Katrin Ehlers

Reversible Calcium-Regulated Stopcocks in Legume Sieve Tubes

Sieve tubes of legumes (Fabaceae) contain characteristic P-protein crystalloids with controversial function. We studied their behavior by conventional light, electron, and confocal laser scanning microscopy. In situ, crystalloids are able to undergo rapid (<1 sec) and reversible conversions from the condensed resting state into a dispersed state, in which they occlude the sieve tubes. Crystalloid dispersal is triggered by plasma membrane leakage induced by mechanical injury or permeabilizing substances. Similarly, abrupt turgor changes imposed by osmotic shock cause crystalloid dispersal. Because chelators generally prevent the response, divalent cations appear to be the decisive factor in crystalloid expansion. Cycling between dispersal and condensation can be induced in opened cells by repetitive exchange of bathing media containing either Ca2+ or chelators. Sr2+ and Ba2+, but not Mg2+, are equally active. In conclusion, the fabacean P-protein crystalloids represent a novel class of mechanically active proteinaceous structures, which provide an efficient mechanism with which to control sieve tube conductivity.

Primary and secondary plasmodesmata: structure, origin, and functioning.

SummaryIn the multicellular organisms of higher plants, plasmodesmata provide pathways for intimate symplasmic communication between neighboring cells. The arguments summarized in the present review demonstrate that plasmodesmata are diverse and highly dynamic structures. Differences in the plasmodesmal origin and modifications of the plasmodesmal structure and functioning at the various cell interfaces are the basic means which give rise to a complicated and flexibile symplasmic network. This complex communication system is discussed to serve a significant role in the coordinated development and in the concerted physiological functioning of the cells within the plant tissues, organs, and organisms.

Sieve elements caught in the act

Phloem is a puzzling plant tissue owing to the unique natural defence responses of the sieve elements to any kind of mechanical manipulation. Recent non-invasive studies have enabled real-time observation of events in intact sieve tubes, including mass transport, sieve-pore sealing and conformational changes of structural proteins. These studies further highlighted the importance of the symplasmic setting for development and functioning of the sieve elements. Exchange of macromolecules between companion cells and sieve elements is indispensable for the survival of the sieve element, but also seems to be involved in long-distance communication. How the branched plasmodesmata between sieve element and companion cell function as corridors for the passage of macromolecules is an intriguing but unresolved story.

Ultrastructural features of well-preserved and injured sieve elements: minute clamps keep the phloem transport conduits free for mass flow.

SummaryAfter chemical fixation following two different preparation procedures, the ultrastructure of mature sieve elements (SEs) was systematically compared in the transport phloem ofVicia faba leaves andLycopersicon esculentum internodes. The SEs in samples obtained by gentle preparation were well preserved, while those in conventionally prepared samples were generally injured. (1) In well-preserved SEs, parietal P-proteins were associated with cisternae of the SE endoplasmic reticulum (ER). Additionally, theV. faba SEs had crystalline P-proteins, and a homogeneous network of filamentous P-proteins occurred in the lumen of theL. esculentum SEs. In injured SEs, all P-proteins were dispersed. (2) In well-preserved SEs, stacked ER cisternae associated with P-proteins lay also on the sieve-plate walls, but passages were kept free in front of the sieve pores. Injured SEs lacked these orderly arranged deposits. Instead, irregular filamentous and membranous materials occluded the sieve pores. (3) In well-preserved SEs, the sieve-pore lumen was free of obstructions, apart from small, lateral coatings of P-proteins. Sieve pores in injured SEs were always occluded. (4) The SE organelles and, in tomato SEs, also the parietal ER located at the longitudinal walls were firmly attached in the SE periphery and stayed in place after injury. The stable parietal attachment is likely exerted by minute, clamplike structures which link the outer membranes of the SE components with one another or to the SE plasma membrane. Single, straight clamps with a length of about 7 nm anchored the SE components directly to the SE plasma membrane. The connections between adjacent SE organelles and/or parietal ER cisternae were mostly twice as long (about 15 nm) and often were branched. Presumably, the long, branched clamps were constituted by the interaction of opposite short clamps. The ultrastructural results are discussed with respect to SE functioning.

Formation of branched plasmodesmata in regenerating Solanum nigrum-protoplasts

During the early stages of culture, discontinuous branched half-plasmodesmata were found randomly scattered in the newly formed outer cell walls of regenerating Solanum nigrum L. protoplasts. During later culture stages, most of these “outer-wall plasmodesmata”, which had been exposed to the culture medium, disappeared, except for those near the periphery of division walls between daughter cells and those near non-division walls between secondarily associated unrelated cells. Moreover, in the peripheral parts of older division walls, there were continuous branched plasmodesmata which showed the typical morphological characteristics of secondary cell connections: several cytoplasmic strands joined in the median plane of the cell wall and were often linked by so-called median cavities. Evidence is presented that this type of continuous plasmodesma originates from the fusion of the half-plasmodesmata which persisted in the outer walls adjacent to the division wall. Due to growth of the cells after division, opposite parts of the outer walls of the daughter cells come into close contact and fuse, elongating the original division wall peripherally. Opposite half-plasmodesmata remaining in these parts of the outer wall may thereby also be brought into contact and fuse to form a continuous secondary cell connection in the secondarily coalesced wall part. Our assumption is supported by further experiments: (i) longterm video observations of living cells showed differences in the development of the shapes of regenerating cells and (ii) electron-microscopical investigations showed differences in the frequency of the, presumably secondary, cell connections in the peripheral parts of the division walls — both related to the firmness of the embedding medium. In the central parts of division walls, unbranched primary cell connections were found as well as a second type of continuous branched plasmodesma showing an entirely different branching pattern: the region of the middle lamella was always traversed by straight, unbranched parts of these plasmodesmata and the branches occurred exclusively within the younger wall layers. Evidence is given that these branches are modifications of originally unbranched primary plasmodesmata, developing during subsequent thickening of the division wall.

