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Plants, Volume 5, Issue 1 (March 2016) – 15 articles

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1228 KiB  
Review
Functional Analysis of Jasmonates in Rice through Mutant Approaches
by Rohit Dhakarey, Preshobha Kodackattumannil Peethambaran and Michael Riemann
Plants 2016, 5(1), 15; https://doi.org/10.3390/plants5010015 - 18 Mar 2016
Cited by 22 | Viewed by 9895
Abstract
Jasmonic acid, one of the major plant hormones, is, unlike other hormones, a lipid-derived compound that is synthesized from the fatty acid linolenic acid. It has been studied intensively in many plant species including Arabidopsis thaliana, in which most of the enzymes [...] Read more.
Jasmonic acid, one of the major plant hormones, is, unlike other hormones, a lipid-derived compound that is synthesized from the fatty acid linolenic acid. It has been studied intensively in many plant species including Arabidopsis thaliana, in which most of the enzymes participating in its biosynthesis were characterized. In the past 15 years, mutants and transgenic plants affected in the jasmonate pathway became available in rice and facilitate studies on the functions of this hormone in an important crop. Those functions are partially conserved compared to other plant species, and include roles in fertility, response to mechanical wounding and defense against herbivores. However, new and surprising functions have also been uncovered by mutant approaches, such as a close link between light perception and the jasmonate pathway. This was not only useful to show a phenomenon that is unique to rice but also helped to establish this role in plant species where such links are less obvious. This review aims to provide an overview of currently available rice mutants and transgenic plants in the jasmonate pathway and highlights some selected roles of jasmonate in this species, such as photomorphogenesis, and abiotic and biotic stress. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Graphical abstract

Graphical abstract
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<p>Biosynthesis of JA, major enzymes involved and mutants in the pathway. The biosynthesis occurs in chloroplasts (green) and peroxisomes (brown). In brief, after cleavage of linolenic acid from a membrane lipid it is converted to OPDA in three enzymatic steps. OPDA is a functional signaling compound but can be transported to peroxisomes specifically where it is further metabolized to JA by the action of OPR and subsequent β-oxidation steps. For further explanation, refer to the text. Mutant names are shown in light blue boxes. <span class="html-italic">Extra glume 1</span> (<span class="html-italic">eg1</span>) is a mutant of a plastidic lipase involved in flower specific JA synthesis. <span class="html-italic">OsHI-LOX</span> antisense plants were found to be impaired in JA-dependent insect responses. A mutant of <span class="html-italic">OsAOS1</span>, <span class="html-italic">coleoptile photomorphogenesis 1</span> (<span class="html-italic">cpm1</span>), was isolated as photomorphogenic rice mutant like <span class="html-italic">hebiba</span> and <span class="html-italic">cpm2</span>, which are mutated in <span class="html-italic">OsAOC</span>. OsOPR7 carries a point-mutation in <span class="html-italic">unclosed glumes</span> (<span class="html-italic">ucgl</span>). Abbreviations: 13-LOX: 13-lipoxygenase, 13-HPOT: (13S)-hydroperoxyoctadecatrienoic, 13-AOS: 13-allene oxide synthase, AOC: allene oxide cyclase, 12-OPDA: 12-oxo-phytodienoic acid, OPR: OPDA reductase, OPC-8:0: 3-oxo-2(2’(Z)-pentenyl)-cyclopentane-1-octanoic acid.</p>
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<p>Activation, inactivation, perception and signaling of JA. Names of mutants and transgenic lines are shown in light blue boxes. JAR1 catalyzes the conjugation of JA to isoleucine (JA-Ile). Several alleles of rice <span class="html-italic">jar1</span> mutants are available because it is a hotspot of Tos17 retrotransposon insertion. Further GH3 enzymes may contribute to the biosynthesis of JA-Ile in rice. JA-Ile is recognized by its receptor COI1, which functions as E3 ubiquitin ligase in a SCF complex. Subsequently, JAZ proteins are recognized by the hormone receptor complex, poly-ubiquitinated and degraded in the 26S proteasome. <span class="html-italic">Extra glume 2 (eg2)</span> is a mutant of <span class="html-italic">OsJAZ1</span>. MYC transcription factors are released from repression by JAZ proteins and can activate transcription of early response genes. JA-Ile can be inactivated by CYP94 enzymes or amidohydrolases. In transgenic approaches, signaling has been affected by overexpressing CYP84C2b. Abbreviations: JA-Ile: jasmonoyl-isoleucine, JAR1: JASMONATE RESISTANT 1, COI1: CORONATINE INSENSITIVE 1, JAZ: JASMONATE ZIM-domain, CYP94: CYTOCHROME P450 CYP94 subfamily, AH: AMIDOHYDROLASES.</p>
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<p>Function of JA in incompatible interaction of rice and <span class="html-italic">Magnaporthe oryzae</span>. Leaf sheath segments of rice plants were inoculated with an incompatible strain of <span class="html-italic">Magnaporthe oryzae</span>. The wild type (WT) produces JA and JA-Ile, probably in response to appressorium penetration. Dependent on the production of JA-Ile, the plant produces phytoalexins such as sakuranetin, momilactones and phytocassanes. The mutant <span class="html-italic">cpm2</span> and <span class="html-italic">hebiba</span>, which are impaired in JA biosynthesis, do not accumulate sakuranetin and less momilactones. Correlating with lower JA-Ile and sakuranetin levels, fungal hyphae spread more easily in the mutants.</p>
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661 KiB  
Review
Beyond the Canon: Within-Plant and Population-Level Heterogeneity in Jasmonate Signaling Engaged by Plant-Insect Interactions
by Dapeng Li, Ian T. Baldwin and Emmanuel Gaquerel
Plants 2016, 5(1), 14; https://doi.org/10.3390/plants5010014 - 16 Mar 2016
Cited by 10 | Viewed by 7165
Abstract
Plants have evolved sophisticated communication and defense systems with which they interact with insects. Jasmonates are synthesized from the oxylipin pathway and act as pivotal cellular orchestrators of many of the metabolic and physiological processes that mediate these interactions. Many of these jasmonate-dependent [...] Read more.
Plants have evolved sophisticated communication and defense systems with which they interact with insects. Jasmonates are synthesized from the oxylipin pathway and act as pivotal cellular orchestrators of many of the metabolic and physiological processes that mediate these interactions. Many of these jasmonate-dependent responses are tissue-specific and translate from modulations of the canonical jasmonate signaling pathway. Here we provide a short overview of within-plant heterogeneities in jasmonate signaling and dependent responses in the context of plant-insect interactions as illuminated by examples from recent work with the ecological model, Nicotiana attenuata. We then discuss means of manipulating jasmonate signaling by creating tissue-specific jasmonate sinks, and the micrografting of different transgenic plants. The metabolic phenotyping of these manipulations provides an integrative understanding of the functional significance of deviations from the canonical model of this hormonal pathway. Additionally, natural variation in jasmonate biosynthesis and signaling both among and within species can explain polymorphisms in resistance to insects in nature. In this respect, insect-guided explorations of population-level variations in jasmonate metabolism have revealed more complexity than previously realized and we discuss how different “omic” techniques can be used to exploit the natural variation that occurs in this important signaling pathway. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Figure 1
<p>Schematic model of modulations in jasmonate perception, signaling and corresponding tissue-specific herbivory-elicited responses. New discoveries in jasmonate signaling in the ecological model, <span class="html-italic">Nicotiana attenuata</span> are presented. The small circle at the top represents jasmonate biosynthesis after herbivore attack; the inner circle represents jasmonate perception and signaling, the outer larger circle represents outputs of tissue-specific responses. The six different types of modulations from the canonical model of jasmonate signaling are presented in different colors. White, jasmonate biosynthesis deviates in different tissue types of leaves; orange, other jasmonate derivatives that are likely to serve as ligands need to be discovered; purple, other F-box proteins may serve as functional groups; green, tissue-specific <span class="html-italic">JAZ</span> expressions need to be fully investigated; pink, tissue-specific transcription factors are yet to be discovered; blue, deviations of hormonal crosstalk in different tissues. Red texts indicate areas where modulations are likely to take place. <span class="html-italic">TPL, TOPLESS.</span></p>
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<p>Population-level quantitative variations in herbivore-elicited metabolites only partly overlaps with jasmonate accumulation polymorphisms. The figure is modified from [<a href="#B40-plants-05-00014" class="html-bibr">40</a>]. Density distribution plots of JA and JA-Ile (x axis, area of intensities and y axis, fitted density with histogram) (123 samples) illustrate the patterns of natural variation in JA and JA-Ile levels as analyzed by targeted LC-MS/MS/MS workflows for leaf samples collected 1 h after simulated herbivory from glasshouse-grown accessions of <span class="html-italic">N. attenuata</span>. Heatmaps of pairwise Pearson correlation coefficients (PCCs) (only PCCs with either JA or JA-Ile &gt;0.3 are shown) illustrate significant co-regulation patterns between metabolite relative levels and JA and JA-Ile levels. Examples of known and unknown metabolites are depicted in density plots and scatter plots (colored with different color boxes accordingly). The herbivory-inducible defense compound, Nicotianoside IV, correlates significantly with JA whereas N-caffeoylputrescine shows significant correlation with JA-Ile. Unknown <span class="html-italic">m/z</span> 848.68 and 245.07 exhibit poor correlations with JA or JA-Ile. Discovering the identity of these and other unknown compounds exhibiting significant correlation scores with JA and JA-Ile levels will be the topic of future research to uncover novel defensive metabolites in <span class="html-italic">N. attenuata</span>.</p>
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2177 KiB  
Article
Effect of Drought on Herbivore-Induced Plant Gene Expression: Population Comparison for Range Limit Inferences
by Gunbharpur Singh Gill, Riston Haugen, Steven L. Matzner, Abdelali Barakat and David H. Siemens
Plants 2016, 5(1), 13; https://doi.org/10.3390/plants5010013 - 11 Mar 2016
Cited by 4 | Viewed by 5767
Abstract
Low elevation “trailing edge” range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, [...] Read more.
