes dynamically during embryo development. We examined the patterns of Phaseolus vulgaris (common bean) seed membrane protein phosphorylation from the mid-maturation stage until two days after germination. Serine and threonine phosphorylation declined during seed maturation while tyrosine phosphorylation remained relatively constant. We discovered that the aquaporin PvTIP3;1 is the primary seed membrane phosphoprotein, and PvTIP3;2 shows a very low level of expression. The level of phosphorylated Ser7 in PvTIP3;1 increased four-fold after seed maturation. Since phosphorylation increases water channel activity, we infer that water transport by PvTIP3;1 is highest in dry and germinating seeds, which would be optimal for seed imbibition. By the use of isoform-specific, polyclonal peptide antibodies, we found that PvTIP3;2 is expressed in a developmental pattern similar to PvTIP3;1. Unexpectedly, PvTIP3;2 is tyrosine phosphorylated following seed maturation, which may suggest a mechanism for the regulation of PvTIP3;2 following seed germination. Analysis of protein secondary structure by circular dichroism spectroscopy indicated that the amino-terminal domain of PvTIP3;1 is generally unstructured, and phosphorylation increases polyproline II (PPII) helical structure. The carboxy-terminal domain also gains PPII character, but in a pH-dependent manner. These structural changes are a first step to understand TIP3 aquaporin regulation. Full article

The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple differentially phosphorylated isoforms. In this study, temporal changes in the phosphorylation status of patatin isoforms and their involvement in patatin mobilization are investigated using phosphoproteomic methods based on targeted two-dimensional electrophoresis (2-DE). High-resolution 2-DE profiles of patatin isoforms were obtained in four sequential tuber life cycle stages of Kennebec cultivar: endodormancy, bud break, sprouting and plant growth. In-gel multiplex identification of phosphorylated isoforms with Pro-Q Diamond phosphoprotein-specific stain revealed an increase in the number of phosphorylated isoforms after the tuber endodormancy stage. In addition, we found that the phosphorylation status of patatin isoforms significantly changed throughout the tuber life cycle (P < 0.05) using the chemical method of protein dephosphorylation with hydrogen fluoride-pyridine (HF-P) coupled to 2-DE. More specifically, patatin phosphorylation increased by 32% from endodormancy to the tuber sprouting stage and subsequently decreased together with patatin degradation. Patatin isoforms were not randomly mobilized because highly phosphorylated Kuras-isoforms were preferably degraded in comparison to less phosphorylated non-Kuras isoforms. These results lead us to conclude that patatin is mobilized by a mechanism dependent on the phosphorylation status of specific isoforms. Full article

Dormancy is the mechanism that allows seeds to become temporally quiescent in order to select the right time and place to germinate. Like in other species, in barley, grain dormancy is gradually reduced during after-ripening. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphoproteins to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different dormancy levels. The LC-MS/MS analysis identified 2346 phosphopeptides in barley embryos, with 269 and 97 of them being up- or downregulated during imbibition, respectively. A number of phosphopeptides were differentially regulated between FH and AR samples, suggesting that phosphoproteomic profiles were quite different between FH and AR grains. Motif analysis suggested multiple protein kinases including SnRK2 and MAPK could be involved in such a difference between FH and AR samples. Taken together, our results revealed phosphosignaling pathways in barley grains during the water imbibition process. Full article

Seed storage proteins play a fundamental role in plant reproduction and human nutrition. They accumulate during seed development as reserve material for germination and seedling growth and are a major source of dietary protein for human consumption. Storage proteins encompass multiple isoforms encoded by multi-gene families that undergo abundant glycosylations and phosphorylations. Two-dimensional electrophoresis (2-DE) is a proteomic tool especially suitable for the characterization of storage proteins because of their peculiar characteristics. In particular, storage proteins are soluble multimeric proteins highly represented in the seed proteome that contain polypeptides of molecular mass between 10 and 130 kDa. In addition, high-resolution profiles can be achieved by applying targeted 2-DE protocols. 2-DE coupled with mass spectrometry (MS) has traditionally been the methodology of choice in numerous studies on the biology of storage proteins in a wide diversity of plants. 2-DE-based reference maps have decisively contributed to the current state of our knowledge about storage proteins in multiple key aspects, including identification of isoforms and quantification of their relative abundance, identification of phosphorylated isoforms and assessment of their phosphorylation status, and dynamic changes of isoforms during seed development and germination both qualitatively and quantitatively. These advances have translated into relevant information about meaningful traits in seed breeding such as protein quality, longevity, gluten and allergen content, stress response and antifungal, antibacterial, and insect susceptibility. This review addresses progress on the biology of storage proteins and application areas in seed breeding using 2-DE-based maps. Full article