TY - JOUR
AU - Riddle, Kimberly A.
AB - Abstract Apoplastic phloem loaders have an apoplastic step in the movement of the translocated sugar, prototypically sucrose, from the mesophyll to the companion cell–sieve tube element complex. In these plants, leaf apoplastic sucrose becomes concentrated in the guard cell wall to nominally 150 mM by transpiration during the photoperiod. This concentration of external sucrose is sufficient to diminish stomatal aperture size in an isolated system and to regulate expression of certain genes. In contrast to apoplastic phloem loaders and at the other extreme, strict symplastic phloem loaders lack an apoplastic step in phloem loading and mostly transport raffinose family oligosaccharides (RFOs), which are at low concentrations in the leaf apoplast. Here, the effects of the phloem-loading mechanism and associated phenomena on the immediate environment of guard cells are reported. As a first step, carbohydrate analyses of phloem exudates confirmed basil (Ocimum basilicum L. cv. Minimum) as a symplastic phloem-loading species. Then, aspects of stomatal physiology of basil were characterized to establish this plant as a symplastic phloem-loading model species for guard cell research. [14C]Mannitol fed via the cut petiole accumulated around guard cells, indicating a continuous leaf apoplast. The (RFO+sucrose+hexoses) concentrations in the leaf apoplast were low, <0.3 mM. Neither RFOs (<10 mM), sucrose, nor hexoses (all, P >0.2) were detectable in the guard cell wall. Thus, differences in phloem-loading mechanisms predict differences in the in planta regulatory environment of guard cells. Apoplast, guard cell, Ocimum basilicum L., phloem, raffinose family oligosaccharides, stomata, sucrose Introduction The evolution of adjustable stomata was an important adaptation for survival in a terrestrial environment because they isolate the plant's water status from that of the surroundings. Aperture size change is effected by the osmotic gain or loss of water by the guard cell symplast (Roelfsema and Hedrich, 2005) as well as by interactions of guard cells with other cells (Buckley, 2005). In turn, water movement is driven by fluctuations in the symplastic concentrations of potassium salts and sucrose (Outlaw, 2003; Roelfsema and Hedrich, 2005). The sucrose that accumulates in guard cells potentially comes from three sources: starch degradation; guard cell photosynthesis; and uptake of apoplastic sucrose (Tallman and Zeiger, 1988). However, starch degradation and guard cell photosynthesis can only provide a limited amount of sucrose (Outlaw, 2003; Vavasseur and Raghavendra, 2005), implying that bulk leaf apoplastic sucrose is the most important source for the sucrose that accumulates in guard cells during stomatal opening. Lines of evidence to support this interpretation include kinetic studies of sucrose movement to guard cells in planta (Lu et al., 1997), sucrose uptake by guard cells in vitro (Reddy and Das, 1986; Outlaw, 1995; Ritte et al., 1999), sugar transporters in guard cells (Stadler et al., 2003), and guard cell enzyme patterns typical of a sucrose sink (Hite et al., 1993). Ultimately, bulk leaf apoplastic sucrose is from recent photosynthesis in leaf mesophyll cells; indeed, following pulse labelling, the peak in sucrose specific activity in the apoplast precedes that in the mesophyll (Lu et al., 1997). In addition to serving as a source for guard cell symplastic sucrose, guard cell apoplastic sucrose itself plays an osmotic role in regulating stomatal aperture size. A model for apoplastic phloem loaders (Outlaw, 2003) includes the symplastic transport of photosynthetically produced sucrose from the mesophyll to the phloem, where it is released to the apoplast. From there, sucrose is loaded into the companion cell–sieve tube element complex for translocation. Our model proposed that the transpiration stream arriving at the bulk leaf apoplast mingles with this veinal apoplastic sucrose transport pool and carries a portion of it to the guard cell apoplast; however, it is not possible to distinguish between the sucrose moving to sieve tubes and that which might have been loaded and leaked within the leaf from them. Evaporation of the transpiration stream causes sucrose accumulation in the guard cell apoplast, which diminishes stomatal aperture size, and thus serves as a physiological