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1.  Chromium Stress Mitigation by Polyamine-Brassinosteroid Application Involves Phytohormonal and Physiological Strategies in Raphanus sativus L. 
PLoS ONE  2012;7(3):e33210.
Brassinosteroids (BRs) and polyamines (PAs) are well-established growth regulators playing key roles in stress management among plants. In the present study, we evaluated the effects of epibrassinolide (EBL, an active BR) and spermidine (Spd, an active PA) on the tolerance of radish to oxidative stress induced by Cr (VI) metal. Our investigation aimed to study the impacts of EBL (10−9 M) and/or Spd (1 mM) on the biochemical and physiological responses of radish (Raphanus sativus L.) under Cr-stress. Applications of EBL and/or Spd were found to improve growth of Cr-stressed seedlings in terms of root length, shoot length and fresh weight. Our data also indicated that applications of EBL and Spd have significant impacts, particularly when applied together, on the endogenous titers of PAs, free and bound forms of IAA and ABA in seedlings treated with Cr-stress. Additionally, co-applications of EBL and Spd modulated more remarkably the titers of antioxidants (glutathione, ascorbic acid, proline, glycine betaine and total phenol) and activities of antioxidant enzymes (guaicol peroxidase, catalase, superoxide dismutase and glutathione reductase) in Cr-stressed plants than their individual applications. Attenuation of Cr-stress by EBL and/or Spd (more efficient with EBL and Spd combination) was also supported by enhanced values of stress indices, such as phytochelatins, photosynthetic pigments and total soluble sugars, and reduction in malondialdehyde and H2O2 levels in Cr-treated seedlings. Diminution of ROS production and enhanced ROS scavenging capacities were also noted for EBL and/or Spd under Cr-stress. However, no significant reduction in Cr uptake was observed for co-application of EBL and Spd when compared to their individual treatments in Cr-stressed seedlings. Taken together, our results demonstrate that co-applications of EBL and Spd are more effective than their independent treatments in lowering the Cr-induced oxidative stress in radish, leading to improved growth of radish seedlings under Cr-stress.
doi:10.1371/journal.pone.0033210
PMCID: PMC3315560  PMID: 22479371
2.  Brassinosteroids-Induced Systemic Stress Tolerance was Associated with Increased Transcripts of Several Defence-Related Genes in the Phloem in Cucumis sativus 
PLoS ONE  2013;8(6):e66582.
Brassinosteroids (BRs), a group of naturally occurring plant steroidal compounds, are essential for plant growth, development and stress tolerance. Recent studies showed that BRs could induce systemic tolerance to biotic and abiotic stresses; however, the molecular mechanisms by which BRs signals lead to responses in the whole plant are largely unknown. In this study, 24-epibrassinosteroid (EBR)-induced systemic tolerance in Cucumis sativus L. cv. Jinyan No. 4 was analyzed through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation. Expression of defense/stress related genes were induced in both treated local leaves and untreated systemic leaves by local EBR application. With the suppressive subtractive hybridization (SSH) library using cDNA from the phloem sap of EBR-treated plants as the tester and distilled water (DW)-treated plants as the driver, 14 transcripts out of 260 clones were identified. Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) validated the specific up-regulation of these transcripts. Of the differentially expressed transcripts with known functions, transcripts for the selected four cDNAs, which encode an auxin-responsive protein (IAA14), a putative ankyrin-repeat protein, an F-box protein (PP2), and a major latex, pathogenesis-related (MLP)-like protein, were induced in local leaves, systemic leaves and roots after foliar application of EBR onto mature leaves. Our results demonstrated that EBR-induced systemic tolerance is accompanied with increased transcript of genes in the defense response in other organs. The potential role of phloem mRNAs as signaling components in mediating BR-regulated systemic resistance is discussed.
doi:10.1371/journal.pone.0066582
PMCID: PMC3686678  PMID: 23840504
3.  Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways 
BMC Plant Biology  2010;10:151.
Background
Brassinosteroids (BRs) play crucial roles in plant development and also promote tolerance to a range of abiotic stresses. Although much has been learned about their roles in plant development, the mechanisms by which BRs control plant stress responses and regulate stress-responsive gene expression are not fully known. Since BR interacts with other plant hormones, it is likely that the stress tolerance conferring ability of BR lies in part in its interactions with other stress hormones.
Results
Using a collection of Arabidopsis mutants that are either deficient in or insensitive to abscisic acid (ABA), ethylene (ET), jasmonic acid (JA) and salicylic acid (SA), we studied the effects of 24-epibrassinloide (EBR) on basic thermotolerance and salt tolerance of these mutants. The positive impact of EBR on thermotolerance in proportion to wild type was evident in all mutants studied, with the exception of the SA-insensitive npr1-1 mutant. EBR could rescue the ET-insensitive ein2 mutant from its hypersensitivity to salt stress-induced inhibition of seed germination, but remained ineffective in increasing the survival of eto1-1 (ET-overproducer) and npr1-1 seedlings on salt. The positive effect of EBR was significantly greater in the ABA-deficient aba1-1 mutant as compared to wild type, indicating that ABA masks BR effects in plant stress responses. Treatment with EBR increased expression of various hormone marker genes in both wild type and mutant seedlings, although to different levels.
