Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element-specific proteins reveal differentiation of the endomembrane system.

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Diversity of funnel plasmodesmata in angiosperms: the impact of geometry on plasmodesmal resistance.

In most plant tissues, threads of cytoplasm, or plasmodesmata, connect the protoplasts via pores in the cell walls. This enables symplasmic transport, for instance in phloem loading, transport and unloading. Importantly, the geometry of the wall pore limits the size of the particles that may be transported, and also (co-)defines plasmodesmal resistance to diffusion and convective flow. However, quantitative information on transport through plasmodesmata in non-cylindrical cell wall pores is scarce. We have found conical, funnel-shaped cell wall pores in the phloem-unloading zone in growing root tips of five eudicot and two monocot species, specifically between protophloem sieve elements and phloem pole pericycle cells. 3D reconstructions by electron tomography suggested that funnel plasmodesmata possess a desmotubule but lack tethers to fix it in a central position. Model calculations showed that both diffusive and hydraulic resistance decrease drastically in conical and trumpet-shaped cell wall pores compared with cylindrical channels, even at very small opening angles. Notably, the effect on hydraulic resistance was relatively larger. We conclude that funnel plasmodesmata generally are present in specific cell-cell interfaces in angiosperm roots, where they appear to facilitate symplasmic phloem unloading. Interestingly, cytosolic sleeves of most plasmodesmata reported in the literature do not resemble annuli of constant diameter but possess variously shaped widenings. Our evaluations suggest that widenings too small for unambiguous identification on electron micrographs may drastically reduce the hydraulic and diffusional resistance of these pores. Consequently, theoretical models assuming cylindrical symmetries will underestimate plasmodesmal conductivities.

Sieve elements rapidly develop 'nacreous walls' following injury - a common wounding response?

Thick glistening cell walls occur in sieve tubes of all major land plant taxa. Historically, these 'nacreous walls' have been considered a diagnostic feature of sieve elements; they represent a conundrum, though, in the context of the widely accepted pressure-flow theory as they severely constrict sieve tubes. We employed the cucurbit Gerrardanthus macrorhizus as a model to study nacreous walls in sieve elements by standard and in situ confocal microscopy and electron microscopy, focusing on changes in functional sieve tubes that occur when prepared for microscopic observation. Over 90% of sieve elements in tissue sections processed for microscopy by standard methods exhibit nacreous walls. Sieve elements in whole, live plants that were actively transporting as shown by phloem-mobile tracers, lacked nacreous walls and exhibited open lumina of circular cross-sections instead, an appropriate structure for Münch-type mass flow of the cell contents. Puncturing of transporting sieve elements with micropipettes triggered the rapid (<1 min) development of nacreous walls that occluded the cell lumen almost completely. We conclude that nacreous walls are preparation artefacts rather than structural features of transporting sieve elements. Nacreous walls in land plants resemble the reversibly swellable walls found in various algae, suggesting that they may function in turgor buffering, the amelioration of osmotic stress, wounding-induced sieve tube occlusion, and possibly local defence responses of the phloem.

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness.

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein-protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.

What actually is the Münch hypothesis? A short history of assimilate transport by mass flow.

In the 1920s, the German forestry scientist Ernst Münch postulated that photo-assimilate transport is a mass flow driven by osmotically induced pressure gradients between source organs (high turgor) and sink organs (lower turgor). Two crucial components of Münch's hypothesis, the translocation by mass flow from sources to sinks and the osmotic mechanism of pressure flow, were established notions at the time, but had been developed by two institutionally separated groups of scholars. A conceptual separation of whole-plant biology from cellular physiology had followed the institutional separation of forestry science from botany in German-speaking central Europe during the so-called Humboldtian reforms, and was reinforced by the delayed institutionalization of plant physiology as an academic discipline. Münch did not invent a novel concept, but accomplished an integration of the organism-focused and the cell-focused research traditions, reducing the polarization that had evolved when research universities emerged in central Europe. Post-Münch debates about the validity of his hypothesis focused increasingly on the suitability of available research methodologies, especially the electron microscope and the proper interpretation of the results it produced. The present work reconstructs the influence of the dynamic scientific and non-scientific context on the history of the Münch hypothesis.

