Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

An NADPH-Oxidase/Polyamine Oxidase Feedback Loop Controls Oxidative Burst Under Salinity.

The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O2·-) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O2·--specific probe benzene sulfonyl-So showed that O2·- levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O2·- accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.

Potential of plants to produce recombinant protein products.

Plants have great potential as photosynthetic factories to produce pharmaceutically important and commercially valuable biomedicines and industrial proteins at low cost. The U.S. Food and Drug Administration (U.S. FDA) has approved the drug Elelyso (taliglucerase alfa) produced by carrot cells for treatment of type 1 Gaucher's disease in 2012. The commercial potential of biomedicines produced by molecular farming has dramatically improved due to the success of an experimental drug called ZMapp, which has immunological activity in Ebola patients. A cocktail of three monoclonal antibodies was produced in tobacco (Nicotiana benthamiana) plants (Chen and Davis 2016). At present, very few drugs made by this technology have been approved by worldwide authorities such as the U.S. FDA. However, plants have been proposed as a novel paradigm for commercial production of proteins over the next decade. In recent years, leading researchers on molecular farming have given more priority to the area of animal-free therapeutic proteins such as parenteral and oral vaccines. Although plant-based platforms have considerable advantages over traditional systems such as bacterial and animal systems, there are several obstacles to commercial-scale production, especially with regards to improving the quality and quantity of plant-produced biologics and industrial materials. One of the biggest barriers to commercialization of this technology is the intense scrutiny of these new plant varieties by regulatory agencies and the public as well as the high costs associated with their regulatory approval.

Constitutive S-adenosylmethionine decarboxylase gene expression increases drought tolerance through inhibition of reactive oxygen species accumulation in Arabidopsis.

Using subtractive hybridization analysis, the S-adenosylmethionine decarboxylase (SAMDC) gene from Capsicum annuum was isolated and renamed CaSAMDC. We generated independent transgenic Arabidopsis (Arabidopsis thaliana) lines constitutively expressing a 35S::CaSAMDC construct. Drought tolerance was significantly enhanced in Arabidopsis T4 transgenic homozygous lines as compared to wild-type (WT) plants. The levels of main polyamines (PAs) were more significantly increased in CaSAMDC-overexpressing transgenic plants after 6 h of drought stress as compared to stressed WT plants. Basal transcription of polyamine oxidase (PAO) showed at a much higher level in unstressed-transgenic plants as compared to unstressed WT plants. However, the difference in PAO transcription level between WT and transgenic plants was reduced after drought stress. Cellular accumulation of reactive oxygen species (ROS) was significantly reduced following drought stress in transgenic Arabidopsis plants as compared to WT plants. These results were in agreement with additional observations that stress-induced ROS generation, as determined by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), was significantly suppressed while transcription of ROS-detoxifying enzymes was notably elevated in transgenic lines in response to drought stress. Further, ROS-induced transcription of the metacaspase II gene was remarkably inhibited in transgenic plants. Collectively, these results suggest that drought stress tolerance due to reduction of ROS production and enhancement of ROS detoxification can be attributed to elevation of PAs.

Synergistic biosynthesis of biphasic ethylene and reactive oxygen species in response to hemibiotrophic Phytophthora parasitica in tobacco plants.

