Distorted phytochrome action spectra in green plants

An evaluation was made of the extent which a Münch-type pressure flow mechanism (i.e., osmotically-generated pressure flow) might contribute to phloem transport in soybean. Estimates of sucrose concentrations in source (leaf) and sink (root) sieve tubes were obtained by a negativestaining procedure. Water potential measurements of the leaf and of the nutrient solution allowed calculation of the turgor pressures in source and sink sieve tubes. The turgor difference between source and sink sieve tubes was compared to that required to drive translocation at the observed velocity between the source and sink, as measured by [¹⁴C] photosynthate movement. Sieve-tube conductivity was calculated from the sieve-tube dimensions, assuming an essentially unobstructed pathway. In three experiments, the sucrose concentration was consistently higher in source sieve tubes (an average of 11.5%) than in sink sieve tubes (an average of 5.3%). The ratio of these values (2.3:1) agreed reasonably well with an earlier ratio for source/sink sieve tube concentrations of 1.8:1, obtained by quantitative microautoradiography. The resulting calculated turgor difference (an average of 4.1 bars) was adequate to drive a pressure flow mechanism at the observed translocation velocities (calculated to require a turgor difference of 1.2 to 4.6 bars). No other force need be presumed to be involved.This work was presented in part at a joint U.S.-Australian Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 15–20, 1975; see Fisher (1976)     

Seasonal Variations of Secondary Phloem Development in Pterocarya Stenoptera and Its Relation to Feeding of Kerria yunnanensis

Early in April of 1987, cells in an undifferentiated state which overwintered on the phloem side of the cambial zone in the branch of Pterocarya stenoptera began to differentiate into merebets of phloem. Cambium divided actively in mid-April and ceased to decide by early-Novembet. Five to eleven bands of fibers alternating with the bands of sieve tubes, companion cells and phloem parenchyma cells produced every year. By mid to late April, new xylem differentiation began. Phloem and xylem differentiation ceased almost simultaneously. Functional sieve tube elements were present all the year round in the phloem. During winter, most sieve tubes produced in the current year ceased functioning, leaving only the zone of functional sieve tube of several rows of cells in width with open pores in the sieve plates. These sieve tubes did not collapse until mid-May. In October, several rows of partially differentiated sieve elements appeared near the cambial zone. They still possessed nuclei. The companion cells had produced but no P-protein. They matured during April of the following year and collapsed by July to September. The life span of sieve elements extended for 8 months at the most. In winter, there were less functional sieve tubes in the branch. This may be one of the reasons that only few Kerria yunnanensis survive on the branch of Pterocarya stenoptera.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.     

A symplasmic flow of sucrose contributes to phloem loading in Ricinus cotyledons

External sucrose, supplied by the endosperm in vivo, is the physiological source of sucrose for Ricinus communis L. seedlings. It is taken up by the cotyledons and exported via the sieve tubes to the growing hypocotyl and root. Two parallel pathways of external sucrose to the sieve tubes, directly via the apoplasm and indirectly after transit through the mesophyll, have already been established (G. Orlich and E. Komor, 1992). In this study, we analysed whether a symplasmic flow of sucrose contributes to phloem loading. Uptake of external sucrose into the mesophyll and into the sieve tubes, and export of total sucrose were measured with intact and exuding seedlings in the presence of p-chloromercuribenzenesulfonic acid (PCMBS). Sucrose uptake into the mesophyll and into the sieve tubes was inhibited by 80–90%. Consequently, export of total sucrose slowed down. However, after the addition of PCMBS, sucrose was transiently exported in such a high amount that could not be accounted for by the residual uptake activity nor by the amount of sucrose confined to the sieve element-companion cell complex (seccc). From the results, we conclude that most of the sucrose exported transiently had moved to the sieve tubes from a symplasmic domain larger than the seccc, comprising at least all the cells of the bundle including the bundle sheath. We suggest that the symplasmic flow of sucrose observed is a mass flow driven by a turgor pressure. As a structural prerequisite for a symplasmic flow, plasmodesmata interconnect all the cells from the bundle sheath to the sieve tubes and also occur between the bundle sheath and the mesophyll. The phloem loading pathway of Ricinus cotyledons can thus be classified as a combination of three different routes. Received: 17 October 1997 / Accepted: 9 March 1998     

Estimation of Osmotic Gradients in Soybean Sieve Tubes by Quantitative Autoradiography: Qualified Support for the MUnch Hypothesis

An attempt was made to evaluate Münch's hypothesis of osmotically generated pressure flow in soybean (Glycine max L.) sieve tubes from velocity measurements and calculations of pressure potentials and sieve tube resistances. Pressure potential was estimated from values for water potentials and osmotic potential. Leaf water potential measurements were made by isopiestic thermocouple psychrometry, while the water potential of the nutrient solution was made with a vapor pressure osmometer. Osmotic potential was measured by first bringing the sucrose pools in the entire plant to the same specific radioactivity by steady-state-labeling of the shoot with constant specific radioactivity ¹⁴CO₂ for 5 to 8 hours. Sucrose concentrations in sieve tubes were calculated from the disintegration rate per unit volume in sieve elements as measured by absolute quantitative microautoradiography of freeze-substituted, Eponembedded source (leaf) and sink (root) tissues.     

