Aart J. E. van Bel

Effects of temperature on the conformation of the endoplasmic reticulum and on starch accumulation in leaves with the symplasmic minor-vein configuration

The phloem-loading-related effects of temperature on leaf ultrastructure were studied in seven species having numerous plasmodesmatal connections between the mesophyll and phloem (symplasmic minor-vein configuration). The response to temperature (between 5 and 30 °C) was characterized by drastic changes in the endoplasmic-reticulum labyrinth (ER labyrinth) of intermediary cells, in the position of the vacuole in bundle-sheath cells, and in the starch content in the chloroplasts of bundle-sheath cells and mesophyll cells. At temperatures above 20 °C, the ER system in the intermediary cells reached its maximal volume, while the vacuole in bundlesheath cells was positioned centripetally (proximal to the intermediary cell). With decreasing temperature, the ER labyrinth in intermediary cells gradually contracted till the ER was fully collapsed at 10 °C and the vacuole in bundle-sheath cells moved to a more centrifugal position. The apparent elimination of photosynthate transport via the ER and plasmodesmata at temperatures lower than 10 °C led to starch accumulation in the chloroplasts of bundle-sheath cells and mesophyll cells. All of these changes were fully temperature-reversible and probably reflect changes in the balance between photosynthate transport and storage. The ultrastructural shifts appear to be correlated with the passage of photosynthate through the intermediary cells and, as a consequence, with the rate of phloem loading at various temperatures. A contraction of the ER/plasmodesmata system imposed by cytoskeletal reorganisation is discussed as the reason for the blockage of phloem loading at low temperatures in association with the general chilling sensitivity of these species.

Thermodynamic Battle for Photosynthate Acquisition between Sieve Tubes and Adjoining Parenchyma in Transport Phloem

In transport phloem, photoassimilates escaping from the sieve tubes are released into the apoplasmic space between sieve element (SE)/companion cell (CC) complexes (SE/CCs) and phloem parenchyma cells (PPCs). For uptake respective retrieval, PPCs and SE/CCs make use of plasma membrane translocators energized by the proton motive force (PMF). Their mutual competitiveness, which essentially determines the amount of photoassimilates translocated through the sieve tubes, therefore depends on the respective PMFs. We measured the components of the PMF, membrane potential and ΔpH, of SE/CCs and PPCs in transport phloem. Membrane potentials of SE/CCs and PPCs in tissue slices as well as in intact plants fell into two categories. In the first group including apoplasmically phloem-loading species (e.g. Vicia, Solanum), the membrane potentials of the SEs are more negative than those of the PPCs. In the second group including symplasmically phloem-loading species (e.g. Cucurbita, Ocimum), membrane potentials of SEs are equal to or slightly more positive than those of PPCs. Pure sieve tube sap collected from cut aphid stylets was measured with H+-selective microelectrodes. Under our experimental conditions, pH of the sieve tube saps was around 7.5, which is comparable to the pH of cytoplasmic compartments in parenchymatous cells. In conclusion, only the membrane potential appears to be relevant for the PMF-determined competition between SE/CCs and PPCs. The findings may imply that the axial sinks along the pathway withdraw more photoassimilates from the sieve tubes in symplasmically loading species than in apoplasmically loading species.

Dissimilar phloem loading in leaves with symplasmic or apoplasmic minor-vein configurations

Plant species were selected on the basis of abundant or no symplasmic continuity between sieveelement-companion-cell (SE-CC) complexes and adjacent cells in the minor veins. Symplasmic continuity and discontinuity are denoted, respectively, as symplasmic and apoplasmic minor-vein configurations. Discs of predarkened leaves from which the lower epidermis had been removed, were exposed to 14CO2. After 2 h of subsequent incubation, phloem loading in control discs and discs treated with p-chloromercuribenzenesulfonic acid (PCMBS) was recorded by autoradiography. Phloem loading was strongly suppressed by PCMBS in minor veins with symplasmically isolated SE-CC complexes (Centaurea, Impatiens, Ligularia, Pelargonium, Pisum, Symphytum). No significant inhibition of phloem loading by PCMBS was observed in minor veins containing sieve elements with abundant symplasmic connections (Epilobium, Fuchsia, Hydrangea, Oenothera, Origanum, Stachys). Phloem loading in minor veins with both types of SE-CC complex (Acanthus) had apoplasmic features. The results provide strong evidence for coincidence between the mode of phloem loading and the minor-vein configuration. The widespread occurrence of a symplasmic mode of phloem loading is postulated.