We observed the biphasic production of ethylene and reactive oxygen species (ROS) in susceptible tobacco (Nicotiana tabacum 'Wisconsin 38') plants after shoot inoculation with Phytophthora parasitica var nicotianae. The initial transient increase in ROS and ethylene at 1 and 3 h (phase I), respectively, was followed by a second massive increase at 48 and 72 h (phase II), respectively, after pathogen inoculation. This biphasic pattern of ROS production significantly differed from the hypersensitive response exhibited by cryptogein-treated wild-type tobacco plants. The biphasic increase in ROS production was mediated by both NADPH oxidase isoforms, respiratory burst oxidase homolog (Rboh) D and RbohF. Conversely, different 1-aminocyclopropane-1-carboxylic acid synthase members were involved in specific phases of ethylene production: NtACS4 in the first phase and NtACS1 in the second phase. Biphasic production of ROS was inhibited in transgenic antisense plant lines expressing 1-aminocyclopropane-1-carboxylic acid synthase/oxidase or ethylene-insensitive3 as well as in transgenic plants impaired in ROS production. All tested transgenic plants were more tolerant against P. parasitica var nicotianae infection as determined based on trypan blue staining and pathogen proliferation. Further, silencing of NtACS4 blocked the second massive increase in ROS production as well as pathogen progression. Pathogen tolerance was due to the inhibition of ROS and ethylene production, which further resulted in lower activation of ROS-detoxifying enzymes. Accordingly, the synergistic inhibition of the second phase of ROS and ethylene production had protective effects against pathogen-induced cell damage. We conclude that the levels of ethylene and ROS correlate with compatible P. parasitica proliferation in susceptible plants.

Phytosphingosine induces systemic acquired resistance through activation of sphingosine kinase.

Phytosphingosine (PHS) is a naturally occurring bioactive sphingolipid molecule. Intermediates such as sphingolipid long-chain bases (LCBs) in sphingolipid biosynthesis have been shown to have important roles as signaling molecules. PHS treatment caused rapid cell damage and upregulated the generation of reactive oxygen species (ROS) and ethylene in tobacco plants. These events were followed by the induction of sphingosine kinase (SphK) in a biphasic manner, which metabolized PHS to phytosphingosine-1-phosphate (PHS-1-P). On the other hand, a PHS treatment with a virulent pathogen, Phytophthora parasitica var. nicotianae (Ppn), alleviated the pathogen-induced cell damage and reduced the growth of Ppn. A Ppn infection increased the PHS and PHS-1-P levels significantly in the upper part of the leaves at the infection site at the later stage. In addition, Ppn increased the transcription levels of serine palmitoyltransferase (LCB1 and LCB2) for sphingolipid biosynthesis at the later stage, which was enhanced further by PHS. Moreover, the PHS treatment increased the transcription and activity of SphK, which was accompanied by prominent increases in the transcription levels of ROS-detoxifying enzymes and PR proteins in the later phase of the pathogen infection. Overall, the PHS-induced resistant effects were prominent during the necrotic stage of this hemibiotrophic infection, indicating that it is more beneficial for inhibiting the pathogenicity on necrotic cell death. Phosphorylated LCBs reduced the pathogen-induced cell damage significantly in this stage. These results suggest that the selective channeling of sphingolipids into phosphorylated forms has a pro-survival effect on plant immunity.

Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis.

Rae1 performs multiple functions in animal systems, acting in interphase as an mRNA export factor and during mitosis as a mitotic checkpoint and spindle assembly regulator. In this study we characterized multiple functions of Rae1 in plants. Virus-induced gene silencing of Nicotiana benthamiana Rae1, NbRae1, which encodes a protein with four WD40 repeats, resulted in growth arrest and abnormal leaf development. NbRae1 was mainly associated with the nuclear envelope during interphase, and NbRae1 deficiency caused accumulation of poly(A) RNA in the nuclei of leaf cells, suggesting defective mRNA export. In the shoot apex, depletion of NbRae1 led to reduced mitotic activities, accompanied by reduced cyclin-dependent kinase (CDK) activity and decreased expression of cyclin B1, CDKB1-1, and histones H3 and H4. The secondary growth of stem vasculature was also inhibited, indicating reduced cambial activities. Differentiated leaf cells of NbRae1-silenced plants exhibited elevated ploidy levels. Immunolabeling in BY-2 cells showed that NbRae1 protein localized to mitotic microtubules and the cell plate-forming zone during mitosis, and recombinant NbRae1 directly bound to microtubules in vitro. Inhibition of NbRae1 expression in BY-2 cells using a beta-estradiol-inducible RNAi system resulted in severe defects in spindle organization and chromosome alignment and segregation, which correlated with delays in cell cycle progression. Together, these results suggest that NbRae1 plays a dual role in mRNA export in interphase and in spindle assembly in mitosis.