signal in the sensing of the rates of transpiration, photosynthesis, and translocation. In addition to the cited evidence on sucrose per se, the effect of apoplastic sucrose has been mimicked by petiolar-fed [14C]mannitol accumulation in the guard cell apoplast (Ewert et al., 2000), indicating that the osmotic effect on guard cells in not overridden by effects on other cells. Although the model has been validated in its broad form, it has not been proposed as exclusive (Lu et al., 1995, 1997) and does not explain all stomatal responses to transpiration (Buckley, 2005) and photosynthesis rate (Kaiser and Legner, 2007). Bulk leaf apoplastic sucrose concentrations (i.e. those obtained with the pressure chamber) range widely among plants and correlate with different phloem-loading patterns. In typical apoplastic phloem loaders, the bulk leaf apoplastic sucrose concentration is 2–6 mM (López-Millán et al., 2000; Voitsekhovskaja et al., 2000; Lohaus et al., 2001), whereas in symplastic phloem loaders, the bulk leaf apoplastic sucrose concentration is usually much less than 1 mM (Voitsekhovskaja et al., 2000). This difference possibly results from the absence of an apoplastic intermediate transport pool in strict symplastic phloem loaders, in which photoassimilate moves symplastically via plasmodesmata into the companion cell–sieve tube element complex (or, as mentioned, diminished leakage). As a means of maintaining the sucrose gradient for passive transport into the complex, sucrose there is used to synthesize RFOs (raffinose family oligosaccharides), which are the predominant export species in symplastic phloem loaders (Turgeon, 2006). The absence of an apoplastic step in photosynthate movement would seem to limit transpiration rate-dependent regulatory alteration of the guard cell apoplastic photosynthates by leaf photosynthesis, as discussed in the preceding paragraph for apoplastic phloem loaders. An alternative explanation, though with the same mechanistic consequence for the model, posits that the prototypical transport sugar in apoplastic loaders, sucrose, leaks from the phloem whereas RFOs do not. Typical apoplastic phloem loaders such as broad bean (Vicia faba; Gamalei, 1991) and arabidopsis (Arabidopsis thaliana; Haritatos et al., 2000) have been used extensively in guard cell studies (e.g. Kang et al., 2007, and Leonhardt et al., 2004, respectively). However, strict symplastic phloem loaders have not been used as guard cell models, although consideration of these plants has merit. First, it is hypothesized that these plants differ in stomatal modulation by apoplastic factors (Roelfsema and Hedrich, 2002). Secondly, the low photosynthate concentration in the bulk leaf apoplast of symplastic phloem loaders resembles that of important crop plants under conditions of low photosynthesis, assuming that they follow the pattern found in V. faba (Kang et al., 2007). The focused aim here was to determine whether bulk leaf apoplastic sugars accumulate in the guard cell apoplast and exert osmotic and other effects on guard cells in symplastic phloem loaders during the photoperiod. As a necessary step toward this goal, a model symplastic phloem loader, a dwarf cultivar of basil (Ocimum basilicum cv. Minimum), was identified, selected, and characterized with regard to stomatal physiology (diurnal stomatal aperture changes, guard cell starch, and potassium content changes), transport physiology (sugars and derivatives in the bulk leaf apoplast and in phloem exudates), and functional anatomy (continuity of the bulk leaf apoplast). The key finding confirmed our hypothesis: the guard cell apoplastic photosynthate concentration was much lower than that in apoplastic phloem loaders and probably exerted no biologically significant osmotic effect on guard cells. Materials and methods Plant material Seeds of a dwarf cultivar of basil (O. basilicum cv. Minimum) were purchased from Harris Seeds Inc. (Rochester, NY, USA). The seeds were germinated in Fafard No. 2 soil-less potting medium (Conrad Fafard, Inc., Agawam, MA, USA) in plastic market packs (∼100 ml per unit) under a fluorescent light bank (PAR, ∼200 μmol m−2 s−1). When the plants were 2 weeks old, they were individually transplanted to 1-litre pots and further culture was in a growth cabinet (for details, see Ewert et al., 2000). In brief, the cabinet was programmed