Microelectrode-recorded development of the symplasmic autonomy of the sieve element/companion cell complex in the stem phloem of Lupinus luteus L.

From the cambial stage onwards, the symplasmic autonomy of sieve element/companion cell complexes (SE/CC-complexes) was followed in stems of Lupinus luteus L. by microinjection techniques. The membrane potential and the symplasmic autonomy of the mature SE/CC-complex was measured in successive internodes. A microelectrode was inserted into SE/CC-complexes or phloem parenchyma cells (PPs) and, after stabilization of the membrane potential, the membrane-impermeant fluorescent dye Lucifer Yellow CH (LYCH) was injected intracellullary. The plasmodesmata of the cambial SE/ CC precursor were gradually shut off at all interfaces beginning at the walls to be transformed into sieve plates. In the course of maturation, symplasmic discontinuity was maintained at the longitudinal walls of the complex. In the transverse walls of the SE, wide sieve pores were formed giving rise to longitudinal multicellular symplasmic domains of SE/CC-complexes. Symplasmic isolation of the files of mature SE/CC-complexes was demonstrated in several ways: (i) the membrane potential of the SE/CC-complexes (between -100 mV and -130 mV) was consistently more negative than that of the PPs (between-50 and -100 mV), (ii) No exchange of LYCH was observed between SE/CC-complexes and the PPs. Lucifer Yellow CH injected into the SEs exclusively moved to the associated CCs and to other SE/CC-complexes whereas LYCH injected into the PPs was only displaced to other PPs. (iii) The electrical coupling ratio between adjacent PPs was ten times higher than that between SE/CC-complex and PP. A gradient in the membrane potential of the SE/CC-complexes along the stem was not conclusively demonstrated.

Symplastic isolation of the sieve element-companion cell complex in the phloem of Ricinus communis and Salix alba stems

The anatomical and physiological isolation of the sieve element-companion cell complex (se-cc complex) was investigated in stems of Ricinus communis L. and Salix alba L. In Ricinus, the plasmodesmatal frequencies were in the proportions 8∶1∶2∶30, in the order given, at the interfaces between sieve tube-companion cell, sieve tube-phloem parenchyma cell, companion cellphloem parenchyma cell, and phloem parenchyma cellphloem parenchyma cell. The membrane potentials of the se-cc complex and the surrounding phloem-parenchyma cells sharply contrasted: the membrane potential of the se-cc complex was about twice as negative as that of the phloem parenchyma. Lucifer Yellow CH injected into the sieve element or into the companion cell remained within the se-cc complex. Dye introduced into phloem parenchyma only moved (mostly poorly) to other phloem-parenchyma cells. The distribution of the plasmodesmatal frequencies, the differential dye-coupling and the sharp discontinuities in membrane potentials indicate that the se-cc complexes constitute symplast domains in the stem phloem. Symplastic autonomy is discussed as a basic necessity for the functioning of the se-cc complex in the stem.

The transport phloem. Specifics of its functioning

Translocation of photoassimilates from the source leaves to the heterotrophic parts of the plant demands three different functions executed by the phloem system. The photosynthate is collected in the source leaves (phloem loading), translocated from source to sink (phloem transport), and delivered in the sink tissues (phloem unloading). It is expected that anatomy and physiology of the particular phloem sections reflect the specific tasks to be carried out. Investigations over the past 10 years revealed a diversity of anatomical settings in sources and sinks which seem to correspond with a multiplicity of mechanisms. Several reviews and opinion papers were recently devoted to the phloem sections engaged in loading (Delrot 1987; Van Bel 1987, 1989, 1992; Gamalei 1989, 1990, 1991; Turgeon 1989; Turgeon and Beebe 1991; Van Bel and Gamalei 1991) and unloading (Murray 1987; Turgeon 1989; Patrick 1990; Oparka 1990; Wolswinkel 1990). The functioning of the transport phloem has gained much less attention, although some silent conceptual progress has been made over the past decade. Only aspects such as the photosynthate unloading from the transport phloem (Patrick 1990) and the relation between transport phloem and solute transfer through rays (Van Bel 1990) have been reviewed recently.

