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Plants remember what they have experienced and are thereby able to confront repeated stresses more promptly and strongly. A subset of the drought responsive genes, called stress memory genes, displayed greatly elevated levels under recurrent drought conditions. To screen for a set of drought stress memory genes in soybean (Glycine max L.), we designed a 180K DNA chip comprising 60-bp probes synthesized in situ to examine 55,589 loci. Through microarray analysis using the DNA chip, we identified 2,162 and 2,385 genes with more than fourfold increases or decreases in transcript levels, respectively, under initial (first) drought stress conditions, when compared with the non-treated control. The transcript levels of the drought-responsive genes returned to basal levels during recovery (watered) states, and 392 and 613 genes displayed more than fourfold elevated or reduced levels, respectively, under subsequent (second) drought conditions, when compared to those observed under the first drought stress conditions. Gene Ontology and MapMan analyses classified the drought-induced memory genes exhibiting elevated levels of transcripts into several functional categories, including those involved in tolerance responses to abiotic stresses, which encode transcription factors, protein phosphatase 2Cs, and late embryogenesis abundant proteins. The drought-repressed memory genes exhibiting reduced levels of transcripts were classified into categories including photosynthesis and primary metabolism. Co-expression network analysis revealed that the soybean drought-induced and -repressed memory genes were equivalent to 172 and 311 Arabidopsis genes, respectively. The soybean drought stress memory genes include genes involved in the dehydration memory responses of Arabidopsis.

The herb holy basil (Ocimum tenuiflorum) is rich in essential oil, which is important in pharmaceutical, nutraceutical, cosmetic, and food industries. O. tenuiflorum grows well in tropical, subtropical, and warm temperate regions. To induce essential oil biosynthesis, holy basil plants were exposed to cold or drought stress for 5 days. Gas chromatography-mass spectrometry (GC–MS) analyses revealed that essential oil components including eugenol, methyl eugenol, and β-caryophyllene were significantly accumulated in 30-day-old juvenile and 130-day-old pre-flowering plants. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that expressional levels of OtEOMT transcript increased by almost 5-fold following the juvenile stage, and by 16.78- to 66.02-fold following the pre-flowering stage among cold-treated plants. These results suggest a biotechnological strategy to increase essential oil content by treating holy basil plants with cold or drought stress at the juvenile and pre-flowering stages.

AtMYB44 transcripts accumulate non-specifically under diverse stress conditions and with various phytohormone treatments in Arabidopsis thaliana. We investigated the chromatin modifications caused by various signals to uncover the induction mechanism of AtMYB44 transcription. Bisulfite sequencing confirmed a previous database illustrating that the AtMYB44 promoter and gene-body regions are completely DNA methylation-free. Chromatin immunoprecipitation (ChIP) assays revealed that the nucleosome density is remarkably low at the AtMYB44 promoter region. Thus, the promoter region appears to be highly accessible for various trans-acting factors. ChIP assays revealed that osmotic stress (mannitol treatment) lowered the nucleosome density at the gene-body regions, while abscisic acid (ABA) or jasmonic acid (JA) treatment did so at the proximal transcription start site (TSS) region. In response to mannitol treatment, histone H3 lysine 4 trimethylation (H3K4me3) and H3 acetylation (H3ac) levels within the promoter, TSS, and gene-body regions of AtMYB44 were significantly increased. However, occupancy of histone variant H2A.Z was not affected by the mannitol treatment. We previously reported that salt stress triggered a significant decrease in H2A.Z occupation without affecting the H3K4me3 and H3ac levels. In combination, our data suggest that each signal transduced to the highly accessible promoter induces a different chromatin modification for AtMYB44 transcription.

We transformed rice (Oryza sativa L. Japonica cv. Ilmi) calli with the Arabidopsis transcription factor gene AtMYB44 using Agrobacterium-mediated transformation. The T-DNA construct to be transformed contained tflA cDNA (encoding a toxoflavin lyase) as a selectable marker. Since toxoflavin is a photosensitizing phytotoxin, transgenic plantlets were selected based on their capacity for root development on medium containing this toxin in the light. Homozygous lines were selected by determining the segregation patterns, expression levels, and copy numbers of AtMYB44. Intergenic genomic locations of the inserted T-DNA in the three transgenic lines were confirmed by adaptor-ligation polymerase chain reaction and analysis using FSTVAL ( http://bioinfo.mju.ac.kr/fstval/ ), an open-access web tool used to localize the flanking sequences of the transgene. Drought tolerance of young seedlings of the transgenic lines was determined based on the recovery of wilted leaves by re-watering after 3 days of water deprivation in a 105-well (35W × 35L × 45D mm/per well) plate. The three transgenic lines showed average survival rates of 80.4, 93.5, and 72.6%, respectively, whereas wild-type plants failed to recover after re-watering. Thus, the transgenic rice plants exhibited significantly enhanced tolerance to drought stress, as was shown previously in AtMYB44-overexpressing transgenic Arabidopsis and soybean. These results suggest that AtMYB44 activates a drought tolerance mechanism that is conserved in both monocotyledonous and dicotyledonous plants.

