Reduced expression of Sytl 1 and Ccdc21 and impaired induction of Mt I by oxidative stress in SII-K1 knockout mice.

SII-K1 is a member of the transcription elongation factor S-II family. In the mouse, SII-K1 is expressed exclusively in the liver, kidney, heart, and skeletal muscle. Here, we report that deletion of the SII-K1 gene in mice resulted in the downregulation of the synaptotagmin-like 1 (Sytl 1) gene in liver and of the coiled-coil domain-containing 21 (Ccdc21) gene in liver and kidney. Moreover, the induction of the metallothionein I (Mt I) gene in SII-K1-deficient mice liver was impaired in diethyl maleate-induced oxidative stress conditions. Our results suggest that SII-K1 regulates these genes in vivo.

Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes.

Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR - detection and expression analysis of gene(s) in real-time - has revolutionized the 21(st) century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant.

High-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: drastic reductions/fragmentation of ribulose-1,5-bisphosphate carboxylase/oxygenase and induction of stress-related proteins.

Employing high-resolution two-dimensional electrophoresis (2-DE), we studied changes in the rice leaf protein patterns, in response to applied heavy and alkaline metals, important environmental pollutants in our surroundings. Drastic changes in 2-DE protein patterns after treatment with copper, cadmium, and mercury, over control were found, including changes in the morphology of the leaf segments. Changes in the major leaf photosynthetic protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, both suppression and fragmentation), and induction of proteins are reported. A total of 33 proteins, which were highly reproducible in repeated experiments, were visually identified as changed over the control, and taken for N-terminal or internal amino acid sequencing. Among these, nine proteins were N-terminally blocked, and six proteins could not be sequenced. Most of the proteins showed homology to RuBisCO protein, and some to defense/stress-related proteins, like the pathogenesis related class 5 protein (OsPR5), the probenazole-inducible protein (referred to as the OsPR10), superoxide dismutase, and the oxygen evolving protein. Results presented here strongly indicate a highly specific action of some of these metals in disturbing the photosynthetic machinery, as evidenced by prominent reductions/fragmentation of the major photosynthetic protein, RuBisCO, and resulting in stress.

Molecular cloning and characterization of a novel Jasmonate inducible pathogenesis-related class 10 protein gene, JIOsPR10, from rice (Oryza sativa L.) seedling leaves.

A novel rice (Oryza sativa L.) gene, homologous to a sorghum pathogenesis-related class 10 protein gene, was cloned from a cDNA library prepared from 2-week-old jasmonic acid-treated rice seedling leaves, and named as JIOsPR10 (jasmonate inducible). JIOsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17,173.23 Da and a pI of 5.84. JIOsPR10 was highly similar (77%) to the sorghum PR10 protein, but showed less than 55% similarity with other identified PR10s at the amino acid level. Genomic Southern analyses indicated the presence of related genes in the rice genome. The JIOsPR10 transcript was not detected in the healthy leaves, and was not induced after cut. Further expression analysis revealed that the signaling components of defense/stress pathways, jasmonate, salicylate, and H(2)O(2) significantly up-regulated the JIOsPR10 mRNA over the cut control, whereas two other stress regulators, ethylene and abscisic acid, failed to induce its expression. Interestingly the protein phosphatase (PP) inhibitors, cantharidin, endothall, and okadaic acid, rapidly and potently up-regulated the JIOsPR10 expression, suggesting involvement of the phosphorylation/dephosphorylation events. Additionally, the inducible expression of the JIOsPR10 gene was influenced by light signal(s). Finally, the blast pathogen (Magnaporthe grisea) also specifically elicited the accumulation of JIOsPR10 mRNA in leaves. Induction of the JIOsPR10 gene expression by signaling molecules, PP inhibitors and pathogen attack, strongly indicate a role for this novel gene in rice self-defense/stress response(s).

Characterization of a rice (Oryza sativa L.) Bowman-Birk proteinase inhibitor: tightly light regulated induction in response to cut, jasmonic acid, ethylene and protein phosphatase 2A inhibitors.

