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Aphids, which constitute one of the most important groups of agricultural pests, ingest nutrients from sieve tubes, the photoassimilate transport conduits in plants. Aphids are able to successfully puncture sieve tubes with their piercing mouthparts (stylets) and ingest phloem sap without eliciting the sieve tubes' normal occlusion response to injury. Occlusion mechanisms are calcium-triggered and may be prevented by chemical constituents in aphid saliva injected into sieve tubes before and during feeding. We recorded aphid feeding behavior with the electrical penetration graph (EPG) technique and then experimentally induced sieve tube plugging. Initiation of sieve tube occlusion caused a change in aphid behavior from phloem sap ingestion to secretion of watery saliva. Direct proof of \"unplugging\" properties of aphid saliva was provided by the effect of aphid saliva on forisomes. Forisomes are proteinaceous inclusions in sieve tubes of legumes that show calcium-regulated changes in conformation between a contracted state (below calcium threshold) that does not occlude the sieve tubes and a dispersed state (above calcium threshold) that occludes the sieve tubes. We demonstrated in vitro that aphid saliva induces dispersed forisomes to revert back to the nonplugging contracted state. Labeling Western-blotted saliva proteins with ⁴⁵Ca²⁺ or ruthenium red inferred the presence of calcium-binding domains. These results demonstrate that aphid saliva has the ability to prevent sieve tube plugging by molecular interactions between salivary proteins and calcium. This provides aphids with access to a continuous flow of phloem sap and is a critical adaptation instrumental in the evolutionary success of aphids.

Aphids feed from sieve tubes deep inside the host plant. Therefore, aphids must be able to recognize their host plant(s) and to direct their stylets which must be long and thin enough to reach and puncture the sieve tubes at a particular site. Sieve tubes in angiosperms are longitudinal arrays of sieve element/companion cell modules which are highly sensitive to disturbance of any kind. The sieve tubes dispose of elaborate sealing mechanisms such as protein plugging and callose sealing which are triggered by a rise in calcium in the sieve tubes. Aphids seem to have developed a range of physical and chemical measures to limit the amount of calcium influx in response to stylet puncturing. Loss of sieve-element turgor pressure induced by stylet insertion is minimized by the minute stylet volume. Turgor-dependent Ca2+ influx, possibly mediated by mechano sensitive Ca2+ channels, must therefore be limited. The components of the sheath and watery saliva play a pivotal role in establishing the physical and chemical constraints on the rise of calcium. Most likely, sheath saliva prevents the influx of calcium from the apoplast by sealing the stylet puncture site while watery saliva may prevent plugging and sealing of sieve plates by potential interaction with SE sap ingredients.

$\\bullet$There is growing evidence that multivesicular bodies and cell wall-associated paramural bodies participate in the enhanced vesicle trafficking induced by pathogen attack.$\\bullet$Here, we performed transmission electron microscopy in combination with cytochemical localization of H2O 2to investigate multivesicular compartments during establishment of compatible interaction in susceptible barley (Hordeum vulgare) and during hypersensitive response in resistant MLA12-barley infected by the barley powdery mildew fungus (Blumeria graminis f. sp. hordei).$\\bullet$Multivesicular bodies, intravacuolar vesicle aggregates and paramural bodies proliferated in the penetrated epidermal cell during development of the fungal haustorium. These vesicular structures also proliferated at the periphery of intact cells, which were adjacent to the hypersensitive dying cells and deposited cell wall appositions associated with H2O 2accumulation. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions.$\\bullet$These results suggest that multivesicular compartments participate in secretion of building blocks for cell wall appositions not only to arrest fungal penetration but also to contain hypersensitive cell death through blocking plasmodesmata. They may also participate in internalization of damaged membranes, deleterious materials, nutrients, elicitors and elicitor receptors.

The relationships between damage-induced electropotential waves (EPWs), sieve tube occlusion, and stop of mass flow were investigated in intact Cucurbita maxima plants. After burning leaf tips, EPWs propagating along the phloem of the main vein were recorded by extra- and intracellular microelectrodes. The respective EPW profiles (a steep hyperpolarization/depolarization peak followed by a prolonged hyperpolarization/depolarization) probably reflect merged action and variation potentials. A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring ~10 min after burning. Early stop of mass flow, well before completion of callose deposition, pointed to an occlusion mechanism preceding callose deposition. This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids. The early occlusion is probably due to proteins, as indicated by a dramatic drop in soluble sieve element proteins and a simultaneous coagulation of sieve element proteins shortly after the burning stimulus. Mass flow resumed 30-40 min after burning, as demonstrated by carboxyfluorescein movement and aphid activities. Stop of mass flow by Ca²⁺-dependent occlusion mechanisms is attributed to Ca²⁺ influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca²⁺ ions.

Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.