In situ microscopy reveals reversible cell wall swelling in kelp sieve tubes: one mechanism for turgor generation and flow control?

Kelps, brown algae (Phaeophyceae) of the order Laminariales, possess sieve tubes for the symplasmic long-distance transport of photoassimilates that are evolutionarily unrelated but structurally similar to the tubes in the phloem of vascular plants. We visualized sieve tube structure and wound responses in fully functional, intact Bull Kelp (Nereocystis luetkeana [K. Mertens] Postels & Ruprecht 1840). In injured tubes, apparent slime plugs formed but were unlikely to cause sieve tube occlusion as they assembled at the downstream side of sieve plates. Cell walls expanded massively in the radial direction, reducing the volume of the wounded sieve elements by up to 90%. Ultrastructural examination showed that a layer of the immediate cell wall characterized by circumferential cellulose fibrils was responsible for swelling and suggested that alginates, abundant gelatinous polymers of the cell wall matrix, were involved. Wall swelling was rapid, reversible and depended on intracellular pressure, as demonstrated by pressure-injection of silicon oil. Our results revive the concept of turgor generation and buffering by swelling cell walls, which had fallen into oblivion over the last century. Because sieve tube transport is pressure-driven and controlled physically by tube diameter, a regulatory role of wall swelling in photoassimilate distribution is implied in kelps.

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates.

BACKGROUND AND AIMS: In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system. METHODS: The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy. KEY RESULTS: Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates. CONCLUSIONS: The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks.

SEORious business: structural proteins in sieve tubes and their involvement in sieve element occlusion.

The phloem provides a network of sieve tubes for long-distance translocation of photosynthates. For over a century, structural proteins in sieve tubes have presented a conundrum since they presumably increase the hydraulic resistance of the tubes while no potential function other than sieve tube or wound sealing in the case of injury has been suggested. Here we summarize and critically evaluate current speculations regarding the roles of these proteins. Our understanding suffers from the suggestive power of images; what looks like a sieve tube plug on micrographs may not actually impede translocation very much. Recent reports of an involvement of SEOR (sieve element occlusion-related) proteins, a class of P-proteins, in the sealing of injured sieve tubes are inconclusive; various lines of evidence suggest that, in neither intact nor injured plants, are SEORs determinative of translocation stoppage. Similarly, the popular notion that P-proteins serve in the defence against phloem sap-feeding insects is unsupported by empirical facts; it is conceivable that in functional sieve tubes, aphids actually could benefit from inducing a plug. The idea that rising cytosolic Ca(2+) generally triggers sieve tube blockage by P-proteins appears widely accepted, despite lacking experimental support. Even in forisomes, P-protein assemblages restricted to one single plant family and the only Ca(2+)-responsive P-proteins known, the available evidence does not unequivocally suggest that plug formation is the cause rather than a consequence of translocation stoppage. We conclude that the physiological roles of structural P-proteins remain elusive, and that in vivo studies of their dynamics in continuous sieve tube networks combined with flow velocity measurements will be required to (hopefully) resolve this scientific roadblock.

Long-distance translocation of photosynthates: a primer.

The storage of light energy in chemical form through photosynthesis is the key process underlying life as we know it. To utilize photosynthates efficiently as structural materials or as fuel to drive endergonic processes, they have to be transported from where they are produced to where they are needed. In this primer, we provide an overview of basic biophysical concepts underlying our current understanding of the mechanisms of photosynthate long-distance transport, and briefly discuss current developments in the field.

Cell wall is a dead metaphor.