Low elevation “trailing edge” range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, but that antagonistic crosstalk prevents their simultaneous co-option. To test this hypothesis, we compared high and low elevation populations of Boechera stricta that have diverged with respect to constitutive levels of glucosinolate defenses and root:shoot ratios; neither population has high levels of both traits. If constraints imposed by antagonistic signaling underlie this divergence, one would predict that high constitutive levels of traits would coincide with lower plasticity. To test this prediction, we compared the genetically diverged populations in a double challenge drought-herbivory growth chamber experiment. Although a glucosinolate defense response to the generalist insect herbivore Spodoptera exigua was attenuated under drought conditions, the plastic defense response did not differ significantly between populations. Similarly, although several potential drought tolerance traits were measured, only stomatal aperture behavior, as measured by carbon isotope ratios, was less plastic as predicted in the high elevation population. However, RNAseq results on a small subset of plants indicated differential expression of relevant genes between populations as predicted. We suggest that the ambiguity in our results stems from a weaker link between the pathways and the functional traits compared to transcripts. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Figure 1
<p>Flat weights (<b>a</b>) just after and (<b>b</b>) before or between watering for the control and drought treatments. All flats in each treatment were watered to the same weight, so there are no error bars for the after watering flat weights. For the before watering flat weights, error bars are ± 1SE across four flats for each watering treatment.</p>
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<p>Genetic divergence between high and low elevation populations in basal dry mass root:shoot ratio and total glucosinolate concentration. Values are least squares means. Statistical analyses in <a href="#plants-05-00013-t003" class="html-table">Table 3</a> and <a href="#plants-05-00013-t004" class="html-table">Table 4</a>. Error bars are ±1SE, total sample size <span class="html-italic">n</span> = 175.</p>
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<p>Effects of herbivory and drought on the carbon isotope ratio. See <a href="#plants-05-00013-t005" class="html-table">Table 5</a>B for statistical analysis. The effect of herbivory was only significant in the drought treated plants (LSD multiple comparisons <span class="html-italic">post hoc</span> test: <span class="html-italic">p</span>’s &lt; 0.05). Error bars are ±1 SE, total sample size <span class="html-italic">n</span> = 116.</p>
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<p>Effect of drought on the herbivore-induced responses of (<b>A</b>) ratio of branch chain (BCGS) to Methionine-derived straight-chain glucosinolates (METGS), (<b>B</b>) METGS, and (<b>C</b>) BCGS. Statistical analysis in <a href="#plants-05-00013-t006" class="html-table">Table 6</a>. Error bars are ±1SE, total sample size <span class="html-italic">n</span> = 175.</p>
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<p>Venn diagrams for number of significantly up- and down-regulated genes (direction of blue arrows indicate up- or down-regulation). Red circle is Drought <span class="html-italic">vs.</span> control (no stress), Yellow circle = Herbivory <span class="html-italic">vs.</span> control (no stress), Green circle is double challenge Drought + Herbivory <span class="html-italic">vs.</span> control (no stress). Number of biological replicates, <span class="html-italic">n</span>, in comparisons: red circle, drought <span class="html-italic">vs.</span> control (<span class="html-italic">n</span> = 8); yellow circle, herbivory <span class="html-italic">vs.</span> control (<span class="html-italic">n</span> = 6), green circle, double challenge drought + herbivory <span class="html-italic">vs.</span> control (<span class="html-italic">n</span> = 8).</p>
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<p>Number of significantly up- and down-regulated genes by defense and drought-tolerance functional categories. Herbivory (Drought <span class="html-italic">vs.</span> Control) means that both drought and control-watered treatments were fed upon by herbivores. Similarly, Drought (Herbivory <span class="html-italic">vs.</span> Control) means that plants in the presence and absence of herbivores were under drought stress. Number of biological replicates for the comparisons ranged between 6 and 10.</p>
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<p>Number of differentially expressed genes of relevant biological processes between populations. The number of biological replicates for the Black Hills population comparisons was <span class="html-italic">n</span> = 4 and for the Big Horn population, <span class="html-italic">n</span> = 5.</p>
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1655 KiB  
Article
Detection of Leptosphaeria maculans and Leptosphaeria biglobosa Causing Blackleg Disease in Canola from Canadian Canola Seed Lots and Dockage
by W. G. Dilantha Fernando, Xuehua Zhang and Chami C. Amarasinghe
Plants 2016, 5(1), 12; https://doi.org/10.3390/plants5010012 - 1 Mar 2016
Cited by 24 | Viewed by 7503
Abstract
Blackleg, caused by Leptosphaeria maculans, is a major threat to canola production in Canada. With the exception of China, L. maculans is present in areas around the world where cruciferous crops are grown. The pathogen can cause trade barriers in international [...] Read more.
Blackleg, caused by Leptosphaeria maculans, is a major threat to canola production in Canada. With the exception of China, L. maculans is present in areas around the world where cruciferous crops are grown. The pathogen can cause trade barriers in international canola seed export due to its potential risk as a seed contaminant. The most recent example is China restricting canola seeds imported from Canada and Australia in 2009. Therefore, it is important to assess the level of Blackleg infection in Canadian canola seed lots and dockage (seeds and admixture). In this study, canola seed lots and dockage samples collected from Western Canada were tested for the presence of the aggressive L. maculans and the less aggressive L. biglobosa. Results showed that both L. maculans and L. biglobosa were present in seed lots and dockage samples, with L. biglobosa being predominant in infected seeds. Admixture separated from dockage had higher levels of L. maculans and L. biglobosa infection than samples from seed lots. Admixture appears to harbour higher levels of L. maculans infection compared to seeds and is more likely to be a major source of inoculum for the spread of the disease than infected seeds. Full article
(This article belongs to the Special Issue Selected/Extended Full Papers of 14th International Rapeseed Congress)
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<p>Canola seeds infected with <span class="html-italic">Leptosphaeria</span> species. Dark dots on and around seeds represents pycnidia.</p>
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<p>Blackleg isolates identified using species-specific polymerase chain reaction (PCR) assay. Lanes: M-100bp DNA ladder; 1, 2, 4, 5, 7, 9––<span class="html-italic">Leptosphaeria biglobosa</span> “brassicae” (444 bp); 3, 6, 8, 10, 11, 12––<span class="html-italic">Leptosphaeria maculans</span> “brassicae” (331 bp).</p>
Full article ">Figure 3
<p>Percentage of <span class="html-italic">Leptosphaeria maculans</span> infected seeds in canola samples with different level of Blackleg resistance. Level of resistance: S––susceptible; MS––moderately susceptible; R––resistant; MR––moderately resistant. Westar: S; Q2: MS; 72-65RR: R; Defender: MR; InVigor 5440: R.</p>
Full article ">Figure 4
<p>Reactions of Westar seedlings to inoculum produced by admixture. Arrows indicate (<b>a</b>) lesions starting to form seven days after wounding, and (<b>b</b>) advanced tissue collapse accompanied by pycnidia formation 14 days after wounding.</p>
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509 KiB  
Communication
Jasmonate Signalling and Defence Responses in the Model Legume Medicago truncatula—A Focus on Responses to Fusarium Wilt Disease
by Louise F. Thatcher, Ling-Ling Gao and Karam B. Singh
Plants 2016, 5(1), 11; https://doi.org/10.3390/plants5010011 - 5 Feb 2016
Cited by 13 | Viewed by 6909
Abstract
Jasmonate (JA)-mediated defences play important roles in host responses to pathogen attack, in particular to necrotrophic fungal pathogens that kill host cells in order to extract nutrients and live off the dead plant tissue. The root-infecting fungal pathogen Fusarium oxysporum initiates a necrotrophic [...] Read more.
Jasmonate (JA)-mediated defences play important roles in host responses to pathogen attack, in particular to necrotrophic fungal pathogens that kill host cells in order to extract nutrients and live off the dead plant tissue. The root-infecting fungal pathogen Fusarium oxysporum initiates a necrotrophic growth phase towards the later stages of its lifecycle and is responsible for devastating Fusarium wilt disease on numerous legume crops worldwide. Here we describe the use of the model legume Medicago truncatula to study legume–F. oxysporum interactions and compare and contrast this against knowledge from other model pathosystems, in particular Arabidopsis thaliana–F. oxysporum interactions. We describe publically-available genomic, transcriptomic and genetic (mutant) resources developed in M. truncatula that enable dissection of host jasmonate responses and apply aspects of these herein during the M. truncatula-–F. oxysporum interaction. Our initial results suggest not all components of JA-responses observed in M. truncatula are shared with Arabidopsis in response to F. oxysporum infection. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Figure 1
<p>JA-inducible gene expression following <span class="html-italic">F. oxysporum</span> infection. <span class="html-italic">M. truncatula</span> A17 seedlings were inoculated with <span class="html-italic">F. oxysporum</span> (<span class="html-italic">Fom</span>-5190a) and root and shoot tissues harvested separately at 1, 2, 4 and 7 days post inoculation (dpi). Gene expression values were determined relative to the internal control <span class="html-italic">Beta-tubulin</span> gene for each mock or <span class="html-italic">Fusarium</span> treated sample. Values shown are fold-inductions in <span class="html-italic">Fusarium</span> treated samples relative to mock treated samples at the same time-point from the average of eight pooled plants.</p>
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<p>Susceptibility of JA-related <span class="html-italic">Tnt1</span>-insertion mutants to <span class="html-italic">F. oxysporum</span>. Seedlings were inoculated with <span class="html-italic">F. oxysporum</span> (<span class="html-italic">Fom</span>-5190a) and survival rates monitored over 35-days. Values are averages ± SE (n = 10). The <span class="html-italic">Tnt1</span>-insertion mutants are in the R108 background and their details noted in <a href="#plants-05-00011-t002" class="html-table">Table 2</a>. A17 is included as a resistant control. Asterisks indicate values that are significantly different (** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05, Student’s t-test) from R108. Similar results were obtained in an independent experiment.</p>
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297 KiB  
Editorial
Acknowledgement to Reviewers of Plants in 2015
by Plants Editorial Office
Plants 2016, 5(1), 10; https://doi.org/10.3390/plants5010010 - 21 Jan 2016
Viewed by 3360
Abstract
The editors of Plants would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2015. [...] Full article
572 KiB  
Review
How Microbes Twist Jasmonate Signaling around Their Little Fingers
by Selena Gimenez-Ibanez, Andrea Chini and Roberto Solano
Plants 2016, 5(1), 9; https://doi.org/10.3390/plants5010009 - 19 Jan 2016
Cited by 61 | Viewed by 9668
Abstract
Plant immunity relies on a complex network of hormone signaling pathways in which jasmonic acid (JA) plays a central role. Successful microbial pathogens or symbionts have developed strategies to manipulate plant hormone signaling pathways to cause hormonal imbalances for their own benefit. These [...] Read more.