Conclusions
These results together indicate that the redox-sensitive protein NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1), a master regulator of SA-mediated defense genes, is likely a critical component of EBR-mediated increase in thermotolerance and salt tolerance, but it is not required for EBR-mediated induction of PR-1 (PATHOGENESIS-RELATED1) gene expression; that BR exerts anti-stress effects independently as well as through interactions with other hormones; that ABA inhibits BR effects during stress; and that BR shares transcriptional targets with other hormones.
doi:10.1186/1471-2229-10-151
PMCID: PMC3095295  PMID: 20642851
4.  Identification of MicroRNA 395a in 24-Epibrassinolide-Regulated Root Growth of Arabidopsis thaliana Using MicroRNA Arrays 
Brassinosteroids (BRs) are endogenous plant hormones and are essential for normal plant growth and development. MicroRNAs (miRNAs) of Arabidopsis thaliana are involved in mediating cell proliferation in leaves, stress tolerance, and root development. The specifics of BR mechanisms involving miRNAs are unknown. Using customized miRNA array analysis, we identified miRNAs from A. thaliana ecotype Columbia (Col-0) regulated by 24-epibrassinolide (EBR, a highly active BR). We found that miR395a was significantly up-regulated by EBR treatment and validated its expression under these conditions. miR395a was over expressed in leaf veins and root tissues in EBR-treated miR395a promoter::GUS plants. We integrated bioinformatics methods and publicly available DNA microarray data to predict potential targets of miR395a. GUN5—a multifunctional protein involved in plant metabolic functions such as chlorophyll synthesis and the abscisic acid (ABA) pathway—was identified as a possible target. ABI4 and ABI5, both genes positively regulated by ABA, were down-regulated by EBR treatment. In summary, our results suggest that EBR regulates seedling development and root growth of A. thaliana through miR395a by suppressing GUN5 expression and its downstream signal transduction.
doi:10.3390/ijms140714270
PMCID: PMC3742243  PMID: 23839095
brassinosteroids; miR395a; root growth; Arabidopsis thaliana; microRNA array
5.  Role of brassinosteroids in alleviation of phenanthrene–cadmium co-contamination-induced photosynthetic inhibition and oxidative stress in tomato 
Journal of Experimental Botany  2012;64(1):199-213.
Heavy metal pollution often occurs together with organic contaminants. Brassinosteroids (BRs) induce plant tolerance to several abiotic stresses, including phenanthrene (PHE) and cadmium (Cd) stress. However, the role of BRs in PHE+Cd co-contamination-induced stress amelioration is unknown. Here, the interactive effects of PHE, Cd, and 24-epibrassinolide (EBR; a biologically active BR) were investigated in tomato plants. The application of Cd (100 µM) alone was more phytotoxic than PHE applied alone (100 µM); however, their combined application resulted in slightly improved photosynthetic activity and pigment content compared with Cd alone after a 40 d exposure. Accumulation of reactive oxygen species and membrane lipid peroxidation were induced by PHE and/or Cd; however, the differences in effect were insignificant between Cd and PHE+Cd. The foliar application of EBR (0.1 µM) to PHE- and/or Cd-stressed plants alleviated photosynthetic inhibition and oxidative stress by causing enhancement of the activity of the enzymes and related transcript levels of the antioxidant system, secondary metabolism, and the xenobiotic detoxification system. Additionally, PHE and/or Cd residues were significantly decreased in both the leaves and roots after application of EBR, more specifically in PHE+Cd-stressed plants when treated with EBR, indicating a possible improvement in detoxification of these pollutants. The findings thus suggest a potential interaction of EBR and PHE for Cd stress alleviation. These results advocate a positive role for EBR in reducing pollutant residues for food safety and also strengthening phytoremediation.