Functional switching of NPR1 between chloroplast and nucleus for adaptive response to salt stress.

Salt stress causes rapid accumulation of nonexpressor of pathogenesis-related genes 1 (NPR1) protein, known as the redox-sensitive transcription coactivator, which in turn elicits many adaptive responses. The NPR1 protein transiently accumulates in chloroplast stroma under salt stress, which attenuates stress-triggered down-regulation of photosynthetic capability. We observed that oligomeric NPR1 in chloroplasts and cytoplasm had chaperone activity, whereas monomeric NPR1 in the nucleus did not. Additionally, NPR1 overexpression resulted in reinforcement of morning-phased and evening-phased circadian clock. NPR1 overexpression also enhanced antioxidant activity and reduced stress-induced reactive oxygen species (ROS) generation at early stage, followed with transcription levels for ROS detoxification. These results suggest a functional switch from a molecular chaperone to a transcriptional coactivator, which is dependent on subcellular localization. Our findings imply that dual localization of NPR1 is related to proteostasis and redox homeostasis in chloroplasts for emergency restoration as well as transcriptional coactivator in the nucleus for adaptation to stress.

Increasing Polyamine Contents Enhances the Stress Tolerance via Reinforcement of Antioxidative Properties.

The diamine putrescine and the polyamines (PAs), spermidine (Spd) and spermine (Spm), are ubiquitously occurring polycations associated with several important cellular functions, especially antisenescence. Numerous studies have reported increased levels of PA in plant cells under conditions of abiotic and biotic stress such as drought, high salt concentrations, and pathogen attack. However, the physiological mechanism of elevated PA levels in response to abiotic and biotic stresses remains undetermined. Transgenic plants having overexpression of SAMDC complementary DNA and increased levels of putrescine (1.4-fold), Spd (2.3-fold), and Spm (1.8-fold) under unstressed conditions were compared to wild-type (WT) plants in the current study. The most abundant PA in transgenic plants was Spd. Under salt stress conditions, enhancement of endogenous PAs due to overexpression of the SAMDC gene and exogenous treatment with Spd considerably reduces the reactive oxygen species (ROS) accumulation in intra- and extracellular compartments. Conversely, as compared to the WT, PA oxidase transcription rapidly increases in the S16-S-4 transgenic strain subsequent to salt stress. Furthermore, transcription levels of ROS detoxifying enzymes are elevated in transgenic plants as compared to the WT. Our findings with OxyBlot analysis indicate that upregulated amounts of endogenous PAs in transgenic tobacco plants show antioxidative effects for protein homeostasis against stress-induced protein oxidation. These results imply that the increased PAs induce transcription of PA oxidases, which oxidize PAs, which in turn trigger signal antioxidative responses resulting to lower the ROS load. Furthermore, total proteins from leaves with exogenously supplemented Spd and Spm upregulate the chaperone activity. These effects of PAs for antioxidative properties and antiaggregation of proteins contribute towards maintaining the physiological cellular functions against abiotic stresses. It is suggested that these functions of PAs are beneficial for protein homeostasis during abiotic stresses. Taken together, these results indicate that PA molecules function as antisenescence regulators through inducing ROS detoxification, antioxidative properties, and molecular chaperone activity under stress conditions, thereby providing broad-spectrum tolerance against a variety of stresses.

Real-Time In Vivo Monitoring of Reactive Oxygen Species in Guard Cells.

