Proteome analysis of rice root proteins regulated by gibberellin

To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. In previous studies, sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid （ABA）, and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic in the GA-induced growth of roots. In among GA, aldolase, and root growth. pathway and may play an important role this review, we discuss the relationship among GA, aldolase, and root growth.     

Identification of rice root proteins regulated by gibberellin using proteome analysis

Gibberellin (GA) promoted rice (Oryza sativa L.) root elongation in a concentration-dependent manner with roots grown in 0.1 µm GA₃ being 13.8% longer than controls. On the other hand, the roots of Tan-ginbozu, a semi-dwarf cultivar were 69.8% shorter in comparison with Nipponbare, a normal cultivar. Treatments with 10 µm uniconazole-P and 10 µm abscisic acid (ABA) caused decreases in root length in Tan-ginbozu by 44.6 and 79.2%, respectively. To investigate how GA influences rice root growth, proteome analysis techniques were applied. Extracted proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analysed using an automated protein sequencer and mass spectrometer. Sixteen proteins show differences in accumulation levels as a result of treatment with GA₃, uniconazole-P and ABA treatment and/or the difference between the semi-dwarf cultivar, Tan-ginbozu, and normal cultivars. Among these proteins, fructose-bisphosphate aldolase (EC 4.1.2.13) increased in roots treated with GA₃, occurred in low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole-P or ABA. Moreover, roots from seedlings grown in 100 µm glucose were 9.1% longer than controls. These results indicate that increases in fructose-bisphosphate aldolase activity stimulate the glycolytic pathway and may play an important role in the GA-induced growth of roots.     

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA₃ promoted an increase in aldolase accumulation when 0.1 M GA₃ was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 M GA₃, FBPA activity increased very little by 0.1 M GA₃ treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H⁺-ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA₃. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA₃-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.     

Functional proteomics to identify critical proteins in signal transduction pathways

Reversible protein phosphorylation plays a crucial role in the regulation of signaling pathways that control various biological responses, such as cell growth, differentiation, invasion, metastasis and apoptosis. Proteomics is a powerful research approach for fully monitoring global molecular responses to the activation of signal transduction pathways. Identification of different phosphoproteins and their phosphorylation sites by functional proteomics provides informational insights into signaling pathways triggered by all kinds of factors. This review summarizes how functional proteomics can be used to answer specific questions related to signal transduction systems of interest. By examining our own example on identifying the novel phosphoproteins in signaling pathways activated by EB virus-encoded latent membrane protein 1 (LMP1), we demonstrated a functional proteomic strategy to elucidate the molecular activity of phosphorylated annexin A2 in LMP1 signaling pathway. Functional profiling of signaling pathways is promising for the identification of novel targets for drug discovery and for the understanding of disease pathogenesis.     

Comparison of two PCR techniques used in amplification of microdissected plant chromosomes from rice and wheat

Linker adaptor-mediated PCR (LAM-PCR) and degenerate oligonucleotide primed PCR (DOP-PCR) are major ways to generate chromosome-specific libraries. In this study, these two PCR techniques were used to amplify the microdissected mitotic and meiotic chromosomes from rice and wheat. The results of amplifications were compared, and the advantages and limitations between the two techniques are presented.     

A proteomics approach to identify the ubiquitinated proteins in mouse heart

There is a growing need for the large-scale identification of the ubiquitinated proteins and ubiquitin attachment sites. As part of this effort, we generated a transgenic mouse expressing a tagged ubiquitin in the heart. We found that the majority of ubiquitinated proteins in mouse heart are insoluble in detergent-free buffer and were chemically cleaved after methionine with CNBr. CNBr cleaved the proteins into smaller polypeptides while preserving the ubiquitin chains. Ubiquitin-conjugated polypeptides were then purified under denaturing conditions, digested with Lys-C and trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. We identified 121 proteins that were ubiquitinated in mouse heart, and we detected 33 ubiquitination sites in 21 of the proteins. Components of cardiac muscle and many mitochondrial proteins were identified as substrates for ubiquitination, strongly suggesting that proteins related to major heart functions such as contraction and energy production are under continuous quality control by the ubiquitin system.     

A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2′-aminohexylcarbamoyl-c-di-GMP (2′-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2′-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.     