Type 2C protein phosphatases (PP2Cs) counteract protein kinases, thereby inhibiting the abscisic acid (ABA)-mediated response to abiotic stress in Arabidopsis thaliana. In the absence of stress, the promoters of PP2C genes (e.g., ABI1, ABI2, and HAI1) are negatively regulated by repressors that suppress gene transcription in a signal-independent manner. Quantitative reverse transcription PCR (RT-qPCR) and chromatin immunoprecipitation (ChIP) assays revealed that the levels of PP2C gene transcripts and RNA polymerase II (RNAPII) that stalled at the transcription start sites (TSS) of PP2C gene loci were increased under salt stress. The salt-induced increases in RNA polymerase-mediated transcription were reduced in 35S:AtMYB44 plants, confirming that AtMYB44 acts as a repressor of PP2C gene transcription. ChIP assays revealed that AtMYB44 repressors are released and nucleosomes are evicted from the promoter regions in response to salt stress. Under these conditions, histone H3 acetylation (H3ac) and methylation (H3K4me3) around the TSS regions significantly increased. The salt-induced increases in PP2C gene transcription were reduced in abf3 plants, indicating that ABF3 activates PP2C gene transcription. Overall, our data indicate that salt stress converts PP2C gene chromatin from a repressor-associated suppression status to an activator-mediated transcription status. In addition, we observed that the Arabidopsis mutant brm-3, which is moderately defective in SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) activity, produced more PP2C gene transcripts under salt stress conditions, indicating that BRM ATPase contributes to the repression of PP2C gene transcription.

AtLEA4-5 is a member of the group 4 late embryogenesis abundant (LEA) proteins, which are involved in the tolerance of water deficit in Arabidopsis thaliana. Chromatin immunoprecipitation assays revealed that the transcription factor AtMYB44 bound directly to the AtLEA4-5 gene promoter region under normal conditions, but was eliminated in response to osmotic stress (mannitol treatment). A quantitative reverse transcription PCR assay revealed that transcription of the AtLEA4-5 gene was induced in response to either salt (salinity) or mannitol (osmosis) treatment. The abiotic stress-induced increase in AtLEA4-5 transcripts was reduced in 35S:AtMYB44 transgenic plants, indicating that the transcription factor AtMYB44 represses gene transcription. More RNA polymerase II stalled at the transcription start site (TSS) of the AtLEA4-5 gene loci under osmotic stress, but the increment was reduced in the 35S:AtMYB44 plants. Histones are evicted from the promoter region under osmotic stress; however, histone eviction was hampered in the 35S:AtMYB44 plants. Under osmotic stress, the acetylated histones remaining at the TSS region was significantly lower in the 35S:AtMYB44 plants compared with wild-type plants. These results indicate that AtMYB44 suppresses polymerase-mediated transcription of the AtLEA4-5.

The plant hormone abscisic acid (ABA) triggers cellular tolerance responses to osmotic stress caused by drought and salinity. ABA controls the turgor pressure of guard cells in the plant epidermis, leading to stomatal closure to minimize water loss. However, stomatal apertures open to uptake CO2 for photosynthesis even under stress conditions. ABA modulates its signaling pathway via negative feedback regulation to maintain plant homeostasis. In the nuclei of guard cells, the clade A type 2C protein phosphatases (PP2Cs) counteract SnRK2 kinases by physical interaction, and thereby inhibit activation of the transcription factors that mediate ABA-responsive gene expression. Under osmotic stress conditions, PP2Cs bind to soluble ABA receptors to capture ABA and release active SnRK2s. Thus, PP2Cs function as a switch at the center of the ABA signaling network. ABA induces the expression of genes encoding repressors or activators of PP2C gene transcription. These regulators mediate the conversion of PP2C chromatins from a repressive to an active state for gene transcription. The stress-induced chromatin remodeling states of ABA-responsive genes could be memorized and transmitted to plant progeny; i.e., transgenerational epigenetic inheritance. This review focuses on the mechanism by which PP2C gene transcription modulates ABA signaling.

Transgenic overexpression of the Arabidopsis gene for jasmonic acid carboxyl methyltransferase (AtJMT) is involved in regulating jasmonate-related plant responses. To examine its role in the compositional profile of soybean (Glycine max), we compared the seeds from field-grown plants that over-express AtJMT with those of the non-transgenic, wild-type (WT) counterpart. Our analysis of chemical compositions included proximates, amino acids, fatty acids, isoflavones, and antinutrients. Overexpression of AtJMT in the seeds resulted in decreased amounts of tryptophan, palmitic acid, linolenic acid, and stachyose, but increased levels of gadoleic acid and genistein. In particular, seeds from the transgenic soybeans contained 120.0-130.5% more genistein and 60.5-82.1% less stachyose than the WT. A separate evaluation of ingredient values showed that all were within the reference ranges reported for commercially available soybeans, thereby demonstrating the substantial equivalence of these transgenic and non-transgenic seeds.