doi:10.1093/jxb/ers323
PMCID: PMC3528031  PMID: 23201830
Brassinosteroids; food safety; heavy metal; photosynthesis; phytoremediation; polycyclic aromatic hydrocarbons (PAHs)
6.  Hydrogen peroxide functions as a secondary messenger for brassinosteroids-induced CO2 assimilation and carbohydrate metabolism in Cucumis sativus *  
Brassinosteroids (BRs) are potent regulators of photosynthesis and crop yield in agricultural crops; however, the mechanism by which BRs increase photosynthesis is not fully understood. Here, we show that foliar application of 24-epibrassinolide (EBR) resulted in increases in CO2 assimilation, hydrogen peroxide (H2O2) accumulation, and leaf area in cucumber. H2O2 treatment induced increases in CO2 assimilation whilst inhibition of the H2O2 accumulation by its generation inhibitor or scavenger completely abolished EBR-induced CO2 assimilation. Increases of light harvesting due to larger leaf areas in EBR- and H2O2-treated plants were accompanied by increases in the photochemical efficiency of photosystem II (ΦPSII) and photochemical quenching coefficient (q P). EBR and H2O2 both activated carboxylation efficiency of ribulose-1,5-bisphosphate oxygenase/carboxylase (Rubisco) from analysis of CO2 response curve and in vitro measurement of Rubisco activities. Moreover, EBR and H2O2 increased contents of total soluble sugar, sucrose, hexose, and starch, followed by enhanced activities of sugar metabolism such as sucrose phosphate synthase, sucrose synthase, and invertase. Interestingly, expression of transcripts of enzymes involved in starch and sugar utilization were inhibited by EBR and H2O2. However, the effects of EBR on carbohydrate metabolisms were reversed by the H2O2 generation inhibitor diphenyleneodonium (DPI) or scavenger dimethylthiourea (DMTU) pretreatment. All of these results indicate that H2O2 functions as a secondary messenger for EBR-induced CO2 assimilation and carbohydrate metabolism in cucumber plants. Our study confirms that H2O2 mediates the regulation of photosynthesis by BRs and suggests that EBR and H2O2 regulate Calvin cycle and sugar metabolism via redox signaling and thus increase the photosynthetic potential and yield of crops.
doi:10.1631/jzus.B1200130
PMCID: PMC3468824  PMID: 23024048
Metabolism; Photosynthesis; Reactive oxygen species; Rubisco; Sucrose
7.  Brassinosteroids are involved in response of cucumber (Cucumis sativus) to iron deficiency 
Annals of Botany  2012;110(3):681-688.
Background and Aims
Brassinosteroids (BR) are a class of plant polyhydroxysteroids with diverse functions in plant growth and development. However, there is little information on the role of BRs played in the response to nutrient deficiency.
Methods
To evaluate the role of BR in the response of plants to iron (Fe) deficiency, the effect of 24-epibrassinolide (EBR) on ferric reductase (FRO) activity, acidification of the rhizosphere and Fe content in cucumber (Cucumis sativus) seedlings under Fe-deficient (1 µm FeEDTA) and Fe-sufficient (50 µm FeEDTA) conditions were investigated.
Key Results
There was a significant increase in FRO activity upon exposure of cucumber seedlings to an Fe-deficient medium, and the Fe deficiency-induced increase in FRO activity was substantially suppressed by EBR. In contrast, application of EBR to Fe-sufficient seedlings stimulated FRO activity. Ethylene production evoked by Fe deficiency was suppressed by EBR, while EBR induced ethylene production from Fe-sufficient seedlings. Fe contents in shoots were reduced by treatment with EBR, while Fe contents in roots were markedly increased under both Fe-deficient and Fe-sufficient conditions. The reductions in Fe contents of shoots were independent of chlorophyll (CHL) contents under Fe-sufficient conditions, but they were positively correlated with CHL contents under Fe-deficient conditions. At the transcriptional level, transcripts encoding FRO (CsFRO1) and Fe transporter (CsIRT1) were increased upon exposure to the Fe-deficient medium, and the increases in transcripts were reversed by EBR.
Conclusions
The results demonstrate that BRs are likely to play a negative role in regulating Fe-deficiency-induced FRO, expressions of CsFRO1 and CsIRT1, as well as Fe translocation from roots to shoots.
doi:10.1093/aob/mcs126
PMCID: PMC3400454  PMID: 22684685
Brassinosteroids; iron deficiency; cucumber; Cucumis sativus; ferric reductase activity; Fe translocation
8.  A role of brassinosteroids in early fruit development in cucumber 
Journal of Experimental Botany  2008;59(9):2299-2308.
Brassinosteroids (BRs) are essential for many biological processes in plants, however, little is known about their roles in early fruit development. To address this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, cell division, and expression of cyclin and cyclin-dependent kinases (CDKs) genes were examined in two cucumber cultivars that differ in parthenocarpic capacity. The application of EBR induced parthenocarpic growth accompanied by active cell division in Jinchun No. 4, a cultivar without parthenocarpic capacity, whereas Brz treatment inhibited fruit set and, subsequently, fruit growth in Jinchun No. 2, a cultivar with natural parthenocarpic capacity, and this inhibitory effect could be rescued by the application of EBR. RT-PCR analysis showed both pollination and EBR induced expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB) after anthesis. cDNA sequences for CsCycD3;1 and CsCycD3;2 were isolated through PCR amplification. Both CsCycD3;1 and CsCycD3;2 transcripts were up-regulated by EBR treatment and pollination but strongly repressed by Brz treatment. Meanwhile, BR6ox1 and SMT transcripts, two genes involved in BR synthesis, exhibited feedback regulation. These results strongly suggest that BRs play an important role during early fruit development in cucumber.
doi:10.1093/jxb/ern093
PMCID: PMC2423651  PMID: 18515830
Brassinosteroids; cell division; Cucumis sativus; cyclin; flow cytometry; parthenocarpy
9.  Transcriptome Profiling of Radish (Raphanus sativus L.) Root and Identification of Genes Involved in Response to Lead (Pb) Stress with Next Generation Sequencing 
PLoS ONE  2013;8(6):e66539.