A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of 14CO2-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate 14C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.

Physiochemical Determinants of Phloem Transport

Publisher Summary This chapter identifies several questions as to the physiochemical determinants of phloem transport. There is a general quantitative unawareness with regard to physiochemical parameters. Moreover, one must be thoughtful of potentially large differences between plant species due to disparate structural/functional conditions. The steepness of the hydraulic gradient depends on the physiological activities along the whole sieve tube stretch. Thus, diversity in modes of phloem loading, release/retrieval along the transport pathway, and in modes of phloem unloading, may have a strong impact on generation of the hydraulic pressure gradient. This recognition requires detailed studies on deployment and functioning of proteins involved in shuttling osmotic equivalents and water through the sieve element/companion cell (SE/CC) plasma membrane of the successive phloem zones and the structural frame of the SE/CCs in various plant groups. Furthermore, the reciprocal feedback regulation of sources and sinks and the compensatory and buffering activities of transport phloem must be investigated on the cell-biological level.

Analysis of valine uptake by Commelina mesophyll cells in a biphasic active and a diffusional component

Valine uptake by isolated Commelina benghalensis L. mesophyll cells was measured over a wide concentration range (10-6–4·10-2 mol l-1). The uptake data were subjected to iterative fitting. Experiments with carbonyl cyanide mchlorophenyl hydrazone (CCCP), diethylstilbestrol (DES), and p-chloromercuriphenylsulphonic acid (PCMBS) provided evidence that the biphasic uptake kinetics of valine consists of a diffusional component and a biphasic active uptake. The data from the control experiments, were also best fitted to one diffusional component and two Michaelis-Menten systems. The presence of two carrier systems in the plasmalemma, however, was considered to be virtual for the following reasons: (1) Both phases of active uptake were equally decreased by high concentrations of K+-ions. (2) Fusicoccin stimulated the active uptake in both phases to the same extent. (3) Inhibitors of the proton-driven uptake (CCCP, DES, PCMBS) similarly inhibited the active uptake at all concentrations. (4) The active uptake equally responded in both phases to changes in the pH. (5) Light also promoted the active uptake over the whole concentration range. These results strongly indicate that, despite its biphasic character, the active uptake is due to one proton-driven carrier system.

Light-promoted diffusional amino acid efflux from Commelina leaf disks Indirect control by proton pump activities

The release (=the measured loss) of amino acids was studied in Commelina benghalensis leaf disks. The release is assumed to be the result of influx and efflux, therefore, both movements were investigated.The uptake of 14C-labeled valine exhibited a biphasic isotherm. The uptake was pH-dependent, especially at low substrate concentrations (pH optimum 4.8). Signals for amino acid/proton co-transport were observed: stimulation of the uptake by fusicoccin (FC), inhibition by diethylstilbestrol (DES) or by high K+ concentrations. In the light, the ATP level of the disks was maintained during the uptake period (2 h), in darkness the ATP content decreased from 87 to 24 nmol g−1 fr. wt. However, light-promoted uptake, which is explained in the proton pump concept by an intensified proton extrusion as the result of high ATP production, was lacking.The release of amino acids was increased by washing with p-chloromercuriphenyl sulphonic acid (PCMBS), nystatin, 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or KCN. The release (Q10 about 1.5) was independent of the external pH and was linearly related to the intracellular amino acid concentration. Light enhanced the rate of release to the same extent at all intracellular concentrations. The present results suggest that the release is balanced by a, at least partially, proton-driven influx and a diffusional ligh-promoted efflux. A provisional model shows how the diffusional effulx can be indirectly controlled by a counter-flow fueled by the metabolism.