SE protoplasts promise to be a powerful tool in studying the membrane …

• Abstract
• Results and discussion
• Materials and Methods
• Acknowledgments
• Literature cited
• Figures

Biology Articles » Cell biology » Functional Sieve Element Protoplasts » Results and discussion

Results and discussion

- Functional Sieve Element Protoplasts

Isolation and Identification of V. faba SE Protoplasts

Following an incubation period of 10 h in the enzymatic digestionmedium, formation of phloem protoplasts released from disintegratingV. faba phloem strands was observed using light microscopy andconfocal laser scanning microscopy (CLSM). The enzymatic treatmentliberates protoplasts of various phloem cell types, which arethen tracked and identified by screening the phloem strandsunder light microscopy. Phloem protoplasts are always producedin low but sufficient numbers for patch-clamp studies, or generalbiophysical and physiological studies (see section on functionalityof SE protoplasts below).

Phloem protoplasts are highly variable in shape, size, and structure.Two types of SE protoplasts were found. Simple SE protoplastsarise from more central parts of disintegrating SEs and areidentified by inclusion of a large protein body named the forisome(Fig. 1A ). Forisomes are responsible for sieve plate occlusiondue to a turgor- and damage-induced, calcium-dependent conformationchange in intact SEs of legumes (Knoblauch et al., 2001, 2003).Thus, protoplasts containing a forisome must inevitably be SEprotoplasts.

 
In addition, composite SE protoplasts, which arise from thesieve plate area (the joint between the SEs), showed up. Hence,composite SE protoplasts include a sieve plate, which providesan unequivocal means of identification (Fig. 1, B, E, and F).Composite protoplasts possess a protoplast at either side ofthe sieve plate ("twin protoplasts"; Fig. 1, B and E). One ofthem usually includes a forisome. SE protoplasts with forisomesin both compartments were seldom observed (Fig. 1B).

Companion cell protoplasts (CCPs) were often found adhered toSEs (Fig. 1, C and D). CCPs typically contain chloroplasts aggregatedat one side; the cytoplasmic compartment occupies 20% to 25%of the total protoplast volume. A quick calculation shows thatthe spindle-shaped CCs with a diameter of 3 to 5 µm anda length of 200 to 300 µm would indeed produce sphericalCCPs with a diameter of 10 to 20 µm.

In contrast to CCPs, which have a diameter of 10 to 20 µm,prosenchymatic phloem parenchyma cells form large protoplastswith an average diameter of 40 to 60 µm. They can readilybe distinguished from CCPs by the equal distribution of thecytoplasm at the margin, matching about 1% of the total protoplastvolume (Fig. 1B).

Membrane Integrity of V. faba SE Protoplasts

As a test for membrane integrity, SE protoplasts were loadedwith the colorless CFDA-AM ester as described for intact phloemtissue (e.g. Knoblauch and van Bel, 1998). During incubation,the ester was cleaved by intracellular SE esterases (Oparkaand Read, 1994) into the membrane-impermeant and fluorescentform carboxyfluorescein (CF). Containment of CF inside the protoplastsafter washing demonstrates the integrity of the SE protoplastmembrane (Fig. 1E).

Information on the intactness of the connection between bothprotoplasts in a twin protoplast was obtained by labeling SEprotoplasts with RH-414, a membrane-soluble fluorescent probe(Fig. 1F). The CLSM pictures of RH-414 staining show labelingof a membrane system enveloping the large forisome-containingand the small protoplast of SE twin protoplasts. Continuingfluorescent striping in the sieve plate area indicates thatthe extensive plasma membrane system lining the sieve poreshad remained intact during the isolation procedure (Fig. 1F).

Mechanism of SE Protoplast Formation

The mechanism of SE protoplast formation is not yet understoodin detail. Precursors of the composite SE twin protoplast emergenear the sieve plates (Fig. 1, B and G). At either side of asieve plate, the SE membrane collapses in such a way that taperingends of SE membranes, situated at both sides of the sieve plate,seem to coalesce and form filamentous plasma membrane tail ends(Fig. 1G). As a result, a longish membrane compartment appearsat either side of the sieve plate (Fig. 1, G and J). At thisstage, the twin protoplast precursor is already sealed as evidencedby CF accumulation in both protoplasts (Fig. 1I). After constrictionof the membranous tails, the composite SE protoplast startsrounding off before it gradually detaches from the phloem strand.

Formation of simple SE protoplasts (Fig. 1A) also depends onmembrane tail formation (Fig. 1H). Thus, both simple and compositeSE protoplasts rely on coalescence and constriction of membranoustails as a crucial step in protoplast formation. The amalgamationmechanism is obscure but deserves further (electron microscopic)studies.