for a 16 h day (25/20 °C day/night temperature; PAR, 500 μmol m−2 s−1) at a constant 60% relative humidity. The first youngest fully expanded pairs of simple opposite leaves of 24- to 28-d-old plants were used in all experiments. Chemicals Enzymes used for guard cell sucrose histochemical assays were purchased from Boehringer Mannheim Corp. (Mannheim, Germany). Malic dehydrogenase used for the detection of cell content contamination in bulk leaf apoplastic sap was purchased from F. Hoffmann-La Roche Ltd (Indianapolis, IN, USA). Mannitol (Sigma-Aldrich Co., St Louis, MO, USA), used for petiolar feeding, was pre-washed with methanol to remove possible abscisic acid (ABA) contamination as described in Ewert et al. (2000). [14C]Mannitol, 2.1 GBq mmol−1, was from PerkinElmer, Inc. (Shelton, CT, USA). HPLC standards for sucrose (Fisher Chemicals, Fairlawn, NJ, USA), and glucose, fructose, stachyose, raffinose, and galactinol (Sigma-Aldrich Co., St Louis, MO, USA) were obtained commercially. Phloem exudation Phloem exudate collection was according to Büchi et al. (1998). At 4 h into the light period, leaves were excised by cutting through the petiole, which was submerged in water; then, the petiole was re-cut under phloem exudation buffer [5 mM KH2PO4/K2HPO4, pH 7.5, containing 5 mM EDTA (disodium salt)]. Incubation was at 100% relative humidity in darkness (to avoid transpiration) and at room temperature with the petiole submerged in phloem exudation buffer (two leaves per 0.8 ml centrifuge tube containing 0.6 ml of phloem exudation buffer). Exudates collected during the initial 2 h were discarded. Exudates collected during three successive 3 h periods were pooled for analysis by HPLC. Leaf conductance Leaf conductance was measured with a LI-1600 steady-state porometer (Li-Cor Inc., Lincoln, NE, USA). Semi-quantitative histochemical estimates of guard cell starch contents and of guard cell potassium contents Guard cell starch was stained with iodine–phenol–potassium iodide (I2KI) reagent as in Heath (1949) and scored zero to five semi-quantitatively. Guard cell potassium was stained with the hexanitrocobaltate [Na3Co(NO2)6] reagent (Sigma-Aldrich Co., St Louis, MO, USA) and scored zero to five semi-quantitatively according to Green et al. (1990). Quantitative histochemical assay of guard cell sucrose content Quantitative histochemical procedures for single-cell sucrose analysis were according to Lu et al. (1997). (For a general description of these methods, see Outlaw and Zhang, 2001.) In brief, tissue was frozen in liquid nitrogen slurry and stored at –80 °C until freeze-drying at –35 °C and <10 μm Hg. Then, guard cell pairs were individually dissected in a climate-controlled environment and the sucrose content of each was measured with oil-well (initial volume 1 μl) and enzymatic-cycling techniques (0.1–1 pmol). Guard cells that were dissected from whole leaf fragments contained both symplastic and apoplastic sucrose. Guard cells that were dissected from epidermis that had been washed before freezing contained only symplastic sucrose. Apoplastic contents were calculated by subtraction; SEs were calculated by an algorithm that provided a maximum apparent variance in the apoplastic pool (Outlaw and De Vlieghere-He, 2001). Bulk leaf apoplastic sap collection Petioles of excised leaves were quickly wrapped with parafilm and then inserted immediately through the sealing grommet of a pressure chamber (Model 1000, PMS Instrument Co., Albany, OR, USA). The first droplet to be extruded, ∼2 μl, was removed by blotting with tissue paper and then discarded. The next aliquot, 8–10 μl, was collected with a restriction pipette and stored at –80 °C until analysis. The maximum pressure exerted was 1.1 MPa. Malic dehydrogenase activity (according to López-Millán et al., 2000) in the bulk leaf apoplastic sap was <1.5% of that in the leaf homogenate (mass basis), a determination made to ensure that cells were not ruptured at high pressures. Importantly, note that the leaf apoplast is heterogeneous (Ewert et al., 2000) and the exact source of the extruded sap is not assigned with the pressure chamber. Bulk leaf apoplastic water content measurement The pressure chamber was also used to determine the leaf aqueous apoplastic volume, expressed as a percentage of the total leaf aqueous volume (Ewert et al., 2000). The tops of well-watered