[show abstract][hide abstract] ABSTRACT: Salt stress inhibits plant growth and development and plants activate kinase-dependent survival pathways in response to salt stress. However, the role of soybean mitogenactivated protein kinases (MAPKs) in salt stress response has yet to be characterized. In this study, we found that salt stress activates Glycine max MAP kinase 1 (GMK1), a soybean MAPK. The activity of GMK1 induced with increasing salt concentrations, up to 300 mM NaCl, after 5 min of the treatment and was regulated by post-translational modification. We found that mastoparan, a heteromeric G-protein activator, also activated GMK1, and that n-butanol, a phospholipase D inhibitor, and neomycin, a phospholipase C inhibitor, inhibited its activity. Moreover, GMK1 activity was reduced by suramin, a heteromeric G-protein inhibitor, and by two inhibitors of phosphatidic acid (PA) generation after 5 min of 300 mM NaCl treatment. Endogenous PA levels were highest 5 min after induction of salt stress, and exogenous PA directly activated GMK1. From these data, we propose that salt stress signaling is transduced from heteromeric G-protein to GMK1 via phospholipases in the early stages of the response to salt stress in soybean.
Journal of Plant Biology 08/2013; 55(4). · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mitogen-activated protein kinase (MAPK) is activated by various biotic and abiotic stresses. Salt stress induces two well-characterized MAPK activating signaling molecules, phosphatidic acid (PA) via phospholipase D and phospholipase C, and reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. In our previous study, the activity of soybean MAPK, GMK1 was strongly induced within 5 min of 300 mM NaCl treatment and this early activity was regulated by PA. In this study, we focused on the regulation of GMK1 at the later stage of the salt stress, because its activity was strongly persistent for up to 30 min. H(2)O(2) activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of the treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H(2)O(2) 10 min after the treatment. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor, but was increased in protoplast overexpressing TESD-GMKK1. However, these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Taken together, we propose that soybean GMK1 is dually regulated by PA and H(2)O(2) at a time dependant manner and translocated to the nucleus by the salt stress signal.
Molecules and Cells 08/2012; 34(3):271-8. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bamboo is one of the fastest growing plants in the world and is an economically important crop species in Asia. To identify
the genes involved in fast shoot growth, an expressed sequence tag analysis was performed on Bambusa edulis Murno fast-growing shoots. Sequencing of the cDNA clones generated 1,402 5′-end high-quality expressed sequence tags (JG296384-JG297785,
average length 655bp), of which 1,101 clusters (143 consensus and 958 singletons) were revealed by sequence comparison to
be unique and 597 (54% of total clusters) of them have a putative ATG start codon. A Basic Local Alignment Search Tool X analysis
showed that 995 of these genes were similar to genes present in the National Center for Biotechnology database. A total of
868 genes were most similar to rice genes. The most abundant genes were three thionin-coding genes, which have 27, 17, or
10 clones, respectively, followed by aminocyclopropanecarboxylate oxidase and cysteine protease. Thionin and putative cell
elongation-associated genes, xyloglucan endotransglycosylase/hydrolase, expansin, cellulose synthase, and pectin esterase
were analyzed by real-time reverse transcription polymerase chain reaction using gene-specific primers. These results suggest
that this high-quality library could be a good resource for understanding molecular events of bamboo shoot elongation, and
the full-length clones could be used for crop improvement studies in the future.
KeywordsBamboo–cDNA library–Real-time RT-PCR–Shoot growth
Journal of Plant Biology 04/2012; 54(6):402-408. · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mitogen-activated protein kinase (MAPK) plays an important role in mediating the intracellular transmission and amplification
of extracellular stimuli. We examined whether MAPK is involved in the signaling process during the early step of nodule formation.
A genistein induced culture filtrate (GCF) ofBradyrhizobium japonicum was prepared for inducing an early response by soybean root hairs via Nod factor. Upon treatment, several types of deformations
were seen, demonstrating that GCF contains active Nod factor molecules. In-gel kinase assays showed that treating soybean
roots with GCF induced the rapid activation of two protein kinases (molecular masses of 47 kD and 44 kD), which phosphorylate
myelin basic protein (MBP). To identify the activated kinase, we prepared an antibody againstGMK1 (Glycine max MAP kinase 1), based on information from SIMK (an alfalfa MAP kinase) and a soybean EST database. An immunocomplex kinase
assay with the GMK1-specific antibody revealed that the 47-kD kinase in GCF-treated seedlings is indeed GMK1. Consistent with
many other MAP kinases, GMK1 is likely to be under post-translational regulation. Considering these results and previous reports
from soybean, GMK1 seems to be a signaling mediator with a broad range of stimuli, including a fungal elicitor, wounding,
and the symbiotic interaction between soybean and B.japonicum.
