Modulation of harpin-triggered apoplastic alkalinisation by different auxins.

<p>Cells were treated with 9 μg ml^-1 harpin (hrp, closed circles) as a positive control, harpin combined with 10 μM (open triangles) or 50 μM auxins (IAA, NAA, or 2, 4-D, closed triangles), or ethanol used as solvent control (con, open circles) in <i>V</i>. <i>rupestris</i> (<b>A</b>, <b>C</b>, and <b>E</b>) and cv. ‘Pinot Noir’ (<b>B</b>, <b>D</b>, and <b>F</b>). Representative experiments from five biological replicas are depicted. Harpin and auxin were added at time 0, if measured isolated, for the combinations, auxins were added 1 h prior to harpin.</p

Effect of Latrunculin and auxins on harpin-induced changes of cell viability.

<p>Cells were treated with Latrunculin B (Lat B, 2 μM), with harpin (hrp, 9 μg ml^-1), or with LatB in combination with harpin and auxin (IAA, NAA or 2, 4-D, 50 μM) following subculture weekly, versus ethanol as solvent control (con) in <i>V. rupestris</i> (A) and cv. ‘Pinot Noir’ (B). Data show mean and standard errors from three independent experiments with 500 cells. Significance levels of differences was analyzed using ANOVA with * significant at P = 5%, and ** significant at P = 1%.</p

Effect of auxins on the induction of defence genes by harpin.

<p>Expression of selected defence genes was conducted by semiquantitative RT-PCR in response to harpin (hrp, 9 μg ml^-1), auxins (IAA, NAA or 2, 4-D, 50 μM), or auxins combined with harpin as compared to ethanol as solvent control (con) in <i>V. rupestris</i> (<b>A</b>) and cv. ‘Pinot Noir’ (<b>B</b>). Transcript abundance was analysed 30 min after addition of harpin preceded by incubation for 60 min with the respective auxins. Genes included <i>StSy</i>, stilbene synthase; <i>PAL</i>, phenylalanine ammonia lyase 1; <i>PR</i>5, <i>PR</i>10, pathogenesis-related proteins 5 and 10). Quantification of transcripts were calculated relative to elongation factor 1α (EF1 α) as internal standard. The data represent mean values from three independent experimental series; error bars show standard errors. Expression difference of defence gene as compared to solvent control were analyzed using ANOVA with * significant at <i>P</i> = 5%, and ** significant at <i>P</i> = 1%.</p

Working model on the antagonistic interaction of signalling triggered by harpin and auxin.

<p>To reduce complexity, only the earliest events are depicted, omitting ROS activation of calcium influx and effects of rac1-signalling on auxin transport. ① harpin activates the NADPH-dependent oxidase RboH leading to the production of superoxide that can spread in the apoplast. ② Superoxide can penetrate through the plasma membrane (probably by aquaporins) and glutathionylate actin in residue Cys374. This will sequester G-actin from being integrated into the growing end of actin filaments. ③ Alternatively, superoxide can be recruited to transduce the effect of auxin (perceived via the auxin-binding protein, ABP) upon the activation of phospholipase D (PLD) through the small G-protein Rac. ④ PLD will generate phosphatidic acids (PA) that can sequester actin capping proteins (cap) to the membrane, such that elongation of actin filaments is enabled. Alternatively, PA can be partitioned to recruit Rac for the activation of the RboH complex. In this case, the capping proteins will not be recruited to the membrane and constrain the elongation of actin filaments leading as secondary consequence to the formation of thick cables through the activity of severing proteins in combination with free G-actin the formation. ⑤ As third alternative, PA can be converted to PIP2, which will recruit actin-depolymerization factor (ADF) to the membrane. Since ADF is sustaining the monomer turnover at the minus end of actin filaments, this recruitment results in a higher stability of fine cortical actin filaments. The molecular targets for the inhibitors diphenyliodonium (DPI), and <i>n</i>-butanol are inserted in red. Hypothetical aspects of the model that have not been addressed experimentally in plant cells, are indicated by blue question marks: The interaction of Harpin with RboH (①) has not been addressed experimentally so far. Also the glutathionylation of actin in consequence of superoxide penetration (②), so far has been shown for animal systems, but not been addressed in plant cells.</p

Quantification of actin responses by measuring apparent width (w) of actin bundles.