The intra-/intercellular homeostasis of reactive oxygen species (ROS), and especially of superoxides (O2.-) and hydrogen peroxide (O2.-) participate in signalling cascades which dictate developmental processes and reactions to biotic/abiotic stresses. Polyamine oxidases terminally oxidize/back convert polyamines generating H2O2. Recently, an NADPH-oxidase/Polyamine oxidase feedback loop was identified to control oxidative burst under salinity. Thus, the real-time localization/monitoring of ROS in specific cells, such as the guard cells, can be of great interest. Here we present a detailed description of the real-time in vivo monitoring of ROS in the guard cells using H2O2- and O2.- specific fluorescing probes, which can be used for studying ROS accumulation generated from any source, including the amine oxidases-dependent pathway, during development and stress.

Deregulation of apoplastic polyamine oxidase affects development and salt response of tobacco plants.

Polyamine (PA) homeostasis is associated with plant development, growth and responses to biotic/abiotic stresses. Apoplastic PA oxidase (PAO) catalyzes the oxidation of PAs contributing to cellular homeostasis of reactive oxygen species (ROS) and PAs. In tobacco, PAs decrease with plant age, while apoplastic PAO activity increases. Our previous results with young transgenic tobacco plants with enhanced/reduced apoplastic PAO activity (S-ZmPAO/AS-ZmPAO, respectively) established the importance of apoplastic PAO in controlling tolerance to short-term salt stress. However, it remains unclear if the apoplastic PAO pathway is important for salt tolerance at later stages of plant development. In this work, we examined whether apoplastic PAO controls also plant development and tolerance of adult plants during long-term salt stress. The AS-ZmPAO plants contained higher Ca2+ during salt stress, showing also reduced chlorophyll content index (CCI), leaf area and biomass but taller phenotype compared to the wild-type plants during salt. On the contrary, the S-ZmPAO had more leaves with slightly greater size compared to the AS-ZmPAO and higher antioxidant genes/enzyme activities. Accumulation of proline in the roots was evident at prolonged stress and correlated negatively with PAO deregulation as did the transcripts of genes mediating ethylene biosynthesis. In contrast to the strong effect of apoplastic PAO to salt tolerance in young plants described previously, the effect it exerts at later stages of development is rather moderate. However, the different phenotypes observed in plants deregulating PAO reinforce the view that apoplastic PAO exerts multifaceted roles on plant growth and stress responses. Our data suggest that deregulation of the apoplastic PAO can be further examined as a potential approach to breed plants with enhanced/reduced tolerance to abiotic stress with minimal associated trade-offs.

A proteomic analysis identifies glutathione S-transferase isoforms whose abundance is differentially regulated by ethylene during the formation of early root epidermis in Arabidopsis seedlings.

The plant hormone ethylene has been shown to play an important role in root hair development in Arabidopsis. With the aid of proteomic analysis, we identified three distinct glutathione S-transferase (GST) isoforms, AtGSTF2, AtGSTF8, and AtGSTU19, expressed early in root epidermal establishment in Arabidopsis seedlings. The AtGSTF2 protein was specifically up-regulated by ethylene. A subsequent RNA expression study revealed that the AtGSTF2 gene was highly sensitive to ethylene, whereas the transcripts for AtGSTF8 and AtGSTU19 were constitutively present in new root tissue of 4-day-old seedlings. The steady-state level of AtGSTF2 mRNA was greatly reduced in the roots of ethylene-insensitive mutants, while mutation at the CTR1 locus, which confers an ectopic root hair phenotype, resulted in a markedly elevated level of AtGSTF2 transcript in young root tissue. Although the physiological function of ethylene-induced AtGSTF2 is not yet clear, there are several possibilities for its role during early root development.

Effects of abiotic stresses on cell cycle progression in tobacco BY-2 cells.

Mild stresses such as high temperature (30 degrees C) or a low H2O2 concentration induced transient cell cycle arrest at G1/S or G2/M depending on the cell cycle stage at which the stress was applied. When stresses were introduced during G0 or G1, the G1/S checkpoint was mainly used; when stresses were introduced after S phase, G2/M was the primary checkpoint. The slowing of cell cycle progression was associated with transient delays in expression of A-, B-, and D-type cyclins. The delay in expression of NtcycA13, one of the A-type cyclins, was most pronounced. The levels of expression of Ntcyc29 (a cyclin B gene) and of CycD3-1 differed most depending on the applied stress, suggesting that different cellular adjustments to mild heat and a low concentration of H2O2 are reflected in the expression of these two cyclins.