The plant-parasitic nematode Heterodera schachtii stimulates plant root cells to form syncytial feeding structures which synthesize all nutrients required for successful nematode development. Cellular re-arrangements and modified metabolism of the syncytia are accompanied by massive intra- and intercellular solute allocations. In this study the expression of all genes annotated as sugar transporters in the Arabidopsis Membrane Protein Library was investigated by Affymetrix gene chip analysis in young and fully developed syncytia compared with non-infected Arabidopsis thaliana roots. The expression of three highly up-regulated (STP12, MEX1, and GTP2) and three highly down-regulated genes (SFP1, STP7, and STP4) was analysed by quantitative RT-PCR (qRT-PCR). The most up-regulated gene (STP12) was chosen for further in-depth studies using in situ RT-PCR and a nematode development assay with a T-DNA insertion line revealing a significant reduction of male nematode development. The specific role of STP12 expression in syncytia of male juveniles compared with those of female juveniles was further shown by qRT-PCR. In order to provide evidence for sugar transporter activity across the plasma membrane of syncytia, fluorescence-labelled glucose was used and membrane potential recordings following the application of several sugars were performed. Analyses of soluble sugar pools revealed a highly specific composition in syncytia. The presented work demonstrates that sugar transporters are specifically expressed and active in syncytia, indicating a profound role in inter- and intracelluar transport processes.

Sieve tubes in legumes contain forisomes, which are spindle-like bodies that are composed of ATP-independent, mechanically active proteins. Upon injury, forisomes occlude sieve tubes by dispersion and thus, help to prevent loss of nutrient-rich transport sap. Forisome enlargement by dispersion is brought about by Ca 2+-induced conformational changes that confer radial expansion and longitudinal contraction. Forisomes recontract upon Ca 2+ removal. In vitro, forisomes reversibly disperse and contract in the presence or absence of Ca 2+, respectively, and at distinct pHs. Recently, forisomes have received renewed attention because of their unique capacity to convert chemical into mechanical energy independent of high-energy organic compounds. Forisome-based ‘smart’ materials can be used to produce self-powered monitoring and diagnostic systems. Here, we focus on physiological, chemical and physical aspects of forisomes and discuss their potential as biomimetic devices.

Vascular tissue was recently shown to be capable of producing nitric oxide (NO), but the production sites and sources were not precisely determined. Here, NO synthesis was analysed in the phloem of Vicia faba in response to stress-and pathogen defence-related compounds. The chemical stimuli were added to shallow paradermal cortical cuts in the main veins of leaves attached to intact plants. NO production in the bare-lying phloem area was visualized by real-time confocal laser scanning microscopy using the NO-specific fluorochrome 4,5-diaminofluorescein diacetate (DAF-2 DA). Abundant NO generation in companion cells was induced by 500 pm salicylic acid (SA) and 10 μm hydrogen peroxide (H₂O₂), but the fungal elicitor chitooctaose was much less effective. Phloem NO production was found to be dependent on Ca²⁺ and mitochondrial electron transport and pharmacological approaches found evidence for activity of a plant NO synthase but not a nitrate reductase. DAF fluorescence increased most strongly in companion cells and was occasionally observed in phloem parenchyma cells. Significantly, accumulation of NO in sieve elements could be demonstrated. These findings suggest that the phloem perceives and produces stress-related signals and that one mechanism of distal signalling involves the production and transport of NO in the phloem.

Forisomes are contractile protein bodies that appear to control flux rates in the phloem of faboid legumes by reversibly plugging the sieve tubes. Plugging is triggered by Ca2+ which induces an anisotropic deformation of forisomes, consisting of a longitudinal contraction and a radial expansion. By conventional light microscopy and confocal laser-scanning microscopy, the three-dimensional geometry of the forisome–sieve element–sieve plate complex in intact sieve tubes of leaflets of Vicia faba L. was reconstructed. Forisomes were mostly located close to sieve plates, and occasionally were observed drifting unrestrainedly along the sieve element, suggesting that they might be utilized as internal markers of flow direction. The diameter of forisomes in the resting state correlated with the diameter of their sieve elements, supporting the idea that radial expansion of forisomes is the geometric basis of reversible sieve tube plugging. Comparison of the present results regarding forisome geometry in situ with previously published data on forisome reactivity in vitro makes it questionable, however, whether forisomes are capable of completely sealing sieve tubes in V. faba leaves.

According to an established concept, injury of the phloem triggers local sieve plate occlusion including callose-mediated constriction and, possibly, protein plugging of the sieve pores. Sieve plate occlusion can also be achieved by distant stimuli, depends on the passage of electropotential waves (EPWs), and is reversible in intact plants. The time-course of the wound response was studied in sieve elements of main veins of intact Vicia faba plants using confocal and multiphoton microscopy. Only 15-45 s after burning a leaf tip, forisomes (giant protein bodies specific for legume sieve tubes) suddenly dispersed, as observed at 3-4 cm from the stimulus site. The dispersion was reversible; the forisomes had fully re-contracted 7-15 min after burning. Meanwhile, callose appeared at the sieve pores in response to the heat shock. Callose production reached a maximum after similar to 20 min and was also reversible; callose degraded over the subsequent 1-2 h. The heat induction of both modes of occlusion coincided with the passage of an EPW visualized by electrophysiology or the potential-sensitive dye RH-414. In contrast to burning, cutting of the leaf tip induced neither an EPW nor callose deposition. The data are consistent with a remote-controlled occlusion of sieve plates depending on the longitudinal propagation of an EPW releasing Ca super(2+) into the sieve element lumen. It is hypothesized that forisome plugs and callose constriction are removed once the cytosolic calcium level has returned to the initial level in those sieve tubes.