Lead (Pb), one of the most toxic heavy metals, can be absorbed and accumulated by plant roots and then enter the food chain resulting in potential health risks for human beings. The radish (Raphanus sativus L.) is an important root vegetable crop with fleshy taproots as the edible parts. Little is known about the mechanism by which radishes respond to Pb stress at the molecular level. In this study, Next Generation Sequencing (NGS)–based RNA-seq technology was employed to characterize the de novo transcriptome of radish roots and identify differentially expressed genes (DEGs) during Pb stress. A total of 68,940 assembled unique transcripts including 33,337 unigenes were obtained from radish root cDNA samples. Based on the assembled de novo transcriptome, 4,614 DEGs were detected between the two libraries of untreated (CK) and Pb-treated (Pb1000) roots. Gene Ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Pb stress are predominately involved in defense responses in cell walls and glutathione metabolism-related processes, while downregulated DEGs were mainly involved in carbohydrate metabolism-related pathways. The expression patterns of 22 selected genes were validated by quantitative real-time PCR, and the results were highly accordant with the Solexa analysis. Furthermore, many candidate genes, which were involved in defense and detoxification mechanisms including signaling protein kinases, transcription factors, metal transporters and chelate compound biosynthesis related enzymes, were successfully identified in response to heavy metal Pb. Identification of potential DEGs involved in responses to Pb stress significantly reflected alterations in major biological processes and metabolic pathways. The molecular basis of the response to Pb stress in radishes was comprehensively characterized. Useful information and new insights were provided for investigating the molecular regulation mechanism of heavy metal Pb accumulation and tolerance in root vegetable crops.
doi:10.1371/journal.pone.0066539
PMCID: PMC3688795  PMID: 23840502
10.  Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt stress: a comparison with responses to drought 
Soil salinity affects a large proportion of rural area and limits agricultural productivity. To investigate differential adaptation to soil salinity, we studied salt tolerance of 18 varieties of Oryza sativa using a hydroponic culture system. Based on visual inspection and photosynthetic parameters, cultivars were classified according to their tolerance level. Additionally, biomass parameters were correlated with salt tolerance. Polyamines have frequently been demonstrated to be involved in plant stress responses and therefore soluble leaf polyamines were measured. Under salinity, putrescine (Put) content was unchanged or increased in tolerant, while dropped in sensitive cultivars. Spermidine (Spd) content was unchanged at lower NaCl concentrations in all, while reduced at 100 mM NaCl in sensitive cultivars. Spermine (Spm) content was increased in all cultivars. A comparison with data from 21 cultivars under long-term, moderate drought stress revealed an increase of Spm under both stress conditions. While Spm became the most prominent polyamine under drought, levels of all three polyamines were relatively similar under salt stress. Put levels were reduced under both, drought and salt stress, while changes in Spd were different under drought (decrease) or salt (unchanged) conditions. Regulation of polyamine metabolism at the transcript level during exposure to salinity was studied for genes encoding enzymes involved in the biosynthesis of polyamines and compared to expression under drought stress. Based on expression profiles, investigated genes were divided into generally stress-induced genes (ADC2, SPD/SPM2, SPD/SPM3), one generally stress-repressed gene (ADC1), constitutively expressed genes (CPA1, CPA2, CPA4, SAMDC1, SPD/SPM1), specifically drought-induced genes (SAMDC2, AIH), one specifically drought-repressed gene (CPA3) and one specifically salt-stress repressed gene (SAMDC4), revealing both overlapping and specific stress responses under these conditions.
doi:10.3389/fpls.2014.00182
PMCID: PMC4021140  PMID: 24847340
polyamines; salt stress; drought stress; gene expression; rice; natural variety
11.  Polyamine metabolic canalization in response to drought stress in Arabidopsis and the resurrection plant Craterostigma plantagineum 
Plant Signaling & Behavior  2011;6(2):243-250.
In this work, we have studied the transcriptional profiles of polyamine biosynthetic genes and analyzed polyamine metabolic fluxes during a gradual drought acclimation response in Arabidopsis thaliana and the resurrection plant Craterostigma plantagineum. The analysis of free putrescine, spermidine and spermine titers in Arabidopsis arginine decarboxylase (adc1–3, adc2–3), spermidine synthase (spds1–2, spds2–3) and spermine synthase (spms-2) mutants during drought stress, combined with the quantitative expression of the entire polyamine biosynthetic pathway in the wild-type, has revealed a strong metabolic canalization of putrescine to spermine induced by drought. Such canalization requires spermidine synthase 1 (SPDS1) and spermine synthase (SPMS) activities and, intriguingly, does not lead to spermine accumulation but to a progressive reduction in spermidine and spermine pools in the wild-type. Our results suggest the participation of the polyamine back-conversion pathway during the drought stress response rather than the terminal catabolism of spermine. The putrescine to spermine canalization coupled to the spermine to putrescine back-conversion confers an effective polyamine recycling-loop during drought acclimation. Putrescine to spermine canalization has also been revealed in the desiccation tolerant plant C. plantagineum, which conversely to Arabidopsis, accumulates high spermine levels which associate with drought tolerance. Our results provide a new insight to the polyamine homeostasis mechanisms during drought stress acclimation in Arabidopsis and resurrection plants.