A leader intron and 115-bp promoter region necessary for expression of the carnation S-adenosylmethionine decarboxylase gene in the pollen of transgenic tobacco.

The expression of CSDC9 encoding S-adenosylmethionine decarboxylase (SAMDC) is developmentally and spatially regulated in carnation. To examine the regulation of the SAMDC gene, we analyzed the spatial expression of CSDC9 with a 5'-flanking beta-glucuronidase fusion in transgenic tobacco plants. GUS was strongly expressed in flower, pollen, stem and vein of cotyledons. Expression in both anther and stigma was under developmental control; analysis of a series of mutants with deletions of the 5'-flanking region demonstrated differential activation in petal, anther, stigma and pollen grains. All the major cis-regulatory elements required for pollen-specific transcription were located in the upstream region between -273 and -158. This region contains four putative elements related to gibberellin induction (pyrimidine boxes, TTTTTTCC and CCTTTT) and pollen-specific expression (GTGA and AGAAA). In addition, the first 5'-leader intron was necessary for tissue-specific expression.

Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants.

The amount of polyamines (such as putrescine, spermidine, and spermine) increased under environmental stress conditions. We used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Because there is a metabolic competition for S-adenosylmethionine as a precursor between polyamine (PA) and ethylene biosyntheses, it was expected that the antisense-expression of ethylene biosynthetic genes could result in an increase in PA biosynthesis. Antisense constructs of cDNAs for senescence-related 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (CAS) and ACC oxidase (CAO) were isolated from carnation flowers that were introduced into tobacco by Agrobacterium-mediated transformation. Several transgenic lines showed higher PA contents than wild-type plants. The number and weight of seeds also increased. Stress-induced senescence was attenuated in these transgenic plants in terms of total chlorophyll loss and phenotypic changes after oxidative stress with hydrogen peroxide (H2O2), high salinity, acid stress (pH 3.0), and ABA treatment. These results suggest that the transgenic plants with antisense CAS and CAO cDNAs are more tolerant to abiotic stresses than wild-type plants. This shows a positive correlation between PA content and stress tolerance in plants.

The putative transcriptional activator MSN1 promotes chromium accumulation in Saccharomyces cerevisiae.

Yeast is a good system for studying molecular mechanisms of metal tolerance. Using a mini-Tn mutagenized yeast pool, we isolated a chromate-tolerant mutant, CrT9, that displayed metal-specific tolerance since it was only tolerant to Cr(VI), not to Cr(III), Cd, As, or Fe. The Cr-tolerance of CrT9 appeared to be due to reduced Cr accumulation as it accumulated only 56% as much as WT (Y800). Using IPCR (inverse PCR), we found that the mini-Tn had been inserted at nt 741 of the transcriptional activator, MSN1. MSN1 is a multifunctional protein involved in invertase activity, iron uptake, starch degradation, pseudohyphal growth, and osmotic gene expression. We found that there was only one mini-Tn insertion in CrT9 since MSN1 and mini-Tn probes hybridized to the same DNA fragment, and the MSN1 probe detected an enlarged MSN1 mRNA. When we over-expressed MSN1 in CrT9 and WT, both accumulated larger amounts of Cr. We conclude that Cr accumulation in S. cerevisiae is promoted by the transcriptional activator MSN1.

Characterization of arginine decarboxylase from Dianthus caryophyllus.

Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.

Lysophosphatidylcholine enhances susceptibility in signaling pathway against pathogen infection through biphasic production of reactive oxygen species and ethylene in tobacco plants.

