Ankie Ammerlaan

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.

Phloem unloading in developing seeds ofVicia faba L. : The effect of several inhibititors on the release of sucrose and amino acids by the seed coat.

Several characteristics of the process of phloem unloading in the seed coat of developing seeds ofVicia faba L. were investigated. Prior to the procedure of measuring the release of solutes by the seed coat, the embryo was removed from each ovule studied. First, a window was made in the pod wall and, subsequently, a rectangular incision was made in the seed coat of several ovules within the same pod. After the removal of the embryo from a developing seed, the “empty” seed coat was filled with a solution (pH 5.5) with or without inhibitors. Following another procedure excised seed-coat halves were incubated in a “wash-out” solution. The effects ofp-chloromercuribenzenesulfonic acid, low temperature, NaN3, carbonylcyanide-m-chlorophenylhydrazone and 25 mM K+ were measured. The pattern of solute unloading from the seed coat was not clearly influenced by 25 mM K+. All other treatments strongly inhibited the release of sucrose ana amino acids (L-valine, L-asparagine, α-aminoisobutyric acid). In many experiments, the amount of label entering “empty” ovules filled with a solution without inhibitor did not differ much from the amount of label entering an intact ovule within the same pod. Moreover, in some experiments in which the absolute amounts of sucrose and asparagine were measured, the release of these solutes by the seed coat of an “empty” ovule was remarkably constant over a period of several hours after the surgical treatment. From these data it can be concluded that, at least in short-term experiments (6 h or somewhat longer), the presence of the developing embryo is not necessary for intensive phloem unloading. “Empty” ovules of legumes represent a valuable system for detailed studies on the regulation of phloem unloading.

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.

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.

Comparison of the uptake kinetics of valine and sucrose and their light-reactivity in mesophyll cells of Commelina benghalensis

Summary Eadie-Hofstee plots demonstrate that valine and sucrose uptake by isolated Commelina benghalensis L. mesophyll cells obeyed biphasic kinetics. The uptake of sucrose is presumably performed by two saturable systems (K m1 , K m2 : 10 −4 and 2,3 × 10 −4 M, respectively; V m1 , V m2 : 4.8 and 928 nmol h −1 /10 6 cells), valine uptake by two saturable systems (K m1 , K m2 : 3.8 × 10 −4 and 7 × 10 −3 M, respectively; V m1 , V m2 : 111 and 171 nmol h 1 /10 6 cells) imposed on a linear term. The differences between the uptake parameters of the sucrose uptake systems (ratios K m2 /K m1 , 1900–2700; V m2 /V m1 , 180–550) surpass by far those of valine uptake (K m2 /K m1 , 25–100; V m2 /V m1 , 1.8–7.5). Light did not affect the K m -values , but exerted a differential effect on the V m -values. Of valine uptake V m1 was promoted more by illumination than V m2 -valine whereas V m2 -sucrose reacted more strongly to light than V m1 -sucrose.