Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7ko plants is accompanied by enhanced expression of carbohydrate transporters.

MAIN CONCLUSION: Loss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport. Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant's response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Species-Specific and Distance-Dependent Dispersive Behaviour of Forisomes in Different Legume Species.

Forisomes are giant fusiform protein complexes composed of sieve element occlusion (SEO) protein monomers, exclusively found in sieve elements (SEs) of legumes. Forisomes block the phloem mass flow by a Ca2+-induced conformational change (swelling and rounding). We studied the forisome reactivity in four different legume species-Medicago sativa, Pisum sativum, Trifolium pratense and Vicia faba. Depending on the species, we found direct relationships between SE diameter, forisome surface area and distance from the leaf tip, all indicative of a developmentally tuned regulation of SE diameter and forisome size. Heat-induced forisome dispersion occurred later with increasing distance from the stimulus site. T. pratense and V. faba dispersion occurred faster for forisomes with a smaller surface area. Near the stimulus site, electro potential waves (EPWs)-overlapping action (APs), and variation potentials (VPs)-were linked with high full-dispersion rates of forisomes. Distance-associated reduction of forisome reactivity was assigned to the disintegration of EPWs into APs, VPs and system potentials (SPs). Overall, APs and SPs alone were unable to induce forisome dispersion and only VPs above a critical threshold were capable of inducing forisome reactions.

Sieve element biology provides leads for research on phytoplasma lifestyle in plant hosts.

Phytoplasmas reside exclusively in sieve tubes, tubular arrays of sieve element-companion cell complexes. Hence, the cell biology of sieve elements may reveal (ultra)structural and functional conditions that are of significance for survival, propagation, colonization, and effector spread of phytoplasmas. Electron microscopic images suggest that sieve elements offer facilities for mobile and stationary stages in phytoplasma movement. Stationary stages may enable phytoplasmas to interact closely with diverse sieve element compartments. The unique, reduced sieve element outfit requires permanent support by companion cells. This notion implies a future focus on the molecular biology of companion cells to understand the sieve element-phytoplasma inter-relationship. Supply of macromolecules by companion cells is channelled via specialized symplasmic connections. Ca2+-mediated gating of symplasmic corridors is decisive for the communication within and beyond the sieve element-companion cell complex and for the dissemination of phytoplasma effectors. Thus, Ca2+ homeostasis, which affects sieve element Ca2+ signatures and induces a range of modifications, is a key issue during phytoplasma infection. The exceptional physical and chemical environment in sieve elements seems an essential, though not the only factor for phytoplasma survival.

Filamentous sieve element proteins are able to limit phloem mass flow, but not phytoplasma spread.

In Fabaceae, dispersion of forisomes-highly ordered aggregates of sieve element proteins-in response to phytoplasma infection was proposed to limit phloem mass flow and, hence, prevent pathogen spread. In this study, the involvement of filamentous sieve element proteins in the containment of phytoplasmas was investigated in non-Fabaceae plants. Healthy and infected Arabidopsis plants lacking one or two genes related to sieve element filament formation-AtSEOR1 (At3g01680), AtSEOR2 (At3g01670), and AtPP2-A1 (At4g19840)-were analysed. TEM images revealed that phytoplasma infection induces phloem protein filament formation in both the wild-type and mutant lines. This result suggests that, in contrast to previous hypotheses, sieve element filaments can be produced independently of AtSEOR1 and AtSEOR2 genes. Filament presence was accompanied by a compensatory overexpression of sieve element protein genes in infected mutant lines in comparison with wild-type lines. No correlation was found between phloem mass flow limitation and phytoplasma titre, which suggests that sieve element proteins are involved in defence mechanisms other than mechanical limitation of the pathogen.

Aphid salivary proteases are capable of degrading sieve-tube proteins.