The Bowman-Birk (BB) family of proteinase inhibitors (PI), initially reported from legume seeds, and thereafter also from wounded alfalfa and maize leaves appear to be regulated in similar ways as the extensively characterized PI I and PI II family from dicots. Here, we report a first characterization of the expression profiles of a rice (Oryza sativa L. cv. Nipponbare) BBPI gene, OsBBPI, which is part of a multigene family as demonstrated by genomic Southern hybridization. OsBBPI was found to be rapidly induced in rice seedling leaf in response to cut, exogenous jasmonic acid (JA), and two potent protein phosphatase 2A (PP2A) inhibitors, cantharidin (CN) and endothall (EN), in a light/dark-, time- and dose-dependent manner; this induction was completely inhibited by cycloheximide (CHX), indicating a requirement for de novo protein synthesis in its induction. Surprisingly, dark strongly up regulated cut-, JA-, CN-, and EN-induced OsBBPI expression, with the strongest enhancement observed with JA. A simultaneous application of a serine/threonine protein kinase inhibitor staurosporine (ST) did not affect significantly the JA-, CN-, and EN-induced OsBBPI transcript. Besides JA, it was found that the ethylene generator ethephon (ET) also had an enhancing effect on OsBBPI transcript, suggesting a direct effect of ethylene on OsBBPI expression. However, a simultaneous application of salicylic acid (SA) and abscisic acid (ABA), with JA, respectively, completely blocked OsBBPI gene expression, whereas kinetin (KN) was only partially effective. To the best of our knowledge, complete inhibition of JA-induced OsBBPI expression by SA is the first report in monocots, and with ABA in plants. Taken together, these results suggest that among the phytohormones tested here, JA and ethylene play important role(s) in regulating OsBBPI expression, with an intimate interaction with light signals. Finally, that the induced OsBBPI expression follows a kinase-signaling cascade is implied by the use of PP2A inhibitors.

Rice (Oryza sativa L.) OsPR1b gene is phytohormonally regulated in close interaction with light signals.

Strategies evolved by plants to counteract a variety of biotic/abiotic stresses include induction of genes encoding pathogenesis-related (PR) protein, in particular the PR class 1 (PR1) gene family, widely used in stress response studies. In spite of its immense importance as a PR family member, and an accepted gene marker in plant disease/defense in dicots, little is known about rice PR1 genes. Recently, we cloned and characterized the first OsPR1a (rice acidic PR1) gene (Agrawal et al. (2000) Biochem. Biophys. Res. Commun. 274, 157-165). Here, we report characterization of a rice basic PR1 (OsPR1b) gene, identified from screening a cDNA library prepared from jasmonic acid (JA)-treated rice seedling leaf, providing detailed and valuable insights into rice PR1 gene expression. The deduced amino acid sequence of OsPR1b reveals only 63.1% homology with the OsPR1a protein, whereas Southern blot analyses indicate that OsPR1b is a multigene family. The JA-inducible OsPR1b gene was also up-regulated by salicylic acid (SA), abscisic acid (ABA), and kinetin (KN). Furthermore, protein phosphatase inhibitors, cantharidin (CN) and endothall (EN) strongly induced the OsPR1b transcript. However, OsPR1b was not cut-responsive, diagrammatically opposite to cut inducibility of OsPR1a. This induction was light-, time-, and dose-dependent, as demonstrated by using, JA, CN, and EN, and completely inhibited by cycloheximide, but not by tetracycline. The simultaneous application of SA, and ABA, with JA, respectively, showed almost complete inhibition of the JA-induced OsPR1b transcript by 200 microM SA or ABA, but not by 100 microM concentrated solutions, suggesting a potential interaction among JA, SA, and ABA, whereas KN dramatically enhanced JA-induced OsPR1b transcript upon simultaneous application. Moreover, a simultaneous application of staurosporine enhances JA-, CN-, and EN-induced OsPR1b transcript, in particular with CN. Finally, a comparative analysis with the OsPR1a gene gives us insight into the differential regulation of the PR1 gene family, while proposing OsPR1 genes as important gene markers in rice, with potential use(s) in analyzing plant defense responses.

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 novel rice (Oryza sativa L.) acidic PR1 gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors.

A novel rice acidic pathogenesis-related (PR) class 1 cDNA (OsPR1a) was isolated from jasmonic acid (JA)-treated rice seedling leaf. The OsPR1a cDNA is 830 bp long and contains an open reading frame of 507 nucleotides encoding 168 amino acid residues with a predicted molecular mass of 17,560 and pI of 4.4. The deduced amino acid sequence of OsPR1a has a high level of identity with acidic and basic PR1 proteins from plants. Southern analysis revealed that OsPR1a is a member of a multigene family. The OsPR1a gene was found to be cut-inducible, whereas the phytohormones JA, salicylic acid (SA), 3-indoleacetic acid, gibberellin, and ethylene (using ethylene generator ethephon, ET) enhanced accumulation of OsPR1a transcript, as well as the protein phosphatase inhibitors cantharidin (CN) and endothall (EN). Induced expression of OsPR1a gene by JA, CN or EN, and ET was light/dark- and dose-dependent and was almost completely inhibited by cycloheximide. Dark downregulated CN-, EN-, and ET-induced OsPR1a gene expression, whereas it was further enhanced with JA. SA and abscisic acid blocked JA-induced OsPR1a transcript. Simultaneous application of staurosporine (ST) enhances CH- or EN-induced OsPR1a transcript, but not with JA. This is the first report on cloning of a rice acidic PR1 gene (OsPR1a), which is regulated by phytohormones, phosphorylation/dephosphorylation event(s), and light.