plants were cut off and discarded. Then the shoots were cut off under water near the crown, and subsequently transferred to individual flasks with the bases submerged in water to ensure hydration. After a 4 h incubation at 100% relative humidity in darkness, one leaf of each pair of simple opposite leaves was used for determination of fresh weight, area, and dry weight. The other leaf (with the intact petiole) was fixed into the pressure chamber as described above. Pressure was increased in a stepwise manner (0.1–0.2 MPa per step) and extruded sap was collected at each step. Pressure–volume curves were constructed, permitting extrapolation to the aqueous bulk leaf symplastic volume (for details, see Turner, 1988). Transmission electron microscopy (TEM) and basil aqueous guard cell wall volume and guard cell volume calculation Microscopy was generally according to Ewert et al. (2000). Mid-section images of guard cells were used to obtain averages for guard cell dimensions and guard cell wall thickness. The guard cell wall volume estimation was based on a cylinder [h, 36.3 μm, from light microscope images (n=10); r (outer dimension), 4.6 μm, from TEM images (n=4)]. The average thickness (0.8 μm) of the guard cell wall was calculated assuming equal wall thickness from TEM images (n=4). The final calculation was corrected for a 15% shrinkage rate in each dimension (Ewert et al., 2000). As before (Ewert et al., 2000), the aqueous guard cell wall volume was assumed to be half of the total wall volume. Estimated thus, the aqueous space in the wall of one basil guard cell pair was 1.2×10−15 m3, and the guard cell pair volume was 4.5×10−15 m3. Movement of extra-foliar xylem source mannitol through the leaf As a means of determining whether the bulk leaf apoplast of basil is uninterrupted, two types of experiments based on feeding the plasma membrane-impermeant solute mannitol to excised leaves were conducted (Ewert et al., 2000). In both types of experiments, leaves were excised and then re-cut under water. Then, the leaf petioles were transferred to plastic tubes (2.7×0.5 cm) containing either 5 mM mannitol, 5 mM 14C-labelled mannitol, or water, and were kept in the growth cabinet. Solutions were replenished every 20 min for up to 3 h. In the first type of experiment, the accumulation of mannitol in the guard cell apoplast was inferred by the diminution of the transpiration rate. In the second type of experiment, the mannitol content of the guard cell apoplast was calculated directly from the 14C content of guard cells that were dissected from freeze-dried [14C]mannitol-fed leaves (as for sucrose analysis above). Contents were converted to concentration using estimates for the guard cell aqueous apoplastic volume, as determined above. Guard cell sugar extraction for HPLC Fragments of basil leaf or washed epidermis were sampled at 11.00 h and freeze-dried at –35 °C according to Lu et al. (1997), then were stored under vacuum at –20 °C until dissection of guard cells. Guard cells were dissected from washed epidermis (containing symplastic sugars) or from leaf fragments (containing both symplastic and apoplastic sugars) as for histochemical analysis. Guard cells were pooled (up to 1000 pairs for RFOs, and up to 120 pairs for sucrose and reducing sugars) for extraction in 30 μl of ice-cold water, and the temperature was immediately elevated to 95 °C, where it remained for 30 min. The extraction solution was then stored at –20 °C before being used for HPLC analysis of sugars. HPLC analysis of sugars in phloem exudates, bulk leaf apoplastic sap, and guard cell extracts Sugars and galactinol were analysed with a Waters 2695 Alliance Separation Module with a temperature-controlled column chamber and autosampler (Waters Co., Milford, MA, USA). The column used was a 250×4.1 mm Hamilton RCX-10 anion exchange HPLC column (Hamilton Co., Reno, NV, USA). Samples were injected in a volume of 20 μl and the mobile phase was 150 mM NaOH running at a speed of 1 ml min−1. The detector was an ESA Coulochem II electrochemical detector with a gold electrode (ESA Biosciences Inc., Chelmsford, MA, USA). Peaks were identified by comparing retention times with those of standard sugars with Millenium32 data-analysis software from Waters Co. Internal standards to insure recovery (>90%) were added to guard cell extracts before injection. The detection