It was previously reported that the amounts of lysophosphatidylcholines (lysoPCs), which are naturally occurring bioactive lipid molecules, significantly increase following pathogen inoculation, as determined using ultraperformance liquid chromatography-quadrupole-time of flight/mass spectrometry analyses. Here, real-time quantitative RT-PCR was performed for the phospholipase A2 (PLA2) genes, Nt1PLA2 and Nt2PLA2, which are responsible for LysoPCs generation. The transcription level of Nt2PLA2 in pathogen-infected tobacco plants transiently peaked at 1h and 36 h, whereas induction of Nt1PLA2 transcription peaked at 36 h. A prominent biphasic ROS accumulation in lysoPC (C18:1(9Z))-treated tobacco leaves was also observed. Transcription of NtRbohD, a gene member of NADPH oxidase, showed biphasic kinetics upon lysoPC 18:1 treatment, as evidenced by an early transient peak in phase I at 1h and a massive peak in phase II at 12h. Each increase in NtACS2 and NtACS4 transcription, gene members of the ACC synthase family, was followed by biphasic peaks of ethylene production after lysoPC 18:1 treatment. This suggested that lysoPC (C18:1)-induced ethylene production was regulated at the transcriptional level of time-dependent gene members. LysoPC 18:1 treatment also rapidly induced cell damage. LysoPC 18:1-induced cell death was almost completely abrogated in ROS generation-impaired transgenic plants (rbohD-as and rbohF-as), ethylene production-impaired transgenic plants (CAS-AS and CAO-AS), and ethylene signaling-impaired transgenic plants (Ein3-AS), respectively. Taken together, pathogen-induced lysoPCs enhance pathogen susceptibility accompanied by ROS and ethylene biosynthesis, resulting in chlorophyll degradation and cell death. Expression of PR genes (PR1-a, PR-3, and PR-4b) and LOX3 was strongly induced in lysoPC 18:1-treated leaves, indicating the involvement of lysoPC 18:1 in the defense response. However, lysoPC 18:1 treatment eventually resulted in cell death, as evidenced by metacaspase gene expression. Therefore, a hypothesis is proposed that the antipathogenic potential of lysoPC 18:1 is dependent on how quickly it is removed from cells for avoidance of lysoPC toxicity.

Metabolic survey of defense responses to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae, in ethylene signaling-impaired tobacco.

Reactive oxygen species (ROS) and ethylene play an important role in determining the resistance or susceptibility of plants to pathogen attack. A previous study of the response of tobacco cultivar ( Nicotiana tabacum L. cv. Wisconsin 38) to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae (Ppn) showed that biphasic bursts of ROS and ethylene are positively associated with disease severity. The levels of ethylene and ROS might influence the susceptibility of plants to pathogens, with changing levels of metabolite related to disease resistance or susceptibility. In this study, to obtain more detailed information on the interaction of ROS and ethylene signaling related to resistance and/or susceptibility of plants to pathogen, Ppn-induced metabolic profiles from wild type (WT) and ethylene signaling-impaired transgenic plants that expressed Ein3 antisense (Ein3-AS) were compared using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Nonredundant mass ions (576 in ESI+ mode and 336 in ESI- mode) were selected, and 56 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Two-way hierarchical clustering analysis of the selected mass ions revealed that nicotine and phenylpropanoid-polyamine conjugates, such as caffeoyl-dihydrocaffeoyl-spermidine, dicaffeoyl-spermidine, caffeoyl-feruloyl-spermidine, and two bis(dihydrocaffeoyl)-spermine isomers, and their intermediates, such as arginine and putrecine, were present at lower levels in Ein3-AS transgenic plants during Ppn interaction than in WT, whereas galactolipid and oxidized free fatty acid levels were higher in Ein3-AS transgenic plants. Taken together, these results reveal a function for ethylene signaling in tobacco defense responses during Ppn interaction.

Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1.

A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H(2)O(2). The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenously-applied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H(2)O(2). Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.