Naringenin 7-O-methyltransferase involved in the biosynthesis of the flavanone phytoalexin sakuranetin from rice (Oryza sativa L.).

An inducible S-adenosyl-L-methionine:naringenin 7-O-methyltransferase (NOMT) catalyzing the methylation of naringenin to sakuranetin, a major rice phytoalexin was purified approximately 985-fold from ultraviolet (UV)-irradiated rice leaves. The enzyme is not found in healthy tissues and was purified to a nearly homogeneous preparation in one step using adenosine-agarose affinity chromatography, with 1 g rice leaves (UV-irradiated) as starting material. Gel filtration chromatography resulted in an almost pure enzyme, as evidenced by a major band migrating to a position corresponding to a molecular mass of approximately 41 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified NOMT was strongly inhibited by Mn(2+), Ni(2+), Cu(2+), Zn(2+), Hg(2+), and Cd(2+), and to a low degree by Co(2+), Mg(2+), Ba(2+), Ca(2+) and ethylenediamine tetraacetic acid. The amino acid sequence of a NOMT cyanogen bromide (CNBr)-cleavage peptide was highly homologous to that of a caffeic acid 3-O-methyltransferase from maize, and about 70% of the amino acid sequence was obtained after sequencing the peptides generated by CNBr and/or formic acid hydrolysis. NOMT was also shown to be induced in a time-dependent manner, and purified from rice leaves treated with jasmonic acid and copper chloride.

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.

Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.): towards a rice proteome.

Proteins extracted from green and etiolated shoots of rice were separated by two-dimensional polyacrylamide gel electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino acid sequences of 21 out of 85 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced. The internal amino acid sequences of proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method and compared with those of known plant and animal protein sequences to understand the nature of the proteins. Green shoots revealed the presence of photosynthetic proteins as expected; however, as etiolated shoots were not photosynthetic, only precursors of the photosynthetic proteins were identified. Interestingly, the presence of L-ascorbate peroxidase only in etiolated shoots suggests a cellular protectant function for this antioxidant enzyme in the etiolating shoots. Using this experimental approach, we could identify the major proteins involved in growth regulation in photosynthetic green shoots as well as in etiolating rice seedlings.

Phytoalexin production elicited by exogenously applied jasmonic acid in rice leaves (Oryza sativa L.) is under the control of cytokinins and ascorbic acid.

Jasmonic acid (JA) has been shown to be a signaling compound which elicits the production of secondary metabolites including phytoalexins in plants. It has been shown that the phytoalexin production is elicited by exogenously applied JA in rice leaves. We now show that this phytoalexin production by exogenously applied JA is significantly counteracted by cytokinins, kinetin and zeatin. Kinetin and zeatin also inhibit the induction of naringenin-7-O-methyltransferase (a key enzyme in rice phytoalexin production) by JA. A natural free radical scavenger, ascorbic acid (AsA) shows both counteractive and enhancing effects on JA-inducible phytoalexin production, depending on its concentration. This effect of AsA suggests that active oxygen species (AOS) may play important roles in phytoalexin production by JA in rice leaves.

Phytoalexin production by amino acid conjugates of jasmonic acid through induction of naringenin-7-O-methyltransferase, a key enzyme on phytoalexin biosynthesis in rice (Oryza sativa L.).

Amino acid conjugates of jasmonic acid are found to elicit production of the flavonoid phytoalexin, sakuranetin in rice leaves. The elicitation is shown to arise from induction of naringenin 7-O-methyltransferase, a key enzyme of sakuranetin biosynthesis. The (-)-phenylalanine conjugate, one of the active compounds, is characterized by high activity for both sakuranetin and enzyme induction and low phytotoxicity against rice growth. Both (+)-enantiomers of the conjugates and free amino acids do not show any activity. The amino acid conjugate of jasmonic acid is speculated to be the later component in the signaling transduction chain in stressed rice plants.

A methyltransferase for synthesis of the flavanone phytoalexin sakuranetin in rice leaves.

Rice (Oryza sativa L.) leaves irradiated with short wave UV light accumulated the major rice phytoalexin, flavanone sakuranetin. The extracts from these leaves catalyzed the methylation of the hydroxyl group at position 7 of naringenin to yield sakuranetin, with S-adenosyl-L-methionine as the methyl donor. Activity of the naringenin 7-O-methyltransferase was not found in healthy rice leaves but increased upon irradiation with UV light in parallel with increase in sakuranetin. The autoradiogram of the enzymatic product on 2D-TLC was found to be sakuranetin. The enzyme did not methylate sakuranetin, but methylated other flavanones, but not isoflavanones.