doi:10.4161/psb.6.2.14317
PMCID: PMC3121985  PMID: 21330782
Arabidopsis; Craterostigma plantagineum; drought; polyamines; polyamine oxidase; abiotic stress
12.  Characterization of transgenic mice with overexpression of spermidine synthase 
Amino Acids  2011;42(2-3):495-505.
A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) in order to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to 9-fold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5 to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.
doi:10.1007/s00726-011-1028-6
PMCID: PMC3245749  PMID: 21809077
polyamine; aminopropyltransferase; transgenic mice; S-adenosylmethionine decarboxylase; spermidine; spermine
13.  The Relationship between Polyamines and Hormones in the Regulation of Wheat Grain Filling 
PLoS ONE  2013;8(10):e78196.
The grain weight of wheat is strongly influenced by filling. Polyamines (PA) are involved in regulating plant growth. However, the effects of PA on wheat grain filling and its mechanism of action are unclear. The objective of the present study was to investigate the relationship between PAs and hormones in the regulation of wheat grain filling. Three PAs, spermidine (Spd), spermine (Spm), and putrescine (Put), were exogenously applied, and the grain filling characteristics and changes in endogenous PA and hormones, i.e., indole-3-acetic acid (IAA), zeatin (Z) + zeatin riboside (ZR), abscisic acid (ABA), ethylene (ETH) and gibberellin 1+4 (GAs), were quantified during wheat grain filling. Exogenous applications of Spd and Spm significantly increased the grain filling rate and weight, but exogenous Put had no significant effects on these measures. Exogenous Spd and Spm significantly increased the endogenous Spd, Spm, Z+ZR, ABA, and IAA contents and significantly decreased ETH evolution in grains. The endogenous Spd, Spm and Z+ZR contents were positively and significantly correlated with the grain filling rate and weight of wheat, and the endogenous ETH evolution was negatively and significantly correlated with the wheat grain filling rate and weight. Based upon these results, we concluded that PAs were involved in the balance of hormones that regulated the grain filling of wheat.
doi:10.1371/journal.pone.0078196
PMCID: PMC3812141  PMID: 24205154
14.  Insecticide Resistance Mechanisms in the Green Peach Aphid Myzus persicae (Hemiptera: Aphididae) II: Costs and Benefits 
PLoS ONE  2012;7(6):e36810.
Background
Among herbivorous insects that have exploited agro-ecosystems, the peach-potato aphid, Myzus persicae, is recognized as one of the most important agricultural pests worldwide. Uses over 400 plant species and has evolved different insecticides resistance mechanisms. As M. persicae feeds upon a huge diversity of hosts, it has been exposed to a wide variety of plant allelochemicals, which probably have promoted a wide range of detoxification systems.
Methodology/Principal Findings
In this work we (i) evaluated whether insecticide resistance mutations (IRM) in M. persicae can give an advantage in terms of reproductive fitness when aphids face two hosts, pepper (Capsicum annuum) a suitable host and radish (Raphanus sativus) the unfavorable host and (ii) examined the transcriptional expression of six genes that are known to be up-regulated in response to insecticides. Our results show a significant interaction between host and IRM on the intrinsic rate of increase (rm). Susceptible genotypes (not carrying insensitivity mutations) had a higher rm on pepper, and the transcriptional levels of five genes increased on radish. The rm relationship was reversed on the unfavorable host; genotypes with multiple IRM exhibited higher rm, without altering the transcriptional levels of the studied genes. Genotypes with one IRM kept a similar rm on both hosts, but they increased the transcriptional levels of two genes.
Conclusions/Significance
Although we have studied only nine genotypes, overall our results are in agreement with the general idea that allelochemical detoxification systems could constitute a pre-adaptation for the development of insecticide resistance. Genotypes carrying IRM exhibited a higher rm than susceptible genotypes on radish, the more unfavorable host. Susceptible genotypes should be able to tolerate the defended host by up-regulating some metabolic genes that are also responding to insecticides. Hence, our results suggest that the trade-off among resistance mechanisms might be quite complex, with a multiplicity of costs and benefits depending on the environment.
doi:10.1371/journal.pone.0036810
PMCID: PMC3369902  PMID: 22685539
15.  Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots 
Journal of Experimental Botany  2013;64(14):4271-4287.
MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.
doi:10.1093/jxb/ert240
PMCID: PMC3808317  PMID: 24014874
Cadmium stress; degradome; high-throughput sequencing; microRNAs; Raphanus sativus; transcriptome.