Gel and gel-free proteomics to identify Saccharomyces cerevisiae cell surface proteins

The study of Saccharomyces cerevisiae cell surface proteins was performed because of their important role in cell wall biogenesis and in the physiology of the yeast. Two different proteomic approaches were carried out. First, proteins loosely associated or S–S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol, separated by 2D-PAGE and identified by mass spectrometry. Second, cell surface-exposed proteins (surfome) were digested with trypsin and DTT from whole intact cells, and analyzed by LC–MS/MS. Ninety-nine different proteins were identified: 67 with DTT treatment and 52 with DTT and trypsin digestion. These proteins were classified in different cellular processes: control of cell wall organization, cell rescue, defence, and virulence, protein fate, protein synthesis and metabolism. Most of the proteins have already been reported as present on the cell surface showing that the yeast cell surface is composed not only by typical but also by atypical cell wall proteins. “Bona fide” cell wall proteins were identified by both protocols but a higher number with the non-gel strategy. However, only 20% of the proteins identified were common to both protocols, thus, for a complete knowledge of the cell surface proteome, several strategies have to be used.     

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.     

An approach to identify cold-induced low-abundant proteins in rice leaf

A proteomic approach has been adopted to investigate the low-abundant proteins in rice leaf in response to cold stress. Rice seedlings were exposed to different temperatures, such as 5 or 10 degrees C, and samples were collected after different time course. To eliminate the high-abundant proteins in leaf tissues such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), proteins were fractionated by polyethylene glycol (PEG). The elimination of Rubisco from the protein samples was confirmed by Western blot analysis. The PEG fractionated protein samples were separated by 2-DE and visualized by silver or CBB staining. A total 12 up-regulated protein spots were identified using the analysis of MALDI-TOF mass spectrometry or ESI MS/MS. We identified some novel proteins such as cysteine proteinase, thioredoxin peroxidase, a RING zinc finger protein-like, drought-inducible late embryogenesis abundant, and a fibrillin-like protein that had not yet been reported in the earlier reports on cold proteomic analysis. The identification of some novel low-abundant proteins in response to cold stress may provide a new homeostasis to develop enhanced cold tolerance transgenic plants. Thus, we propose that a PEG fractionation system can be used as an influential protein extraction method from the leaf samples, which can lead to knowledge of the expression pattern of low-abundant proteins in response to various biotic or abiotic stresses.     

Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth.     

Proteomics to identify pathogenesis-related proteins in rice roots under water deficit

Upland and lowland rice (Oryza sativa L.) showed different mechanisms of water stress resistance. Hydroponically grown 3-week-old seedlings of a lowland variety IR64 and an upland variety were exposed to 15% polyethylene glycol (PEG-6000). After 7 d of treatment, IR64 maintained high relative water content and developed a well-branched root. Therefore, IR64 had better water-deficit tolerance than Azucena under PEG treatment. To identify water-deficit-responsive proteins associated with the tolerance differences between two ecotypes, a comparative proteomic analysis of roots was conducted. Out of 700 proteins reproducibly detected on two-dimensional electrophoresis gels, 65 proteins exhibited significant changes in at least one ecotype at 48 h of water deficit. Only 15 proteins showed different responses to water deficit between the two ecotypes. Twelve proteins were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry, which involved in energy and metabolism, protein processing and degradation, detoxification and pathogenrelated (PR) proteins, i.e. PR-1a, RSOsPR10 and JIOsPR10. All three PR proteins were induced more strongly in IR64 than in Azucena by water deficit at both protein and mRNA level. The results suggested that PR-1a, RSOsPR10 and JIOsPR10 may play important roles in protecting root cells against water deficit in rice.     

A proteomics approach for identifying osmotic-stress-related proteins in rice

Osmotic stress can endanger the survival of plants. To investigate the mechanisms of how plants respond to osmotic stress, rice protein profiles from mannitol-treated plants, were monitored using a proteomics approach. Two-week-old rice seedlings were treated with 400mM mannitol for 48h. After separation of proteins from the basal part of leaf sheaths by two-dimensional polyacrylamide gel electrophoresis, 327 proteins were detected. The levels of 12 proteins increased and the levels of three proteins decreased with increasing concentration or duration, of mannitol treatment. Levels of a heat shock protein and a dnaK-type molecular chaperone were reduced under osmotic, cold, salt and drought stresses, and ABA treatment, whereas a 26S proteasome regulatory subunit was found to be responsive only to osmotic stress. Furthermore, proteins whose accumulation was sensitive to osmotic stress are present in an osmotic-tolerant cultivar. These results indicate that specific proteins expressed in the basal part of rice leaf sheaths show a coordinated response to cope with osmotic stress.     

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.     

Identification of kaempferol‐regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.