Adventitious rooting declines with the vegetative to reproductive switch and involves a changed auxin homeostasis

Adventitious rooting, whereby roots form from non-root tissues, is critical to the forestry and horticultural industries that depend on propagating plants from cuttings. A major problem is that age of the tissue affects the ability of the cutting to form adventitious roots. Here, a model system has been developed using Pisum sativum to differentiate between different interpretations of ageing. It is shown that the decline in adventitious rooting is linked to the ontogenetic switch from vegetative to floral and is mainly attributed to the cutting base. Using rms mutants it is demonstrated that the decline is not a result of increased strigolactones inhibiting adventitious root formation. Monitoring endogenous levels of a range of other hormones including a range of cytokinins in the rooting zone revealed that a peak in jasmonic acid is delayed in cuttings from floral plants. Additionally, there is an early peak in indole-3-acetic acid levels 6h post excision in cuttings from vegetative plants, which is absent in cuttings from floral plants. These results were confirmed using DR5:GUS expression. Exogenous supplementation of young cuttings with either jasmonic acid or indole-3-acetic acid promoted adventitious rooting, but neither of these hormones was able to promote adventitious rooting in mature cuttings. DR5:GUS expression was observed to increase in juvenile cuttings with increasing auxin treatment but not in the mature cuttings. Therefore, it seems the vegetative to floral ontogenetic switch involves an alteration in the tissue’s auxin homeostasis that significantly reduces the indole-3-acetic acid pool and ultimately results in a decline in adventitious root formation

The Silver Lining of a Viral Agent: Increasing Seed Yield and Harvest Index in Arabidopsis by Ectopic Expression of the Potato Leaf Roll Virus Movement Protein1

Ectopic expression of viral movement proteins (MPs) has previously been shown to alter plasmodesmata (PD) function and carbon partitioning in transgenic plants, giving rise to the view of PD being dynamic and highly regulated structures that allow resource allocation to be adapted to environmental and developmental needs. However, most work has been restricted to solanaceous species and the potential use of MP expression to improve biomass and yield parameters has not been addressed in detail. Here we demonstrate that MP-mediated modification of PD function can substantially alter assimilate allocation, biomass production, and reproductive growth in Arabidopsis (Arabidopsis thaliana). These effects were achieved by constitutive expression of the potato leaf roll virus 17-kD MP (MP17) fused to green fluorescent protein (GFP) in different Arabidopsis ecotypes. The resulting transgenic plants were analyzed for PD localization of the MP17:GFP fusion protein and different lines with low to high expression levels were selected for further analysis. Low-level accumulation of MP17 resulted in enhanced sucrose efflux from source leaves and a considerably increased vegetative biomass production. In contrast, high MP17 levels impaired sucrose export, resulting in source leaf-specific carbohydrate accumulation and a strongly reduced vegetative growth. Surprisingly, later during development the MP17-mediated inhibition of resource allocation was reversed, and final seed yield increased in average up to 30% in different transgenic lines as compared to wild-type plants. This resulted in a strongly improved harvest index. The release of the assimilate export block was paralleled by a reduced PD binding of MP17 in senescing leaves, indicating major structural changes of PD during leaf senescence

Regulation of Cell Wall-Bound Invertase in Pepper Leaves by Xanthomonas campestris pv. vesicatoria Type Three Effectors

Xanthomonas campestris pv. vesicatoria (Xcv) possess a type 3 secretion system (T3SS) to deliver effector proteins into its Solanaceous host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that Xcv may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient Xcv strain showed a higher level of cw-Inv mRNA and enzyme activity relative to Xcv wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the pathogenesis-related gene PRQ. These results suggest that Xcv suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify Xcv T3Es that regulate cw-Inv activity, a screen was performed with eighteen Xcv strains, each deficient in an individual T3E. Seven Xcv T3E deletion strains caused a significant change in cw-Inv activity compared to Xcv wild type. Among them, Xcv lacking the xopB gene (Xcv ΔxopB) caused the most prominent increase in cw-Inv activity. Deletion of xopB increased the mRNA abundance of PRQ in Xcv ΔxopB-infected pepper leaves, but not of Pti5 and Acre31, two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited Xcv-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity in planta to suppress sugar-enhanced defense responses during Xcv infection