16.  Physiological and biochemical effect of 24-epibrassinoslide on cold tolerance in maize seedlings 
Germination and early seedling growth are important for establishment of maize because maize is chilling sensitive crop and low temperature during early period of growth can be detrimental to subsequent crop growth and productivity. Therefore, it is important to protect maize seedling from cold stress. A study was conducted on induced cold tolerance by 24-epibrassinoslide (EBR) at the Indian Agricultural Research Institute, New Delhi, India. Maize seedlings were raised in green house condition (25/18 °C day-night temperatures). Ten days old seedlings were treated with EBR (0.0, 0.01, 0.1, 1.0 and 10 μM) and then divided into two sets, one set was kept in greenhouse (25/18 °C day-night temperatures) and another was transferred to net house (cold stress). Data on various morpho-physiological traits was recorded after 7, 14 and 21 days of treatment. Exogenous application of 1.0 μM EBR had significant effect on growth and morpho-physiological traits under both conditions. The maize seedlings treated with EBR were more tolerant to cold stress than the untreated one. Significant increase in plant height, dry matter accumulation, chlorophyll content, total soluble proteins and starch contents was observed under both conditions, however, the results were more pronounced under cold stress. 1.0 μ M concentration being the most effective under both conditions. Maintenance of high tissue water content, reduced membrane injury index, increased total chlorophyll, soluble sugar and protein content were taken as the possible indicators of EBR induced chilling tolerance.
doi:10.1007/s12298-012-0122-x
PMCID: PMC3550514  PMID: 23814437
24-epibrassinoslide; Cold tolerance; Maize; Glycine betaine; Total chlorophyll; Membrane injury index
17.  BZR1 and BES1 participate in regulation of glucosinolate biosynthesis by brassinosteroids in Arabidopsis  
Journal of Experimental Botany  2013;64(8):2401-2412.
The effect of 24-epibrassinolide (EBR) on glucosinolate biosynthesis in Arabidopsis thaliana was investigated in the present study by using mutants and transgenic plants involved in brassinosteroid (BR) biosynthesis and signal transduction, as well as glucosinolate biosynthesis. The results showed that EBR significantly decreased the contents of major aliphatic glucosinolates including glucoiberin (S3), glucoraphanin (S4), and glucoerucin (T4), as well as the indolic glucosinolates glucobrassicin (IM) and neoglucobrassicin (1IM). In addition, a significantly higher level of glucosinolates accumulated in the BR-deficient mutant cpd and a dramatically lower glucosinolate content in the transgenic plant DWF4-ox overexpressing the BR biosynthetic gene DWF4 compared with their related wild-types, confirmed the repressing effect of BR on glucosinolate biosynthesis. BRI1, the receptor of BR signal transduction, was involved in regulation of glucosinolate biosynthesis by BR. Furthermore, the observation of reduced content of glucosinolates and lower expression levels of glucosinolate biosynthetic genes in 35S-BZR1/bzr1-1D and bes1-D plants compared with the corresponding wild-types suggested that BZR1 and BES1, two important components in BR signal transduction, are responsible for the inhibiting role of BR in glucosinolate biosynthesis. The disappearance of the repressing effect of BR on glucosinolate content in the myb28, myb34, and myb122 mutants indicated that these three MYB factors are important for the regulation of BR in glucosinolate biosynthesis.
doi:10.1093/jxb/ert094
PMCID: PMC3654425  PMID: 23580754
Arabidopsis thaliana; brassinosteroids; BZR1; BES1; glucosinolates; MYB.
18.  Does polyamine catabolism influence root development and xylem differentiation under stress conditions? 
Plant Signaling & Behavior  2011;6(11):1844-1847.
Amine oxidases (AOs) oxidize polyamines (PAs) to aldehydes, simultaneously producing the removed amine moiety and hydrogen peroxide (H2O2). AOs, which include copper-containing amine oxidases (CuAOs) and flavin-containing amine oxidases (PAOs), are stress-inducible enzymes involved in both PA homeostasis and H2O2 production. Here, we suggest that H2O2 derived from PAO-mediated PA catabolism has a role in inducing root xylem differentiation during plant stress responses, whereas its involvement in this event during plant development under physiological conditions is not suitably supported by the currently available data. Moreover, we show that spermidine (Spd) supply leads to a higher induction of cell death in wild-type (WT) tobacco (Nicotiana tabacum) plants as compared to tobacco plants over-expressing maize (Zea mays) PAO (S-ZmPAO) in the cell wall, in apparent contradiction with the already reported results obtained by the analysis of the corresponding WT and S-ZmPAO Spd-untreated plants. Considering this last observation, we propose that PAs  diversely affect plant development and stress responses depending on the expression levels of AOs, which in turn may lead to different plant responses by altering the PAs/H2O2 balance.
doi:10.4161/psb.6.11.17640
PMCID: PMC3329365  PMID: 22057326
copper amine oxidase; hydrogen peroxide; PCD; polyamine; polyamine oxidase; xylem
19.  The Photoprotective Role of Spermidine in Tomato Seedlings under Salinity-Alkalinity Stress 
PLoS ONE  2014;9(10):e110855.
