Michael Knoblauch

Sieve Tubes in Action

A method was designed for in vivo observation of sieve element/companion complexes by using confocal laser scanning microscopy. A leaf attached to an intact fava bean plant was mounted upside down on the stage of a confocal microscope. Two shallow paradermal cortical cuts were made in the major vein. The basal cortical window allowed us to observe the phloem intact. The apical window at 3 cm from the site of observation was used to apply phloem-mobile fluorochromes, which identified living sieve elements at the observation site. In intact sieve tubes, the sieve plates did not present a barrier to mass flow, because the translocation of fluorochromes appeared to be unhindered. Two major occlusion mechanisms were distinguished. In response to intense laser light, the parietal proteins detached from the plasma membrane and formed a network of minute strands and clustered material that aggregated and pressed against the sieve plate. In response to mechanical damage, the evenly distributed P plastids exploded, giving rise to the formation of a massive plug against the sieve plate. In case of mechanical damage, the parietal proteins transformed into elastic threads (strands) that extended throughout the sieve element lumen. Our observations cover the phenomena encountered in previous microscopic and electron microscopic studies and provide a temporal disentanglement of the events giving rise to the confusing mass of structures observed thus far.

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.

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes.

A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 μm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow–dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA cantaining its gene into individual chloroplasts.

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.

Sieve tube geometry in relation to phloem flow

This work describes a novel method for imaging cell anatomy and cell wall features by scanning electron microscopy. The method was used to image sieve plates and sieve element geometry at high resolution to correlate sieve tube–specific conductivity with phloem flow measurements. Sieve elements are one of the least understood cell types in plants. Translocation velocities and volume flow to supply sinks with photoassimilates greatly depend on the geometry of the microfluidic sieve tube system and especially on the anatomy of sieve plates and sieve plate pores. Several models for phloem translocation have been developed, but appropriate data on the geometry of pores, plates, sieve elements, and flow parameters are lacking. We developed a method to clear cells from cytoplasmic constituents to image cell walls by scanning electron microscopy. This method allows high-resolution measurements of sieve element and sieve plate geometries. Sieve tube–specific conductivity and its reduction by callose deposition after injury was calculated for green bean (Phaseolus vulgaris), bamboo (Phyllostachys nuda), squash (Cucurbita maxima), castor bean (Ricinus communis), and tomato (Solanum lycopersicum). Phloem sap velocity measurements by magnetic resonance imaging velocimetry indicate that higher conductivity is not accompanied by a higher velocity. Studies on the temporal development of callose show that small sieve plate pores might be occluded by callose within minutes, but plants containing sieve tubes with large pores need additional mechanisms.

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.

Phloem Ultrastructure and Pressure Flow: Sieve-Element-Occlusion-Related Agglomerations Do Not Affect Translocation

Several protocols and methods were developed to study the in vivo structure, ultrastructure, and physiology of sieve tubes. The ultrastructure of sieve tubes differs significantly from what has been shown so far. The impact on phloem function is discussed. Since the first ultrastructural investigations of sieve tubes in the early 1960s, their structure has been a matter of debate. Because sieve tube structure defines frictional interactions in the tube system, the presence of P protein obstructions shown in many transmission electron micrographs led to a discussion about the mode of phloem transport. At present, it is generally agreed that P protein agglomerations are preparation artifacts due to injury, the lumen of sieve tubes is free of obstructions, and phloem flow is driven by an osmotically generated pressure differential according to Münch’s classical hypothesis. Here, we show that the phloem contains a distinctive network of protein filaments. Stable transgenic lines expressing Arabidopsis thaliana Sieve-Element-Occlusion-Related1 (SEOR1)–yellow fluorescent protein fusions show that At SEOR1 meshworks at the margins and clots in the lumen are a general feature of living sieve tubes. Live imaging of phloem flow and flow velocity measurements in individual tubes indicate that At SEOR1 agglomerations do not markedly affect or alter flow. A transmission electron microscopy preparation protocol has been generated showing sieve tube ultrastructure of unprecedented quality. A reconstruction of sieve tube ultrastructure served as basis for tube resistance calculations. The impact of agglomerations on phloem flow is discussed.

Sieve element and companion cell: the story of the comatose patient and the hyperactive nurse

Sieve elements and companion cells constitute the modules of the conducting elements in the phloem ofAngiosperms. Consequently, phloem transport basically relies on the concerted action of the sieve element/companion cell complexes. Sieve elements and companion cells are highly interactive units and show an extreme division of labour as exemplified by their state of life. Whereas the sieve element is almost ‘clinically’ dead, the companion cell is a paragon of bubbling activity. In the course of evolution, the sieve element has sacrificed all of its genetic and most of its metabolic equipment to serve photoassimilate translocation. A small part of the structural and metabolic outfit has been retained for a proper accomplishment of its function. In contrast, the cells bordering the sieve element have gained metabolic weight during evolution. With reference to their evolutionary descent, the peculiarities of sieve elements and companion cells are discussed in the light of recent cell-biological and molecular-biological findings. Emphasis is focused on their interaction, which is the secret of the success of the sieve element/companion cell complex.

GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.

The structure of the phloem - still more questions than answers

Long-distance assimilate distribution in higher plants takes place in the enucleate sieve-tube system of the phloem. It is generally accepted that flow of assimilates is driven by an osmotically generated pressure differential, as proposed by Ernst Münch more than 80 years ago. In the period between 1960 and 1980, the pressure flow hypothesis was challenged when electron microscopic images suggested that sieve tubes contain obstructions that would prevent passive flow. This led to the proposal of alternative translocation mechanisms. However, most investigators came to the conclusion that obstructions in the sieve-tube path were due to preparation artifacts. New developments in bioimaging have vastly enhanced our ability to study the phloem. Unexpectedly, in vivo studies challenge the pressure-flow hypothesis once again. In this review we summarize current investigations of phloem structure and function and discuss their impact on our understanding of long-distance transport in the phloem.