Regulation of Cell Wall-Bound Invertase in Pepper Leaves by <em>Xanthomonas campestris</em> pv. <em>vesicatoria</em> Type Three Effectors

<div><p><em>Xanthomonas campestris</em> pv. <em>vesicatoria (Xcv)</em> possess a type 3 secretion system (T3SS) to deliver effector proteins into its <em>Solanaceous</em> host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that <em>Xcv</em> may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient <em>Xcv</em> strain showed a higher level of cw-Inv mRNA and enzyme activity relative to <em>Xcv</em> wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the <em>pathogenesis-related</em> gene <em>PRQ.</em> These results suggest that <em>Xcv</em> suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify <em>Xcv</em> T3Es that regulate cw-Inv activity, a screen was performed with eighteen <em>Xcv</em> strains, each deficient in an individual T3E. Seven <em>Xcv</em> T3E deletion strains caused a significant change in cw-Inv activity compared to <em>Xcv</em> wild type. Among them, <em>Xcv</em> lacking the <em>xopB</em> gene (<em>Xcv</em> Δ<em>xopB</em>) caused the most prominent increase in cw-Inv activity. Deletion of <em>xopB</em> increased the mRNA abundance of <em>PRQ</em> in <em>Xcv</em> Δ<em>xopB-</em>infected pepper leaves, but not of <em>Pti5</em> and <em>Acre31,</em> two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited <em>Xcv</em>-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity <em>in planta</em> to suppress sugar-enhanced defense responses during <em>Xcv</em> infection.</p> </div

Content of soluble sugars in susceptible pepper leaves after infection with <i>Xcv</i> wild type or with the TTSS<i>-</i>deficient <i>Xcv</i> Δ<i>hrpB1</i> strain.

<p>Contents of glucose, fructose and sucrose were determined following inoculation of pepper leaves with <i>Xcv</i> wild type (wt) or the <i>Xcv</i> Δ<i>hrpB1</i> using a concentration of 5×10⁸ cfu ml⁻¹ and compared to 10 mM MgCl₂ infiltrated control leaves. Samples were taken before (0 h), 24 and 48 hours post infection (hpi). Each value represents the mean ± SE of four different experiments each with four to six individual samples. Statistically significant differences to Mock-inoculated control plants were determined using two-tailed t-test assuming normal distribution and are indicated by asterisks (*p<0.05).</p

Screening for <i>Xcv</i> T3Es involved in regulation of cw-Inv activity.

<p>Leaves of pepper plants were infiltrated with wild type and mutant <i>Xcv</i> strains at 10⁹ cfu ml⁻¹ and cw-Inv activity was measured 2 and 3 days post infection (dpi) in independent experiments. Graphs represent values calculated relative to the <i>Xcv</i> wild type (wt) response which was set to 100% for each individual experiment. Mean cw-Inv activities after infection with <i>Xcv</i> wild type were 20.96 µmol min⁻¹ m⁻²±8.43 (100% ±40.2) and 57.57 µmol min⁻¹ m⁻²±23.92 (100% ±41.6) at 2 and 3 dpi, respectively. The variance of <i>Xcv</i> wild type response (ca. 42%) is illustrated as a dashed line. Values are the mean response (as percentage to <i>Xcv</i> wild type) ± SD from three to nine different experiments. Statistically significant differences from <i>Xcv</i> wild type response were determined using two-tailed t-test assuming normal distribution and are indicated by asterisks (**p<0.01); (***p<0.001).</p

Inducible <i>xopB</i> expression in transgenic tobacco plants causes severe leaf abnormality.