Sieve tubes serve as transport conduits for photo-assimilates and other resources in angiosperms and are profitable targets for piercing-sucking insects such as aphids. Sieve-tube sap also contains significant amounts of proteins with diverse functions, for example in signalling, metabolism, and defence. The identification of salivary proteases in Acyrthosiphon pisum led to the hypothesis that aphids might be able to digest these proteins and by doing so suppress plant defence and access additional nitrogen sources. Here, the scarce knowledge of proteases in aphid saliva is briefly reviewed. In order to provide a better platform for discussion, we conducted a few tests on in vitro protease activity and degradation of sieve-tube sap proteins of Cucurbita maxima by watery saliva. Inhibition of protein degradation by EDTA indicates the presence of different types of proteases (e.g. metalloproteses) in saliva of A. pisum. Proteases in the watery saliva from Macrosiphum euphorbiae and A. pisum were able to degrade the most abundant phloem protein, which is phloem protein 1. Our results provide support for the breakdown of sieve-element proteins by aphid saliva in order to suppress/neutralize the defence responses of the plant and to make proteins of sieve-tube sap accessible as a nitrogen source, as is discussed in detail. Finally, we discuss whether glycosylation of sieve-element proteins and the presence of protease inhibitors may confer partial protection against the proteolytic activity of aphid saliva.

Involvement of the sieve element cytoskeleton in electrical responses to cold shocks.

This study dealt with the visualization of the sieve element (SE) cytoskeleton and its involvement in electrical responses to local cold shocks, exemplifying the role of the cytoskeleton in Ca(2+)-triggered signal cascades in SEs. High-affinity fluorescent phalloidin as well as immunocytochemistry using anti-actin antibodies demonstrated a fully developed parietal actin meshwork in SEs. The involvement of the cytoskeleton in electrical responses and forisome conformation changes as indicators of Ca(2+) influx was investigated by the application of cold shocks in the presence of diverse actin disruptors (latrunculin A and cytochalasin D). Under control conditions, cold shocks elicited a graded initial voltage transient, ΔV1, reduced by external La(3+) in keeping with the involvement of Ca(2+) channels, and a second voltage transient, ΔV2. Cytochalasin D had no effect on ΔV1, while ΔV1 was significantly reduced with 500 nm latrunculin A. Forisome dispersion was triggered by cold shocks of 4°C or greater, which was indicative of an all-or-none behavior. Forisome dispersion was suppressed by incubation with latrunculin A. In conclusion, the cytoskeleton controls cold shock-induced Ca(2+) influx into SEs, leading to forisome dispersion and sieve plate occlusion in fava bean (Vicia faba).

Spread the news: systemic dissemination and local impact of Ca²âº signals along the phloem pathway.

We explored the idea of whether electropotential waves (EPWs) primarily act as vehicles for systemic spread of Ca(2+) signals. EPW-associated Ca(2+) influx may trigger generation and amplification of countless long-distance signals along the phloem pathway given the fact that gating of Ca(2+)-permeable channels is a universal response to biotic and abiotic challenges. Despite fundamental differences, both action and variation potentials are associated with a sudden Ca(2+) influx. Both EPWs probably disperse in the lateral direction, which could be of essential functional significance. A vast set of Ca(2+)-permeable channels, some of which have been localized, is required for Ca(2+)-modulated events in sieve elements. There, Ca(2+)-permeable channels are clustered and create so-called Ca(2+) hotspots, which play a pivotal role in sieve element occlusion. Occlusion mechanisms play a central part in the interaction between plants and phytopathogens (e.g. aphids or phytoplasmas) and in transient re-organization of the vascular symplasm. It is argued that Ca(2+)-triggered systemic signalling occurs in partly overlapping waves. The forefront of EPWs may be accompanied by a burst of free Ca(2+) ions and Ca(2+)-binding proteins in the sieve tube sap, with a far-reaching impact on target cells. Lateral dispersion of EPWs may induce diverse Ca(2+) influx and handling patterns (Ca(2+) signatures) in various cell types lining the sieve tubes. As a result, a variety of cascades may trigger the fabrication of signals such as phytohormones, proteins, or RNA species released into the sap stream after product-related lag times. Moreover, transient reorganization of the vascular symplasm could modify cascades in disjunct vascular cells.

Phytoplasma-triggered Ca(2+) influx is involved in sieve-tube blockage.