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.     

Scaling phloem transport: elasticity and pressure-concentration waves

Earlier theoretical analyses of the rate of propagation of pressure-concentration waves in the phloem were performed without adequate attention to the elastic expansion of sieve tube walls. Here, it is shown that the rate of propagation of pressure-concentration waves in phloem sieve tubes is not significantly impeded by wall elasticity, but rather, as previously implicated, by the ratio of sap osmotic pressure to the axial drop in sap hydrostatic pressure. It is also shown that pressure-concentration waves move equally well in both the upstream and downstream directions. These results permit future models to ignore elastic effects, and lend additional theoretical support to the "osmoregulatory flow" hypothesis, which argues that efficient molecular control of the phloem is permitted by maintaining sieve sap hydrostatic pressure at a value that is spatially nearly constant, which in turn permits changes in sieve tube state to be rapidly transmitted throughout the sieve tube via pressure-concentration waves.     

Phloem ultrastructure and pressure flow: Sieve-Element-Occlusion-Related agglomerations do not affect translocation

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.     

Ricerche Sull'infrastruttura del Coleottile di Avena

Abstract

Researches on ultrastructure of Avena coleoptile. 3. The sieve elements. — A study on the ultrastructural organization of the mature sieve elements of Avena coleoptile has been carried out. Data suggest that functional phloem tubes are alive and remain alive until they are working. Judging on morphological basis, the metabolic activity of sieve elements should be of peculiar type and low in comparison to that of the companion cells. In fact the cytoplasm is located in a narrow parietal strand, mitochondria, Golgi apparatus and endoplasmic reticulum are present, but they appear very modified; plastids and nucleus are absent. The cytoplasm is bounded externally by a normal plasmalemma, whilst the vacuole has no visible limits: a tonoplast is, therefore not identifiable.

The strands connecting the superimposed sieve elements with one another through the sieve plate result to be made by a double membrane system very similar to the endoplasmic reticulum, which we believe to realize cytoplasmic continuity between phloem tubes.

The data reported are more favorable to the existence in the sieve tubes of an active mechanism of translocation of organic solutes than a passive mass-flow.

The collaboration of companion cells in the translocation mechanism has been discussed.     

A Model for the Translocation of Photosynthate in the Soybean

The transport of photosynthetically incorporated carbon downthe soybean stem is discussed in terms of flow from a reservoirin the upper part of the plant. A comparison of this model withthe experimental data from the preceding paper leads to an estimateof translocation velocity of about 60 cm. per hr. and to leakageof material from the sieve tubes of about o.8 per cent. percm. of stem traversed. The effects of velocity distributionsand delays in the translocation system are considered.     

Analysis of the driving forces of phloem transport in Ricinus seedlings: sucrose export and volume flow are determined by the source

The aim of this study was to reveal the factors determining sucrose export and volume flow through the sieve tubes in Ricinus communis L. seedlings. The cotyledons take up sucrose from the apoplasm in vivo, and export most of it to the growing sinks, hypocotyl and root. This simple source-sink system allowed sucrose uptake and export to be studied under controlled conditions with respect to apoplasmic sucrose concentrations. From the additional knowledge of the sucrose concentrations in the mesophyll and the sieve tubes, transmembrane concentration differences were calculated. The volume flow rate along the sieve tubes could be calculated from the export rate and the sucrose concentration in the sieve tubes. While the export rate exhibited saturation kinetics, the volume flow rate decreased at high external sucrose concentrations. The export rate correlated with the sucrose uptake rate, the volume flow rate correlated with the sucrose concentration (osmotic pressure) difference across the sieve-tube plasma membrane, the driving force for transmembrane water flux. From these data it can be concluded that sucrose export and the volume flow through the sieve tubes are determined by activities of the source. Export out of Ricinus cotyledons was considerably higher than export out of green source leaves of different species. The concomitant comparatively low sucrose concentration in the sieve-tube sap of the seedlings can thus be attributed to a very high water flux into and along the sieve tubes associated with the high sucrose flux. Received: 28 November 1997 / Accepted: 4 April 1998     

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.     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.     

