Unravelling the Helianthus tuberosus L. (Jerusalem Artichoke,Kiku-Imo) Tuber Proteome by Label-Free Quantitative Proteomics

The present research investigates the tuber proteome of the ‘medicinal’ plant Jerusalem artichoke (abbreviated as JA) (Helianthus tuberosus L.) using a high-throughput proteomics technique. Although JA has been historically known to the Native Americans, it was introduced to Europe in the late 19th century and later spread to Japan (referred to as ‘kiku-imo’) as a folk remedy for diabetes. Genboku Takahashi research group has been working on the cultivation and utilization of kiku-imo tuber as a traditional/alternative medicine in daily life and researched on the lowering of blood sugar level, HbA1c, etc., in human subjects (unpublished data). Understanding the protein components of the tuber may shed light on its healing properties, especially related to diabetes. Using three commercially processed JA tuber products (dried powder and dried chips) we performed total protein extraction on the powdered samples using a label-free quantitate proteomic approach (mass spectrometry) and catalogued for the first time a comprehensive protein list for the JA tuber. A total of 2967 protein groups were identified, statistically analyzed, and further categorized into different protein classes using bioinformatics techniques. We discussed the association of these proteins to health and disease regulatory metabolism. Data are available via ProteomeXchange with identifier PXD030744.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Gel-based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama

We examined responses of cultivated bean (Phaseolus vulgaris L. cv. IDIAP R-3) and maize (Zea mays L. cv. Guarare 8128) plants exposed to ozone (O(3)) using a leaf injury assessment and proteomics approach. Plants grown for 16 days in greenhouse were transferred to an O(3) chamber and exposed continuously to 0.2 ppm O(3) or filtered pollutant-free air for up to 72 h. CBB-stained gels revealed changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) protein. By Western analysis changes in marker proteins for O(3) damage in leaves by 1-DE were checked. In bean leaves, two superoxide dismutase (SOD) protein (19 and 20 kDa) were dramatically decreased, while ascorbate peroxidase (APX, 25 kDa), small heat shock protein (HSP, 33 kDa), and a naringenin-7-O-methyltransferase (NOMT, 42 kDa) were increased by O(3). In maize leaves, expression levels of catalase (increased), SOD (decreased), and APX (increased) were drastically changed by O(3) depending on the leaf stage, whereas crossreacting HSPs (24 and 30 kDa) and NOMT (41 kDa) proteins were strongly increased in O(3)-stressed younger leaves. These results indicated a clear modulation of oxidative stress-, heat shock-, and secondary metabolism-related proteins by O(3). Finally, 2-DE at 72 h after O(3) exposure revealed changes (induction/suppression) in expression levels of 25 and 12 protein spots in bean and maize leaves, respectively. Out of these, ten and nine nonredundant proteins in bean and maize, respectively, were identified by MS. A novel pathogenesis-related protein 2 may serve as a potential marker for O(3) stress in bean.     

Role of jasmonate in the rice (Oryza sativa L.) self-defense mechanism using proteome analysis

Exogenously applied jasmonic acid (JA) was used to study changes in protein patterns in rice (Oryza sativa L.) seedling tissues, to classify these changes, and to assign a role for these changes, in order to define the role of JA in the rice self-defense mechanism. High resolution two-dimensional polyacrylamide gel electrophoretic analysis revealed induction of new proteins in both leaf and stem tissues after JA treatment, with the major protein spots further analyzed through N-terminal and internal amino acid sequencing, purification, antibody production, and immunoblot analysis. JA treatment results in necrosis in these tissues, which is accompanied by drastic reductions in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) subunits, and was confirmed using immunoblotting. Induction of novel proteins was found particularly in the stem tissues, including a new basic 28 kDa Bowman-Birk proteinase inhibitor protein (BBPIN; jasmonate-induced stem protein, JISP 6), and acidic 17 kDa pathogenesis-related class 1 protein (PR-1, JISP 9). This induction of proteins was blocked by a protein synthesis inhibitor cycloheximide (CHX) indicating de novo protein synthesis. Kinetin (KIN), a cytokinin and free radical scavenger reversed RuBisCO decreases, but not induction of proteins. Immunoblot analysis using antibodies generated against these purified proteins revealed a tissue-specific expression pattern and time-dependent induction after JA treatment. Our results indicate that jasmonate affects defense-related gene expression in rice seedlings, as evidenced by de novo synthesis of novel proteins with potential roles in plant defense.     

A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf

By using an in vivo hydroponic rice seedling culture system, we investigated the physiological and biochemical responses of a model rice japonica cultivar Nipponbare to salt stress using proteomics and classical biochemical methods. Yoshida's nutrient solution (YS) was used to grow rice seedlings. YS-grown 18-day-old seedlings manifested highly stable and reproducible symptoms, prominently the wilting and browning of the 3rd leaf, reduced photosynthetic activity, inhibition in overall seedling growth, and failure to develop new (5th) leaf, when subjected to salt stress by transferring them to YS containing 130 mM NaCl for 4 days. As leaf response to salt stress is least investigated in rice by proteomics, we used the 3rd leaf as source material. A comparison of 2-DE protein profiles between the untreated control and salt-stressed 3rd leaves revealed 55 differentially expressed CBB-stained spots, where 47 spots were increased over the control. Of these changed spots, the identity of 33 protein spots (27 increased and 5 decreased) was determined by nESI-LC-MS/MS. Most of these identified proteins belonged to major metabolic processes like photosynthetic carbon dioxide assimilation and photorespiration, suggesting a good correlation between salt stress-responsive proteins and leaf morphology. Moreover, 2-DE immunoblot and enzymatic activity analyses of 3rd leaves revealed remarkable changes in the key marker enzymes associated with oxidative damage to salt stress: ascorbate peroxidase and lipid peroxidation were induced, and catalase was suppressed. These results demonstrate that hydroponic culture system is best suited for proteomics of salt stress in rice seedling.     

