Long-term functioning of enucleate sieve elements: possible mechanisms of damage avoidance and damage repair

Abstract. The sieve-tube elements of long-lived arborescent monocotyledons which lack secondary thickening remain functional for many decades despite lacking a nucleus. A minimal requirement for transport by mass flow powered by the Munch mechanism is maintenance of semi-permeability of the plasmalemma of the sieve tube elements; loading and re-loading could be deputed to nucleate cells symplastically linked to the sieve elements. An additional requirement in the long-term relates to replacement of components damaged by mechanical, chemical or radiation intrusions. Minimizing the damage from radiation and chemical agents can be related to a number of commonly observed features of sieve tubes. Damage from O₂ and radicals derived there from is minimized in these essentially aerobic cells by (1) the absence of intercellular gas spaces in the phloem combined with the lower O₂ solubility and diffusivity in concentrated disaccharide (or sugar alcohol) solutions, (2) the absence of photosynthetic machinery which could generate singlet oxygen, and (3) the presence of at least some components of scavenging mechanisms (glutathione, peroxidase, abscorbate). Non-enzymic glycosylation (exacerbated by O2) of proteins is minimized by the low concentrations of reducing sugars in sieve-tubes. Ultraviolet damage is minimized by UV-absorbing materials between the plant surface and the sieve tubes, including the selerenchymatous cap of fibres on the vascular bundles. The extent to which repair involves symplastic import of polypeptides from nucleate companion cells, thus breaching the −800-Da limit on symplastic transport, is unclear, but it could occur in fully differentiated companion cell-sieve element associations without necessarily upsetting development.     

A critical assessment of standard processing methods for the preparation of palynological samples

Standard processing techniques for the isolation of organic walled dinoflagellate cysts from geological samples are examined, with particular attention to the size and type of sieve mesh used. Variations within the ‘standard’ processing techniques used by different laboratories are identified, and an assessment of the retention capacities of meshes of different sizes and different materials is carried out. Some dinoflagellate cysts and large numbers of Lycopodium spores, used for the calculations of absolute abundance data, were found to pass through 20 μm meshes. This is due to a combination of factors including: the diagonal aperture diameter of a 20 μm mesh measuring over 28 μm; the three-dimensional properties of different mesh weaves (nylon and polyester); and the non-spherical shape of the particles. Experiments demonstrate that the maximum mesh size that should be used in palynological processing is 15 μm. Nylon mesh is more practical to use than polyester as processing time is reduced, but nylon is degraded by contact with acid solutions. Meshes with apertures < 15 μm may be used, though this may be impractical for large samples containing significant quantities of fine siliciclastic or organic material.     

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.     

Electrically recorded feeding behaviour of cassava mealybug on host and non-host plants

The feeding behaviour of cassava mealybug (Phenacoccus manihoti) was analysed by the electrical penetration graph technique (EPG, DC-system) and by light microscopy. This study confirmed a typical phloem-feeding behaviour with an aphid-like predominance of extracellular pathways of stylets. Similarities of EPGs from mealybugs with those of aphids and whiteflies allowed adoption of standard pattern labelling. The main differences with aphid EPGs consisted of fewer but longer intracellular punctures (pd duration of 20 svs 5–7 s for aphids), longer times to the first phloem-ingestion period and a lower motility of stylets within the phloem searching process. Comparison of the feeding behaviour on 6 plant types, including two occasional hosts (Talinum and poinsettia), one cassava hybrid and three common cassava cultivars (Manihot esculenta), allowed differentiation of penetration profiles. Intracellular punctures were shorter on occasional hosts, on which phloem finding and subsequent ingestion were markedly delayed although a great variability within plants persisted. Chemical analysis of some secondary plant substances revealed that none of the plants tested contained detectable amounts of alkaloids, and that cyanides were restricted to true hosts (cassavas andManihot hybrid). Levels of total flavonoids did not differ between hosts and non-hosts, in contrast to those of phenolic acids. Preliminary correlations with EPG paterns are discussed.