Polyamines are small, ubiquitous, nitrogenous compounds that scavenge reactive oxygen species and stabilize the structure and function of the photosynthetic apparatus in response to abiotic stresses. Molecular details underlying polyamine-mediated photoprotective mechanisms are not completely resolved. This study investigated the role of spermidine (Spd) in the structure and function of the photosynthetic apparatus. Tomato seedlings were subjected to salinity-alkalinity stress with and without foliar application of Spd, and photosynthetic and morphological parameters were analyzed. Leaf dry weight and net photosynthetic rate were reduced by salinity-alkalinity stress. Salinity-alkalinity stress reduced photochemical quenching parameters, including maximum photochemistry efficiency of photosystem II, quantum yield of linear electron flux, and coefficient of photochemical quenching (qP). Salinity-alkalinity stress elevated nonphotochemical quenching parameters, including the de-epoxidation state of the xanthophyll cycle and nonphotochemical quenching (NPQ). Microscopic analysis revealed that salinity-alkalinity stress disrupted the internal lamellar system of granal and stromal thylakoids. Exogenous Spd alleviated the stress-induced reduction of leaf dry weight, net photosynthetic rate, and qP parameters. The NPQ parameters increased by salinity-alkalinity stress were also alleviated by Spd. Seedlings treated with exogenous Spd had higher zeaxanthin (Z) contents than those without Spd under salinity-alkalinity stress. The chloroplast ultrastructure had a more ordered arrangement in seedlings treated with exogenous Spd than in those without Spd under salinity-alkalinity stress. These results indicate that exogenous Spd can alleviate the growth inhibition and thylakoid membrane photodamage caused by salinity-alkalinity stress. The Spd-induced accumulation of Z also may have an important role in stabilizing the photosynthetic apparatus.
doi:10.1371/journal.pone.0110855
PMCID: PMC4207769  PMID: 25340351
20.  Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways 
Cell Death & Disease  2012;3(10):e401-.
The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.
doi:10.1038/cddis.2012.139
PMCID: PMC3481127  PMID: 23059820
Drosophila melanogaster; spermidine; paraquat; oxidative stress; activity; starvation
21.  The Enterococcus faecium Enterococcal Biofilm Regulator, EbrB, Regulates the esp Operon and Is Implicated in Biofilm Formation and Intestinal Colonization 
PLoS ONE  2013;8(5):e65224.
Nowadays, Enterococcus faecium is one of the leading nosocomial pathogens worldwide. Strains causing clinical infections or hospital outbreaks are enriched in the enterococcal surface protein (Esp) encoding ICEEfm1 mobile genetic element. Previous studies showed that Esp is involved in biofilm formation, endocarditis and urinary tract infections. In this study, we characterized the role of the putative AraC type of regulator (locus tag EfmE1162_2351), which we renamed ebrB and which is, based on the currently available whole genome sequences, always located upstream of the esp gene, and studied its role in Esp surface exposure during growth. A markerless deletion mutant of ebrB resulted in reduced esp expression and complete abolishment of Esp surface exposure, while Esp cell-surface exposure was restored when this mutant was complemented with an intact copy of ebrB. This demonstrates a role for EbrB in esp expression. However, during growth, ebrB expression levels did not change over time, while an increase in esp expression at both RNA and protein level was observed during mid-log and late-log phase. These results indicate the existence of a secondary regulation system for esp, which might be an unknown quorum sensing system as the enhanced esp expression seems to be cell density dependent. Furthermore, we determined that esp is part of an operon of at least 3 genes putatively involved in biofilm formation. A semi-static biofilm model revealed reduced biofilm formation for the EbrB deficient mutant, while dynamics of biofilm formation using a flow cell system revealed delayed biofilm formation in the ebrB mutant. In a mouse intestinal colonization model the ebrB mutant was less able to colonize the gut compared to wild-type strain, especially in the small intestine. These data indicate that EbrB positively regulates the esp operon and is implicated in biofilm formation and intestinal colonization.
doi:10.1371/journal.pone.0065224
PMCID: PMC3669262  PMID: 23741484
22.  Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression 
Annals of Botany  2010;106(5):791-802.
Background and Aims
It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant–fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil.
Methods
In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal- (Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription–PCR. Free and conjugated foliar PA concentrations were determined in parallel.
Results
On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil.