Journal of Plant Biology 04/2012; 51(4):291-296. · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Arabidopsis CYP51A2 (AtCYP51A2) mediates the sterol 14α-demethylation step inde novo sterol biosynthesis, and is constitutively and highly expressed in all plant tissues (Kim et al., 2005). We exploited the
molecular features of its expression and the fundamental role of sterol biosynthesis in cells to develop a plant-derived promoter.
Our GUS expression analysis between transgenicArabidopsis lines forAtCYP51A2::GUS and35S::GUS revealed that activity of theAtCYP51A2 promoter was comparable to that of the35S promoter, based on enzymatic activities and protein levels. TheAtCYP51A2 promoter was also constitutively active in transgenic tobacco, indicating that 5′ regulatory elements could be conserved
amongCYP51 promoters in dicot plants. A homologue ofAtCYP51A2 was identified from rape seed, a crop species closely related toArabidopsis. Its constitutive tissue expression pattern implies that the application of thisAtCYP51A2 promoter is possible for that species. Based on these results, we present a new binary vector system with the plant-derivedAtCYP51A2 promoter, which is able to constitutively and ectopically drive a transgene in various dicotyledonous plants.
Journal of Plant Biology 04/2012; 51(5):359-365. · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have determined that a nodule-specific cDNA clone (GmCysP1), obtained from a soybean root nodule-specific EST pool, encodes cysteine proteinase. Its amino acid sequence homology, as
well as the conservation of typical motifs and amino acid residues involved in active site formation, shows that GmCysP1 can
be classified as a legumain (C13) family cysteine proteinase, belonging to clan CD. Moreover, based on its expression patterns,GmCysP1 is a nodule-specific cysteine proteinase gene that is possibly associated with nodule development or senescence. Our genomic
Southern analysis also suggests thatGmCysP1 is a member of a multigene family. Therefore, we propose that GmCysP1 is the first to be identified as a nodule-specific
and senescence-related cysteine proteinase that belongs to the legumain family from soybean.
Journal of Plant Biology 04/2012; 47(3):216-220. · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: The nucleotide sequence of a 20.5-kb genomic region harboring nif genes was determined and analyzed. The fragment was obtained from Frankia sp. EuIK1 strain, an indigenous symbiont of Elaeagnus umbellata. A total of 20 ORFs including 12 nif genes were identified and subjected to comparative analysis with the genome sequences of 3 Frankia strains representing diverse host plant specificities. The nucleotide and deduced amino acid sequences showed highest levels of identity with orthologous genes from an Elaeagnus-infecting strain. The gene organization patterns around the nif gene clusters were well conserved among all 4 Frankia strains. However, characteristic features appeared in the location of the nifV gene for each Frankia strain, depending on the type of host plant. Sequence analysis was performed to determine the transcription units and suggested that there could be an independent operon starting from the nifW gene in the EuIK strain. Considering the organization patterns and their total extensions on the genome, we propose that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.
Archives of Microbiology 07/2011; 194(1):29-34. · 1.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14alpha-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.
[show abstract][hide abstract] ABSTRACT: To establish expressed sequence tag databases of the two life stages (the dispersal and propagative stages) of pinewood nematode Bursaphelenchus xylophilus, subtractive EST libraries that were specific to the dispersal 4th larval stage (D4S) and the pine-grown propagative mixed (PGPS) stage were constructed by suppressed subtractive hybridization, and annotated by BLASTx and Gene Ontology (GO). A total of 1112 (57.7%) contigs from the D4S-cDNA library and 1215 (46.7%) contigs from the PGPS-specific cDNA libraries had matched BLASTx hits (E<or=10(-2)), among which 913 (47.4%) and 960 (36.9%) contigs, respectively, were classified into three GO categories. A total of 14 genes were selected on the basis of stage-specific abundances and GO subcategories, and their transcription levels were analyzed by quantitative real-time PCR. We discussed the potentials of some stage-specific genes, such as sorbitol dehydrogenase, cysteine protease, venom allergen-like protein, and FMRFamide-like peptide, as diagnosis markers and novel control targets.
[show abstract][hide abstract] ABSTRACT: Three catalase cDNA clones were isolated from the small radish (Raphanus sativus L.). Their nucleotide and deduced amino acid sequences showed the greatest homology to those of Arabidopsis. Genomic Southern blot analysis, using RsCat1 cDNA as a probe, showed that catalases are encoded by small multigene family in the small radish. Nondenaturing polyacrylamide gels revealed the presence of several catalase isozymes, the levels of which varied among the organs examined. The isozyme activities were assigned the individual catalase genes by Northern analysis using total RNA from different organs. The three catalase genes were differentially expressed in response to treatments such as white light, xenobiotics, osmoticum, and UV. Their expression in seedlings was controlled by the circadian clock under a light/dark cycle and/or in constant light. Interestingly, RsCat1 transcripts peaked in the morning, while those of RsCat2 and RsCat3 peaked in the early evening. Our results suggest that the RsCat enzymes are involved in defense against the oxidative stress induced by environmental changes.