Plants have evolved strategies to cope with drought stress by maximizing physiological capacity and adjusting developmental processes such as flowering time. The WOX13 orthologous group is the most conserved among the clade of WOX homeodomain-containing proteins and is found to function in both drought stress and flower development. In this study, we isolated and characterized OsWOX13 from rice. OsWOX13 was regulated spatially in vegetative organs but temporally in flowers and seeds. Overexpression of OsWOX13 (OsWOX13-ov) in rice under the rab21 promoter resulted in drought resistance and early flowering by 7–10 days. Screening of gene expression profiles in mature leaf and panicles of OsWOX13-ov showed a broad spectrum of effects on biological processes, such as abiotic and biotic stresses, exerting a cross-talk between responses. Protein binding microarray and electrophoretic mobility shift assay analyses supported ATTGATTG as the putative cis-element binding of OsWOX13. OsDREB1A and OsDREB1F, drought stress response transcription factors, contain ATTGATTG motif(s) in their promoters and are preferentially expressed in OsWOX13-ov. In addition, Heading date 3a and OsMADS14, regulators in the flowering pathway and development, were enhanced in OsWOX13-ov. These results suggest that OsWOX13 mediates the stress response and early flowering and, thus, may be a regulator of genes involved in drought escape.

Transcripts of the Arabidopsis transcription factor gene, AtMYB44, accumulate rapidly to mediate a tolerance mechanism in response to salt stress. The AtMYB44 promoter is activated by salt stress, as illustrated in AtMYB44pro::GUS transgenic plants. Chromatin immunoprecipitation (ChIP) assays revealed that RNA polymerases were enriched on the AtMYB44 gene, especially on TSS-proximal regions, and nucleosome density was markedly reduced in the AtMYB44 gene-body region in response to salt stress. In addition, H2A.Z occupation was significantly decreased at the AtMYB44 promoter, transcription start site (TSS), and gene-body regions. Histone modifications including histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 and H4 acetylation (H3ac and H4ac) were not affected under the same stress conditions. We found a decrease in the number of AtMYB44 proteins bound to their own gene promoters in response to salt stress. These results suggest that salt stress induces the eviction of H2A.Z-containing nucleosomes from the AtMYB44 promoter region, which may weaken its affinity for binding AtMYB44 protein that acts as a repressor for AtMYB44 gene transcription under salt stress-free conditions.

A protein binding microarray (PBM) analysis was performed to determine the nucleotide sequence responsible for the specific binding of FUS3, a transcription factor in Arabidopsis that is considered to be a master regulator of seed development. Full-length FUS3 cDNA was fused in-frame with sequences encoding the fluorescent protein DsRed and a polyhistidine tag, and expressed in Escherichia coli. The partially purified fusion protein was incubated with the quadruple 9-mer PBM. An analysis of fluorescent images revealed that the fusion protein recognized and bound to the consensus sequence CATGCA. This result suggests that, among the nucleotides contained within the RY motif (CATGCA[TG]), six nucleotides (CATGCA) are sufficient for FUS3-specific binding. It appears that an additional ‘TG’ at the 3′ end of the RY motif found in seed-specific gene promoters was not necessary for binding specificity. Notably, FUS3 may also bind to the palindromic RY motif in its dimeric form.

The thermostable pectinase gene TmPec isolated from Thermotoga maritima was introduced into the NdeI site of pRSET-B vector and expressed in its intact form in Escherichia coli BL21. The overexpressed intact form of pectinase (TmPecN protein) was partially purified by heat-denaturation procedure. TmPecN showed the highest activity between 85 and 95 o C, and at approximately pH 6.5. Enzyme activity was stably maintained at temperatures below 85 o C. In the presence of Ca 2+ , pectinase activity of TmPecN increased to 128.4% of normal level. In contrast, Ba 2+ , Zn 2+ , and Mn 2+ strongly inhibited TmPecN activity. We conclude that the biochemical properties of the intact form of TmPecN are comparable to those of the recombinant protein TmPec reported previously.

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots. [BMB Reports 2014; 47(1): 27-32]

Tissue extracts were prepared from liquid-cultured Arabidopsis and reacted with UDP-[]-GlcNAc. Phospholipid fractions were then extracted by butanol partitioning. Consecutive thin-layer chromatography identified two glycolipids sensitive to PI-specific phospholipase C, known as early intermediates in glycosylphosphatidylinositol anchor biosynthesis; phosphatidylinositol N-acetylglucosamine and phosphatidylinositol glucosamine.

OsASR1 expression was induced through Abscisic acid (ABA) and stress treatments in leaves. The constitutive overexpression of OsASR1 in rice reduced ABA sensitivity, and increased high salinity and osmotic stress tolerance in early growth stages. These results indicated that OsASR1 has function in abiotic stress tolerance during early growth stages of rice.

The Arabidopsis AtAHL gene encodes a 3′(2′),5′-bisphosphate nucleotidase that catalyzes the conversion of adenosine 3′,5′-bisphosphate (PAP) into adenosine monophosphate and inorganic phosphate. We have generated transgenic Arabidopsis overexpressing this gene under control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Transgenic lines integrating a single copy of the insert DNA and constitutively expressing the AtAHL gene were selected. The transgenic lines of Arabidopsis plants exhibited enhanced resistance to Pectobacterium carotovorum subsp. carotovorum. In general, plant defense responses and sulfur metabolism are linked through jasmonic acid signaling. The expression of sulfur-related defense genes is known to be induced via a jasmonate-mediated signaling pathway. In this work, we observed that the expression of AtAHL was also induced by jasmonate treatment in Arabidopsis. Our data suggest that PAP catabolic activity enhanced by the jasmonate signaling pathway contributes to the rapid flux of the sulfur activation pathway, accelerates the incorporation of activated sulfur into sulfur-containing defense molecules such as defensins, thionins, and glucosinolates, and thereby increases defense resistance in plants.