Phytoplasmas are obligate, phloem-restricted phytopathogens that are disseminated by phloem-sap-sucking insects. Phytoplasma infection severely impairs assimilate translocation in host plants and might be responsible for massive changes in phloem physiology. Methods to study phytoplasma- induced changes thus far provoked massive, native occlusion artifacts in sieve tubes. Hence, phytoplasma-phloem relationships were investigated here in intact Vicia faba host plants using a set of vital fluorescent probes and confocal laser-scanning microscopy. We focused on the effects of phytoplasma infection on phloem mass-flow performance and evaluated whether phytoplasmas induce sieve-plate occlusion. Apparently, phytoplasma infection brings about Ca(2+) influx into sieve tubes, leading to sieve-plate occlusion by callose deposition or protein plugging. In addition, Ca(2+) influx may confer cell wall thickening of conducting elements. In conclusion, phytoplasma effectors may cause gating of sieve-element Ca(2+) channels leading to sieve-tube occlusion with presumptive dramatic effects on phytoplasma spread and photoassimilate distribution.

Interaction of xylem and phloem during exudation and wound occlusion in Cucurbita maxima.

Collection of cucurbit exudates from cut petioles has been a powerful tool for gaining knowledge on phloem sap composition without full notion of the complex exudation mechanism. Only few publications explicitly mentioned that exudates were collected from the basal side of the cut, which exudes more copiously than the apical side. This is surprising since only exudation from the apical side is supposedly driven by phloem pressure gradients. Composition of carbohydrates and pH values at both wounding sides are equal, whereas protein concentration is higher at the basal side. Apparently, exudation is far more complex than just the delivery of phloem sap. Xylem involvement is indicated by lower protein concentrations after elimination of root pressure. Moreover, dye was sucked into xylem vessels owing to relaxation of negative pressure after cutting. The lateral water efflux from the vessels increases turgor of surrounding cells including sieve elements. Simultaneously, detached parietal proteins (PP1/PP2) induce occlusion of sieve plates and cover wound surface. If root pressure is strong enough, pure xylem sap can be collected after removal of the occlusion plug at the wound surface. The present findings provide a mechanism of sap exudation in Cucurbita maxima, in which the contribution of xylem water is integrated.

The sieve-element endoplasmic reticulum: A focal point of phytoplasma-host plant interaction?

The rough endoplasmic reticulum (r-ER) is of paramount importance for adaptive responses to biotic stresses due to an increased demand for de novo synthesis of immunity-related proteins and signaling components. In nucleate cells, disturbance of r-ER integrity and functionality leads to the "unfolded protein response" (UPR), which is an important component of innate plant immune signalling. In contrast to an abundance of reports on r-ER responses to biotic challenges, sieve-element endoplasmic reticulum (SE-ER) responses to phytoplasma infection have not been investigated. We found that morphological SE-ER changes, associated with phytoplasma infection, are accompanied by differential expression of genes encoding proteins involved in shaping and anchoring the reticulum. Phytoplasma infection also triggers an increased release of bZIP signals from the (SE-ER)/r-ER and consequent differential expression of UPR-related genes. The modified expression patterns seem to reflect a trade-off between survival of host cells, needed for the phytoplasmic biotrophic lifestyle, and phytoplasmas. Specialized plasmodesmata between sieve element and companion cell may provide a corridor for transfer of phytoplasma effectors inducing UPR-related gene expression in companion cells.

Forisome performance in artificial sieve tubes.

In the legume phloem, sieve element occlusion (SEO) proteins assemble into Ca(2+)-dependent contractile bodies. These forisomes presumably control phloem transport by forming reversible sieve tube plugs. This function, however, has never been directly demonstrated, and appears questionable as forisomes were reported to be too small to plug sieve tubes, and failed to block flow efficiently in artificial microchannels. Moreover, plugs of SEO-related proteins in Arabidopsis sieve tubes do not affect phloem translocation. We improved existing procedures for forisome isolation and storage, and found that the degree of Ca(2+)-driven deformation that is possible in forisomes of Vicia faba, the standard object of earlier research, has been underestimated substantially. Forisomes deform particularly strongly under reducing conditions and high sugar concentrations, as typically found in sieve tubes. In contrast to our previous inference, Ca(2+)-inducible forisome swelling certainly seems sufficient to plug sieve tubes. This conclusion was supported by 3D-reconstructions of forisome plugs in Canavalia gladiata. For a direct test, we built microfluidics chips with artificial sieve tubes. Using fluorescent dyes to visualize flow, we demonstrated the complete blockage of these biomimetic microtubes by Ca(2+)-induced forisome plugs, and concluded by analogy that forisomes are capable of regulating phloem flow in vivo.

