Figure 1. A to D, Isolation and identification of V. faba SE protoplasts. A, Light-microscopic image of a simple SE protoplast. Simple SE protoplasts can readily be identified by inclusion of a forisome (asterisk), which is typical of fabaceaen SEs. B, An emerging composite SE protoplast (twin protoplast), partially detached from the phloem strand, composed of two small cylindrical protoplast precursors intermitted by a sieve plate (arrow). Both protoplasts enclose a forisome (white asterisks) and tiny P-plastids (arrowheads) near the protoplast membrane. Next to the SE protoplast, a large parenchyma cell protoplast (PPCP) is visible. C, An isolated CCP adhered to a SE during isolation. The forisome of the adjacent intact SE is marked with an asterisk. D, An enzymatically isolated one-layer phloem strand. A CCP is adhered to a collapsed SE (sieve plate marked with an arrow). At the right, the precursor of a large vacuolar parenchyma cell (PPC) is visible. E and F, Membrane integrity of V. faba SE protoplasts. E, CLSM image of a composite SE protoplast loaded with CFDA-AM ester. The twin protoplast consists of a large (large arrowhead) and a small protoplast (small arrowhead) separated by a sieve plate (arrow). Both protoplasts have accumulated fluorescent CF arising from the esterase-mediated cleavage of CFDA-AM. CF is not removed by washing, indicative of an intact membrane system. F, CLSM image of a twin protoplast adhered to a phloem strand. The protoplast was stained using the membrane-soluble fluorescent dye RH-414. Staining shows two protoplasts enclosed by a plasma membrane intermitted by a sieve plate (arrow) in which each sieve pore is lined by a plasma membrane (orange striping). The larger protoplast (large arrowhead) contains a forisome (light-transmission picture not shown). G to J, Mechanism of SE protoplast formation. G, Formation of a SE protoplast precursor in disintegrating phloem tissue. Due to distal collapse of the SE plasma membranes at either side of the sieve plate (arrow), two adjoining SE protoplasts emerge. Note the formation of a membranous tail (arrowhead), composed of appending membranes. Forisomes are marked with asterisks. H, Formation of a simple SE protoplast as a result of constriction from the sieve plate (arrow). Membranous tails (arrowheads) occur at both sides of the SE protoplast precursor. I, CLSM picture of an emerging SE twin protoplast loaded with CFDA. At this stage of formation, the protoplast is already sealed as demonstrated by the accumulation of CF. The large protoplast encloses a forisome (asterisk); the sieve plate is marked with an arrow. J, Transmission picture of I. Clearly visible are SE plastids (arrowheads) in the small protoplast precursor. The protoplast is being formed at a branching point of a sieve tube with two sieve plates. The lower part of the sieve plate separates two large SE protoplast precursors. The small upper sieve plate part may give access to a third tiny flat protoplast sealed with a SE plasma membrane (small arrows). The pictures were taken after 3 h of enzyme incubation with the exception of A and H (taken after 10 h of enzyme incubation).
Figure 2. Dispersion of a forisome in an intact SE protoplast of V. faba in response to a hypo-osmotic shock. A, The composite SE protoplast, stored in a medium containing 600 mol m–3 mannitol and 1 mol m–3 CaCl2, is composed of a larger protoplast (large arrowhead) and a smaller protoplast (small arrowhead, dotted outline) separated by a sieve plate (arrow). The outline of the forisome is delineated by a dotted line, those of the protoplasts by a dashed line. B, After a rapid bath perfusion with a solution containing 50 mol m–3 mannitol and 1 mol m–3 CaCl2, the protoplast swells. C and D, The ready dispersion of the forisome within seconds indicates calcium influx into the protoplast. E to H, A simple SE protoplast, including a forisome (asterisk), in the standard medium containing 600 mol m–3 mannitol and 1 mol m–3 CaCl2 was osmotically shocked by a solution containing 50 mol m–3 mannitol and 4 mol m–3 EGTA (calcium chelator). The calcium-dependent forisome fails to disperse in the absence of free Ca2+ ions. Following medium change, the protoplast swells (E and F) and collapses after exceeding a critical expansion level within a few seconds (G and H). Note that the oval form of the SE protoplast is dictated by the shape of the forisome.