Plant immunity relies on a complex network of hormone signaling pathways in which jasmonic acid (JA) plays a central role. Successful microbial pathogens or symbionts have developed strategies to manipulate plant hormone signaling pathways to cause hormonal imbalances for their own benefit. These strategies include the production of plant hormones, phytohormone mimics, or effector proteins that target host components to disrupt hormonal signaling pathways and enhance virulence. Here, we describe the molecular details of the most recent and best-characterized examples of specific JA hormonal manipulation by microbes, which exemplify the ingenious ways by which pathogens can take control over the plant’s hormone signaling network to suppress host immunity. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Figure 1
<p>Phytotoxins and microbial effectors targeting the JA signaling components. Several microbes produce JA-Ile precursors or the JA-Ile mimic coronatine that are perceived by the JA-Ile receptor complex and induce degradation of JAZ repressors (see <a href="#plants-05-00009-t001" class="html-table">Table 1</a> for specific examples). HopX1 from <span class="html-italic">P. syringae</span> pv. <span class="html-italic">tabaci</span> (<span class="html-italic">Pta</span>) 11528 encodes a cysteine protease that associates with JAZ proteins induces their degradation in a proteasome- and COI1-independent manner, likely via its cysteine protease activity [<a href="#B56-plants-05-00009" class="html-bibr">56</a>]. HopZ1a from <span class="html-italic">P. syringae</span> pv. <span class="html-italic">syringae</span> (<span class="html-italic">Pss</span>) strain A2 encodes a cysteine protease/acetyltransferase that acetylates several JAZ proteins and induces their degradation through an undefined mechanism that is dependent on COI1 [<a href="#B57-plants-05-00009" class="html-bibr">57</a>]. AvrB interacts with [<a href="#B71-plants-05-00009" class="html-bibr">71</a>] and phosphorylates MPK4, which triggers activation of JA signaling [<a href="#B58-plants-05-00009" class="html-bibr">58</a>]. AvrB also induces the degradation of multiple JAZ proteins by positively regulating the PM H+-ATPase AHA1 to enhance bacterial penetration and virulence through RIN4 [<a href="#B59-plants-05-00009" class="html-bibr">59</a>]. The ectomycorrhizal MiSSP7 effector interacts with PtJAZ6 and protects PtJAZ6 from JA-induced degradation, attenuating JA-dependent host defenses to promote fungal colonization and mutualism [<a href="#B61-plants-05-00009" class="html-bibr">61</a>]. The oomycete downy mildew effector RxL44 directly interacts with the mediator subunit MED19a (Mediator19a), resulting in the proteasome-mediated degradation of MED19a, which shifts the balance of defense gene transcription from SA-responsive to JA/ET-mediated defense, enhancing susceptibility to biotrophs [<a href="#B60-plants-05-00009" class="html-bibr">60</a>]. In all cases, degradation of JAZs leads to de-repression of the TFs that initiate the activation of JA-dependent gene expression, suppression of SA responses and plant susceptibility. Black arrows indicate activation of the indicated hormonal pathway. Black bars indicate inhibition of the indicated hormonal pathway. Dashed arrows denote indirect or unclear mechanism leading to the activation of the indicated hormonal pathway. A circled Ac indicates acetylation. A circled P indicates phosphorylation.</p>
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1005 KiB  
Communication
The C2 Protein from the Geminivirus Tomato Yellow Leaf Curl Sardinia Virus Decreases Sensitivity to Jasmonates and Suppresses Jasmonate-Mediated Defences
by Tábata Rosas-Díaz, Alberto P. Macho, Carmen R. Beuzón, Rosa Lozano-Durán and Eduardo R. Bejarano
Plants 2016, 5(1), 8; https://doi.org/10.3390/plants5010008 - 15 Jan 2016
Cited by 33 | Viewed by 8105
Abstract
An increasing body of evidence points at a role of the plant hormones jasmonates (JAs) in determining the outcome of plant-virus interactions. Geminiviruses, small DNA viruses infecting a wide range of plant species worldwide, encode a multifunctional protein, C2, which is essential for [...] Read more.
An increasing body of evidence points at a role of the plant hormones jasmonates (JAs) in determining the outcome of plant-virus interactions. Geminiviruses, small DNA viruses infecting a wide range of plant species worldwide, encode a multifunctional protein, C2, which is essential for full pathogenicity. The C2 protein has been shown to suppress the JA response, although the current view on the extent of this effect and the underlying molecular mechanisms is incomplete. In this work, we use a combination of exogenous hormone treatments, microarray analysis, and pathogen infections to analyze, in detail, the suppression of the JA response exerted by C2. Our results indicate that C2 specifically affects certain JA-induced responses, namely defence and secondary metabolism, and show that plants expressing C2 are more susceptible to pathogen attack. We propose a model in which C2 might interfere with the JA response at several levels. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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<p>Infection of C2-expressing plants with <span class="html-italic">Pseudomonas syringae</span> pv. <span class="html-italic">tomato</span> DC3000. (<b>A</b>) Bacterial growth of wild-type (<span class="html-italic">Pto</span> DC3000) or a deficient strain unable to synthesize coronatine (COR-) in wild-type Col-0 (control) or transgenic C2-expressing plants in dip-inoculation experiments. Samples were taken at 4 dpi. Values are the mean of five plants. Bars represent standard error. One-way ANOVA Tukey’s Multiple comparison tests were used to distinguish differences among samples at <span class="html-italic">p</span>-value &lt;0.05. Different letters indicate statistically significant difference. Results are the mean of three independent biological replicates; (<b>B</b>) Symptoms displayed by dip-inoculated plants. Three different categories are considered: no symptoms, few symptoms or full symptoms, as indicated in the legend. The percentage of leaves in each category is represented. Bars represent standard error. In both (<b>A</b>) and (<b>B</b>), experiments were repeated three times with similar results; results from one representative experiment are shown.</p>
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<p>Venn diagrams showing the number of genes up- or down-regulated (UR or DR, respectively) in C2-TS plants, either JA- or mock-treated (in (<b>A</b>,<b>B</b>), respectively), and JA-treated control plants. The Venn diagrams were constructed using the software Venny (<a href="http://bioinfogp.cnb.csic.es/tools/venny" target="_blank">http://bioinfogp.cnb.csic.es/tools/venny</a>). Total number of genes in each subset is indicated in brackets.</p>
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<p>MapMan visualization of defence-related differentially expressed genes in response to MeJA in control or C2-TS-expressing plants.</p>
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<p>MapMan visualization of defence-related differentially expressed genes in response to MeJA in control or C2-TS-expressing plants.</p>
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<p>Transgenic C2-TS plants are more susceptible to <span class="html-italic">Pseudomonas syringae</span> pv. <span class="html-italic">tomato</span> DC3000 and <span class="html-italic">Potato virus X</span>. (<b>A</b>) Bacterial growth of <span class="html-italic">Pto</span> DC3000 in wild-type or C2-expressing Arabidopsis plants upon inoculation by infiltration. Samples were taken at 4 dpi. Values are the mean of five plants. Bars represent standard error. The asterisk indicates a statistically significant difference from the control sample (* <span class="html-italic">p</span>-value &lt; 0.05) according to a Student’s <span class="html-italic">t</span>-test. Three independent experiments were performed with similar results; results from one representative experiments are shown; (<b>B</b>) Bacterial growth of wild-type <span class="html-italic">Pto</span> DC3000, a Δ<span class="html-italic">hrcC</span> mutant, or a wild-type strain expressing the heterologous effector AvrRpt2 on wild-type or C2-TS-expressing Arabidopsis plants. Values represent the average of five plants. Bars represent standard error. The asterisk indicates a statistically significant difference from the control sample (* <span class="html-italic">p</span>-value &lt; 0.05) according to a Student’s <span class="html-italic">t</span>-test. Three independent experiments were performed with similar results; results from one representative experiments are shown; (<b>C</b>) Infection of wild-type (WT) or C2-TS-expressing <span class="html-italic">N. benthamiana</span> plants with PVX-GFP or TMV-GFP at 10 dpi. Values represent relative expression of viral RNA estimated by semi-quantitative RT-PCR, and are the average of ten infected plants. Bars represent standard error. Asterisks indicate a statistically significant difference from the control sample (** <span class="html-italic">p</span>-value &lt; 0.01) according to a Student’s <span class="html-italic">t</span>-test. Two independent experiments were performed with similar results; results from one representative experiment are shown; (<b>D</b>) Pictures of representative TMV-GFP and PVX-GFP infected plants under UV light.</p>
Full article ">Figure 4 Cont.
<p>Transgenic C2-TS plants are more susceptible to <span class="html-italic">Pseudomonas syringae</span> pv. <span class="html-italic">tomato</span> DC3000 and <span class="html-italic">Potato virus X</span>. (<b>A</b>) Bacterial growth of <span class="html-italic">Pto</span> DC3000 in wild-type or C2-expressing Arabidopsis plants upon inoculation by infiltration. Samples were taken at 4 dpi. Values are the mean of five plants. Bars represent standard error. The asterisk indicates a statistically significant difference from the control sample (* <span class="html-italic">p</span>-value &lt; 0.05) according to a Student’s <span class="html-italic">t</span>-test. Three independent experiments were performed with similar results; results from one representative experiments are shown; (<b>B</b>) Bacterial growth of wild-type <span class="html-italic">Pto</span> DC3000, a Δ<span class="html-italic">hrcC</span> mutant, or a wild-type strain expressing the heterologous effector AvrRpt2 on wild-type or C2-TS-expressing Arabidopsis plants. Values represent the average of five plants. Bars represent standard error. The asterisk indicates a statistically significant difference from the control sample (* <span class="html-italic">p</span>-value &lt; 0.05) according to a Student’s <span class="html-italic">t</span>-test. Three independent experiments were performed with similar results; results from one representative experiments are shown; (<b>C</b>) Infection of wild-type (WT) or C2-TS-expressing <span class="html-italic">N. benthamiana</span> plants with PVX-GFP or TMV-GFP at 10 dpi. Values represent relative expression of viral RNA estimated by semi-quantitative RT-PCR, and are the average of ten infected plants. Bars represent standard error. Asterisks indicate a statistically significant difference from the control sample (** <span class="html-italic">p</span>-value &lt; 0.01) according to a Student’s <span class="html-italic">t</span>-test. Two independent experiments were performed with similar results; results from one representative experiment are shown; (<b>D</b>) Pictures of representative TMV-GFP and PVX-GFP infected plants under UV light.</p>
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<p>Root growth inhibition assays in C2-expressing plants. (<b>A</b>) Relative root length of wild-type Col-0 (Control), C2-TS (plants expressing the <span class="html-italic">C2</span> gene from TYLCSV), and C2-TM (plants expressing the <span class="html-italic">C2</span> gene from TYLCV) <span class="html-italic">Arabidopsis</span> seedlings in increasing concentrations of MeJA (0, 10, 50, and 100 μM). The values are the mean of at least ten seedlings. Bars represent standard error. One-way ANOVA Tukey’s Multiple comparison tests were used to distinguish differences among samples at <span class="html-italic">p</span>-value &lt; 0.05. Different letters indicate statistically significant difference. Experiments were repeated three times with similar results; results from one representative experiment are shown; (<b>B</b>) Pictures of representative seedlings used in (<b>A</b>).</p>
Full article ">Figure 6
<p>Expression levels of JA-regulated and control genes in Arabidopsis transgenic C2-TS and control plants for microarray validation. (<b>A</b>) Relative expression level of <span class="html-italic">AtASN1</span> (At3g47340), <span class="html-italic">AtAOS1</span> (At5g42650), <span class="html-italic">AtGOLS</span> (At2g47180), <span class="html-italic">AtXTH31</span> (At3g44990), <span class="html-italic">AtPRB1</span> (At2g14580), <span class="html-italic">AtTINY</span> (At5g25810), <span class="html-italic">AtFLS2</span>; (At5g46330), <span class="html-italic">AtAIR1</span> (At4g12550) and <span class="html-italic">AtPR1</span> (At2g14610) genes in transgenic C2-TS and control Arabidopsis seedlings, mock- or MeJA-treated, determined by real-time PCR. Values are the mean of three technical replicates. Bars represent standard error; (<b>B</b>) Comparison between microarray and real-time PCR expression data of the MeJA-regulated and control genes in Arabidopsis transgenic C2-TS and control plants used in (<b>A</b>). Up- and down-regulation are shown in red and green, respectively; lack of differential expression or coincidence between the two methods are shown in grey and white, respectively. Values from eFP browser after 3 h of JA treatment are also represented [<a href="#B52-plants-05-00008" class="html-bibr">52</a>] (Arabidopsis eFP Browser. Available online: <a href="http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi" target="_blank">http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi</a>). The numbers represent the expression value in fold change.</p>
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740 KiB  
Review
Control of Carbon Assimilation and Partitioning by Jasmonate: An Accounting of Growth–Defense Tradeoffs
by Nathan E. Havko, Ian T. Major, Jeremy B. Jewell, Elham Attaran, John Browse and Gregg A. Howe
Plants 2016, 5(1), 7; https://doi.org/10.3390/plants5010007 - 15 Jan 2016
Cited by 86 | Viewed by 11424
Abstract
Plant growth is often constrained by the limited availability of resources in the microenvironment. Despite the continuous threat of attack from insect herbivores and pathogens, investment in defense represents a lost opportunity to expand photosynthetic capacity in leaves and absorption of nutrients and [...] Read more.