Conclusions
Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs.
doi:10.1093/aob/mcq170
PMCID: PMC2958786  PMID: 20810743
Arbuscular mycorrhizal fungi; contaminated soil; heavy metals; metallothioneins; polyamines; Populus alba; white poplar
23.  Spermidine affects the transcriptome responses to high temperature stress in ripening tomato fruit* #  
Objective: High temperature adversely affects quality and yield of tomato fruit. Polyamine can alleviate heat injury in plants. This study is aimed to investigate the effects of polyamine and high temperature on transcriptional profiles in ripening tomato fruit. Methods: An Affymetrix tomato microarray was used to evaluate changes in gene expression in response to exogenous spermidine (Spd, 1 mmol/L) and high temperature (33/27 °C) treatments in tomato fruits at mature green stage. Results: Of the 10 101 tomato probe sets represented on the array, 127 loci were differentially expressed in high temperature-treated fruits, compared with those under normal conditions, functionally characterized by their involvement in signal transduction, defense responses, oxidation reduction, and hormone responses. However, only 34 genes were up-regulated in Spd-treated fruits as compared with non-treated fruits, which were involved in primary metabolism, signal transduction, hormone responses, transcription factors, and stress responses. Meanwhile, 55 genes involved in energy metabolism, cell wall metabolism, and photosynthesis were down-regulated in Spd-treated fruits. Conclusions: Our results demonstrated that Spd might play an important role in regulation of tomato fruit response to high temperature during ripening stage.
doi:10.1631/jzus.B1100060
PMCID: PMC3323944  PMID: 22467370
Solanum lycopersicum L.; Spermidine; High temperature; Microarray; Gene expression
24.  Comprehensive analysis of expressed sequence tags from cultivated and wild radish (Raphanus spp.) 
BMC Genomics  2013;14:721.
Background
Radish (Raphanus sativus L., 2n = 2× = 18) is an economically important vegetable crop worldwide. A large collection of radish expressed sequence tags (ESTs) has been generated but remains largely uncharacterized.
Results
In this study, approximately 315,000 ESTs derived from 22 Raphanus cDNA libraries from 18 different genotypes were analyzed, for the purpose of gene and marker discovery and to evaluate large-scale genome duplication and phylogenetic relationships among Raphanus spp. The ESTs were assembled into 85,083 unigenes, of which 90%, 65%, 89% and 89% had homologous sequences in the GenBank nr, SwissProt, TrEMBL and Arabidopsis protein databases, respectively. A total of 66,194 (78%) could be assigned at least one gene ontology (GO) term. Comparative analysis identified 5,595 gene families unique to radish that were significantly enriched with genes related to small molecule metabolism, as well as 12,899 specific to the Brassicaceae that were enriched with genes related to seed oil body biogenesis and responses to phytohormones. The analysis further indicated that the divergence of radish and Brassica rapa occurred approximately 8.9-14.9 million years ago (MYA), following a whole-genome duplication event (12.8-21.4 MYA) in their common ancestor. An additional whole-genome duplication event in radish occurred at 5.1-8.4 MYA, after its divergence from B. rapa. A total of 13,570 simple sequence repeats (SSRs) and 28,758 high-quality single nucleotide polymorphisms (SNPs) were also identified. Using a subset of SNPs, the phylogenetic relationships of eight different accessions of Raphanus was inferred.
Conclusion
Comprehensive analysis of radish ESTs provided new insights into radish genome evolution and the phylogenetic relationships of different radish accessions. Moreover, the radish EST sequences and the associated SSR and SNP markers described in this study represent a valuable resource for radish functional genomics studies and breeding.
doi:10.1186/1471-2164-14-721
PMCID: PMC3816612  PMID: 24144082
Radish; EST; SNP; SSR; Comparative analysis; Whole genome duplication; Phylogenetic relationship
25.  CGI-58, a key regulator of lipid homeostasis and signaling in plants, also regulates polyamine metabolism 
Plant Signaling & Behavior  2014;9:e27723.
Comparative Gene Identification-58 (CGI-58) is an α/β hydrolase-type protein that regulates lipid homeostasis and signaling in eukaryotes by interacting with and stimulating the activity of several different types of proteins, including a lipase in mammalian cells and a peroxisomal ABC transporter (PXA1) in plant cells. Here we show that plant CGI-58 also interacts with spermidine synthase 1 (SPDS1), an enzyme that plays a central role in polyamine metabolism by converting putrescine into spermidine. Analysis of polyamine contents in Arabidopsis thaliana plants revealed that spermidine levels were significantly reduced, and putrescine increased, in both cgi-58 and cgi-58/pxa1 mutant plants, relative to pxa1 mutant or wild-type plants. Evaluation of polyamine-related gene expression levels, however, revealed similar increases in transcript abundance in all mutants, including cgi-58, pxa1, and cgi-58/pxa1, in comparison to wild type. Taken together, the data support a model whereby CGI-58 and PXA1 contribute to the regulation of polyamine metabolism at the transcriptional level, perhaps through a shared lipid-signaling pathway, and that CGI-58 also acts independently of PXA1 to increase spermidine content at a post-transcriptional level, possibly through protein-protein interaction with SPDS1.
doi:10.4161/psb.27723
PMCID: PMC4091556  PMID: 24492485
Arabidopsis; CGI-58; Polyamines; PXA1; SPDS1; Spermidine; Spermidine Synthase

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