Binding sequence specificity of the Arabidopsis transcription factor AtMYB77 was determined by the quadruple 9-mer-based protein binding microarray (Q9-PBM) analysis. The position weight matrix and Wilcoxon-Mann-Whitney test with total 1843 clustered signals revealed that the full size AtMYB77 protein binds specifically to the consensus sequence pAACnG, where p represents T or C; n, A, G, C or T. This sequence is known as the type I binding site for MYB transcription factors. This result indicates that functional diversity among the type I MYB transcription factors is not due to their binding specificity on a particular promoter sequence.

AtMYB44, a member of the subgroup 22 R2R3 MYB transcription factors, positively regulates abscisic acid signaling to induce stomatal closure, thus conferring drought/salt-stress tolerance in Arabidopsis thaliana. In this study, AtMYB44 was transformed into soybean [Glycine max (L.) Merrill] using the cotyledonary-node method. The resulting homozygous lines were shorter than the non-transgenic controls (Bert) throughout the growth period when grown in a greenhouse. The transgenic soybeans exhibited significantly enhanced drought/salt-stress tolerance, as observed in Arabidopsis. In field cultivation studies, the transgenic soybean plants showed reduced growth, but much higher yields upon seed harvest, demonstrating improved environmental stress tolerance. The amino acid and fatty acid compositions were not significantly altered in seeds harvested from the transgenic lines. These results suggest that the interaction of AtMYB44 with specific sequences in target gene promoters and/or specific proteins activates a tolerance mechanism that is conserved in Arabidopsis and soybean.

Jasmonates play important roles in development, stress responses and defense in plants. Here, we report the results of a study using a functional genomics approach that identified a rice basic helix-loop-helix domain gene, OsbHLH148, that conferred drought tolerance as a component of the jasmonate signaling module in rice. OsbHLH148 transcript levels were rapidly increased by treatment with methyl jasmonate (MeJA) or abscisic acid, and abiotic stresses including dehydration, high salinity, low temperature and wounding. Transgenic over-expression of OsbHLH148 in rice confers plant tolerance to drought stress. Expression profiling followed by DNA microarray and RNA gel-blot analyses of transgenic versus wild-type rice identified genes that are up-regulated by OsbHLH148 over-expression. These include OsDREB and OsJAZ genes that are involved in stress responses and the jasmonate signaling pathway, respectively. OsJAZ1, a rice ZIM domain protein, interacted with OsbHLH148 in yeast two-hybrid and pull-down assays, but it interacted with the putative OsCOI1 only in the presence of coronatine. Furthermore, the OsJAZ1 protein was degraded by rice and Arabidopsis extracts in the presence of coronatine, and its degradation was inhibited by MG132, a 26S proteasome inhibitor, suggesting 26S proteasome-mediated degradation of OsJAZ1 via the SCF(OsCOI1) complex. The transcription level of OsJAZ1 increased upon exposure of rice to MeJA. These results show that OsJAZ1 could act as a transcriptional regulator of the OsbHLH148-related jasmonate signaling pathway leading to drought tolerance. Thus, our study suggests that OsbHLH148 acts on an initial response of jasmonate-regulated gene expression toward drought tolerance, constituting the OsbHLH148-OsJAZ-OsCOI1 signaling module in rice.

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the ArabidopsisJMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.

The Arabidopsis thaliana transcription factor gene AtMYB44 was induced within 10 min by treatment with methyl jasmonate (MeJA). Wound-induced expression of the gene was observed in local leaves, but not in distal leaves, illustrating jasmonate-independent induction at wound sites. AtMYB44 expression was not abolished in Arabidopsis mutants insensitive to jasmonate (coi1), ethylene (etr1), or abscisic acid (abi3-1) when treated with the corresponding hormones. Moreover, various growth hormones and sugars also induced rapid AtMYB44 transcript accumulation. Thus, AtMYB44 gene activation appears to not be induced by any specific hormone. MeJA-induced activation of jasmonate-responsive genes such as JR2, VSP, LOXII, and AOS was attenuated in transgenic Arabidopsis plants overexpressing the gene (35S:AtMYB44), but significantly enhanced in atmyb44 knockout mutants. The 35S:MYB44 and atmyb44 plants did not show defectiveness in MeJA-induced primary root growth inhibition, indicating that the differences in jasmonate-responsive gene expression observed was not due to alterations in the jasmonate signaling pathway. 35S:AtMYB44 seedlings exhibited slightly elevated chlorophyll levels and less jasmonate- induced anthocyanin accumulation, demonstrating suppression of jasmonate-mediated responses and enhancement of ABA-mediated responses. These observations support the hypothesis of mutual antagonistic actions between jasmonate- and abscisic acid-mediated signaling pathways.