Plant growth is often constrained by the limited availability of resources in the microenvironment. Despite the continuous threat of attack from insect herbivores and pathogens, investment in defense represents a lost opportunity to expand photosynthetic capacity in leaves and absorption of nutrients and water by roots. To mitigate the metabolic expenditure on defense, plants have evolved inducible defense strategies. The plant hormone jasmonate (JA) is a key regulator of many inducible defenses. Synthesis of JA in response to perceived danger leads to the deployment of a variety of defensive structures and compounds, along with a potent inhibition of growth. Genetic studies have established an important role for JA in mediating tradeoffs between growth and defense. However, several gaps remain in understanding of how JA signaling inhibits growth, either through direct transcriptional control of JA-response genes or crosstalk with other signaling pathways. Here, we highlight recent progress in uncovering the role of JA in controlling growth-defense balance and its relationship to resource acquisition and allocation. We also discuss tradeoffs in the context of the ability of JA to promote increased leaf mass per area (LMA), which is a key indicator of leaf construction costs and leaf life span. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Graphical abstract

Graphical abstract
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<p>Jasmonate (JA) induces the re-budgeting of resources from tissue expansion to the production of defense compounds. (<b>A</b>) Plant growth is achieved using carbon skeletons, ATP, and NADPH from photosynthesis; (<b>B</b>) During the JA-mediated defense response, carbon skeletons, ATP, and NADPH that could otherwise contribute to growth are used for <span class="html-italic">de novo</span> synthesis of defense compounds; (<b>C</b>) In the absence of biotic attack, defense compounds are produced at a low basal level; (<b>D</b>) JA triggers the accumulation of defense compounds accompanied by an arrest of tissue expansion. In defended leaf tissue, cell size is similar to that in undefended leaves but the leaf mass per area (LMA) may increase as a consequence of increase carbon deposition into defense compounds.</p>
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<p>Simplified model depicting interactions between the JA, GA, and phyB signaling pathways. Points of positive and negative regulation are indicated by arrows and perpendicular lines, respectively. bHLH-TFs, basic helix-loop-helix transcription factors that bind G-box <span class="html-italic">cis</span>-regulatory elements typically located in the promoter region of response genes. In full sunlight, PIF transcription factors are inhibited by the active conformer of phyB. See text for details.</p>
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<p>Jasmonate-mediated suppression of leaf area and biomass growth in <span class="html-italic">Arabidopsis thaliana</span> plants. Soil-grown Columbia-0 plants treated with mock (grey) or with 5 µM coronatine (COR, black) solutions [<a href="#B71-plants-05-00007" class="html-bibr">71</a>] were measured for leaf area (<b>A</b>), rosette diameter (<b>B</b>), and dry weight (<b>C</b>) at indicated times after treatment. Data are the mean increase in growth (<span class="html-italic">n</span> = 12 plants) relative to the day of treatment (day 0) from two independent experiments (diamonds and squares distinguish experiments) and lines are second-order polynomial regressions of combined data from both experiments. Projected leaf area and rosette diameter were determined from overhead images, and dry weight was determined from rosettes (without roots) freeze-dried in a lyophilizer. In (<b>D</b>), leaf mass per area (LMA) was calculated from dry weight/leaf area from one experiment in A and C (square points). Error bars are standard deviation and asterisks indicate <span class="html-italic">P</span> &lt; 0.05 between mock and COR treatment from a Student’s t-Test.</p>
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1568 KiB  
Review
The Ubiquitin System and Jasmonate Signaling
by Astrid Nagels Durand, Laurens Pauwels and Alain Goossens
Plants 2016, 5(1), 6; https://doi.org/10.3390/plants5010006 - 9 Jan 2016
Cited by 40 | Viewed by 9830
Abstract
The ubiquitin (Ub) system is involved in most, if not all, biological processes in eukaryotes. The major specificity determinants of this system are the E3 ligases, which bind and ubiquitinate specific sets of proteins and are thereby responsible for target recruitment to the [...] Read more.
The ubiquitin (Ub) system is involved in most, if not all, biological processes in eukaryotes. The major specificity determinants of this system are the E3 ligases, which bind and ubiquitinate specific sets of proteins and are thereby responsible for target recruitment to the proteasome or other cellular processing machineries. The Ub system contributes to the regulation of the production, perception and signal transduction of plant hormones. Jasmonic acid (JA) and its derivatives, known as jasmonates (JAs), act as signaling compounds regulating plant development and plant responses to various biotic and abiotic stress conditions. We provide here an overview of the current understanding of the Ub system involved in JA signaling. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Figure 1
<p>The Ub conjugation pathway. (<b>A</b>) Ubiquitin (Ub) is first activated and bound by E1 in an ATP-dependent reaction, after which it is transferred to E2. Then, E3 interacts simultaneously with the substrate and the E2-Ub conjugate, facilitating transfer of Ub to the substrate. E3 depicted in (A) is a single subunit which interacts via its RING or U-box domain with E2. An example of a RING-type E3 in JA signaling is DAD1-ACTIVATING FACTOR (DAF) [<a href="#B17-plants-05-00006" class="html-bibr">17</a>]. The HECT-type E3s are not shown. (<b>B</b>) E3 can also occur as a multi-subunit E3 consisting of a CULLIN protein serving as scaffold for an adaptor module that binds the substrate, and the RING domain-containing RBX1 protein that binds E2. An example of a CULLIN-RING ubiquitin ligase (CRL) in JA signaling is SCF<sup>COI1</sup>.</p>
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<p>Overview of the core JA-signaling pathway. In the absence of JA-Ile, the activity of the TFs MYC2/3/4, bound to the G-box of JA-responsive promotors, is repressed by interaction with a JAZ protein that binds to the JAZ-interacting domain (JID) of MYC2/3/4 through their Jas domain. Upon perception of the hormone (green dot) by the co-receptor complex composed of a JAZ protein and the F-box component COI1 (SCF<sup>COI1</sup>), which is potentiated by inositol pentakisphosphate (InsP5, blue dot), the CUL-RING E3 ligase (CRL) binds with SCF<sup>COI1</sup> and ubiquitinates the JAZ protein. Subsequently, JAZ proteins are degraded and MYC2/3/4 activate transcription of JA-responsive genes eventually resulting in a JA response. Figure adapted from Pauwels and Goossens [<a href="#B60-plants-05-00006" class="html-bibr">60</a>].</p>
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<p>JAZ proteins repress JA signaling. (<b>A</b>) JAZ proteins interact with bHLH-type TFs, such as MYC2/3/4, mediated by direct binding between the Jas and JAZ-interacting domain (JID) domains, respectively. The ZIM domain of most JAZ proteins can interact with the adapter protein Novel Interactor of JAZ (NINJA). Through an ERF-associated amphiphilic repression (EAR) motif, NINJA is capable of recruiting the co-repressor TOPLESS (TPL) to the complex. (<b>B</b>) The ZIM domains of some JAZ proteins, such as JAZ8 and JAZ13, are unable to bind NINJA, but these JAZ proteins contain an EAR motif themselves. (<b>C</b>) Alternative splicing of certain <span class="html-italic">JAZ</span> transcripts leads to truncated JAZ proteins lacking part of the Jas domain. In the case of JAZ10.4, an N-terminal cryptic MYC2-binding domain (CMID) mediates interaction with bHLH-type TFs such as MYC2/3/4. Both JAZ8/13 and JAZ10.4 have deviating Jas domains and do not associate with SCF<sup>COI1</sup> in a JA-Ile dependent way. (<b>D</b>) The interaction of JAZ proteins with MYC2 is mediated by both the JID and the transcriptional activation domain (TAD). This interaction competes with the binding of this surface with the co-activating mediator subunit MED25. Figure partially adapted from Pauwels and Goossens [<a href="#B60-plants-05-00006" class="html-bibr">60</a>].</p>
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<p>Regulation of SCF<sup>COI1</sup> by Nedd8/RUB. The activity of SCF<sup>COI1</sup> is modulated by cyclic attachment and removal of the ubiquitin-like modifier (UBL) Nedd8/RUB to the CUL1 subunit of the SCF complex. Accordingly, plants carrying mutations in the genes encoding components of the neddylation machinery (E1: ECR1/AXR1/AXL; E2: RCE1/2) or in the COP9 deneddylase signalosome complex are also affected in JA signaling. Defects in the RUB-conjugation pathway can also be mimicked using the E1-specific inhibitor MLN4924 [<a href="#B114-plants-05-00006" class="html-bibr">114</a>].</p>
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1382 KiB  
Article
Defense Priming and Jasmonates: A Role for Free Fatty Acids in Insect Elicitor-Induced Long Distance Signaling
by Ting Li, Tristan Cofer, Marie Engelberth and Jurgen Engelberth
Plants 2016, 5(1), 5; https://doi.org/10.3390/plants5010005 - 8 Jan 2016
Cited by 20 | Viewed by 7299
Abstract
Green leaf volatiles (GLV) prime plants against insect herbivore attack resulting in stronger and faster signaling by jasmonic acid (JA). In maize this response is specifically linked to insect elicitor (IE)-induced signaling processes, which cause JA accumulation not only around the damage site, [...] Read more.