An aphid's Odyssey--the cortical quest for the vascular bundle.

Sensing pH and sucrose concentration (with a preference for pH values of 7.0-7.5 and sucrose concentrations of approximately 400 mmol l(-1)) enables aphids to recognise sieve tubes inside vascular bundles. However, it is still unclear how aphids find their way to the vascular bundles. Membrane potentials in the cortex of Vicia faba stems were measured along a radial transect from the epidermis to the sieve elements and there was no gradient detected that could be used by aphids to guide their stylets to the sieve elements. Additionally, aphids did not demonstrate a preference between artificial diets with low or high levels of dissolved oxygen, making it unlikely that oxygen gradients in the cortex assist orientation towards the phloem. Tracks of salivary sheaths indicate that aphids search for vascular bundles in a radial direction (perpendicular from the stem surface to the vascular bundle) with regular side punctures in a pre-programmed fashion. Optical examination and electrical penetration graph (EPG) recordings suggest that aphids (Megoura viciae) probe the vacuolar sap of cortex cells. Acidic pH (5.0-5.5) and low sucrose concentrations in vacuoles, therefore, may provoke aphids to retract their stylets and probe the next cell until a favourable cell sap composition is encountered. The importance of sucrose as a cue was demonstrated by the experimental manipulation of Ricinus communis plants that cause them to transport hexoses instead of sucrose. Aphids (Aphis fabae) ingested less phloem sap of plants transporting hexoses compared with plants transporting the normal sucrose. The proposed rejection-acceptance behaviour provides a universal plant-directed mode of how aphids orientate their stylets towards the phloem.

The plant axis as the command centre for (re)distribution of sucrose and amino acids.

Along with the increase in size required for optimal colonization of terrestrial niches, channels for bidirectional bulk transport of materials in land plants evolved during a period of about 100 million years. These transport systems are essentially still in operation - though perfected over the following 400 million years - and make use of hydrostatic differentials. Substances are accumulated or released at the loading and unloading ends, respectively, of the transport channels. The intermediate stretch between the channel termini is bifunctional and executes orchestrated release and retrieval of solutes. Analyses of anatomical and physiological data demonstrate that the release/retrieval zone extends deeper into sources and sinks than is commonly thought and covers usually much more than 99% of the translocation stretch. This review sketches the significance of events in the intermediate stretch for distribution of organic materials over the plant body. Net leakage from the channels does not only serve maintenance and growth of tissues along the pathway, but also diurnal, short-term or seasonal storage of reserve materials, and balanced distribution of organic C- and N-compounds over axial and terminal sinks. Release and retrieval are controlled by plasma-membrane transporters at the vessel/parenchyma interface in the contact pits along xylem vessels and by plasma-membrane transporters at the interface between companion cells and phloem parenchyma along sieve tubes. The xylem-to-phloem pathway vice versa is a bifacial, radially oriented system comprising a symplasmic pathway, of which entrance and exit are controlled at specific membrane checkpoints, and a parallel apoplasmic pathway. A broad range of specific sucrose and amino-acid transporters are deployed at the checkpoint plasma membranes. SUCs, SUTs, STPs, SWEETs, and AAPs, LTHs, CATs are localized to the plasma membranes in question, both in monocots and eudicots. Presence of Umamits in monocots is uncertain. There is some evidence for endo- and exocytosis at the vessel/parenchyma interface supplementary to the transporter-mediated uptake and release. Actions of transporters at the checkpoints are equally decisive for storage and distribution of amino acids and sucrose in monocots and eudicots, but storage and distribution patterns may differ between both taxa. While the majority of reserves is sequestered in vascular parenchyma cells in dicots, lack of space in monocot vasculature urges "outsourcing" of storage in ground parenchyma around the translocation path. In perennial dicots, specialized radial pathways (rays) include the sites for seasonal alternation of storage and mobilization. In dicots, apoplasmic phloem loading and a correlated low rate of release along the path would favour supply with photoassimilates of terminal sinks, while symplasmic phloem loading and a correlated higher rate of release along the path favours supply of axial sinks and transfer to the xylem. The balance between the resource acquisition by terminal and axial sinks is an important determinant of relative growth rate and, hence, for the fitness of plants in various habitats. Body enlargement as the evolutionary drive for emergence of vascular systems and mass transport propelled by hydrostatic differentials.