limit (P <0.001) of sugars with this method was 4 pmol (2×10−7 M, 20 μl), i.e. given a total of 1000 pairs of guard cells pooled in each 30 μl extraction solution, the detection limit of each sugar was 0.006 pmol pair−1. Results Sugar analysis of the phloem exudates confirmed that basil is a typical symplastic phloem loader Basil was selected as a guard cell model for symplastic phloem loaders after evaluation of a taxonomically diverse range of candidates. Basil is in the Lamiaceae, a family generally recognized as symplastic phloem loaders (Gamalei, 1991) and indeed the one in which symplastic phloem loading was discovered (Weisberg et al., 1988). Here (Fig. 1) the carbohydrate profile of phloem exudates of this species are reported to be typical of a symplastic phloem loader (Turgeon and Medville, 2004). RFOs accounted for 76% (molar basis) of total sugars, with stachyose being predominant. In contrast, sucrose was the least abundant sugar (∼4% of the total) whereas it accounted for 90% of total sugars in exudates of the apoplastic phloem loader broad bean, which lacked detectable RFOs (data not shown). Fig. 1. Open in new tabDownload slide The sugar composition (mole per cent, mean ±SE, n=4 plants) of basil phloem exudates. The exudates were collected over several hours in darkness following a 4 h illumination period. Sugars were analysed by HPLC. Fig. 1. Open in new tabDownload slide The sugar composition (mole per cent, mean ±SE, n=4 plants) of basil phloem exudates. The exudates were collected over several hours in darkness following a 4 h illumination period. Sugars were analysed by HPLC. Diurnal kinetics of basil leaf conductance, stomatal aperture size, and guard cell starch and potassium contents was typical Basic stomatal physiological parameters of basil were examined and compared with those of broad bean (Fig. 2). Leaf conductance and stomatal aperture size (determined on different growth lots) increased in the morning, reached a maximum near noon, and decreased before the end of the photoperiod. The maximum stomatal aperture size was ∼7 μm, which is ∼2 μm smaller than the correlate value of broad bean (Lu et al., 1995; Outlaw and De Vlieghere-He, 2001) and is consistent with the somewhat smaller guard cells in basil (36×11 μm). However, the maximum leaf conductance of basil (∼0.5 mol m−2 s−1) was higher than that reported for broad bean (∼0.14 mol m−2 s−1, Lu et al., 1995; Ewert et al., 2000) and may be accounted for by the higher stomatal density of basil (11 000 cm−2 versus ∼6000 cm−2 on the lower epidermis, not shown). Fig. 2. Open in new tabDownload slide Patterns of leaf conductance, stomatal aperture size, potassium content, and starch content of basil over the course of a day. (A) Leaf conductance (mean ±SE, n=7 plants). (B) Stomatal aperture size, guard cell potassium content, and guard cell starch content (means ±SEs, n=180 guard cell pairs from plants from three independent experiments). Fig. 2. Open in new tabDownload slide Patterns of leaf conductance, stomatal aperture size, potassium content, and starch content of basil over the course of a day. (A) Leaf conductance (mean ±SE, n=7 plants). (B) Stomatal aperture size, guard cell potassium content, and guard cell starch content (means ±SEs, n=180 guard cell pairs from plants from three independent experiments). The increase in stomatal aperture size following the onset of illumination corresponded to an increase in guard cell potassium content and a decrease in guard cell starch content. The extremes of these parameters occurred at 11.00 h (Fig. 2B), after which the potassium content declined faster than the stomatal aperture size. Thus, between 11.00 h and 14.00 h, stomatal aperture size only decreased moderately, from 6.7±0.3 μm to 5.9±0.2 μm, but guard cell potassium content decreased from a score of 3.7±0.1 to 2±0.1, a decrease of 46% of the original value. The decline in potassium content without observation of a commensurate decrease in stomatal aperture size is reminiscent of the situation in broad bean (Talbott and Zeiger, 1996). Guard cell starch content appeared to be inversely correlated to stomatal aperture size throughout the photoperiod, which is often observed, but with many exceptions (reviewed in Heath, 1949). Basil bulk leaf apoplastic sugar and galactinol concentrations were in the micromolar range Mature guard cells