Green leaf volatiles (GLV) prime plants against insect herbivore attack resulting in stronger and faster signaling by jasmonic acid (JA). In maize this response is specifically linked to insect elicitor (IE)-induced signaling processes, which cause JA accumulation not only around the damage site, but also in distant tissues, presumably through the activation of electrical signals. Here, we present additional data further characterizing these distal signaling events in maize. Also, we describe how exposure to GLV increases free fatty acid (fFA) levels in maize seedlings, but also in other plants, and how increased fFA levels affect IE-induced JA accumulation. Increased fFA, in particular α-linolenic acid (LnA), caused a significant increase in JA accumulation after IE treatment, while JA induced by mechanical wounding (MW) alone was not affected. We also identified treatments that significantly decreased certain fFA level including simulated wind and rain. In such treated plants, IE-induced JA accumulation was significantly reduced when compared to un-moved control plants, while MW-induced JA accumulation was not significantly affected. Since only IE-induced JA accumulation was altered by changes in the fFA composition, we conclude that changing levels of fFA affect primarily IE-induced signaling processes rather than serving as a substrate for JA. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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<p>Effect of <span class="html-italic">Z</span>-3-hexenol (GLV) priming on local and distal jasmonic acid (ng·gFW<sup>−1</sup>) accumulation after treatment with insect elicitor (IE). Plants were exposed to 10 μg of GLV for 16 h and then IE for 1 h. Controls were treated similarly, but without GLV. Jasmonic acid was analyzed by GC/MS as described in [<a href="#B19-plants-05-00005" class="html-bibr">19</a>,<a href="#B34-plants-05-00005" class="html-bibr">34</a>,<a href="#B45-plants-05-00005" class="html-bibr">45</a>]. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by Tukey tests where appropriate (<span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">N</span> = 4, error bars represent standard deviation.</p>
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<p>Characterization of distal signaling speed induced by mechanical wounding (MW) and insect elicitors (IE) treatment at 2 min, 5 min, 10 min, and 30 min. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by Tukey tests where appropriate (<span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">N</span> = 4, error bars represent standard deviation.</p>
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<p>(<b>A</b>) Effect of cut stem application of phenidone on local and distal signaling after treatment with insect elicitor (IE); (<b>B</b>) Effect of local application of phenidone on local and distal signaling after treatment with IE. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by Tukey tests where appropriate (<span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">N</span> = 3, error bars represent standard deviation.</p>
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<p>Comparison of insect elicitor (IE) and alamethicin (ALM) on local and distal jasmonic acid accumulation. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by Tukey tests where appropriate (<span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">N</span> = 4, error bars represent standard deviation.</p>
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<p>(<b>A</b>) Increases in free fatty acids in different plant species after exposure to <span class="html-italic">Z</span>-3-hexenol as our model GLV. Plants were exposed to 20 µg of <span class="html-italic">Z</span>-3-hexenol (HOL) for 15 min and free fatty acids analyzed by GC/MS. Note that <span class="html-italic">Zea</span> and <span class="html-italic">Solanum</span> show increased linolenic acid (LnA) levels, whereas in <span class="html-italic">Pisum</span> and <span class="html-italic">Oryza</span> palmitoleic acid was increased. Relative amounts are displayed with free fatty acid levels in control plants set at 100%. * denotes significant differences (<span class="html-italic">t-</span>test, <span class="html-italic">p</span> ≤ 0.05). (<b>B</b>–<b>D</b>) Effects of prolonged GLV treatments on free linolenic acid levels in maize leaves. Maize plants were exposed to 20 μg of GLV for the time indicated. (<b>B</b>) Amounts of free LnA after 2, 6, and 16 h; (<b>C</b>) Amounts of free stearic acid (StA) after 2, 6, and 16 h; (<b>D</b>) Ratios of LnA to StA after 2, 6, and 16 h. * denotes significant differences (<span class="html-italic">t-</span>test, <span class="html-italic">p</span> ≤ 0.05). <span class="html-italic">N</span> = 5, error bars represent standard deviation.</p>
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<p>(<b>A</b>) Linolenic acid (LnA) accumulation after treatment with 300 µM LnA overnight; (<b>B</b>) Ratio of free LnA to free stearic acid (StA) after treatment with 300 µM LnA overnight. * denotes significant differences (<span class="html-italic">t-</span>test, <span class="html-italic">p</span> ≤ 0.05). <span class="html-italic">N</span> = 4, error bars represent standard deviation.</p>
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<p>(<b>A</b>) Effects of free linolenic acid (LnA) on insect elicitor (IE) induced jasmonic acid accumulation; (<b>B</b>) Effects of free linolenic acid (LnA) on mechanical wounding (MW) induced jasmonic acid accumulation; (<b>C</b>) Comparison of volatiles release from corn seedlings pre-treated LnA and then with IE. Controls were treated similarly, but without LnA. Plant were treated with 300 μM aqueous solution of the respective fatty acid overnight and then treated by either MW or application of IE. JA was measured 1 and 2 h after treatment. JA was analyzed by GC/MS. Plant volatiles were collected between 4 and 5 h after treatment with IE [<a href="#B34-plants-05-00005" class="html-bibr">34</a>]. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by Tukey tests where appropriate (<span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">N</span> = 5, error bars represent standard deviation. Statistical analysis was done for each volatile group independently.</p>
Full article ">Figure 7 Cont.
<p>(<b>A</b>) Effects of free linolenic acid (LnA) on insect elicitor (IE) induced jasmonic acid accumulation; (<b>B</b>) Effects of free linolenic acid (LnA) on mechanical wounding (MW) induced jasmonic acid accumulation; (<b>C</b>) Comparison of volatiles release from corn seedlings pre-treated LnA and then with IE. Controls were treated similarly, but without LnA. Plant were treated with 300 μM aqueous solution of the respective fatty acid overnight and then treated by either MW or application of IE. JA was measured 1 and 2 h after treatment. JA was analyzed by GC/MS. Plant volatiles were collected between 4 and 5 h after treatment with IE [<a href="#B34-plants-05-00005" class="html-bibr">34</a>]. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by Tukey tests where appropriate (<span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">N</span> = 5, error bars represent standard deviation. Statistical analysis was done for each volatile group independently.</p>
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<p>Free linolenic acid (LnA) levels in corn plants treated with salicylic acid (SA) overnight. Corn seedlings were treated overnight with 50 mL of a 100 μM salicylic acid solution added to the soil. * denotes significant differences (<span class="html-italic">t-</span>test, <span class="html-italic">p</span> ≤ 0.05). <span class="html-italic">N</span> = 4, error bars represent standard deviation.</p>
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<p>Effects of plant movements on free fatty acid levels and jasmonic acid accumulation. (<b>A</b>) Effect of shaking on free fatty acid levels in corn leaves. Plants were shaken for 15 s (upper panel); Free fatty acids were extracted and measured at times indicated in upper panel after treatment; (<b>B</b>) Effect of shaking on jasmonic acid accumulation after treatment with insect elicitor (IE, volicitin) or mechanical wounding (MW) for 1 h. Plants were shaken as above and then left standing for 30 min before treatment with IE or MW. (<b>C</b>) Effect of simulated rain on free fatty acid levels in corn leaves. Plants were treated with simulated rain for 30 s Free fatty acids were analyzed 30 min after rain treatment. Displayed are the ratios between linolenic acid (LnA) and stearic acid (StA). StA levels did not change upon treatments described herein. (<b>D</b>) Effects of rain on jasmonic acid accumulation after treatment with insect elicitor (IE) or mechanical wounding (MW) for 60 min. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by Tukey tests where appropriate (<span class="html-italic">p</span> &lt; 0.05). * denotes significant differences (<span class="html-italic">t-</span>test, <span class="html-italic">p</span> ≤ 0.05). <span class="html-italic">N</span> = 6, error bars represent standard deviation.</p>
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1365 KiB  
Article
Dynamics of Jasmonate Metabolism upon Flowering and across Leaf Stress Responses in Arabidopsis thaliana
by Emilie Widemann, Ekaterina Smirnova, Yann Aubert, Laurence Miesch and Thierry Heitz
Plants 2016, 5(1), 4; https://doi.org/10.3390/plants5010004 - 6 Jan 2016
Cited by 28 | Viewed by 7346
Abstract
The jasmonic acid (JA) signaling pathway plays important roles in adaptation of plants to environmental cues and in specific steps of their development, particularly in reproduction. Recent advances in metabolic studies have highlighted intricate mechanisms that govern enzymatic conversions within the jasmonate family. [...] Read more.