Molecules and Cells 09/2007; 24(1):37-44. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.
Molecules and Cells 03/2007; 23(1):115-21. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR: mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the full-length cDNA, glutamate treatment triggered greater Ca2+ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated Ca2+ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis.
Molecules and Cells 07/2006; 21(3):418-27. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: For high throughput screening of root nodule-enhanced genes, cDNA libraries specific for three different developmental stages of soybean root nodules were constructed after inoculation with Bradyrhizobium japonicum USDA110. 5,469 cDNA clones were sequenced and grouped into 2,511 non-redundant (nr) ESTs consisting of 769 contigs and 1,742 singletons. Using similarity searches against several public databases we constructed a functional classification of the ESTs into root nodule-specific nodulin genes, stress-responsive genes and genes related to carbon and nitrogen metabolism. We also constructed a cDNA microarray with 382 selected clones that appeared to be up-regulated in the root nodule. Using the microarray we compared the transcript levels of uninfected roots and root nodules from four developmental stages. We identified 81 genes that were differentially expressed, and grouped them into seven clusters according to the similarity of their expression profiles, using a hierarchical clustering algorithm. Clusters 1, 2, 3, and 6, comprised of 58 genes, showed root nodule-enhanced expression. The information from this study will be used to analyze the roles of root nodule-specific genes and signaling pathways during root nodule development.
Molecules and Cells 09/2004; 18(1):53-62. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hemoglobins (Hbs) are heme proteins found in all five kingdoms of living organisms. In plants, three different classes of
Hbs have been identified-nonsymbiotic Hbs from diverse species, symbiotic Hbs from nitrogen-fixing plants, and so-called 2-on-2
Hbs. Here, we report the cloning and expression analysis of the 2-on-2 Hb gene,GmGLB3, from soybean. TheGmGLB3 cDNA clone encodes a protein for 172 amino acid residues. Its deduced amino acid sequence shows the highest identity (74%)
with 2-on-2 Hb fromMedicago truncatula. Multiple sequence alignment confirms the conserved and signature amino acid residues previously reported with plant 2-on-2
Hbs. Genomic Southern hybridization demonstrates thatGmGLB3 has two copies in the soybean genome. Based on our northern hybridization, theGmGLB3 gene is specifically expressed in root nodules, with levels increasing in the late stage during nodule development. Its transcript
level is also increased under flooding and kinetin treatments in the roots, or under flooding and 2-iP treatments in the stems.
However, no transcript is detected in the leaves regardless of treatment. Therefore, we propose that theGmGLB3 gene is specifically expressed in root nodules and that its expression in other plant organs is regulated by cytokinin and/or
Journal of Plant Biology 05/2004; 47(2):92-98. · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: We analyzed a cDNA clone encoding cytosolic glutamine synthetase,EuNOD-GS1, isolated from a root nodule cDNA library ofElaeagnus umbellata. This clone has an insert size of 1359 bp and encodes a protein for 355 amino-acid residues, with a molecular weight of 39.2
kDa. Its expression is slightly higher in the root nodules than in the leaves or uninfected roots. Analysis of the deduced
amino acid sequences and phytogeny revealed thatEuNOD-GS1 is clustered with cytosolic GS-α isoenzymes. Therefore, based on this and previous results, we propose that the main physiological
role ofEuNOD-GS1 is the assimilation of ammonia from secondary and, in part, primary sources.
Journal of Plant Biology 01/2004; 47(4):401-406. · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have used the hybridization-competition method to isolateEuNOD-CHI from a root nodule cDNA library ofElaeagnus umbellate. This cDNA clone encodes chalcone isomerase (CHI) for a protein of 256 amino-acid residues and a mature molecular mass of
28 kDa. Multiple sequence alignment and phylogenetic analysis have demonstrated that EuNOD-CHI can be classified as Type I.
Moreover, northern hybridization shows that theEuNOD-CHI gene is highly expressed in root nodules, with levels increasing during nodule development The highest level of expression
is at 6 to 8 weeks after inoculation, decreasing thereafter. Genomic Southern hybridization also demonstrates thatEuNOD-CHI has as many as two copies in theE umbellate genome. Taken together with the previous results, we propose that the higher expression level of theEuNOD-CHI gene in root nodules is likely associated with this species’ defense mechanism against infection byFrankia.
Journal of Plant Biology 01/2003; 46(4):263-270. · 0.99 Impact Factor