It was recently suggested to end the usage of the term cell wall in microbiology (including prokaryotes and fungi) because it represented an inappropriate and potentially misleading metaphor (Casadevall and Gow, 2022). The analysis of the arguments for such a move from the viewpoint of the plant physiologist indicates that the suggestion is based on misunderstandings, first, of the early history of cell biology and its terminology; second, of the development of modern concepts of cell wall function since the late 19th century; and third, of the nature of metaphors and their role in scientific communication. We conclude that misconceptions concerning cell walls may arise due to pedagogical shortcomings in introducing students to our technical terminology rather than from the fact that part of this terminology originated from metaphors.

Locally optimal geometry for surface-enhanced diffusion.

Molecular diffusion in bulk liquids proceeds according to Fick's law, which stipulates that the particle current is proportional to the conductive area. This constrains the efficiency of filtration systems in which both selectivity and permeability are valued. Previous studies have demonstrated that interactions between the diffusing species and solid boundaries can enhance or reduce particle transport relative to bulk conditions. However, only cases that preserve the monotonic relationship between particle current and conductive area are known. In this paper, we expose a system in which the diffusive current increases when the conductive area diminishes. These examples are based on the century-old theory of a charged particle interacting with an electrical double layer. This surprising discovery could improve the efficiency of filtration and may advance our understanding of biological pore structures.

Pavement cells distinguish touch from letting go.

A micro-cantilever technique applied to individual leaf epidermis cells of intact Arabidopsis thaliana and Nicotiana tabacum synthesizing genetically encoded calcium indicators (R-GECO1 and GCaMP3) revealed that compressive forces induced local calcium peaks that preceded delayed, slowly moving calcium waves. Releasing the force evoked significantly faster calcium waves. Slow waves were also triggered by increased turgor and fast waves by turgor drops in pressure probe tests. The distinct characteristics of the wave types suggest different underlying mechanisms and an ability of plants to distinguish touch from letting go.

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.

How Münch's adaptation of Pfeffer's circulating water flow became the pressure-flow theory, and the resulting problems - A historical perspective.

Long-distance transport of photoassimilates in the phloem of vascular plants occurs as bulk flow in sieve tubes. These tubes are arrays of cells that lose nuclei, cytoskeleton, and some organelles when they differentiate into mature sieve elements. Symplasmic continuity is achieved by perforations that turn the cell walls between adjoining sieve elements into sieve plates. These structural features are interpreted as adaptations that reduce the resistance sieve tubes offer to cytoplasmic bulk flow. According to the common reading of Ernst Münch's pressure-flow theory, the driving forces for these flows are osmotically generated gradients of hydrostatic pressure along the sieve tubes. However, the significance of pressure gradients in the flow direction has also been questioned. Münch himself stated that no detectable pressure gradients existed between the linked osmotic cells that he used to demonstrate the validity of his ideas, and the earliest explanation of osmotically driven flows by Wilhelm Pfeffer, on which Münch based his theory, explicitly claimed the absence of pressure gradients. To resolve the apparent contradiction, we here reconstruct the history of the idea that osmotically driven transport processes in organisms necessarily require steps or gradients of hydrostatic pressure along the transport route. Our analysis leads us to conclude that some defects of overly simplifying interpretations of Münch's ideas (such as the sieve plate fallacy) could be avoided if our descriptions of his theory in textbooks and the scientific literature would follow the logics of the theory's earliest formulations more closely.

Plasmodesmata and the problems with size: Interpreting the confusion.

Plant tissues exhibit a symplasmic organization; the individual protoplasts are connected to their neighbors via cytoplasmic bridges that extend through pores in the cell walls. These bridges may have diameters of a micrometer or more, as in the sieve pores of the phloem, but in most cell types they are smaller. Historically, botanists referred to cytoplasmic bridges of all sizes as plasmodesmata. The meaning of the term began to shift when the transmission electron microscope (TEM) became the preferred tool for studying these structures. Today, a plasmodesma is widely understood to be a 'nano-scale' pore. Unfortunately, our understanding of these nanoscopic channels suffers from methodological limitations. This is exemplified by the fact that state-of-the-art EM techniques appear to reveal plasmodesmal pore structures that are much smaller than the tracer molecules known to diffuse through these pores. In general, transport processes in pores that have dimensions in the size range of the transported molecules are governed by different physical parameters than transport process in the macroscopic realm. This can lead to unexpected effects, as experience in nanofluidic technologies demonstrates. Our discussion of problems of size in plasmodesma research leads us to conclude that the field will benefit from technomimetic reasoning - the utilization of concepts developed in applied nanofluidics for the interpretation of biological systems.