The jasmonic acid (JA) signaling pathway plays important roles in adaptation of plants to environmental cues and in specific steps of their development, particularly in reproduction. Recent advances in metabolic studies have highlighted intricate mechanisms that govern enzymatic conversions within the jasmonate family. Here we analyzed jasmonate profile changes upon Arabidopsis thaliana flower development and investigated the contribution of catabolic pathways that were known to turnover the active hormonal compound jasmonoyl-isoleucine (JA-Ile) upon leaf stress. We report a rapid decline of JA-Ile upon flower opening, concomitant with the massive accumulation of its most oxidized catabolite, 12COOH-JA-Ile. Detailed genetic analysis identified CYP94C1 as the major player in this process. CYP94C1 is one out of three characterized cytochrome P450 enzymes that define an oxidative JA-Ile turnover pathway, besides a second, hydrolytic pathway represented by the amido-hydrolases IAR3 and ILL6. Expression studies combined with reporter gene analysis revealed the dominant expression of CYP94C1 in mature anthers, consistent with the established role of JA signaling in male fertility. Significant CYP94B1 expression was also evidenced in stamen filaments, but surprisingly, CYP94B1 deficiency was not associated with significant changes in JA profiles. Finally, we compared global flower JA profiles with those previously reported in leaves reacting to mechanical wounding or submitted to infection by the necrotrophic fungus Botrytis cinerea. These comparisons revealed distinct dynamics of JA accumulation and conversions in these three biological systems. Leaf injury boosts a strong and transient JA and JA-Ile accumulation that evolves rapidly into a profile dominated by ω-oxidized and/or Ile-conjugated derivatives. In contrast, B. cinerea-infected leaves contain mostly unconjugated jasmonates, about half of this content being ω-oxidized. Finally, developing flowers present an intermediate situation where young flower buds show detectable jasmonate oxidation (probably originating from stamen metabolism) which becomes exacerbated upon flower opening. Our data illustrate that in spite conserved enzymatic routes, the jasmonate metabolic grid shows considerable flexibility and dynamically equilibrates into specific blends in different physiological situations. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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<p>Proposed model for interconversion routes between jasmonic acid (JA) and its Ile-conjugated/ω-oxidized derivatives upon leaf stress responses or flower maturation, with emphasis on quantitative aspects as explained in main text. Upon leaf stress responses (wounding or <span class="html-italic">B. cinerea</span> infection) or flower maturation JA synthesis is activated to different extents, mechanical wounding providing the strongest burst. JA can be modified via different metabolic routes, one being the formation of jasmonoyl-isoleucine (JA-Ile) by JAR1 or related conjugating enzymes. JA-Ile is then either ω-oxidized by the action of CYP94 family enzymes to its hydroxy- (12OH-JA-Ile) or carboxy- (12COOH-JA-Ile) derivatives. In addition, JA-Ile and 12OH-JA-Ile can be hydrolyzed by the amidohydrolases IAR3 and ILL6, leading to the formation of JA and 12OH-JA, respectively. The thickness of arrows for the main JA metabolic pathway (from JA to 12COOH-JA-Ile) reflects the abundance of a given reaction product in each physiological context as detailed in Discussion. Comparative analysis of jasmonate profiles in the three biological systems as described in this paper shows that adequate JA-Ile levels can be achieved with distinct upstream and downstream metabolite pool sizes through the action of common enzymes. Blue—flower maturation, green—leaf wounding, purple—leaf <span class="html-italic">B. cinerea</span> infection. Predominant enzymes performing a given reaction are mentioned as follows: CYP94C1: C1, CYP94B1: B1, CYP94B3: B3, ST2a—sulfotransferase 2a. The compounds are shown in (3,7)-cis stereochemistry.</p>
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<p>(<b>A</b>) <span class="html-italic">A. thaliana</span> flower development stages corresponding to that indicated in [<a href="#B32-plants-05-00004" class="html-bibr">32</a>]. Stages 1–12 further referred to closed buds, stages 13 and 14—to open flowers (as indicated above). Scale bar corresponds to 3 mm. (<b>B</b>) UPLC-MS/MS quantification of five JAs content in <span class="html-italic">Col0</span> closed flower buds and open flowers. Note the different scale for 12COOH-JA-Ile on the right axis. Values are presented as means with SD of three independent biological repeats. (<b>C</b>, <b>D</b>) Relative expression levels of 4 <span class="html-italic">CYP94</span> genes <span class="html-italic">(–B1, –B3, –C1, –D2)</span> in <span class="html-italic">Col0</span> flowers distributed by development stages (C) or flower organs (stade 14)(D). Expression levels are normalized by expression of two reference genes. Values are presented as means with SD of three technical repeats. (<b>E</b>) Typical GUS staining pattern of flowers of <span class="html-italic">pCYP94C1::GUS</span>, <span class="html-italic">pCYP94B1::GUS</span> and <span class="html-italic">pCYP94D2::GUS</span> transgenic lines at different magnitude (from whole inflorescence to a separate anther). Two independent lines were analyzed for each construct. Scale bar corresponds to 3 mm. (<b>F</b>) Relative expression levels of 4 <span class="html-italic">AH</span> genes <span class="html-italic">(ILL2, ILL5, ILL6, IAR3)</span> in <span class="html-italic">Col0</span> closed flower buds or open flowers. Expression levels are normalized by expression of two reference genes. Values are presented as means with SD of three technical repeats.</p>
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<p>UPLC-MS/MS analysis of conjugated JAs (JA-Ile, 12OH-JA-Ile and 12COOH-JA-Ile) abundance in closed buds or open flowers of <span class="html-italic">Col0</span> control plants and of simple mutants <span class="html-italic">cyp94b1, –b3 and –c1</span>, double mutants <span class="html-italic">cyp94b1b3, –b1c1 and –b3c1</span>, triple mutant <span class="html-italic">cyp94b1b3c1</span>, and <span class="html-italic">CYP94C1</span> over-expressor mutant <span class="html-italic">(C1-OE).</span> Values are presented as means with SD of three independent biological repeats. Statistical analysis was applied separately to the pool of data corresponding to either closed buds or open flowers. Columns are labelled with different letters indicating a significant difference in given jasmonate content between genotypes as determined by Kruskal-Wallis one-way analysis of variance (ANOVA) (<span class="html-italic">p</span> &lt; 0.05). Absence of letters corresponds to the absence of significant difference in a given JA abundance in different analyzed genotypes.</p>
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<p>Pie diagrams of five JAs content in three biological systems<b>:</b> In left column, the number indicates cumulated amounts (nmol·g·FW<sup>−1</sup>) of JA, 12OH-JA, JA-Ile, 12OH-JA-Ile and 12COOH-JA-Ile as means with SD of three independent biological repeats. The left column pies indicate the proportions of conjugated (JA-Ile + 12OH-JA-Ile + 12COOH-JA-Ile) <span class="html-italic">versus</span> un-conjugated (JA + 12OH-JA). The right column pies indicate the proportions of non-ω-oxidized (JA + JA-Ile) <span class="html-italic">versus</span> ω-oxidized (12OH-JA + 12OH-JA-Ile + 12COOH-JA-Ile) JAs. (<b>A</b>) flower maturation: closed buds or open flowers (<b>B</b>) leaf wounding: 1 h post-wounding (hpw) or 4 hpw (<b>C</b>) leaf <span class="html-italic">B. cinerea</span> infection: 3 days post-inoculation (dpi).</p>
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1965 KiB  
Article
Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members
by Markus Otto, Christin Naumann, Wolfgang Brandt, Claus Wasternack and Bettina Hause
Plants 2016, 5(1), 3; https://doi.org/10.3390/plants5010003 - 6 Jan 2016
Cited by 25 | Viewed by 7834
Abstract
Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of [...] Read more.
Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles. Full article
(This article belongs to the Special Issue The Jasmonate Pathway)
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Figure 1
<p>Recombinant Arabidopsis allene oxide cyclase 2 (AOC2) is a trimer. His-tagged proteins were purified on Ni-NTA, treated with SDS at given temperatures for 5 min and separated by SDS-PAGE. Detection of proteins was performed by immunolabeling using an anti-His-tag antibody. (<b>A</b>) Recombinant AOC2 protein with N-terminal His-tag; (<b>B</b>) recombinant AOC2 protein with C-terminal His-tag; (<b>C</b>) recombinant AOC2 protein with N-terminal His-tag cross-linked or not with 200 μM glycol-bis(succinic acid <span class="html-italic">N</span>-hydroxysuccinimide ester) (EGS) for 40 min. Note that only treatment at 96 °C resulted in the predominant occurrence of monomers (~26 kDa), whereas trimers resisted treatments at lower temperatures. Accordingly, cross-linking with EGS prevented separation of trimers by SDS treatment at 96 °C (C). M = size marker.</p>
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<p>Identification of amino acids at the interaction sites between AOC2 monomers within the trimer. (<b>A</b>) Surface representation of the AOC2 trimer (PDB Code 2Q4I). The view is along the trimer axis. The three monomers are given in different gray scales. The hydrophobic core (detailed in (<b>B</b>)) and the salt bridges (detailed in (<b>C</b>)) are shown in green and red, respectively. Salt bridges on the back are detailed in (<b>D</b>); see <a href="#app1-plants-05-00003" class="html-app">Figure S1</a>. (B) Location of Leu40, Leu50, Leu53 and Ile79 of all three monomers building the hydrophobic core of the trimer. (C) Salt bridge (yellow dashed line) between Lys152 of one monomer and Glu128 of the neighboring monomer. The distance between atoms building the salt bridge is given in Å. (D) Hydrogen bonds between Arg34 and Asn193 of one monomer and salt bridges (yellow dashed lines) to Glu80 of the neighboring monomer. The distances between atoms building a salt bridge are given in Å. Interacting faces are shown in stick representation (oxygen, red; nitrogen, blue; carbon, green; atoms not involved in the interaction, gray).</p>
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<p>Monomers of AOC2 do not exhibit enzymatic activity. AOC2 was mutated to prevent trimer formation by the exchange of amino acids involved in salt bridge formation (K152A/E80A = “AB”), the formation of the hydrophobic core (L53S = “C”) or both (K152A/E80A/L53S = “ABC”) (see <a href="#plants-05-00003-t001" class="html-table">Table 1</a>). (<b>A</b>) His-tagged recombinant proteins were treated with SDS at 42 °C for 5 min and separated on SDS-PAGE. Note that all mutant proteins are predominantly detectable as monomers. M = size marker. (<b>B</b>) AOC enzyme activity of recombinant wild-type AOC2 and mutant proteins. Each value is given as nmol of enzymatically-formed <span class="html-italic">cis</span>-(+)-12-oxophytodienoic acid (OPDA) per μg protein and min and is represented by the mean of three independent replicates ± SD. Different letters designate statistically-different values (one-way ANOVA with Tukey’s HSD test, <span class="html-italic">p</span> &lt; 0.01). co = control done without the addition of protein to the reaction mixture.</p>
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<p>Heteromerization of AOC results in altered activities. (<b>A</b>) Purification of His-tagged homomers was done using Ni-NTA (left). Heteromers were purified using Ni-NTA (for His-tag) and StrepTactin (for Strep-tag) subsequently. Immunoblots of purified recombinant proteins treated with SDS at 96 °C show homomers with His-tag (hAOC) and heteromers exhibiting one isoform with His-Tag (hAOC) and the other with Strep-tag (sAOC). Note that the heteromers were detectable by both immuno-decorations. (<b>B</b>) Activity of recombinant homomeric and heteromeric AOCs. Each value is given as nmol of enzymatically-formed OPDA per μg protein and min and is represented by the mean of three independent replicates (±SD) obtained from independent protein preparations. Different letters designate statistically-different values (one-way ANOVA with Tukey’s HSD test, <span class="html-italic">p</span> &lt; 0.05).</p>
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2479 KiB  
Article
Response of Arabidopsis thaliana Roots with Altered Lipid Transfer Protein (LTP) Gene Expression to the Clubroot Disease and Salt Stress
by Sabine Jülke and Jutta Ludwig-Müller
Plants 2016, 5(1), 2; https://doi.org/10.3390/plants5010002 - 24 Dec 2015
Cited by 34 | Viewed by 9199
Abstract
The clubroot disease of Brassicaceae is caused by the obligate biotrophic protist Plasmodiophora brassicae. The disease is characterized by abnormal tumorous swellings of infected roots that result in reduced drought resistance and insufficient distribution of nutrients, leading to reduced crop yield. It [...] Read more.
