Furch et al., 2007:Forisome reaction in the sieve element of Vicia faba in response to cutting the main vein (at a distance of 3 cm upstream from the observation window) as recorded by confocal laser scanning microscopy. The phloem tissue was double-stained with the voltage-sensitive membrane dye RH-414 (red) and CMEDA/CMFDA (green) to stain the forisome. (Top) Bare-lying phloem tissue of an intact Vicia faba plant briefly before the wounding showing a condensed forisome in the SE (asterisk). Note the forked end of the forisome indicative of the presence of subunits. (Middle) The forisome disperses in response to cutting despite the apparent absence of an electrical potential wave. An increase of RH-414 fluorescence as with distant burning was never observed. (Bottom) The forisome recondenses some minutes after cutting. The bright fluorescent spots represent chloroplasts in the CCs and parenchyma cells around the SE. The arrowheads mark the sieve plate. The direction of translocation is from top to bottom. SE, sieve element; CC, companion cell
Actual main research and methods: Using diverse microscopic techniques, biochemical and molecular biological methods
I) The role of sieve-element occlusion proteins in root pathogenesis and phloem signalling
After MAMP application to Arabidopsis thaliana roots the first and most rapid reaction is the transmission of an electrical signal along the plasma membrane of phloem cells. The membrane depolarization leads to a Ca2+ increase in sieve elements, resulting in filament formation of P-proteins and, later on, callose deposition at sieve plates and pore plasmodesma units (PPUs). This topic will be investigated by Dr. Matthias R Zimmermann in cooperation with Prof. Dr. Dirk Prüfer (Fraunhofer Aachen) and Dr. Gundula Noll (Uni Münster).
Furch et al., unpubl.: Confocal images of AtSEO:hrGFP fluorescence in intact Arabidopsis roots using the multiphoton laser.
A) Small agglomerates occurred in sieve elements, especially at sieve plates as described before (Froelich et al. 2011). B) Application of flg22 (1.25 μM) to the root induced the formation of fluorescent apparent protein fibrils which initiate sieve-tube occlusion. Plugs at sieve plates are marked with white arrows and occlusion of whole sieve element with a white circle. The red color represents the autofluorescence and green represents the GFP fluorescence.
II) The impact of phytoplasma infection on phloem function
Phytoplasmas are prokaryotic plant pathogens of the class Mollicutes, inhabit almost exclusively the phloem sieve elements and are transferred by insect vectors. Phytoplasma infection severely impairs photoassimilate translocation in plants and might be responsible for changes in phloem physiology. Infections brings about Ca2+ influx into sieve elements, changes in phytohormone distribution and sieve element occlusion (Pagliari et al., 2017). This topic will be investigated by Dr. George Ochieng Asudi (Humboldt Fellowship, Kenja) in cooperation with the Julius Kühn Institute Dossenheim and the University Udine, Italy.
Zimmermann et al., 2015: Phytoplasma life cycle
III) Vascular exudation process
The typical response of cucurbit plants to mechanical wounding is an immediate occurrence of several, rapidly increasing, single droplets at wound surfaces that merge to one single drop and cover the entire wound surface area at the end. The wound-induced exudation reflects the first, rapid defence response to mechanical damage of plant tissue with an orchestrated loss of vascular content for plant health and tissue integrity. We will analyse quantitatively diverse parameter influencing the exudation process respectively exudates volume and allows deeper insights into the exudation physiology and the cooperation of phloem and xylem during the exudation process.
Zimmermann et al., 2013: Model of the exudation mechanism in cucurbits.
Xylem and phloem are parallel orientated vascular tissues in which pressure and tension gradients are built up in sieve tubes and xylem vessels (X), respectively. In intact plants, the negative water potential in X (ΨW (X)) is in balance with that inside sieve tubes (ΨW (SE)) (a). At once, the water columns in the X are destroyed by cutting, the hydrostatic potential in the X will approach atmospheric positive pressure. As result, water is withdrawn from the vessels by the living adjacent cells including the sieve tubes. The incoming water dilutes the sieve tube sap and increases the turgor of the sieve tubes. By implication, exudation is promoted and the exudates represent diluted phloem sap. Furthermore, the increased flow rate and water influx possibly stimulate detachment of the PP1 and PP2 (b). The latter coagulate by oxygenation at the wound surface being exposed to air. Exuding droplets are covered by a hardening layer and occlude the sieve tubes, while air is sucked into the X bringing about some degree of sealing. In the next stage, the exuding droplet extends and starts covering and occluding the entire vascular surface. Hence, the exudation comes to a stop (c).When the gelous droplets are removed (after more than 2 min, when the internal sieve tube occlusion is completed), exudation of pure xylem sap resumes after recovery of the root pressure. Occasionally, root pressure is strong enough to burst the remained gelled crust to drive xylem exudation (d; see a). CC, companion cell; SE, sieve element; SP, sieve plate; PC, parenchyma cell; X, xylem vessels.
IV) The interaction of xylem- and phloem-compounds during pathogen attack
The lateral exchange of phytohormones between xylem and phloem after treatment with pathogens like Pseudomonas syringae, Golovinomyces orontii and Turnip Crincle Virus and with the beneficial fungus Piriformospora indica shall illustrate a collaboration during short- and long-distance signalling. (In cooperation with PD Dr. Axel Mithöfer; MPI, Jena)
Furch et al., 2014: Analysis of phytohormones in phloem and xylem exudates of unstressed Cucurbita maxima plants.The concentrations (nM) of the phytohormones cis-12-oxo-phytodienoic acid (cis-OPDA), jasmonic acid (JA), (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), salicylic acid (SA), and abscisic acid (ABA) are individually determined in phloem (■) and xylem (□) exudates from the same site of a stem-cut plant (nplant = 8). The dilution of phloem exudates with xylem water is considered with a factor of 8 (■) to respect the in vivo situation (Zimmermann et al., 2013). The statistical significance is tested with one-way ANOVA by pairwise comparison of xylem and phloem exudates (also phloem 8x) with Student´s t-test (p < 0.05) and marked with different letters (superscript apostrophe for phloem 8x). Vertical lines represent standard deviations of eight replicates.
V) Mechanisms for maintaining the diversity of host races in the pea aphid
There are more species of herbivorous insects on our planet than almost any other group of animals outside of microbes. To explain this enormous proliferation of species, we are studying how insect herbivore species diversify by host plant shifting using the pea aphid (Acyrthosiphon pisum) as our model. PhD student Maria Paulmann will explore potential plant-aphid interactions, will measure the phloem mass flow, forisome configuration, hormone levels and defense metabolites and genes in selected combinations of pea aphid host races and legume host plants.
Supervisors: Grit Kunert and Jonathan Gershenzon (Department of Biochemistry, MPICE); Alexandra CU Furch and Ralf Oelmüller (Friedrich-Schiller-University, Jena)