Münch, morphology, microfluidics - our structural problem with the phloem.

The sieve tubes of the phloem are enigmatic structures. Their role as channels for the distribution of assimilates was established in the 19th century, but their sensitivity to disturbations has hampered the elucidation of their transport mechanisms and its regulation ever since. Ernst Münch's classical monograph of 1930 is generally regarded as the first coherent theory of phloem transport, but the 'Münchian' pressure flow mechanism had been discussed already before the turn of the century. Münch's impact rather rested on his simple physical models of the phloem that visualized pressure flow in an intuitive way, and we argue that the downscaling of such models to realistic, low-Reynolds-number sizes will boost our understanding of phloem transport in this century just as Münch's models did in the previous one. However, biologically meaningful physical models that could be used to test predictions of the many existing mathematical models would have to be designed in analogy with natural phloem structures. Unfortunately, the study of phloem anatomy seems in decline, and we still lack basic quantitative data required for evaluating the plausibility of our theoretical deductions. In this review, we provide a subjective overview of unresolved problems in angiosperm phloem structure research within a functional context.

The diffusive injection micropipette (DIMP).

The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern cell biology. Microinjection techniques include the penetration of the plasma membrane and, if present, the cell wall with micropipettes, and the application of pressure or electrical currents to drive the micropipette contents into the cell. These procedures interfere with cellular functions and therefore may induce artifacts. We designed the diffusive injection micropipette (DIMP) that avoids most of the possible artifacts due to the drastically reduced volume of its fluid contents and the utilization of diffusion for cargo delivery into the target cell. DIMPs were successfully tested in plant, fungal, and animal cells. Using the continuity of cytoplasmic dynamics over ten minutes after impalement of Nicotiana trichome cells as a criterion for non-invasiveness, we found DIMPs significantly less disruptive than conventional pressure microinjection. The design of DIMPs abolishes major sources of artifacts that cannot be avoided by other microinjection techniques. Moreover, DIMPs are inexpensive, easy to produce, and can be applied without specific equipment other than a micromanipulator. With these features, DIMPs may become the tool of choice for studies that require the least invasive delivery possible of materials into live cells.

Size-dependent locomotory performance creates a behaviorally mediated prey size refuge in the marine snail Olivella semistriata: a study in the natural habitat.

The effects of the variability of individual prey locomotory performance on the vulnerability to predation are poorly understood, partly because individual performance is difficult to determine in natural habitats. To gain insights into the role(s) of individual variation in predatory relationships, we study a convenient model system, the neotropical sandy beach gastropod Olivella semistriata and its main predator, the carnivorous snail Agaronia propatula. The largest size class of O. semistriata is known to be missing from A. propatula's spectrum of subdued prey, although the predator regularly captures much larger individuals of other taxa. To resolve this conundrum, we analyzed predation attempts in the wild. While A. propatula attacked O. semistriata of all sizes, large prey specimens usually escaped by 'sculling', an accelerated, stepping mode of locomotion. Olivella semistriata performed sculling locomotion regardless of size, but sculling velocities determined in the natural environment increased strongly with size. Thus, growth in size as such does not establish a prey size refuge in which O. semistriata is safe from predation. Rather, a behaviorally mediated size refuge is created through the size-dependence of sculling performance. Taken together, this work presents a rare quantitative characterization in the natural habitat of the causal sequence from the size-dependence of individual performance, to the prey size-dependent outcome of predation attempts, to the size bias in the predator's prey spectrum.