The clubroot disease of Brassicaceae is caused by the obligate biotrophic protist Plasmodiophora brassicae. The disease is characterized by abnormal tumorous swellings of infected roots that result in reduced drought resistance and insufficient distribution of nutrients, leading to reduced crop yield. It is one of the most damaging diseases among cruciferous crops worldwide. The acquisition of nutrients by the protist is not well understood. Gene expression profiles in Arabidopsis thaliana clubroots indicate that lipid transfer proteins (LTPs) could be involved in disease development or at least in adaptation to the disease symptoms. Therefore, the aim of the study was to examine the role of some, of the still enigmatic LTPs during clubroot development. For a functional approach, we have generated transgenic plants that overexpress LTP genes in a root specific manner or show reduced LTP gene expression. Our results showed that overexpression of some of the LTP genes resulted in reduced disease severity whereas the lipid content in clubs of LTP mutants seems to be unaffected. Additional studies indicate a role for some LTPs during salt stress conditions in roots of A. thaliana. Full article
(This article belongs to the Special Issue Plant Root Development)
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<p>Expression of selected <span class="html-italic">LTP</span> genes in clubroot infected <span class="html-italic">A. thaliana</span> roots. The expression for selected <span class="html-italic">LTP</span> genes during clubroot infection between seven and 26 days after inoculation is shown compared to control roots of the same age. For infection, the single spore isolate e<sub>3</sub> was used. At least two technical replicates were done to confirm the results. For each analyzed time point, approximately 30–50 plants were used for RNA extraction. The asterisk marks an unspecific PCR product. C = control (not inoculated); I = inoculated (infected).</p>
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<p>Regulation of <span class="html-italic">LTP</span> gene expression due to hormonal treatment according to Genevestigator [<a href="#B49-plants-05-00002" class="html-bibr">49</a>] data. The elongation factor 1B gamma is shown as a reference gene in comparison to the analyzed <span class="html-italic">LTP</span> genes. Green color indicates repression and red induction of gene expression.</p>
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<p>Typical disease symptoms from club-rooted <span class="html-italic">A. thaliana</span> for the different disease classes. Disease classes range from 0 = no symptoms visible to 4 = no fine root system present, but one large root gall. For detailed description of symptoms, see <a href="#sec3dot2-plants-05-00002" class="html-sec">Section 3.2</a>. Bar = 1 cm.</p>
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<p>Clubroot development in plants that overexpress a <span class="html-italic">LTP</span> gene. The Disease Index (<b>A</b>), distribution of disease classes (<b>B</b>), and Shoot Index (<b>C</b>) for mutants that overexpress (OX) the indicated <span class="html-italic">LTP</span> gene in comparison to wild type (WT) are shown. Each mutant line was tested in single experiments with wild type as comparison. Therefore, each mutant line is shown in comparison with its own wild type graph to evaluate the disease development. Each disease index bar value shows the mean value ± SE. The Shoot Index also represents the mean value, but because all analyzed plants were pooled for measuring fresh weight and the calculation of the shoot index, the calculation of the SE was not possible. Mean values were calculated from at least two independent experiments with approximately 100 plants per line. Asterisks indicate a significant difference (for <b>**</b> <span class="html-italic">p</span> &lt; 0.01; <b>***</b> <span class="html-italic">p</span> &lt; 0.001). The growth comparison of the aboveground parts of <span class="html-italic">P. brassicae</span> infected <span class="html-italic">A. thaliana</span> plants 26 days after inoculation and control (not inoculated) plants is shown in (<b>D</b>). The pictures show plants from one representative experiment for all tested mutant lines and wild type. OX: overexpression of the indicated <span class="html-italic">LTP</span> gene (<span class="html-italic">LTP1</span>, <span class="html-italic">LTP3</span>, <span class="html-italic">LTP4</span>, <span class="html-italic">AT1G12090</span>, <span class="html-italic">LTP8</span>), WT: wild type, EPG: empty vector control, bar = 1 cm.</p>
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<p>Clubroot development in plants with reduced <span class="html-italic">LTP</span> gene expression. The Disease Index (<b>A</b>), distribution of disease classes (<b>B</b>), and shoot index (<b>C</b>), for T-DNA insertion mutants (KO) and antisense lines (AS) for the indicated <span class="html-italic">LTP</span> gene in comparison to wild type plants (WT) are shown. Each mutant line was tested in single experiments with wild type as comparison. Therefore, each mutant line is shown in comparison with its own wild type graph to evaluate the disease development. Each disease index bar value shows the mean value ± SE. The Shoot Index also represents the mean value but because all analyzed plants were pooled for measuring fresh weight and the calculation of the shoot index, the calculation of the SE was not possible. Mean values were calculated from at least two independent experiments with approximately 100 plants per line. Asterisks indicate a significant difference (for *** <span class="html-italic">p</span> &lt; 0.001). The growth comparison of aboveground parts of <span class="html-italic">P. brassicae</span> infected <span class="html-italic">A. thaliana</span> plants at 26 days after inoculation and not inoculated plants is shown in (<b>D</b>). The pictures show plants from one representative experiment for all tested mutant lines and wild type. KO: T-DNA insertion in the indicated <span class="html-italic">LTP</span> gene (<span class="html-italic">AT1G12090</span>, <span class="html-italic">LTP8</span>, <span class="html-italic">AT3G22620</span>, <span class="html-italic">AT5G05960</span>, <span class="html-italic">LTP3</span>, <span class="html-italic">LTP4</span>), AS: silencing of the indicated gene (<span class="html-italic">AT4G33550</span>, <span class="html-italic">AT1G62510</span>) using the antisense technique, WT: wild type, bar = 1 cm.</p>
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<p>Lipid composition of control roots and galls of <span class="html-italic">A. thaliana</span>. Results of thin layer chromatography from non-polar lipids isolated from equal amounts of infected roots (galls) 30 days after inoculation or of healthy root material (roots) of the same age. Plant material was from wild type (WT) and LTP mutants that overexpress <span class="html-italic">LTP3</span> (<span class="html-italic">LTP3-OX</span>) and from plants with reduced <span class="html-italic">LTP3</span> expression (<span class="html-italic">LTP3-KO</span>). Two biological replicates with approximately 25 plants each were analyzed. The small differences in the fractions (<b>a</b>) and (<b>b</b>) were not consistent between biological replicates. Triacylglycerol (TAG) was used as a standard.</p>
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<p>Regulation of <span class="html-italic">LTP</span> gene expression due to salt, osmotic and drought stress based on microarray data (data taken from Genevestigator [<a href="#B49-plants-05-00002" class="html-bibr">49</a>]).</p>
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<p>Growth reduction due to salt stress conditions. Root growth and whole plant fresh weight from wild type (WT) and LTP overexpressor and mutant lines (<span class="html-italic">LTP1-OX</span>, <span class="html-italic">LTP3-OX</span>, <span class="html-italic">AT4G33550-AS</span> and <span class="html-italic">AT1G62510-AS</span>) in response to salt stress are shown. To calculate the root growth and fresh weight (in %), the values from unstressed plants (0 mM NaCl) were set to 100%. Therefore, the graphs show the growth reduction due to salt stress. Since these graphs show a ratio based on the mean value, the standard deviation could not be plotted on this graph. n ≥ 50. Asterisks indicate a significant difference (for * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001). bar = 1 cm.</p>
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<p>Overview of effects caused by a single LTP mutation or overexpression of one <span class="html-italic">LTP</span> gene. All effects found for the different analyzed LTP mutants are shown. OX: overexpression, KO: knockout or knockdown, AS: antisense.</p>
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1783 KiB  
Article
A Comparative Study of Proteolytic Mechanisms during Leaf Senescence of Four Genotypes of Winter Oilseed Rape Highlighted Relevant Physiological and Molecular Traits for NRE Improvement
by Alexandra Girondé, Marine Poret, Philippe Etienne, Jacques Trouverie, Alain Bouchereau, Françoise Le Cahérec, Laurent Leport, Marie-Françoise Niogret and Jean-Christophe Avice
Plants 2016, 5(1), 1; https://doi.org/10.3390/plants5010001 - 22 Dec 2015
Cited by 9 | Viewed by 6033
Abstract
Winter oilseed rape is characterized by a low N use efficiency related to a weak leaf N remobilization efficiency (NRE) at vegetative stages. By investigating the natural genotypic variability of leaf NRE, our goal was to characterize the relevant physiological traits and the [...] Read more.
