The role of endogenous strigolactones and their interaction with ABA during the infection process of the parasitic weed Phelipanche ramosa in tomato plants

The root parasitic plant species Phelipanche ramosa, branched broomrape, causes severe damage to economically important crops such as tomato. Its seed germination is triggered by host-derived signals upon which it invades the host root. In tomato, strigolactones (SLs) are the main germination stimulants for P. ramosa. Therefore, the development of low SL-producing lines may be an approach to combat the parasitic weed problem. However, since SLs are also a plant hormone controlling many aspects of plant development, SL deficiency may also have an effect on post-germination stages of the infection process, during the parasite-host interaction. In this study, we show that SL-deficient tomato plants (Solanum lycopersicum; SlCCD8 RNAi lines), infected with pre-germinated P. ramosa seeds, display an increased infection level and faster development of the parasite, which suggests a positive role for SLs in the host defense against parasitic plant invasion. Furthermore, we show that SL-deficient tomato plants lose their characteristic SL-deficient phenotype during an infection with P. ramosa through a reduction in the number of internodes and the number and length of secondary branches. Infection with P. ramosa resulted in increased levels of abscisic acid (ABA) in the leaves and roots of both wild type and SL-deficient lines. Upon parasite infection, the level of the conjugate ABA-glucose ester (ABA-GE) also increased in leaves of both wild type and SL-deficient lines and in roots of one SL-deficient line. The uninfected SL-deficient lines had a higher leaf ABA-GE level than the wild type. Despite the high levels of ABA, stomatal aperture and water loss rate were not affected by parasite infection in the SL-deficient line, while in wild type tomato stomatal aperture and water loss increased upon infection. Future studies are needed to further underpin the role that SLs play in the interaction of hosts with parasitic plants and which other plant hormones interact with the SLs during this process

Auxin Controls \u3ci\u3eArabidopsis\u3c/i\u3e Adventitious Root Initiation by Regulating Jasmonic Acid Homeostasis

Vegetative shoot-based propagation of plants, including mass propagation of elite genotypes, is dependent on the development of shoot-borne roots, which are also called adventitious roots. Multiple endogenous and environmental factors control the complex process of adventitious rooting. In the past few years, we have shown that the auxin response factors ARF6 and ARF8, targets of the microRNA miR167, are positive regulators of adventitious rooting, whereas ARF17, a target of miR160, is a negative regulator. We showed that these genes have overlapping expression profiles during adventitious rooting and that they regulate each other’s expression at the transcriptional and posttranscriptional levels by modulating the homeostasis of miR160 and miR167. We demonstrate here that this complex network of transcription factors regulates the expression of three auxin-inducible Gretchen Hagen3 (GH3) genes, GH3.3, GH3.5, and GH3.6, encoding acyl-acid-amido synthetases. We show that these three GH3 genes are required for fine-tuning adventitious root initiation in the Arabidopsis thaliana hypocotyl, and we demonstrate that they act by modulating jasmonic acid homeostasis. We propose a model in which adventitious rooting is an adaptive developmental response involving crosstalk between the auxin and jasmonate regulatory pathways

Enzymatic activities in response to external JA application.

<p>Acid phosphatase activities (A), proteolytic activities (B) and activities measured after inhibition with 10 µM cysteine proteinase inhibitor E-64. Activity without inhibitor of given variant is 100 % (C). ** - significant differences at <i>P</i>  =  0.01 (Student's t-test), means ± s.e., n  =  6–7.</p

Double trigger mechanism for protein digestion.

<p>Three control points in prey capture and digestion of Venus flytrap, which ensure effective production of digestive enzymes. For detailed description, see discussion. AP – action potential, the increase of phytohormone level is indicated by arrows: no increase (-), small (↑), moderate (↑↑), high (↑↑↑).</p

An improved strategy to analyse strigolactones in complex sample matrices using UHPLC-MS/MS

Background: Strigolactones represent the most recently described group of plant hormones involved in many aspects of plant growth regulation. Simultaneously, root exuded strigolactones mediate rhizosphere signaling towards beneficial arbuscular mycorrhizal fungi, but also attract parasitic plants. The seed germination of parasitic plants induced by host strigolactones leads to serious agricultural problems worldwide. More insight in these signaling molecules is hampered by their extremely low concentrations in complex soil and plant tissue matrices, as well as their instability. So far, the combination of tailored isolation - that would replace current unspecific, time-consuming and labour-intensive processing of large samples - and a highly sensitive method for the simultaneous profiling of a broad spectrum of strigolactones has not been reported. Results: Depending on the sample matrix, two different strategies for the rapid extraction of the seven structurally similar strigolactones and highly efficient single-step pre-concentration on polymeric RP SPE sorbent were developed and validated. Compared to conventional methods, controlled temperature during the extraction and the addition of an organic modifier (acetonitrile, acetone) to the extraction solvent helped to tailor strigolactone isolation from low initial amounts of root tissue (150 mg fresh weight, FW) and root exudate (20 ml), which improved both strigolactone stability and sample purity. We have designed an efficient UHPLC separation with sensitive MS/MS detection for simultaneous analysis of seven natural strigolactones including their biosynthetic precursors - carlactone and carlactonoic acid. In combination with the optimized UHPLC-MS/MS method, attomolar detection limits were achieved. The new method allowed successful profiling of seven strigolactones in small exudate and root tissue samples of four different agriculturally important plant species - sorghum, rice, pea and tomato. Conclusion: The established method provides efficient strigolactone extraction with aqueous mixtures of less nucleophilic organic solvents from small root tissue and root exudate samples, in combination with rapid single-step pre-concentration. This method improves strigolactone stability and eliminates the co-extraction and signal of matrix-associated contaminants during the final UHPLC-MS/MS analysis with an electrospray interface, which dramatically increases the overall sensitivity of the analysis. We show that the method can be applied to a variety of plant species.</p

Protein pattern, Western blot and zymogram in response to mechanical and chemical stimulation.

<p>Silver-stained SDS-polyacrylamide gel containing proteins in <i>D. muscipula</i> digestive fluid after mechanical (M), P(K) and N(Cl) stimulation (A). The same amount of proteins was electrophoresed in 12% (v/v) SDS-polyacrylamide gel and subjected to Western blot analysis using antibodies against dionain-1 (B). Detection of protease activity in 12% (v/v) SDS-polyacrylamide gel with casein as a substrate (C). The clear bands against background indicate protease activity. Representative gels at least from 4 repetitions are shown.</p

Electrical activity measured by extracellular electrode on trap surface.

<p>Mechanical irritation delivered in 10 minutes intervals (A), detailed view on action potentials (B) and chemical stimulation with NH₄Cl and KH₂PO₄ did not trigger any AP (C).</p

Traps of <i>Dionaea muscipula</i> in response to mechanical stimulation.

<p>An open trap (A), the same trap 1 min after double mechanical stimulation of trigger hairs (B) and 3 hours later after repeated mechanical stimulation in narrowed phase (C).</p

Enzymatic activities in response to mechanical and chemical stimulation after 24 (black bars) and 48 hours (white bars).

<p>Acid phosphatase activity (A) and proteolytic activity (B). Different letters denote significant differences at the same time interval at <i>P</i> < 0.05 (ANOVA, Tukey-test), means ± s.e., n  =  7–9.</p