EREBP, ethylene responsive element binding protein, plays a role in plant tolerance to abiotic stresses such as low temperatures, drought and high salinity. We previously used cDNA microarrays and northern blot analysis to determine the mechanisms responsible for those underlying defenses. These analyses led to identification of CaEREBP-C4, a gene that encodes the ethylene responsive element binding protein from hot pepper (Capsicum annuum). In that study, we demonstrated that the CaEREBP-C4 gene was strongly induced by cold stress. Here, we used Ti-plasmid and Agrobacterium-mediated transformation to engineer CaEREBP-C4 under control of the CaMV 35S promoter for constitutive expression in transgenic tobacco. The resultant CaEREBP-C4 transgenic plants exhibited significantly increased tolerance to low temperature. Moreover, the transgenic plants that showed strong cold tolerance also had greater tolerance to drought stress. In addition, none of the CaEREBP-C4 transgenic plants showed visible phenotypic alteration when compared to wild type plants. Taken together, these results suggest that CaEREBP-C4 plays a biological role in conferring plant abiotic stress tolerance.

The Arabidopsis gene AtLEC (At3g15356) gene encodes a putative 30-kDa protein with a legume lectin-like domain. Likely to classic legume lectin family of genes, AtLEC is expressed in rosette leaves, primary inflorescences, and roots, as observed in Northern blot analysis. The accumulation of AtLEC transcript is induced very rapidly, within 30 min, by chitin, a fungal wall-derived oligosaccharide elictor of the plant defense response. Transgenic Arabidopsis carrying an AtLEC promoter-driven beta-glucuronidase (GUS) construct exhibited GUS activity in the leaf veins, secondary inflorescences, carpel heads, and silique receptacles, in which no expression could be seen in Northern blot analysis. This observation suggests that AtLEC expression is induced transiently and locally during developmental processes in the absence of an external signal such as chitin. In addition, mechanically wounded sites showed strong GUS activity, indicating that the AtLEC promoter responds to jasmonate. Indeed, methyl jasmonate and ethylene exposure induced AtLEC expression within 3-6 h. Thus, the gene appears to play a role in the jasmonate-/ethylene-responsive, in addition to the chitin-elicited, defense responses. However, chitin-induced AtLEC expression was also observed in jasmonate-insensitive (coi1) and ethylene-insensitive (etr1-1) Arabidopsis mutants. Thus, it appears that chitin promotes AtLEC expression via a jasmonate- and/or ethylene-independent pathway.

We developed a quantitative method for the determination of methyl esterase activity, analyzing substrate specificity against three major signal molecules, jasmonic acid methyl ester (MeJA), salicylic acid methyl ester (MeSA), and indole-3-acetic acid methyl ester (MeIAA). We used a silylation reagent for chemical derivatization and used gas chromatography (GC)-mass spectroscopy in analyses, for high precision. To test this method, an Arabidopsis esterase gene, AtME8, was expressed in Escherichia coli, and then the kinetic parameters of the recombinant enzyme were determined for three substrates. Finally, this method was also applied to the direct quantification of phytohormones in petals from lilies and roses.

The jasmonate family comprises lipid-derived oxidation compounds (oxylipins), which function as plant hormones to regulate diverse developmental processes and defense responses. The pleiotropic effects of jasmonates are ascribed to a variety of biologically active derivatives synthesized along different branches in the octadecanoic pathway. Jasmonate biosynthesis occurs in the first round of the signaling pathway, which is initiated by certain external signal molecules or developmental cues connected to the release of fatty acid precursors from membrane lipids. Newly synthesized jasmonate molecules then mediate the second round of that pathway, inducing the expression of related genes. In particular, certain jasmonates produced in a localized site along their biosynthetic pathway act as a long-distance signal that transmits to distal parts of the plant, eliciting an immune response against a broad spectrum of pathogens and herbivores. The jasmonate-signaling pathway is connected to other signaling pathways associated with various phytohormones, all constituting a complex regulatory network linked to ubiquitin/proteasome-mediated protein degradation of repressors that negatively regulate transcription. In this review article, we highlight the pioneering research conducted on signaling pathways for the biosynthesis and action of jasmonates.

We cloned a salicylic acid/benzoic acid carboxyl methyltransferase gene, OsBSMT1, from Oryza sativa. A recombinant OsBSMT1 protein obtained by expressing the gene in Escherichia coli exhibited carboxyl methyltransferase activity in reactions with salicylic acid (SA), benzoic acid (BA), and de-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid (dSM-BTH), producing methyl salicylate (MeSA), methyl benzoate (MeBA), and methyl dSM-BTH (MeBTH), respectively. Compared to wild-type plants, transgenic Arabidopsis overexpressing OsBSMT1 accumulated considerably higher levels of MeSA and MeBA, some of which were vaporized into the environment. Upon infection with the bacterial pathogen Pseudomonas syringae or the fungal pathogen Golovinomyces orontii, transgenic plants failed to accumulate SA and its glucoside (SAG), becoming more susceptible to disease than wild-type plants. OsBSMT1-overexpressing Arabidopsis showed little induction of PR-1 when treated with SA or G. orontii. Notably, incubation with the transgenic plant was sufficient to trigger PR-1 induction in neighboring wild-type plants. Together, our results indicate that in the absence of SA, MeSA alone cannot induce a defense response, yet it serves as an airborne signal for plant-to-plant communication. We also found that jasmonic acid (JA) induced AtBSMT1, which may contribute to an antagonistic effect on SA signaling pathways by depleting the SA pool in plants.