Dynamics of plasmodesmal connectivity in successive interfaces of the cambial zone.

Frequency, density and branching of plasmodesmata were counted in successive tangential and transverse walls in the cambial zone of tomato stems in order to examine development of the plasmodesmal network in a chronological order. Coincident with progress of cell development, plasmodesmal connectivity increased, both at the xylem- and phloem-side. In transverse walls, the number of secondary plasmodesmata enhanced considerably. The same held for tangential walls, with a superimposed plasmodesmal doubling during the first phase of phloem development. This plasmodesmal doubling was interpreted to result from the deposition of wall material between branched plasmodesmal strands. Structural plasmodesmal development was correlated with production of hydroxyl radicals which control local cell wall alterations. Successive phases of plasmodesmal deployment and modification were distinguished which may coincide with differential functional capacities as documented by intracellular injection of fluorochromes. Diffusion-driven symplasmic transport appeared to be transiently interrupted during cell maturation.

Rapid cooling triggers forisome dispersion just before phloem transport stops.

Phloem transport stops transiently within dicot stems that are cooled rapidly, but the cause remains unknown. Now it is known that (1) rapid cooling depolarizes cell membranes giving a transient increase in cytoplasmic Ca(2+), and (2) a rise of free calcium triggers dispersion of forisomes, which then occlude sieve elements (SEs) of fabacean plants. Therefore, we compared the effects of rapid chilling on SE electrophysiology, phloem transport and forisomes in Vicia faba. Forisomes dispersed after rapid cooling with a delay that was longer for slower cooling rates. Phloem transport stopped about 20 s after forisome dispersion, and then transport resumed and forisomes re-condensed within similar time frames. Transport interruption and forisome dispersion showed parallel behaviour--a cooling rate-dependent response, transience and desensitization. Chilling induced both a fast and a slow depolarization of SE membranes, the electrical signature suggesting strongly that the cause of forisome dispersion was the transient promotion of SE free calcium. This apparent block of SEs by dispersed forisomes may be assisted by other Ca(2+)-dependent sealing proteins that are present in all dicots.

Remote-controlled stop of phloem mass flow by biphasic occlusion in Cucurbita maxima.

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 approximately 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 Ca(2+)-dependent occlusion mechanisms is attributed to Ca(2+) influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca(2+) ions.

Plant cues for aphid navigation in vascular tissues.

The ability of aphids to detect and find sieve tubes suggests that aphids receive cues for sieve-tube recognition by taking samples. Specific natural conditions such as pH value, sugar species and concentration, viscosity, and oxygen pressure may enable sieve-tube detection. We tested the preference of Megoura viciae and Myzus persicae for potential plant-borne orientation parameters in artificial choice-chamber systems. Both species preferred sucrose (in comparison with fructose, glucose, raffinose or sorbitol) at concentrations of 15-22.5% (over a tested range of 0-22.5%) and at approximately pH 7 (over a tested range of pH 5-8). This preference matches the composition of the sieve-tube sap of their host plants. Likewise, Rhopalosiphum padi (normally found on barley plants with sucrose in the phloem sap) and Macrosiphum euphorbiae (normally found on pumpkin plants with raffinose-family oligosaccharides in the phloem sap) showed a significant preference for sucrose. In the absence of sucrose, however, M. euphorbiae strongly preferred raffinose. No clear preference for any carbohydrate was observed for Macrosiphum rosae and Aphis pomi (both normally found on plants with various amounts of sorbitol in the phloem sap). Electrical penetration graph (EPG) measurements of M. persicae feeding on artificial diets confirmed that sieve tubes are recognized solely by a combination of carbohydrate abundance and a neutral to slightly alkaline pH.

Season-associated modifications in symplasmic organization of the cambium in Populus nigra.