Yield of V. faba SE Protoplasts and Formation Mechanism

The minute yield of SE protoplasts is regarded to be the aggregateresult of various bottlenecks in SE protoplast preparation asoutlined here.

1. The tight packing of the phloem tissue.

In contrast to the loose packing of parenchymatous tissues,the tight packing of the phloem tissue impedes a quick and uniformdiffusion of the enzyme mixture.

2. The sensitivity of phloem tissue to wounding.

In comparison to other cell types, the SE/CC complex is verysensitive to the slightest injury. Phloem slicing induces massivewound effects and turgor changes and triggers a physiologicaland structural collapse of most SEs in a tissue slice. Thus,the preparation method requires the use of thick phloem strandsto have a few intact SEs and imposes unavoidable artifacts thatminimize SE protoplast yield.

3. Composition of the enzyme mixture.

The composition of the enzyme mixture is a critical factor forthe success of SE protoplast formation in any plant species.Extensive concentration tests (not presented here) evidencedthat only enzyme mixtures in a narrow concentration window leadto successful cell wall degradation and a complete detachmentof SE protoplasts. Higher concentrations turn the phloem tissueinto mash; lower ones only liberate parenchyma protoplasts.

4. Composition of the SE cell wall.

SEs of dicotyledonous plants develop cell walls thicker thanthose of adjacent parenchyma cell walls. In several species,SE cell walls consist of two morphologically distinct layers,a relatively thin outer layer and a thicker inner layer, thenacreous layer with a pearly appearance (Evert, 1990). The complexityof the SE cell wall is a potential ground for cumbersome enzymaticdigestion.

5. Constriction of the membranous tails.

Transformation of parenchyma cells into protoplasts "only" demandsthe removal of cell wall material and the breakage of plasmodesmata.The production of SE protoplasts is more complicated in thatthe large SEs fragment during the isolation process, which requiresconsiderable membrane reconstitution. Presumably, the SE plasmamembrane is collapsing during the enzyme treatment. In a fewcases, the membrane constricts and coalesces at one side (compositeprotoplasts) or at both sides (single protoplasts) of the emergingprotoplast body. Coalescence of free membrane ends to a closedtail (Fig. 1, G and H) is a critical step toward formation ofviable SE protoplasts. In most cases, a mismatch between themembrane ends or an incomplete coalescence of membrane tailsprevents the final formation of SE protoplasts. It should benoted that the emergence of a composite protoplast depends onsuccessful sealing at either side of the sieve plate. In protoplastswith a sieve plate, the creation of the twin configuration isnecessary since otherwise the protoplast is not sealed at bothsides.

Given the numerous handicaps, it may be some time before onecan expect to gain high yields of SE protoplasts.

Functionality of SE Protoplasts; Activity of Calcium Channels

Indicative of membrane integrity of SE protoplasts is theirreaction to osmotic shocks. SE protoplasts were shocked osmoticallyby an abrupt change from 600 to 50 mol m^–3 mannitol inthe external medium by microperfusion, while the external calciumconcentration was maintained constant at 1 mol m^–3 (Fig. 2, A and B ).The sudden decline in the external osmolarity induced a gradualswelling (Fig. 2, B–D) within 30 to 120 s. Further protoplastswelling resulted in a burst (not shown) of the SE protoplast.The forisome inside SE protoplasts dispersed instantaneouslyin response to the osmotic shock (Fig. 2, A–D) in keepingwith the forisome behavior in intact SEs (Knoblauch et al.,2001). The calcium-dependent forisome dispersion (Knoblauchet al., 2003) is ascribed to calcium influx due to activationof mechanosensitive calcium channels.

 
The same osmotic shock (600–50 mol m^–3) in a calcium-freemedium containing 4 mol m^–3 of the calcium chelator EGTAonly induced protoplast swelling without forisome dispersion(Fig. 2, E and F). The expansion resulted in rupture and collapseof the SE protoplasts (Fig. 2, G and H), while the forisomestayed in the condensed conformation due to the absence of calcium.Thus, the forisome dispersion in 1 mol m^–3 Ca²⁺ suggests(Fig. 2, A–D) the presence of functional mechanosensitivecalcium channels in the SE plasma membrane.

The functionality of SE protoplasts was further demonstratedby applying suction to the SE plasma membrane via a microcapillaryconnected to a pressure device (Fig. 3, A–D ). In reactionto suction, the forisome dispersed presumably as a result ofCa²⁺ influx through mechanosensitive channels (Fig. 3, A–D).Forisome dispersion failed to occur (Fig. 3, E–H) in thepresence of the Ca²⁺ channel blocker Gd³⁺ (2 mol m^–3).These results (Fig. 3, A–H) again indicate functionalmechanosensitive calcium channels in the SE plasma membrane.