Winter oilseed rape is characterized by a low N use efficiency related to a weak leaf N remobilization efficiency (NRE) at vegetative stages. By investigating the natural genotypic variability of leaf NRE, our goal was to characterize the relevant physiological traits and the main protease classes associated with an efficient proteolysis and high leaf NRE in response to ample or restricted nitrate supply. The degradation rate of soluble proteins and D1 protein (a thylakoid-bound protein) were correlated to N remobilization, except for the genotype Samouraï which showed a low NRE despite high levels of proteolysis. Under restricted nitrate conditions, high levels of soluble protein degradation were associated with serine, cysteine and aspartic proteases at acidic pH. Low leaf NRE was related to a weak proteolysis of both soluble and thylakoid-bound proteins. The results obtained on the genotype Samouraï suggest that the timing between the onset of proteolysis and abscission could be a determinant. The specific involvement of acidic proteases suggests that autophagy and/or senescence-associated vacuoles are implicated in N remobilization under low N conditions. The data revealed that the rate of D1 degradation could be a relevant indicator of leaf NRE and might be used as a tool for plant breeding. Full article
(This article belongs to the Special Issue Plant Senescence)
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Figure 1
<p>Changes in N and <sup>15</sup>N amounts in the source leaves in response to restricted (LN, 0.375 mM) or ample (HN, 3.75 mM) nitrate applied for 21 days. The N and <sup>15</sup>N amounts, estimated in HN (<b>a</b>,<b>c</b>) and LN (<b>b</b>,<b>d</b>) conditions, are expressed as mg N·leaf<sup>−1</sup> and µg <sup>15</sup>N·leaf<sup>−1</sup>, respectively. The percentage between brackets corresponds to the decrease in N and <sup>15</sup>N at D21 as a percentage of the initial level (D0). D0: day 0; D14: day 14; D21: day 21. Data are expressed as the mean ± standard error (SE). For each genotype, the statistical differences in kinetics are indicated by letters a, b, c and a difference between N treatments is indicated by an asterisk (<span class="html-italic">n</span> = 3, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Changes in the amounts of soluble proteins and amino acids in the source leaves in response to restricted (LN, 0.375 mM) or ample (HN, 3.75 mM) nitrate applied for 21 days. The amounts of soluble proteins and <sup>15</sup>N, estimated in HN (<b>a</b>,<b>c</b>) and LN (<b>b</b>,<b>d</b>) conditions, are expressed as mg·leaf<sup>−1</sup> and µmol·leaf<sup>−1</sup>, respectively. The percentage between brackets corresponds to the remobilization of soluble proteins and amino acids at D21 as a percentage of the initial level (D0). D0: day 0; D14: day 14; D21: day 21. Data are expressed as the mean ± standard error (SE). For each genotype, the statistical differences in kinetics are indicated by letters a, b, c and a difference between N treatments is indicated by an asterisk (<span class="html-italic">n</span> = 3, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Immunodetection of large (LSU; (<b>a</b>)) and small (SSU; (<b>b</b>)) subunits of Rubisco, D1 protein of the photosystem II (<b>c</b>) and lhcb3 of the light harvesting complex (<b>d</b>) in the source leaves in response to restricted (LN, 0.375 mM) or ample (HN, 3.75 mM) nitrate applied for 21 days. The protein extracts of the three biological repetitions were pooled and 15 µg were loaded per lane for immunodetection. The protein abundance was determined with specific antibodies (see <a href="#sec4-plants-05-00001" class="html-sec">Experimental Section</a> for details). D0: day 0; D14: day 14; D21: day 21. In LN conditions, immunodetection was not performed at D21 because there was not enough protein. The variation in protein abundance (ΔD21 or ΔD14) between D0 and D21 or D0 and D14 (when D21 is not available) is expressed as a percentage of the abundance observed at D0 and is given on the right of each immunoblot.</p>
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<p>Total proteolytic activity at pH 5 at day 14 (D14; (<b>a</b>,<b>b</b>)) and the inhibition of cysteine proteases, serine proteases, metalloproteases and aspartic proteases by specific inhibitors (<b>c</b>,<b>d</b>). The plants were supplied with ample (HN, 3.75 mM; (<b>a</b>,<b>c</b>)) or restricted (LN, 0.375 mM; (<b>b</b>,<b>d</b>)) nitrate supply. The proteolytic activity corresponds to degradation of the Rubisco large subunit (LSU) visualized on Stain Free gels and quantified by Image Lab software (Bio-Rad) after incubation at 37 °C (for details see <a href="#sec4-plants-05-00001" class="html-sec">Experimental Section</a>). The total proteolytic activity was determined with protein extract (PE) without protease inhibitor and is expressed as the percentage of degradation after 20 min of incubation. To determine the contribution of the different classes of proteases, the extract was incubated in the presence of specific protease inhibitors to determine the percentage of inhibition of LSU proteolysis observed without inhibitor (% inhibition; (<b>c</b>,<b>d</b>)). The inhibitors used were: iodoacetamide for cysteine proteases (CPI), aprotinin for serine proteases (SPI), 1–10 phenanthroline for metalloproteases (MPI) and pepstatin A for aspartic proteases (API). Due to the need to dissolve 1–10 phenanthroline and pepstatin A in methanol, the total proteolytic activity was also determined with methanol. The detailed gels are presented in <a href="#plants-05-00001-f009" class="html-fig">Figure A1</a>. For a given genotype, an asterisk is indicates if the percentage of degradation or the percentage of inhibition is at least two-fold increased in response to LN treatment compared with HN conditions.</p>
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<p>Zymograms of the proteolytic activities observed at pH 5 in the source leaves in response to ample (HN, 3.75 mM; (<b>a</b>)) or restricted (LN, 0.375 mM; (<b>b</b>)) nitrate applied for 14 days. The soluble proteins of the three biological repetitions at day 14 (D14) were pooled and 75 µg were loaded per lane. The white bands representing proteolytic activities at pH5 were analyzed to identify the proteases responsible under HN (<b>a</b>) and LN (<b>b</b>) conditions. One aspartic protease was successfully identified and is indicated by the red arrows (see <a href="#plants-05-00001-t002" class="html-table">Table A1</a> for details).</p>
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<p>Total proteolytic activity at pH 7.5 at day 14 (D14; (<b>a</b>,<b>b</b>) and the inhibition of cysteine proteases, serine proteases, metalloproteases, aspartic proteases and proteasome by specific inhibitors (<b>c</b>,<b>d</b>)). The plants were supplied with ample (HN, 3.75 mM; (<b>a</b>,<b>c</b>)) or restricted (LN, 0.375 mM; (<b>b</b>,<b>d</b>)) nitrate supply. The proteolytic activity corresponds to the Rubisco large subunit (LSU) degradation visualized on Stain Free gels and quantified by Image Lab software (Bio-Rad) after an incubation at 37 °C. The total proteolytic activity was determined with protein extract (PE) without protease inhibitor and is expressed as the percentage of degradation after 60 min of incubation. To determine the contribution of the different classes of proteases, the extract was incubated in the presence of specific protease inhibitors to determine the percentage inhibition of LSU proteolysis observed without inhibitor (% inhibition; (<b>c</b>,<b>d</b>)). The inhibitors used were: iodoacetamide for cysteine proteases (CPIs), aprotinin for serine proteases (SPIs), 1–10 phenanthroline for metalloproteases (MPIs), pepstatin A for aspartic proteases (APIs) and MG132 (carbobenzoxy-Leu-Leu-leucinal) for proteasome (PI). Due to the need to dissolve 1–10 phenanthroline and pepstatin A in methanol and MG132 in DMSO, the total proteolytic activity was also determined with methanol and DMSO. The detailed gels are presented in <a href="#plants-05-00001-f010" class="html-fig">Figure A2</a>. For a given genotype, an asterisk is indicates if the % of degradation or the % of inhibition is increased at least two-fold in response to LN treatment compared with HN conditions.</p>
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<p>Abundance of FtsH proteases in the source leaf in response to ample (HN, 3.75 mM) or restricted (LN, 0.375 mM) nitrate applied for 21 days. The protein extracts of the three biological repetitions were pooled and 15 µg were loaded per lane for the detection of FtsH. The protein abundance was determined with specific antibodies (see <a href="#sec4-plants-05-00001" class="html-sec">Experimental Section</a> for details). D0: day 0; D14: day 14; D21: day 21. In LN conditions, immunodetection was not performed at D21 because there was not enough protein. The graph below immunoblots represents the changes of protein abundance.</p>
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<p>Principal component analysis (PCA) of the criteria of N remobilization determined in response to ample (HN, 3.75 mM; (<b>a</b>)) and restricted (LN, 0.375 mM; (<b>b</b>)) nitrate applied for 21 days. In both cases the first axis is mainly associated with the total proteolysis at pH 7.5 at D14 and the second axis refers to the N remobilization between D0 and D14. On the individual factor map, the N remobilization in the source leaves is indicated in color for each genotype. Δ<sup>15</sup>N: variation in the amount of <sup>15</sup>N between D0 and D14; ΔAA: variation in the amount of amino acids between D0 and D14; AP: contribution of aspartic proteases at D14; Av: genotype Aviso; Cali: genotype Californium; CP: contribution of cysteine proteases at D14; ΔD1: variation in the amount of D1 protein between D0 and D14; ΔFtsH: variation in the amount of FtsH between D0 and D14; Δlhcb3: variation in the amount of lhcb3 between D0 and D14; ΔLSU: variation in the amount of the Rubisco large subunit between D0 and D14; MP: contribution of metalloproteases at D14; ΔN: variation in the N amount between D0 and D14; P: contribution of the proteasome at D14; Polysis: total proteolysis activity at D14; Sam: genotype Samouraï; SP: contribution of serine proteases at D14; ΔSProt: variation in the amount of soluble protein between D0 and D14; ΔSSU: variation in the amount of the Rubisco small subunit between D0 and D14.</p>
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<p>Total proteolytic activity at pH 5 (PE) and the inhibition of cysteine proteases (PE + CPI), serine proteases (PE + SPI), metalloproteases (PE + Me + MPI) and aspartic proteases (PE + Me + API) by specific inhibitors at day 14 (D14). The plants were supplied with ample (HN, 3.75 mM) or restricted (LN, 0.375 mM) nitrate supply. The proteolytic activity corresponds to degradation of the Rubisco large subunit (LSU) visualized on Stain Free gels and quantified by Image Lab software (Bio-Rad<sup>®</sup>) after incubation at 37 °C. The incubation was 20 min in LN conditions and 45 min in HN for all genotypes except Californium, which required 30 and 55 min, respectively. The percentage degradation is indicated below the gels. The inhibitors used were: iodoacetamide for cysteine proteases (CPI), aprotinin for serine proteases (SPI), 1–10 phenanthroline for metalloproteases (MPI) and pepstatin A for aspartic proteases (API). Due to the need to dissolve 1–10 phenanthroline and pepstatin A in methanol (Me), the total proteolytic activity was also determined with methanol (PE + Me).</p>
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<p>Total proteolytic activity at pH 7.5 (PE) and the inhibition of cysteine proteases (PE + CPI), serine proteases (PE + SPI), metalloproteases (PE + Me + MPI), aspartic proteases (PE + Me + API) and proteasome (PE + DMSO + PI) by specific inhibitors at day (D14). The plants were supplied with ample (HN, 3.75 mM) or restricted (LN, 0.375 mM) nitrate supply. The proteolytic activity corresponds to the Rubisco large subunit (LSU) degradation visualized on Stain Free gels and quantified by Image Lab software (Bio-Rad<sup>®</sup>) after an incubation at 37 °C. The incubation was 90 min in both N conditions for all genotypes except Oase, which was incubated for only 60 min. The percentage of degradation is indicated below the gels. The inhibitors used were: iodoacetamide for cysteine proteases (CPI), aprotinin for serine proteases (SPI), 1–10 phenanthroline for metalloproteases (MPI), pepstatin A for aspartic proteases (API) and MG132 (carbobenzoxy-Leu-Leu-leucinal) for proteasome (PI). Due to the need to dissolve 1–10 phenanthroline and pepstatin A in methanol (Me) and MG132 in DMSO, the total proteolytic activity was also determined with methanol (PE + Me) and DMSO.</p>
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