Thermodynamic battle for photosynthate acquisition between sieve tubes and adjoining parenchyma in transport phloem

Abstract

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 DeltapH, 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.

Figure 1.

A, Fluorescence dispersal pattern after iontophoresis of LYCH into a SE in the stem phloem of V. faba tissue slices; asterisk (*) marks point of injection. The fluorescent dye moves axially to several successive SEs. Remarkable are the strongly fluorescent spheres in the SEs (marked with arrows) due to unspecific coloring of forisomes with LYCH. Bar = 100 μm. B, Fluorescence dispersal pattern after iontophoresis of LYCH into a PPC in stem phloem of V. faba tissue slices with random transport to adjoining PPCs. Asterisk (*) marks point of injection. N, Nucleus of the PPCs marked with arrow.

Figure 2.

Time course of LYCH distribution after injection into a SE of the stem phloem in V. faba tissue slices. A to C, Immediately after injection into the SE, the dye accumulated in the nuclei of the CCs (A), after 0.5 h the dye was evenly distributed over the SE/CC compartments (B), and after 17.5 h LYCH was nearly completely accumulated by the vacuole of the CCs (C). No LYCH traffic into the adjoining PPCs was visible. Arrows point to the nuclei of the CCs. Bar = 100 μm.

Figure 3.

A, Fluorescence dispersal pattern after iontophoresis of LYCH into a SE in the stem phloem of O. basilicum tissue slices; asterisk (*) marks point of injection. The fluorochrome moved to several successive SEs. Arrow indicates nucleus of the CC. Bar = 100 μm. B, LYCH injection (* marks point of injection) into a PPC in the stem phloem of O. basilicum tissue slices with random transport to other PPCs (small arrows). Asterisk (*) marks point of injection. N with arrows, Nuclei of the PPCs.

Figure 4.

Confocal laser scanning microscopy of translocating SEs in intact V. faba (A), S. lycopersicum (B), C. pepo (C), and O. basilicum (D) plants. CFDA was applied to an apical transport window in the main leaf vein. Within a time range of 30 to 120 min depending on the distance to the observation window and on the plant species, fluorochrome translocation (green) through intact sieve tubes was observed. CFDA fluorescence is restricted to the SE/CCs, indicative of a symplasmic disjunction between SE/CCs and PPCs in transport phloem of the plant species tested. Double staining by fluorescent RH414 applied to the window at the site of observation provided a better demarcation of SEs and CCs (Knoblauch and van Bel, 1998).

Figure 5.

A, Resting potentials (±sd) of SE/CCs and adjoining PPCs in excised tissue slices of different species (right). The number of replicates is presented in the bars. Left, Membrane potential ratios defined as ΔΨ_{m SE/CC}/ΔΨ_{m PPC} (striped bars). B, Resting potentials of SE/CCs and adjoining PPCs in transport phloem in main veins of intact plants (right). The number of replicates is presented in the bars. Left, Membrane potential ratios defined as ΔΨ_{m SE/CC}/ΔΨ_{m PPC} (striped bars).

Figure 6.

A, Optical surveillance of microelectrode impalement. Microelectrode (m, microelectrode tip) impaled into a SE in a main vein of an intact V. faba plant is shown. *, Forisome. B, Postinjection of LYCH into a SE in a main vein of an intact V. faba plant as part of a verification procedure to identify the cell of which the membrane potential was measured. Note the tip of the microelectrode (arrowhead, m) and the forisome (*). C, Postinjection of LYCH into a PPC of an intact V. faba plant after membrane potential measurement.

Figure 7.

Micro-pH measurement under silicon oil in a 100-pL droplet exuded from a stylet punctured into a SE in the main vein of an intact V. faba plant. On the left is an ion-selective electrode, on the right a KCl reference electrode inserted into the droplet. The aphid stylet is marked with a dark arrowhead.

Figure 8.

The difference in PMF (ΔPMF or ΔC) between SE/CCs and PPCs (ΔC = ΔΨ_{m SE/CC} − ΔΨ_{m PPC}) and the ratio (R) of theoretical substrate accumulation rates by SE/CCs versus PPCs in transport phloem of apoplasmically (□,▪) and symplasmically (○,•) phloem-loading species. R values were calculated from Nernst equation (Eq. 4). The open symbols represent the values for excised tissues, and the closed symbols those for intact plants. Photoassimilate retrieval by SE/CCs is presumed to be favored when R > 1, and photoassimilate accumulation by PPCs when R < 1. Inset, Magnification of the graph for R values <1.

Figure 9.

Hypothetical models for the impact of the difference in PMF between SE/CCs and PPCs in transport phloem (ΔC = ΔΨ_{m SE/CC} − ΔΨ_{m PPC}) on photoassimilate partitioning in symplasmically (A, B) and apoplasmically phloem-loading (C) species. The size of the horizontal arrows quantifies release from the SE/CCs and retrieval by SE/CCs and uptake by PPCs. In symplasmically phloem-loading species, more photoassimilates are accumulated by the PPCs and adjacent axial sinks, which are invested (A) or temporarily stored (B) in axial sinks. As a consequence, photoassimilates are invested to a lower degree (A) or invested with a delay (dashed line, B) into terminal sinks. The high retrieval by SE/CCs in apoplasmically loading species enables more direct photoassimilate investment into terminal sinks. The vertical arrows quantify the extent of investment in terminal sinks (A–C).