 

Detection of Cell Wall Remnants by Calcofluor White Staining

To test the suitability for patch-clamp studies, SE protoplastswere tested on cellulosic and callosic wall remnants by CalcofluorWhite (CW) staining (Choi and O'Day, 1984; Nakamura et al.,1984). As control experiments, intact phloem tissue (Fig. 4A )and a disintegrating phloem strand (Fig. 4B) were stained withCW. In intact phloem tissue, cell walls, sieve plates, as wellas the pore plasmodesmata units were stained intensively (Fig. 4A).The CW staining gradually disappeared with incubation time inthe digesting medium (Fig. 4B), indicative of dissolution ofthe cell wall. After an incubation time of 10 h, several SEprotoplasts solely showed CW staining of the sieve plate (Fig. 4, C and D).

 

Functionality of SE Protoplasts; Membrane Currents across the Plasma Membrane

Simple patch-clamp measurements were executed using SE protoplaststo merely test whether their membranes were functional. Underprevailing conditions, inward and outward currents were observed.

Inward Currents
At negative membrane voltages, instantaneous and time-dependentcurrents were observed in response to a series of test voltagesbetween –172 and +53 mV. The corresponding steady-statecurrent-voltage relationship recorded in asymmetrical potassium-gluconatesolutions showed that the currents only weakly rectified atmembrane voltages between +53 mV and –172 mV (Fig. 5, B and D ).The I-V plot of the steady-state currents (Fig. 5B) revealeda reversal voltage at –43 ± 2 mV (n = 4), closeto the predicted equilibrium voltage for a 10-fold K⁺ gradient(E_K+ = –58 mV) across the membrane, but was differentfrom that of Mg²⁺ (–9 mV), Cl^– (0 mV), or gluconate(+58 mV). Since the resting potential of the SE plasma membranein V. faba is around –130 mV (Hafke et al., 2005) andthus more negative than E_K+, the observed channel may contributeto K⁺ loading/retrieval into the SE.

 
The observed weak rectifying potassium-selective channel activeat negative membrane voltages shares electric properties withthe AKT2/3 K⁺-channel family found in Arabidopsis (Arabidopsisthaliana) phloem cells (Marten et al., 1999; Lacombe et al.,2000; Deeken et al., 2002). This channel family may be involvedin K⁺ transport accompanying phloem loading (Deeken et al.,2000, 2002) and unloading processes (Lacombe et al., 2000),control of membrane potential (Marten et al., 1999; van Beland Hafke, 2005), and reestablishment of the membrane potentialthat depolarizes during a phloem-propagating action potential(Marten et al., 1999).

Outward Currents
Clamping the SE plasma membrane from a holding potential of–22 mV to test voltages between –172 mV and +153mV results in activation of time-dependent outward currents(Fig. 5, C and D) positive to +70 mV. In addition, instantaneouscurrents were observed. For tail-current analysis (Fig. 5E),the plasma membrane of the SE protoplast was clamped from theholding voltage of –78 mV to a conditioning voltage of+153 mV to activate the time-dependent component. In a subsequentstep, the plasma membrane was clamped to a series of test voltagesbetween –172 mV and +53 mV. During the test pulse, themacroscopic tail currents gradually deactivated (Fig. 5E). Aplot of tail-current amplitude revealed a reversal voltage of–50 mV (Fig. 5F), close to the equilibrium voltage forK⁺ (–58 mV). Under prevailing artificial conditions withpotassium-gluconate on both sides of the membrane, this channelis not active at physiological membrane voltages.

SE Protoplasts from Other Plant Species

Modifications of the preparative steps with respect to enzymeconcentrations, preparation temperatures, and incubation timesyielded SE protoplasts from Nicotiana tabacum (Fig. 6, A and B )and SE protoplast precursors from Cucurbita pepo (Fig. 6C).These SE protoplasts were composed of two protoplasts intermittedby a sieve plate. After countless tests, typical isodiametricround protoplasts separated from the sieve plate have only beenobtained for Nicotiana. Despite a broad range of digestion conditionstested with respect to enzyme composition, osmolarity, durationof incubation, and temperature of the enzyme mixture, solelythe longish precursor of Cucurbita SE protoplasts was produced.This form is ascribed to cell wall remnants residing on theplasma membrane. In view of additional experience acquired withother species, preparation of SE protoplasts seems to be highlyspecies specific and the yield will probably always remain low.

 

Outlook

SE protoplasts are a promising tool for studying phloem biophysics.In the near future, V. faba SE protoplasts may be adopted asa model system for transporter deployment in the SE plasma membranein view of the relatively easy mode of isolation and the resultsobtained with intact Vicia plants by other groups.