Figure 3. Expansion of a forisome in an intact SE protoplast of V. faba in response to suction using a microcapillary connected to a pressure device. A, A simple oval SE protoplast including a forisome in a medium containing 600 mol m–3 mannitol and 1 mol m–3 CaCl2. A microcapillary (arrow) filled with bathing medium and connected to a pressure device is visible in the vicinity of the protoplast. The outline of the forisome is delineated by a dashed line. The oval form of the SE protoplast is dictated by the shape of the forisome. B and C, In response to suction exerted on the plasma membrane of the SE protoplast, the forisome starts to disperse at both ends (marked with arrowheads). As shown in C, the oval form of the protoplast disappears before the midsection of the forisome has dispersed completely. D, The full dispersal is accompanied by the rounding-off of the SE protoplast. E to H, A simple SE protoplast including a forisome (asterisk) stored in the standard medium containing 600 mol m–3 mannitol, 1 mol m–3 CaCl2, and 2 mol m–3 GdCl3 (calcium channel blocker; E) was attached to a microcapillary (arrow) containing the bath solution (F). Strong suction exerted on the protoplast (G and H) demonstrates the absence of the calcium-dependent forisome dispersion, presumably due to inhibition of Ca2+ influx by blocking the mechanosensitive calcium channels localized in the SE plasma membrane.
Figure 4. Detection of cell wall cellulose and callose using CW staining on intact tissue, disintegrating phloem tissue, and isolated SE protoplasts. A, CW staining of an intact SE in the main vein of a V. faba leaf. The sieve plate (arrow), the pore plasmodesmata units (arrowheads), as well as the cell wall of the SE show distinct staining. B, CW staining of a disintegrating phloem strand. Cell wall staining is less intent than under A. The sieve plates are marked with arrows. C, Solely the sieve plate (arrow) shows CW staining in an intact twin protoplast. Cell wall staining has disappeared completely. D, Transmission picture of C. In one protoplast of the twin protoplast, a dispersed forisome is visible (asterisk). The sieve plate is marked with an arrow.
Figure 5. Membrane currents across the plasma membrane measured in the whole-cell configuration. A, Current responses to voltage steps from the holding potential of –22 mV to a series of test voltages of 3.5-s duration in steps of 25 mV between –172 mV and +53 mV recorded in standard bath (6 mol m–3 potassium-gluconate, pH 5.5) and pipette (60 mol m–3 potassium-gluconate, pH 7.5) solutions. B, Current-voltage (I-V) relationship of the steady-state current densities from A. Data points were fitted with a polynomial function of the third order. The black arrow marks the Nernst potential for K+ (EK+). C, Time- and voltage-dependent as well as instantaneous outward currents elicited by applying depolarizing voltages in the whole-cell configuration. Current responses to voltage steps from the holding potential (–22 mV) to a series of test voltages of 3.5-s duration in steps of 25 mV between –172 mV and +153 mV recorded in standard solutions as mentioned in A. D, Current-voltage (I-V) relationship of the steady-state currents (Iss) from C. The black arrow marks the Nernst potential for K+ (EK+). E, Tail-current analysis of the time-dependent outward currents. Deactivation current densities in response to a double-pulse protocol starting from a holding potential of –72 mV to a prepulse voltage of +153 mV for 5 s. Tail currents were obtained by following current deactivation at test voltages between –172 mV and +53 mV. Erev, Reversal potential. F, I-V plot of tail currents from E. Values were calculated as the difference between the instantaneous and the stationary currents 1 s after stepping to each deactivating voltage. The predicted equilibrium voltage (EK+) is indicated for K+ with an arrow.
Figure 6. SE protoplasts from other plant species. A and B, Composite SE protoplasts from N. tabacum as a twin protoplast precursor with two longish compartments (A) and in a nearly round configuration as known for V. faba protoplasts (B). Note that N. tabacum protoplasts contain numerous SE plastids. C, Longish precursor of a composite SE protoplast of C. pepo; the longish shape hints at the presence of cell wall remnants. A to C, Sieve plates are indicated by arrows.
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