<p><b>A</b> Time course of actin bundling in response to 9 μg^.ml^-1 harpin. <b>B</b> Dose-dependency of harpin-induced actin bundling over different concentrations of either the natural auxin indole-3-acetic acid (IAA), or the artificial auxins 1-napthyl acetic acid (NAA), or 2,4-dichlorophenoxy acid (2,4-D) as compared to the control (con) or harpin (hrp) in the absence of supplemental auxins measured after 60 min of incubation in <i>V</i>. <i>rupestris</i> and cv. ‘Pinot Noir’. Concentrations of auxins indicated in μM. "n.d." means "not determined" to indicate that this value was not measured in cv. ‘Pinot Noir’ (to distinguish it from a zero value). <b>C</b> Effect of pharmacological inhibition of phospholipase D by preincubation with 0.5% (v/v) of <i>n</i>-butanol or the inactive analogue <i>sec</i>-butanol for 30 min prior to the indicated treatment for 30 min, and effect of pharmacological inhibition of RboH on harpin-induced bundling by preincubation with 20 μM dephenylene iodonium (DPI) for 30 min prior to the indicated treatment for 30 min. <b>IAA</b> 10 μM indole-3-acetic acid, <b>hrp</b> 9 μg^.ml^-1 harpin. Arrows represent complete elimination of actin, such that no value for bundling could be measured. <b>n</b> represents the number of individual cells per sample, error bars standard errors for a population randomly collected from three biological replicates and 200 cells were used for each experiment. Brackets indicate significance levels of differences with * significant at <i>P</i> = 5%, and ** significant at <i>P</i> = 1%. "n.d." means "not determined" to indicate that this value was not measured (to distinguish it from a zero value).</p

Auxin quells the stimulation of oxidative burst induced by harpin.

<p>Concentrations of the lipid peroxidation product malone dialdehyde (MDA) were determined as readout for superoxide-activity in cells of V. rupestris either treated with harpin alone or following pretreatment with 50 μM IAA for 1 h. The response was scored 30 min after addition of 9 μg ml^-1 harpin. As negative control, cells were incubated with culture medium. Values represent means and standard errors from three independent biological replicas for the increase in MDA over the control. Brackets indicate significance levels of differences with * significant at <i>P</i> = 5%, and ** significant at <i>P</i> = 1% as tested by a paired t-test.</p

Tracking Se Assimilation and Speciation through the Rice Plant – Nutrient Competition, Toxicity and Distribution

<div><p>Up to 1 billion people are affected by low intakes of the essential nutrient selenium (Se) due to low concentrations in crops. Biofortification of this micronutrient in plants is an attractive way of increasing dietary Se levels. We investigated a promising method of Se biofortification of rice seedlings, as rice is the primary staple for 3 billion people, but naturally contains low Se concentrations. We studied hydroponic Se uptake for 0–2500 ppb Se, potential phyto-toxicological effects of Se and the speciation of Se along the shoots and roots as a function of added Se species, concentrations and other nutrients supplied. We found that rice germinating directly in a Se environment increased plant-Se by factor 2–16, but that nutrient supplementation is required to prevent phyto-toxicity. XANES data showed that selenite uptake mainly resulted in the accumulation of organic Se in roots, but that selenate uptake resulted in accumulation of selenate in the higher part of the shoot, which is an essential requirement for Se to be transported to the grain. The amount of organic Se in the plant was positively correlated with applied Se concentration. Our results indicate that biofortification of seedlings with selenate is a successful method to increase Se levels in rice.</p></div

Presentation1.pdf

<p>It is widely known that numerous adaptive responses of drought-stressed plants are stimulated by chemical messengers known as phytohormones. Jasmonic acid (JA) is one such phytohormone. But there are very few reports revealing its direct implication in drought related responses or its cross-talk with other phytohormones. In this study, we compared the morpho-physiological traits and the root proteome of a wild type (WT) rice plant with its JA biosynthesis mutant coleoptile photomorphogenesis 2 (cpm2), disrupted in the allene oxide cyclase (AOC) gene, for insights into the role of JA under drought. The mutant had higher stomatal conductance, higher water use efficiency and higher shoot ABA levels under severe drought as compared to the WT. Notably, roots of cpm2 were better developed compared to the WT under both, control and drought stress conditions. Root proteome was analyzed using the Tandem Mass Tag strategy to better understand this difference at the molecular level. Expectedly, AOC was unique but notably highly abundant under drought in the WT. Identification of other differentially abundant proteins (DAPs) suggested increased energy metabolism (i.e., increased mobilization of resources) and reactive oxygen species scavenging in cpm2 under drought. Additionally, various proteins involved in secondary metabolism, cell growth and cell wall synthesis were also more abundant in cpm2 roots. Proteome-guided transcript, metabolite, and histological analyses provided further insights into the favorable adaptations and responses, most likely orchestrated by the lack of JA, in the cpm2 roots. Our results in cpm2 are discussed in the light of JA crosstalk to other phytohormones. These results together pave the path for understanding the precise role of JA during drought stress in rice.</p

Mean linear combination fitting results & R-factor for Se speciation in plant tissue after treatment with 500, 2000 or 10000 μg/L Se as selenate or selenite.

<p>Mean linear combination fitting results & R-factor for Se speciation in plant tissue after treatment with 500, 2000 or 10000 μg/L Se as selenate or selenite.</p

comparison of rice root growth for the additions of 0, 1000 and 2500 μg/L Se as Na₂SeO₃ or Na₂SeO₄ to agar of the nutrient-free, direct Se exposure experiment.

<p>comparison of rice root growth for the additions of 0, 1000 and 2500 μg/L Se as Na₂SeO₃ or Na₂SeO₄ to agar of the nutrient-free, direct Se exposure experiment.</p