BACKGROUND AND AIMS: Alterations of plasmodesma (PD) connectivity are likely to be very important for plant development. Here, the repetitive division pattern of cambial initials in Populus nigra 'italica' was studied to follow the development of the PD network during maturation. Furthermore, seasonal changes were investigated in order to trace indications for developmental and functional adaptations. METHODS: Cambium samples of P. nigra twigs, collected in summer, autumn and spring, were chemically fixed for transmission electron microscopy. The parameters, PD density (number of PDs per square micrometre cell-wall area) and PD frequency (total number of PDs per average cell-wall area), were determined for radial and tangential cell interfaces deposited in chronological order. KEY RESULTS: Data sets, presented in plasmodesmograms, show a strong variability in the PD network throughout the year. In summer, high PD numbers occur at the division wall which, after PD doubling by longitudinal fission, decline with further development both at the xylem and the phloem side. In autumn, the number of PDs at the division wall is low as they are in subsequent tangential interfaces. In spring, the first cell division coincides with a massive increase in PD numbers, in particular at the division wall. Only the radial walls between initials maintain their PD equipment throughout the year. This feature can be exploited for identification of the initial layer. CONCLUSIONS: PD networks in the cambium go through a strict developmental programme depending on the season, which is associated with changing functional requirements. For instance, PD numbers correlate with proliferative activity and potential pathways for intercellular signalling. Increases in PD numbers are ascribed to longitudinal fission as a major mechanism, whereas the decline in older derivatives is ascribed to PD degradation.

Immunolocalization indicates plasmodesmal trafficking of storage proteins during cambial reactivation in Populus nigra.

BACKGROUND AND AIMS: Cambium reactivation after dormancy and budbreak in deciduous trees requires a supply of mobilized reserve materials. The pathway and mode of transfer of these materials are poorly understood. METHODS: Transport of reserve materials during cambium reactivation in Populus nigra was investigated by conventional and immunocytochemical TEM analyses, SDS-PAGE, western blotting and intracellular microinjection of fluorescent dyes. KEY RESULTS: Proteinaceous compounds stored in vacuoles and protein bodies of vascular cells and ray cells disappeared within 3 weeks after cambial reactivation and budbreak. Some of these proteins (32 kDa, 30 kDa and 15 kDa) were labelled by lectin antibodies in SDS-PAGE. The same antibodies were localized to plasmodesmata (PDs) between phloem parenchyma, ray cells and fusiform cambial cells. In addition, proteinaceous particles were localized inside the cytoplasmic sleeves of these PDs during budbreak. During this period, the functional diameter of PDs was about 2.2 nm which corresponds approximately to the Stokes' radius of the detected 15-kDa protein. CONCLUSIONS: Lectin-like reserve proteins or their degradation products seem to be transferred through PDs of phloem parenchyma and rays during cambial reactivation and budbreak. PD transfer of storage proteins is a novelty which supports the concept of symplasmic nutrient supply to the cambial region.

Pre-symptomatic modified phytohormone profile is associated with lower phytoplasma titres in an Arabidopsis seor1ko line.

The proteins AtSEOR1 and AtSEOR2 occur as conjugates in the form of filaments in sieve elements of Arabidopsis thaliana. A reduced phytoplasma titre found in infected defective-mutant Atseor1ko plants in previous work raised the speculation that non-conjugated SEOR2 is involved in the phytohormone-mediated suppression of Chrysanthemum Yellows (CY)-phytoplasma infection transmitted by Euscelidius variegatus (Ev). This early and long-lasting SEOR2 impact was revealed in Atseor1ko plants by the lack of detectable phytoplasmas at an early stage of infection (symptomless plants) and a lower phytoplasma titre at a later stage (fully symptomatic plants). The high insect survival rate on Atseor1ko line and the proof of phytoplasma infection at the end of the acquisition access period confirmed the high transmission efficiency of CY-phytoplasma by the vectors. Transmission electron microscopy analysis ruled out a direct role of SE filament proteins in physical phytoplasma containment. Time-correlated HPLC-MS/MS-based phytohormone analyses revealed increased jasmonate levels in midribs of Atseor1ko plants at an early stage of infection and appreciably enhanced levels of indole acetic acid and abscisic acid at the early and late stages. Effects of Ev-probing on phytohormone levels was not found. The results suggest that SEOR2 interferes with phytohormonal pathways in Arabidopsis midrib tissues in order to establish early defensive responses to phytoplasma infection.