Separation of proteins from stressed rice (Oryza sativa L.) leaf tissues by two-dimensional polyacrylamide gel electrophoresis: induction of pathogenesis-related and cellular protectant proteins by jasmonic acid, UV irradiation and copper chloride

We have used three kinds of stresses, including the signaling compound jasmonic acid, an environmental stressor, UV irradiation, and a heavy metal salt copper chloride, to study changes in the protein patterns in rice (Oryza sativa L.) leaf tissues using two-dimensional polyacrylamide gel electrophoresis. However, instead of using lysis buffer containing urea (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) for extraction of proteins from rice seedling tissues, we used Tris-HCl buffer (commonly used for extraction of proteins for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for extraction of proteins and resolved these extracted proteins by the usual method of O'Farrell. Furthermore, the induction of a large number of proteins was clearly observed over controls. No spots corresponding to these induced proteins were found in the control experiment, indicating qualitative changes in protein patterns after various stress treatments. A total of 12 out of 13 proteins could be N-terminally sequenced from jasmonic acid-treated rice leaf tissues, and one protein was sequenced from UV-irradiated leaf tissues. These proteins showed high homology to pathogenesis-related (thaumatin-like protein, a PR5 class protein; a beta-1,3-glucanase precursor; an intracellular PR protein encoded by PBZ1 gene, and an antifungal protein) and cellular protectant (glutathione transferase, EC 2.5.1.18; and ascorbate peroxidase) proteins, from plants, including rice. Results presented here suggest a role for jasmonic acid in the self-defense mechanisms of rice plants.     

AC and DC properties of M-type SrCo<Subscript>x</Subscript>Ti<Subscript>x</Subscript>Fe<Subscript>(12−2x)</Subscript>O<Subscript>19</Subscript> hexagonal ferrite

Microwave characterization of SrCo _x Ti _x Fe_(12?2x)O₁₉ (x = 0.0, 0.2, 0.3, 0.5, 0.6, 0.7, 1.0) ferrites has been studied as a function of frequency, substitution and thickness, and static electrical current density-electric field characteristics have been investigated as a function of substitution. Microwave characteristics have been measured using power meter in the rectangular slotted waveguide and current density is measured using electrometer. The microwave absorption is evaluated using the standard available model. The results depict ?11.57 dB reflection loss at 10.38 GHz in composition x = 0.6. The electrical current density decreases at lower substitution and increases at higher substitution. The substitution of Co²⁺ and Ti⁴⁺ ions causes enhancement of electromagnetic and static electrical properties.     

The role of proteasome in yeast Saccharomyces cerevisiae response to sub-lethal high pressure treatment

Hydrostatic pressure is a physical factor that can induce stress in organisms. This stress leads to growth inhibition, cellular arrest, and cellular death, and these effects depend on the degree of pressure, temperature, and sensitivity of the organisms to hydrostatic pressure. Genomics studies of yeast cells under conditions recovering from high pressure-induced cellular damage showed evidence that multiprotein complexes or membrane proteins, and not soluble proteins, are the critical targets. We performed a metabolomic analysis. The metabolomics results suggested that membrane-spanning proteins broke down after high pressure treatment and recovery conditions. We also found 13 genes that were common to essential and pressure-induced gene groups. Among these 13 genes, more than 10 were associated with proteasome structure and functions. This suggests that proteasome structure or functions can be the critical target or a highly important factor. This hypothesis is supported by the fact that yeast cells are sensitive to the proteasome inhibitor MG132 after high pressure treatment.     

Microwave and electrical properties of Co-Ti substituted M-type Ba hexagonal ferrite

The microwave characteristics of Co²⁺ and Ti⁴⁺ ions substituted, BaCo _x Ti _x Fe_(12?2x)O₁₉ (x = 0.1, 0.3, 0.5, 0.7, 0.9) ferrite have been studied as a function of thickness, frequency and substitution. The results depict reflection loss of ? 31.94 dB at 10.47 GHz in x = 0.9. The highest static electrical current is observed at lower substitution. The model accompanying microwave absorption is used to evaluate microwave absorption characteristics. The electromagnetic and static electrical characteristics are improved with the substitution of Co²⁺ and Ti⁴⁺ ions. The compositions for possible electromagnetic applications are also explored.     

Rice proteomics: current status and future perspectives

Rice, the first cereal crop genome to be decoded, has attracted the attention of researchers worldwide because of its immense socio-economic impact on human existence. With the availability of the draft genome sequence of two major types, japonica- and indica-rice, "rice proteomics" has entered into the era of functional genomics. Although during the last decade an important but limited progress (mainly construction of protein datafiles) has been made in the field of rice proteomics, it is only recently that dedicated research groups have taken this challenge to systematically analyze the rice proteome at the cell (and organelle), tissue, and whole plant level. Important gains achieved by the accelerated technological progress in protein separation and identification will help in going beyond the simple cataloguing of rice proteins in realistic terms. In this review, we discuss the progress made in the field of rice proteomics to date and dwell upon the future direction/problems/approaches towards defining the rice proteome.