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Résumé Le comportement alimentaire de la cochenille du manioc (Phenacoccus manihoti) a été étudié par électrographie de pénétration (EPG, analyse en courant continu) et par microscopie optique. Cette étude a permis de confirmer le caractère typiquement phloémophage de l'alimentation de cet Homoptère, présentant une large prédominance du trajet extracellulaire des stylets, comme cela est également observé chez les pucerons. Les similitudes des tracés EPG effectués sur cochenille et sur pucerons ou aleurodes ont permis d'adopter la même typologie du signal. Les principales différences avec les pucerons concernent le nombre moins important et la durée plus longue des ponctions intracellulaires (20 s en moyenne contre 5 à 7 s chez les pucerons), l'allongement du temps d'accès au phloème et une moins grande mobilité des stylets lors de la phase de recherche du phloème. La comparaison du comportement alimentaire sur 6 plantes, parmi lesquelles deux h?tes occasionnels (Talinum et poinsettia), un hybride et trois variétés courantes de manioc (Manihot esculenta), a permis de différencier plusieurs profils de pénétration. Les ponctions intracellulaires se sont révélées plus courtes sur h?tes occasionnels, qui induisent également, malgré la variabilité observée, un allongement net de la période de localisation du phloème et donc un retard à l'ingestion. Aucune des plantes testées ne contient d'alcalo?des, et les composés cyanés se restreignent au genreManihot, h?te naturel de la cochenille. Les flavono?des totaux ne permettent pas de distinguer plantes h?tes et non-h?tes, à la différence des acides phénoliques.

     

Transmission of Electric Signals in Sieve Tubes of Zucchini Plants

Membrane potentials of ?;160 to ?210 mV were recorded with microelectrodes inserted into meta-phloem sieve tubes of intact zucchini plants (Cucurbita pepo L. var. medullosa Alef.). The effects of darkness, white light and colored light on membrane potential were studied. Reference electrodes were in contact with the apoplast via fluid-filled cavities or “drinks”. Electrolyte solutions (100 mM) in the cavities could be quickly replaced by flushing with 100 mM solutions of sucrose, KCl, sorbitol, or EDTA without altering osmolarity. KCl and EDTA caused depolarization of the sieve tube membrane potential, while sucrose caused depolarization or hyperpolarization of the sieve tube membrane potential in mature or growing plant parts respectively. Recovery of the original voltage was recorded when rapid (sucrose) or slow (sorbitol) transients occurred. When two measuring circuits were installed, one in a growing fruit and the other in the petiole of the subtending mature leaf, the alteration of the sieve tube membrane potential at one site was accompanied by an alteration of the potential at the other site after a few seconds. The responses were opposite in the exporting leaf and importing fruit when sucrose was applied. The signal, transmitted via the sieve tubes, reached maximum velocities of 10 cm per second.     

COMPOSITION OF SIEVE TUBE SAP FROM MACROCYSTIS PYRIFERA (PHAEOPHYTA) WITH EMPHASIS ON THE INORGANIC CONSTITUENTS1

A quantitative accounting of the solids in the sieve tube sap from Macrocystis pyrifera (L.) e. Ag. was performed. The major organic compounds (mannitol, amino acids, and protein) and inorganic cations (K⁺, Na⁺, Mg²⁺ and Ca²⁺) were present near previously reported levels. The anions, until now unreported (except for iodide), included chloride as the major inorganic ion, bromide, phosphate, nitrate, and bicarbonate as the major inorganic carbon species. Sulfate and ammonium were below the detection limits of 1.5 ppm and 2 ppm, respectively. The elements B and As were also present although their speciation was not determined.     

(Questions) on phloem biology. 1. Electropotential waves, Ca fluxes and cellular cascades along the propagation pathway

This review explores the relationships between electrical long-distance signalling, Ca²⁺ influx coincident with propagation of electropotential waves, and cellular responses to Ca²⁺ influx including the consequences for sieve-tube conductivity and mass flow. Ca²⁺ influx is inherent to electropotential waves and appears to constitute the key link between rapid physical signals and resultant chemical cascades in sieve tubes and adjacent cells. Members of several channel groups are likely involved the regulation of Ca²⁺ levels in sieve elements. Among them are hyperpolarization-activated, depolarization-activated, and mechanosensitive Ca²⁺ channels located in the plasma membrane and Ca²⁺ dependent Ca²⁺ channels that reside in ER-membranes of sieve elements. These channels collectively determine intracellular Ca²⁺ levels in sieve elements and their neighbour cells. The latter cells react to Ca²⁺ elevation by inducing diverse functional responses dependent on the cell type. If the Ca²⁺ concentration in sieve elements surpasses a threshold level, dual sieve-plate occlusion by proteins and callose deposition is triggered. Occlusion is reversed when Ca²⁺ levels subside. Electrical messages may regulate the degree of sieve plate hydraulic conductivity in intact plants by partial sieve-plate occlusion that has a major impact on volume flow through sieve tubes. Furthermore, complete but temporary occlusion of sieve tubes may modify mass flow patterns in intact plants.