<p>A.) Analysis of <i>xopB</i>-specific transcript accumulation in transgenic tobacco lines. Seven different lines (No. 22, 26, 37, 44, 64, 71, 72) and two control plants (wt) were analysed for <i>xopB</i> expression by Northern blotting. Total RNA was isolated 1 day after watering plants with 1% ethanol to induce <i>xopB</i> expression. Twenty µg of RNA were separated on a formaldehyde-containing agarose gel and analysed by hybridization with a <i>xopB</i>-specific radioactively labelled probe. Ethidium bromide stained rRNA is shown as loading control. B.) Analysis of XopB protein accumulation upon watering with 1% ethanol in selected transgenic lines (#22, #71). XopB migrates at ∼70 kDa, while in tobacco a cross-reactive band appeared at ∼55kDa. Expression of RubisCO as stained by Coomassie Blue is shown as control for protein loading. C.) Phenotypic changes in transgenic tobacco plants caused by <i>xopB</i> expression. Upper panel: symptoms 2 days after ethanol-treatment; lower panel: phenotypic alterations 10 days after induction. Arrows indicate morphological changes of the leaf lamina and cell death of meristematic tissue, respectively. From left to right: control line, lines #22 and #71.</p

Expression of PTI marker genes <i>Pti5</i> and <i>Acre31</i> in susceptible pepper leaves after infection with different <i>Xcv</i> strains.

<p>Leaves of pepper plants were inoculated with <i>Xcv</i> wild type (wt), <i>Xcv</i> Δ<i>hrpB1</i>, <i>Xcv</i> Δ<i>xopB</i> using a concentration of 10⁹ cfu ml⁻¹, and with 10 mM MgCl₂. Total RNA was isolated from samples taken before (0 h), 6 h and 24 h after infiltration and reverse transcribed into cDNA. Abundance of <i>Pti5</i> (A.) and <i>Acre31</i> (B.) mRNA was detected by qPCR. Data were analysed using MxPro software v4.1. The expression levels of <i>Pti5</i> and <i>Acre31</i> were normalized with <i>Actin</i> and displayed relative to the expression level at time point 0 h which was set to a value of 1. The average ± SE of three replicates is shown. Similar results were obtained in an independent experiment. White bars, MgCl₂; light grey, <i>Xcv</i> wild type; black, <i>Xcv</i> Δ<i>hrpB1</i>, dark grey, <i>Xcv</i> Δ<i>xopB</i>.</p

Expression of <i>cw-Inv</i>, <i>PRQ</i> and <i>RbcS</i> in susceptible pepper leaves in response to infection with <i>Xcv</i> Δ<i>xopB</i>.

<p>Leaves of pepper plants were infected with the <i>Xcv</i> wild type (wt), <i>Xcv</i> Δ<i>hrpB1</i>, <i>Xcv</i> Δ<i>xopB</i> using a concentration of 10⁹ cfu ml⁻¹, and as control with 10 mM MgCl₂.Total RNA was isolated from pepper leaves before (0), and 1, 2, 3 days post infection (dpi). Twenty five µg of total RNA was separated per each lane. Northern blots were hybridized with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051763#pone.0051763-Dean1" target="_blank">[³²]</a>P dCTP-labelled cDNA fragments of <i>cw-Inv</i>, <i>PRQ</i> and <i>RbcS.</i> A representative experiment is shown. Similar results were obtained in two other experiments.</p

Inducible <i>xopB</i> expression in transgenic tobacco leaves suppresses cw-Inv activity during <i>Xcv</i> infection.

<p>Control plants and two selected transgenic tobacco lines with inducible <i>xopB</i> expression (#22, #71) were watered with 1% ethanol. After 24 h, plantlets were inoculated with a 10⁹ cfu ml⁻¹ suspension of <i>Xcv</i> wild type. Samples were taken directly before inoculation and 1 and 2 days post inoculation (1dpi +EtOH; 2dpi <i>Xcv</i>+ EtOH). Non-ethanol watered plants were also inoculated with <i>Xcv</i> and samples were taken accordingly (1dpi -EtOH; 2dpi -EtOH). For control purposes ethanol-treated and non-treated plants were inoculated with 10 mM MgCl₂. Cw-Inv activity was determined from four independent samples and fold changes ± SD were calculated for each sample relative to values obtained before <i>Xcv</i> inoculation. The experiment was repeated with similar results.</p