The Arabidopsis AHL gene encodes a 3'(2'),5'-bisphosphate nucleotidase (BPNTase) involved in the reductive sulfate activation pathway. A bacterial expression vector containing AHL cDNA was randomly mutagenized with hydroxylamine and transformed into the E. coli cysteine auxotrophic mutant cysQ. Bacterial colonies that did not show evidence of complementation, i.e. those that exhibited slower growth on cysteine-free medium, were selected for further study. Sequencing of the AHL cDNA in one such clone revealed the conversion of cytosine 635 (C635) to thymine, resulting in an Alanine (A212) to Valine substitution. This microbial complementation procedure is useful in BPNTase structure-activity studies for biotechnological applications.

Using an Affymetrix GeneChip™ containing 8300 oligonucleotide probes, we measured transcript levels in transgenicArabidopsis overexpressing the jasmonate carboxyl methyltransferase gene (AtJMT). When compared with wild-type plants, 5-week-old transgenics exhibited significant alterations (more than a two-fold increase or decrease) in the expression levels of 168 genes. Among them, 80 were up-regulated, including those involved in defense, oxidative stress-tolerance, and senescence. In contrast, the expression of 88 genes, including those that function in photosynthesis and cold/drought-stress responses, was significantly down-regulated. Thus, endogenous generation of methyl jasmonate through AtjMT-overexpression modified the transcript levels of genes previously identified as being jasmonate-responsive. This result confirms that MeJA formation is a key control point for jasmonate-responsive gene expression in plants.

The optimum conditions for producing rice starch enriched in slowly digestible and resistant fractions by citric acid treatment determined by a response surface methodology (RSM) model equation, were: reaction temperature, 128.4 °C; reaction time, 13.8 h; and citric acid, 2.62 mmol/20 g starch. The slowly digestible and resistant starch fractions of the optimally acid-treated rice starch totalled 54.1%, which was 28.1% higher than the control. The slowly digestible and resistant fractions of the acid-treated rice starch did not differ significantly after heat treatment, whereas those of raw rice starch decreased by 49.6–63.8%, depending on the type of heat treatment (cooking at 100 °C or autoclaving). The slowly digestible fraction of the acid-treated starch increased by 8.9–14.2%. After autoclaving, the glucose response of the acid-treated starch was lower than untreated starch, but similar to that of Novelose 330. After heat treatment, the rate of blood glucose decrease was slower for the acid-treated starch than for Novelose 330. Compared to raw rice starch, the acid-treated starch exhibited increases in apparent amylose content, blue value, dextrose equivalent, cold-water solubility and transmittance, and decreases in wavelength of maximum absorbance, viscosity, and gel-forming ability.

We established an effective biolistic transformation procedure fortransferring foreign genes into garlic (Allium sativumL.),which we demonstrated by generating transgenic plants resistant tochlorsulfuron, a sulfonylurea herbicide. We subcultured callus tissue from theapical meristem of garlic cloves and repeatedly selected calli with brittle,non-mucilaginous surfaces for over six months, to increase transformationefficiency. We then constructed recombinant DNA that contained the acetolactatesynthase (ALS) gene from a chlorsulfuron-resistantArabidopsis mutant, the cauliflower mosaic virus 35Spromoter, the β-glucuronidase (GUS) reporter gene, and the hygromycinphosphotransferase (HPT) selectable marker gene. The garlic calli werebombarded twice with tungsten particles coated with the DNA constructs. Transformed calliwere efficiently selected by embedding them in solid agar medium containing 50mg l−1 hygromycin B. Selected propagules wereregenerated into 12 independent plants. We confirmed that the transgenes wereintegrated and expressed in the plants using PCR-Southern and Northern blotanalyses and by β-glucuronidase expression assay forGUS. The regenerated plants survived in the presence of 3mg l−1 chlorsulfuron, demonstrating that theirALS was insensitive to this herbicide. These results illustrate the successfultransformation of foreign genes into garlic plants. The set of proceduresdeveloped in this study is applicable to the generation of transgenic garlicplants with other agronomically beneficial traits.

The tomato geneRSI-1 was previously identified as a molecular marker for auxin-induced lateral root initiation. We have further characterized the expression mode of theRSI-1 gene in tomato andArabidopsis thaliana. Northern blot analyses revealed that the gene was induced specifically by auxin in tomato roots and hypocotyls. For experiments with transgenic plants, the 5′ flanking region of theRSI-1 gene was linked to a GUS reporter gene, then transformed into tomato andArabidopsis. In these transgenic tomato plants, GUS activity was detected at the sites of initiation for lateral and adventitious roots. Expression of the fusion gene was auxin-dependent and tissue-specific. This was consistent with results from the northern blot analyses. In transgenicArabidopsis, the overall expression pattern of theRSI-GUS gene, including tissue specificity and auxin inducibility, was comparable to that in transgenic tomato seedlings. These results indicate that an identical regulatory mechanism for lateral root initiation might be conserved in both plants. Thus, the expression mode of theRSI-CUS gene inArabidopsis mutants defective in lateral root development should be investigated to provide details of this process.