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.     

Measurement of turgor pressure and its gradient in the Phloem of oak

A direct method is described for measuring the pressure in secondary phloem sieve tubes of oak trees. One end of a 26-gauge stainless steel tube was shaped such that when it penetrated the outer bark and transected a few sieve elements, it was stopped by the xylem so that small openings in the end allowed phloem sap to enter the tube. The other end of the stainless tube (phloem needle) was joined to a long glass capillary sealed at its other end to form a manometer for measuring phloem sap pressure. A method for measuring the average osmotic and turgor pressures in cells of leaves is also described. Phloem turgor pressures varied greatly in a series of phloem punctures around the trunk at 1.5 and at 6.3 meters. The variation in turgor pressure was always greater than the variation in osmotic pressure. In a series of turgor pressures arranged in descending order, the values in a sequence for the upper level was usually a little (0-3 atm) larger than the values for the lower level. These results may suggest that translocation of assimilate is favored by a small turgor pressure gradient, but they do more to emphasize the complications in measuring gradients in an elastic low resistance distribution system composed of contiguous longitudinal conduits. The results also imply that the sieve tubes are inflated with assimilate fluid under high pressure which can readily move longitudinally and with less pressure drop than would be necessary if the sieve tubes were rigid.     

Cyanide Inhibition of Phloem Transport along the Stolon of Saxifraga sarmentosa L

Long thin stolons of Saxifraga were treated with cyanide inboth the solution and gaseous forms; the latter was much moreeffective. Cyanide strongly inhibited the transport of ¹³⁷Csand of natural ¹⁴C-assimilates. As judged by a variety of approaches,including the use of ¹⁴C-cyanide, the inhibition was certainlyeffective in the sieve tubes themselves. Callose formation didnot seem to be promoted. The inhibition was completely reversible,and was never accompanied by a build-up of tracer in, or before,the treated zone; failure to traverse it was not due thereforeto membrane damage and consequent leakage. For these reasonsthe results are held to favour a mechanism of mass flow invokingactive pumping in the sieve tubes.     

ASSOCIATION OF THE ENDOPLASMIC RETICULUM AND THE PLASTIDS IN ACER AND PINUS

A close sheathing of the plastids by endoplasmic reticulum has been observed. This is restricted to the companion cells and developing sieve tubes of the phloem of Acer pseudoplatanus and the resin canal cells and leaf callus cells from Pinus pinea. The sheathing is transitory in callus and sieve tubes but is a permanent feature of the companion and the resin canal cells. Possible functional relationships between the two organelles are discussed.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): QUANTITATIVE ASPECTS,AND SOLUTE CONCENTRATIONS OF THE SIEVE-TUBE MEMBERS

The vascular system of the leaf of Populus deltoides Bartr. ex Marsh, was examined quantitatively, and plasmolytic studies were carried out to determine the solute concentrations of sieve-tube members at various locations in the leaf. Both the total number and total crosssectional area of each cell type decreases with decreasing vein size. Although the proportion of phloem occupied by sieve tubes varies considerably from location to location, a linear relationship exists between cross-sectional area of the vascular bundles and both total and mean cross-sectional area of sieve tubes. Collectively, the cross-sectional area of all tertiary and minor veins feeding into a secondary exceeds the total cross-sectional area of sieve tubes at the base of that secondary. Moreover, the total volume of sieve tubes in the “catchment area” of a secondary vein is much greater than the total sieve tube volume of the secondary itself. Both tracheary elements and sieve-tube members undergo a reduction in both total and mean crosssectional area in the constricted zone at the base of the leaf. The plasmolytic studies revealed the presence of positive concentration gradients in sieve tubes of the lamina from the minor veins and tips of the secondaries to the bases of the secondaries and their associated subjacent midvein bundles and from the upper to lower portions of the median bundle of the midvein.     

Structurally,phloem unloading in the maize leaf cannot be symplastic

Developing longitudinal vascular bundles of the leaf blades of maize (Zea mays L. cv. W273) were examined with the transmission electron microscope to determine the frequency of plasmodesmata between the sieve tubes and their neighboring cells. Of particular interest were the protophloem sieve tubes, the first sieve tubes to mature in importing (all large and some intermediate) bundles. The protophloem sieve tubes, most of which lack companion cells, intergrade structurally with the thin-walled metaphloem sieve tubes. Both the protophloem sieve tubes and the thin-walled metaphloem sieve tubes and their companion cells (the sieve tube-companion cell complexes) are virtually isolated symplastically from the rest of the leaf, precluding a symplastic mechanism of phloem unloading in the leaf blade of maize.