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.

Subcellular localization of ubiquitin in plant protoplasts and the function of ubiquitin in selective degradation of outer-wall plasmodesmata in regenerating protoplasts

Immunocytochemical localizations in Vicia faba L. protoplasts and cultures of regenerating Solanum nigrum L. protoplasts support former observations that in plant cells ubiquitin occurs within the cytoplasm, the nucleus, the chloroplasts and at the plasmalemma, but not within the vacuole or the cell wall. Immunoresponses were also observed within mitochondria and associated with the endoplasmic reticulum, which is in accordance with previous findings on animal cells. Moreover, the tonoplast membrane system was found to be labelled. For regenerating S. nigrum protoplasts, evidence is given that ubiquitin plays a role in selective degradation even of whole subcellular structures. Most of the discontinuous plasmodesmata formed in the newly deposited outer cell walls during the early stages of culture disappear later on, except for those near the periphery of division walls or of non-division walls, which are probably used for the formation of continuous cell connections during further culture. Outer-wall plasmodesmata which are destined to disappear show high immunoreactivity to ubiquitin antibody, but no conspicuous immunolabelling was observed with the remaining plasmodesmata. Thus, the selective disintegration of whole plasmodesmatal structures is obviously regulated by ubiquitination of plasmodesmatal proteins. A model for the mechanism of degradation of outer-wall plasmodesmata during extension growth of the cell wall is presented.

Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L.

Abstract. Minor-vein ultrastructure and sugar export were studied in mature summer and winter leaves of the three broadleaf-evergreen species Ajuga reptans var. artropurpurescens L., Aucuba japonica Thunb. and Hedera helix L. to assess temperature effects on phloem loading. Leaves of the perennial herb Ajuga exported substantial amounts of assimilates in form of raffinose-family oligosaccharides (RFOs). Its minor-vein companion cells represent typical intermediary cells (ICs), with numerous small vacuoles and abundant plasmodesmal connectivity to the bundle sheath. The woody plants Hedera and Aucuba translocated sucrose as the dominant sugar species, and only traces of RFOs. Their minor-vein phloem possessed a layer of highly vacuolated cells (VCs) intervening between mesophyll and sieve elements. Depending on their location and ontogeny, VCs were classified either as companion or parenchyma cells. Both cell types showed symplasmic continuity to the adjacent mesophyll tissue although at a lower plasmodesmal frequency compared to the Ajuga ICs. p-Chloromercuribenzenesulfonic acid did not reduce leaf sugar export in any of the plants, indicating a symplasmic mode of phloem loading. Winter leaves did not show symptoms of frost injury, and the vacuolar pattern in ICs and VCs was equally prominent in both seasons. Starch accumulation as a result of reduced phloem loading was not observed to be triggered by low temperature. In contrast, high amounts of starch were found in mesophyll and bundle-sheath cells of summer leaves. Physiological data on season-dependent leaf exudation showed the maintenance of sugar export in cold-acclimated winter leaves.

The formation of symplasmic domains by plugging of plasmodesmata: a general event in plant morphogenesis?

SummaryThe plasmodesmal network was examined in multicellular protoplast-derived calluses of the dicotyledonSolanum nigrum which had not yet formed any visible adventitious organs and in globular proembryogenic structures developed from scutellar calluses of the monocotyledonMolinia caerulea. Electron microscopical analyses revealed that both calluses and proembryos consisted of small, undifferentiated cells. The interconnecting plasmodesmata at many cell interfaces were structurally inconspicuous in both systems; in particular cell walls, however, all plasmodesmata were occluded with an osmiophilic, dense material. As the blocking material was obviously located in the microchannels of the plasmodesmal cytoplasmic sleeves, the plugged plasmodesmata can be assumed to be nonfunctional. Thus, selective occlusion of all the plasmodesmata in specific cell walls resulted in the symplasmic disconnection of particular adjacent cells. Complex patterns of symplasmic continuity and discontinuity were established within the developing tissues. Some cells or groups of cells were entirely symplasmically disconnected from the surrounding cells by plugged plasmodesmata and might function as independent domains. However, blockage of plasmodesmata was achieved by the surrounding cells rather than by those cells belonging to the isolated domains. The demarcation of symplasmic domains might be a general prerequisite for differential morphogenesis, since they were found to be established very early in the course of morphogenetic processes.

CRT1 is a nuclear-translocated MORC endonuclease that participates in multiple levels of plant immunity

Arabidopsis thaliana CRT1 (compromised for recognition of Turnip Crinkle Virus) was previously shown to be required for effector-triggered immunity. Sequence analyses previously revealed that CRT1 contains the ATPase and S5 domains characteristic of Microchidia (MORC) proteins; these proteins are associated with DNA modification and repair. Here we show that CRT1 and its closest homologue, CRH1, are also required for pathogen-associated molecular pattern (PAMP)-triggered immunity, basal resistance, non-host resistance and systemic acquired resistance. Consistent with its role in PAMP-triggered immunity, CRT1 interacted with the PAMP recognition receptor FLS2. Subcellular fractionation and transmission electron microscopy detected a subpopulation of CRT1 in the nucleus, whose levels increased following PAMP treatment or infection with an avirulent pathogen. These results, combined with the demonstration that CRT1 binds DNA, exhibits endonuclease activity, and affects tolerance to the DNA-damaging agent mitomycin C, argue that this prototypic eukaryotic member of the MORC superfamily has important nuclear functions during immune response activation.