TheRSI-1 gene is expressed in pericycle cells just prior to the first round of cell division for lateral root development in tomato. We transformed tomato plants with theRSI-1 gene promoter linked to a GUS reporter gene. GUS activity was detected not only at the sites of initiation for lateral and adventitious roots, but also at the primary root tip. Expression of the fusion gene was also regulated at various stages of tissue development: in particular, during the formation of reproductive organs such as pollen and fruit Overexpression of theRSI-1 gene in either the sense or antisense orientation resulted in arrest of fruit development and seed germination. TheRSI-1 gene product, therefore, may play a role in auxin-induced cell division in various developing tissues. Inter and intramolecular disulfide bridges between cysteines rich in the RSI-1 protein might be involved in cell-wall modifications that are essential for new cell division. These hypotheses for the role of theRSI-1 gene in lateral-root and reproductive-organ development remain to be tested.

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-β-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta-β-glucoside elicitor by binding proteins in soybean cells. Elicitor-binding proteins with properties expected of physiological receptors for the hepta-β-glucoside elicitor have been identified in soybean root membranes. These elicitor-binding proteins co-migrate with a plasma membrane marker (vanadate-sensitive H + -ATPase) on linear sucrose density gradients. Binding of a radio-iodinated derivative of the hepta-β-glucoside elicitor by membrane-localized elicitor-binding proteins is specific, reversible, saturable, and of high affinity (K d 1 mM). After solubilization with the nonionic detergent, n-dodecylsucrose, the elicitor-binding proteins retain their high affinity (K d =1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-β-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta-β-glucoside elicitor-binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta-β-glucoside binding proteins are among the first steps toward elucidating how the hepta-β-glucoside ellicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean

AtMYB44 is a member of the R2R3 MYB subgroup 22 transcription factors and regulates diverse cellular responses in Arabidopsis thaliana. We performed quadruple 9-mer-based protein binding microarray (PBM) analysis, which revealed that full-size AtMYB44 recognized and bound to the consensus sequence AACnG, where n represents A, G, C or T. The consensus sequence was confirmed by electrophoretic mobility shift assay (EMSA) with a truncated AtMYB44 protein containing the N-terminal side R2R3 domain. This result indicates that the R2R3 domain alone is sufficient to exhibit AtMYB44 binding specificity. The sequence AACnG is the type I binding site for MYB transcription factors, including all members of the subgroup 22. EMSA showed that the R2R3 domain protein binds in vitro to promoters of randomly selected Arabidopsis genes that contain the consensus binding sequence. This implies that AtMYB44 binds to any promoter region that contains the consensus sequence, without determining their functional activity or specificity. The C-terminal side transcriptional activation domain of AtMYB44 contains an asparagine-rich fragment, NINNTTSSRHNHNN (aa 215–228), which, among the members of subgroup 22, is unique to AtMYB44. A transcriptional activation assay in yeast showed that this fragment is included in a region (aa 200–240) critical for the ability of AtMYB44 to function as a transcriptional activator. We hypothesize that the C-terminal side of the protein, but not the N-terminal side of the R2R3 domain, contributes to the functional activity and specificity of AtMYB44 through interactions with other regulators generated by each of a variety of stimuli.

We are studying the cellular signalling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-beta-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete, Phytophthora megasperma f. sp. glycinea. Our research has focused on the first step in this signal pathway, namely the specific recognition of the hepta-beta-glucoside elicitor by plasma-membrane-localized binding protein(s) in soybean cells. Binding of a radio-iodinated derivative of the elicitor-active hepta-beta-glucoside by membrane elicitor-binding proteins is specific, reversible, saturable and of high affinity (Kd = 0.75 nM). After solubilization using the non-ionic detergent n-dodecylsucrose, the elicitor-binding proteins retain the binding affinity (Kd = 1.8 nM) for the radiolabelled elicitor and the binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of elicitor-active and structurally related inactive oligoglucosides to displace labelled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-beta-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the elicitor-binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification and cloning of the hepta-beta-glucoside elicitor-binding proteins. Purification and characterization of the hepta-beta-glucoside-binding protein(s) or their corresponding cDNAs is a first step toward elucidating how the hepta-beta-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.

