Plasma membrane proteome in Arabidopsis and rice

Plant cells contain many membrane systems that are specially adapted to perform particular functions. In plant cells, the processing of signals that are involved in responses to biotic and abiotic stressors occurs in the plasma membrane. Therefore, characterization of the plasma membrane proteome can provide new insights into the functions of various plant membrane systems. Plant plasma membrane proteomics can also provide valuable information for plant-specific biological investigations. Despite recent advances in preparative and analytical techniques for plant plasma membrane proteins, the characterization of these proteins, particularly the hydrophobic ones, remains challenging. In this review, plant plasma membrane proteomics data compiled from the literature on Arabidopsis thaliana are presented. Initial attempts to determine the physiological significance of some proteins identified from plasma membrane proteomics in rice and other plants are also described from the results of our research.     

Human lectin-like oxidized low-density lipoprotein receptor-1 functions as a dimer in living cells

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a unique scavenger receptor that plays important roles in atherogenesis and has been thought to function as a monomer. Using coimmunoprecipitation studies, we demonstrate that human LOX-1 (hLOX-1) forms constitutive homo-interactions in vivo. Western blot analysis of cell lysates under nonreducing or reducing conditions revealed one clear immunoreactive species corresponding to the size of a putative receptor dimer or a monomer, respectively, consistent with the presence of disulfide-linked hLOX-1 complexes. Site-directed mutagenesis studies indicated that cysteine 140 has a key role in the formation of these disulfide-linked hLOX-1 dimers. Eliminating this intermolecular disulfide bond markedly impairs the recognition of Escherichia coli by hLOX-1. Furthermore, these dimers can act as a "structural unit" to form noncovalently associated oligomers, as demonstrated by a membrane-impermeant crosslinker, which resulted in immunoreactive species corresponding to the sizes of putative tetramers and hexamers. These results provide the first evidence for the existence of hLOX-1 dimers/oligomers.     

Ozone stress‐induced proteomic changes in leaf total soluble and chloroplast proteins of soybean reveal that carbon allocation is involved in adaptation in the early developmental stage

Considerable soybean yield losses caused by ozone (O₃) stress have been demonstrated by large‐scale meta‐analyses of free‐gas concentration enrichment systems. In this study, comparative proteomic approach was employed to explore the differential changes of proteins in O₃ target structures such as leaf and chloroplasts of soybean seedlings. Acute O₃ exposure (120 parts‐per‐billion) for 3 days did not cause any visible symptoms in developing leaves. However, higher amounts of ROS and lipid peroxidation indicated that severe oxidative burst occurred. Immunoblot analysis of O₃‐induced known proteins revealed that proteins were modulated before symptoms became visible. Proteomic analysis identified a total of 20 and 32 differentially expressed proteins from O₃‐treated leaf and chloroplast, respectively. Proteins associated with photosynthesis, including photosystem I/II and carbon assimilation decreased following exposure to O₃. In contrast, proteins involved in antioxidant defense and carbon metabolism increased. The activity of enzymes involved in carbohydrate metabolism increased following exposure to O₃, which is consistent with the decrease in starch and increase in sucrose concentrations. Taken together, these results suggest that carbon allocation is tightly programmed, and starch degradation probably feeds the tricarboxylic acid cycle while the photosynthesis pathway is severely affected during O₃ stress.     

Mineral Composition of and the Relationships Between Them of Human Basal Ganglia in Very Old Age

Trace elements and the relationships among them were investigated by direct chemical analysis in three basal ganglia regions in very old age individuals and age- and gender-related differences were assessed. After ordinary dissections at Nara Medical University were finished, the caudate nucleus, putamen, and globus pallidus belonging to the basal ganglia were removed from the identical cerebra of the subjects who consisted of 22 men and 23 women, ranging in age from 70 to 101 years (average age?=?83.3?±?7.5 years). After incineration with nitric acid and perchloric acid, the element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the Ca, P, and Mg contents increased significantly in the putamen with aging and the Mg content increased significantly in the globus pallidus with aging, but no elements increased significantly in the caudate nucleus with aging. Regarding the relationships among elements in the basal ganglia, extremely significant direct correlations were found among the Ca, P, and Mg contents in the putamen. These results suggested that slight calcification occurred in the putamen in very old age. With regard to seven elements of Ca, P, S, Mg, Zn, Fe, and Na, it was examined whether there were significant correlations among the caudate nucleus, putamen, and globus pallidus. It was found that there were extremely significant direct correlations among all of the three basal ganglia in the P content. Likewise, with regard to the Fe content, there were extremely or very significant direct correlations among all of the three basal ganglia. Regarding the gender difference in elements, it was found that the Ca content of the caudate nucleus was significantly higher in women than in men.     

Analysis of the Proteome of Common Bean (Phaseolus vulgaris L.) Roots after Inoculation with Rhizobium etli

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.     

PROTEOME ANALYSIS OF GUT AND SALIVARY GLAND PROTEINS OF FIFTH-INSTAR NYMPH AND ADULTS OF THE SUNN PEST, Eurygaster integriceps

In the digestive system of the sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), the salivary gland has a key role in extra oral digestion and the gut is the main site for digestion of food. In this study, proteomics was used to study the role of proteins involved in digestion. The amount of feeding on wheat grain by adult insects increased by comparison to fifth-instar nymphs. Proteins of the gut and salivary gland in adults and fifth-instar nymphs were analyzed 1 day after feeding. The proteins related to digestion, metabolism, and defense against toxins were accumulated in the gut of adult insects. Three plant proteins including serpin, dehydroascorbate reductase, and β-amylase were accumulated in guts of adults. In the salivary gland, phospholipase A2 and arginine kinase were increased in adults. Heat shock protein 70 increased in the gut of fifth-instar nymphs. Proteomic analysis revealed that most of changed proteins in digestive system of sunn pest were increased in adults. This study provided more targets derived from gut and salivary gland for pest management.     

Proteome approach to characterize the methylmalonate-semialdehyde dehydrogenase that is regulated by gibberellin

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths ofslr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA(3). Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice.     

Comprehensive phosphoproteome analysis in rice and identification of phosphoproteins responsive to different hormones/stresses

Phosphoproteins in rice were detected by in vitro protein phosphorylation followed by two-dimensional polyacrylamide gel electrophoresis. Forty-four phosphoproteins were detected on a 2D-gel after in vitro protein phosphorylation of the crude extract from rice leaf sheath. Among the phosphoproteins detected, 42 were identified through analysis by Q-TOF MS/MS and/or MALDI-TOF MS. The largest percentage of the identified phosphoproteins are involved in signaling (30%), while 18% are involved in metabolism. When rice seedlings were treated with various hormones and stresses, it was observed that the phosphorylation of 13 proteins was enhanced differentially by different hormone and stress treatments. Furthermore, when the hormone/stress regulated phosphoproteins are compared in rice leaf sheath, leaf blade and root, only cytoplasmic malate dehydrogenase was found to be phosphorylated in all the tissues. Results suggest that in the phosphorylation cascade of rice, glycolytic metabolism processes and Ca(2+)-signaling seem to be important targets in response to hormones and stresses. Furthermore, the direct visualization of phosphoproteins by (32)P-labeling and their mass spectrometric identification provides an accurate and reliable method of analyzing the rice phosphoproteome.