Jasmonates play important roles in development, stress responses and defense in plants. Here, we report the results of a study using a functional genomics approach that identified a rice basic helix-loop-helix domain gene, OsbHLH148, that conferred drought tolerance as a component of the jasmonate signaling module in rice. OsbHLH148 transcript levels were rapidly increased by treatment with methyl jasmonate (MeJA) or abscisic acid, and abiotic stresses including dehydration, high salinity, low temperature and wounding. Transgenic over-expression of OsbHLH148 in rice confers plant tolerance to drought stress. Expression profiling followed by DNA microarray and RNA gel-blot analyses of transgenic versus wild-type rice identified genes that are up-regulated by OsbHLH148 over-expression. These include OsDREB and OsJAZ genes that are involved in stress responses and the jasmonate signaling pathway, respectively. OsJAZ1, a rice ZIM domain protein, interacted with OsbHLH148 in yeast two-hybrid and pull-down assays, but it interacted with the putative OsCOI1 only in the presence of coronatine. Furthermore, the OsJAZ1 protein was degraded by rice and Arabidopsis extracts in the presence of coronatine, and its degradation was inhibited by MG132, a 26S proteasome inhibitor, suggesting 26S proteasome-mediated degradation of OsJAZ1 via the SCF(OsCOI1) complex. The transcription level of OsJAZ1 increased upon exposure of rice to MeJA. These results show that OsJAZ1 could act as a transcriptional regulator of the OsbHLH148-related jasmonate signaling pathway leading to drought tolerance. Thus, our study suggests that OsbHLH148 acts on an initial response of jasmonate-regulated gene expression toward drought tolerance, constituting the OsbHLH148-OsJAZ-OsCOI1 signaling module in rice.

Arabidopsis RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatases regulate stress-responsive gene expression and plant development via the dephosphorylation of serine (Ser) residues of the CTD. Some of these phosphatases (CTD phosphatase-like 1 (CPL1) to CPL3) negatively regulate ABA and stress responses. Here, we isolated AtCPL5, a cDNA encoding a protein containing two CTD phosphatase domains (CPDs). To characterize AtCPL5, we analyzed the gene expression patterns and subcellular protein localization, investigated various phenotypes of AtCPL5-overexpressors and knockout mutants involved in ABA and drought responses, performed microarray and RNA hybridization analyses using AtCPL5-overexpressors, and assessed the CTD phosphatase activities of the purified AtCPL5 and each CPD of the protein. Transcripts of the nucleus-localized AtCPL5 were induced by ABA and drought. AtCPL5-overexpressors exhibited ABA-hypersensitive phenotypes (increased inhibition of seed germination, seedling growth, and stomatal aperture), lower transpiration rates upon dehydration, and enhanced drought tolerance, while the knockout mutants showed weak ABA hyposensitivity. AtCPL5 overexpression changed the expression of numerous genes, including those involved in ABA-mediated responses. In contrast to Ser-5-specific phosphatase activity of the negative stress response regulators, purified AtCPL5 and each CPD of the protein specifically dephosphorylated Ser-2 in RNAPII CTD. We conclude that AtCPL5 is a unique CPL family protein that positively regulates ABA-mediated development and drought responses in Arabidopsis.

The presence of a specific binding site for a hepta-beta-glucoside elicitor of phytoalexin accumulation has been demonstrated in soybean microsomal membranes. A tyramine conjugate of the elicitor-active hepta-beta-glucoside was prepared and radiolabeled with 125I. The labeled hepta-beta-glucoside-tyramine conjugate was used as a ligand in binding assays with a total membrane fraction prepared from soybean roots. Binding of the radiolabeled hepta-beta-glucoside elicitor was saturable, reversible, and with an affinity (apparent Kd = 7.5 x 10(-10) M) comparable with the concentration of hepta-beta-glucoside required for biological activity. A single class of hepta-beta-glucoside binding sites was found. The binding site was inactivated by proteolysis and by heat treatment, suggesting that the binding site is a protein or glycoprotein. Competitive inhibition of binding of the radiolabeled hepta-beta-glucoside elicitor by a number of structurally related oligoglucosides demonstrated a direct correlation between the binding affinities and the elicitor activities of these oligoglucosides. Thus, the hepta-beta-glucoside-binding protein fulfills criteria expected of a bona fide receptor for the elicitor-active oligosaccharin.

The abilities of a family of chemically synthesized oligo-beta-glucosides, ranging in size from hexamer to decamer, to induce phytoalexin accumulation in soybean cotyledons were investigated to determine which structural elements of the oligoglucosides are important for their biological activity. The results of the biological assays established that the following structural motif is necessary for the oligo-beta-glucosides to have high elicitor activity: [formula; see text] The branched trisaccharide at the nonreducing end of the oligoglucosides was found to be essential for maximum elicitor activity. Substitution of either the nonreducing terminal backbone glucosyl residue or the side-chain glucosyl residue closest to the nonreducing end with glucosaminyl or N-acetylglucosaminyl residues reduced the elicitor activity of the oligoglucosides between 10-fold and 10,000-fold. Elicitor activity was also reduced 1000-fold if the two side-chain glucosyl residues were attached to adjacent backbone glucosyl residues rather than to glucosyl residues separated by an unbranched residue. In contrast, modifications of the reducing terminal glucosyl residue of an elicitor-active hepta-beta-glucoside by conjugation with tyramine and subsequent iodination had no significant effect on the elicitor activity of the hepta-beta-glucoside. These results demonstrate that oligo-beta-glucosides must have a specific structure to trigger the signal transduction pathway, which ultimately leads to the de novo synthesis of phytoalexins in soybean.