are symplastically isolated from surrounding cells (Wille and Lucas, 1984, and references therein) and guard cells have undetectable or limited capacity for photosynthetic carbon reduction (reviewed in Outlaw, 2003). Consequently, the bulk leaf apoplast supplies guard cells with carbohydrates, which in broad bean accumulate in the guard cell apoplast and osmotically diminish the stomatal aperture size (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001; Kang et al., 2007). In addition to sugars themselves, other components, e.g. ABA (Zhang and Outlaw, 2001c) and malate (Hedrich et al., 1994), of the bulk leaf apoplastic sap are signalling substances that target guard cells (see also Roelfsema and Hedrich, 2002). Therefore, characterization of the bulk leaf apoplastic sap of a guard cell model is required. Stachyose and glucose were the most abundant of the five sugars detected at each time assayed (Fig. 3). Their concentration increased nominally 3-fold from the onset of illumination to reach a maximum of ∼90 μM at 11.00 h, after which they, and apparently the other less abundant sugars, decreased (P=0.01). Galactinol was present in the bulk leaf apoplastic sap, but at only ∼20 μM at 11.00 h (not shown). In summary, the combined maximum (total sugar+galactinol) concentration peaked at nearly 300 μM. By way of comparison with an apoplastic phloem loader, RFOs and galactinol are not detectable in the broad bean bulk leaf apoplastic sap, in which sucrose, at ∼2–5 mM, is the most abundant, and glucose and fructose are each <1 mM (Lu et al., 1995, 1997; Voitsekhovskaja et al., 2000; Lohaus et al., 2001; Outlaw and De Vlieghere-He, 2001). Fig. 3. Open in new tabDownload slide Daily pattern of sugar concentrations (mean ±SE, n=3–7) in the basil bulk leaf apoplast, which was collected with a pressure chamber and analysed by HPLC. Fig. 3. Open in new tabDownload slide Daily pattern of sugar concentrations (mean ±SE, n=3–7) in the basil bulk leaf apoplast, which was collected with a pressure chamber and analysed by HPLC. Mannitol fed via the petiole accumulated in the guard cell apoplast, indicating the continuity of the bulk leaf apoplast in basil Membrane-impermeant solutes have been introduced into the transpiration stream as a marker to determine its fate. Some results have been interpreted by Canny (1995) as evidence for barriers within the bulk leaf apoplast that prevent the transpiration stream from reaching stomata. However, [14C]mannitol fed to excised broad bean leaflets via the petiole accumulated in the guard cell apoplast (Ewert et al., 2000), proving continuity of the apoplast, at least in that species. Corroboratively, exogenous ABA provided by petiolar infusion (Zhang and Outlaw, 2001a) or endogenous ABA elevated by water stress (Zhang and Outlaw, 2001b, c) accumulated around broad bean guard cells. Finally, and again with broad bean, sucrose produced by photosynthesis accumulated in the guard cell apoplast (Lu et al., 1997; Kang et al., 2007) and this accumulation depended upon transpiration (Outlaw and De Vlieghere-He, 2001). Altogether, these results with broad bean are consistent with the absence of apoplastic barriers. Here, similar experiments were conducted with basil (Figs 4–6) to test the continuity of the bulk leaf apoplast in that species. Fig. 4. Open in new tabDownload slide Pressure–volume curve of basil leaf to demonstrate utility of the approach with this succulent plant. Basil bulk leaf apoplastic solution was collected in consecutive drops as the pressure inside the pressure chamber increased in a stepwise manner. The intercept of the linear plot with the x-axis indicates the volume of the total leaf symplastic water. The total leaf aqueous volume was calculated by subtracting the dry weight from the fresh weight. Fig. 4. Open in new tabDownload slide Pressure–volume curve of basil leaf to demonstrate utility of the approach with this succulent plant. Basil bulk leaf apoplastic solution was collected in consecutive drops as the pressure inside the pressure chamber increased in a stepwise manner. The intercept of the linear plot with the x-axis indicates the volume of the total leaf symplastic water. The total leaf aqueous volume was calculated by subtracting the dry weight from the fresh weight. Fig. 5. Open in new tabDownload slide Time-course of basil leaf transpiration rate (mean ±SE) when excised leaves were supplied with water (n=6 plants, two growth lots) or 5 mM mannitol (n=7 plants, two growth lots) through the petiole. The experiments were conducted inside an illuminated growth chamber and began at 11.00 h. The transpiration rate was measured directly by replenishing the solution supplied to the petiole. Fig. 5. Open in new tabDownload slide Time-course of basil leaf transpiration rate (mean ±SE) when excised leaves were supplied with water (n=6 plants, two growth lots) or 5 mM mannitol (n=7 plants, two growth lots) through the petiole. The experiments were conducted inside an illuminated growth chamber and began at 11.00 h. The transpiration rate was measured directly by replenishing the solution supplied to the petiole. Fig. 6. Open in new tabDownload slide [14C]Mannitol accumulation (mean ±SE) in the bulk leaf apoplast and in the guard cell apoplast of basil plants after 5 mM [14C]mannitol was fed to the leaf through the petiole for 2 h. The experiment was conducted inside an illuminated growth chamber and began at 11.00 h. [14C]Mannitol of the guard cells (n=15 pairs) was assayed by liquid scintillation counting. The calculation was based on a bulk leaf apoplastic aqueous volume of 39% of the total leaflet water and an aqueous guard cell wall volume of 1.2×10−15 m3. Other details are given in Fig. 5. Fig. 6. Open in new tabDownload slide [14C]Mannitol accumulation (mean ±SE) in the bulk leaf apoplast and in the guard cell apoplast of basil plants after 5 mM [14C]mannitol was fed to the leaf through the petiole for 2 h. The experiment was conducted inside an illuminated growth chamber and began at 11.00 h. [14C]Mannitol of the guard cells (n=15 pairs) was assayed by liquid scintillation counting. The calculation was based on a bulk leaf apoplastic aqueous volume of 39% of the total leaflet water and an aqueous guard cell wall volume of 1.2×10−15 m3. Other details are given in Fig. 5. First, the volumes of the bulk leaf apoplast and of the guard cell apoplast of basil were determined in order to estimate mannitol concentrations from assayed chemical amounts. The bulk leaf apoplastic aqueous volume was 39±3% (n=9) (average=102 μl per leaf) of the total leaflet water, as determined by extrapolation of the pressure–volume curve (Turner, 1988; Fig. 4, a plot to document the utility of this method with basil, a herbaceous plant). In comparison, that of the broad bean is ∼29% (Ewert et al., 2000). The aqueous guard cell wall volume of basil was ∼1.2×10−15 m3 pair−1 calculated based on the geometric model of a cylinder. As support for this estimate, the aqueous space of wall volume of a broad bean guard cell pair was calculated similarly and the value obtained was 3.9×10−15 m3, which is essentially identical to that (∼4.2×10−15 m3) obtained by serial sectioning and 3-D computational analysis (Ewert et al., 2000). The transpiration rate of excised leaves of basil increased (P=0.02) over a 3 h period if the petioles were submerged in water (Fig. 5). (A common observation, this increase results from loss of resistance to water flow due to removal of the roots and shoot.) The typical turnover rate of bulk leaf apoplastic water through transpiration (assuming 3 mmol H2O m−2 s−1) was twice per hour. When the leaves were fed 5 mM mannitol instead of water, the average of the transpiration rates was relatively stable during the first hour then declined steadily over the next 2 h (Fig. 5). The difference in transpiration rate between mannitol- and water-fed leaves was manifested by 120 min (P=0.02) and was highly significant by the end of the time-course (P <0.001), when the transpiration rate of mannitol-fed leaves was only 64% of that of controls. Considered alone, these results provide only equivocal evidence for an open apoplast, i.e. the simplest interpretation is that mannitol accumulated around guard cells and diminished the aperture size (Fig. 5), but plausible and more complex interpretations such as a mannitol-evoked ABA increase are not excluded by these data alone. As a means of directly testing mannitol movement from the petiole to stomata, 5 mM [14C]mannitol was fed to excised leaves via the petiole for 2 h and then [14C]mannitol in guard cells was assayed (Fig. 6). Transpirational water loss during the 2 h of petiolar feeding concentrated the [14C]mannitol in the bulk leaf apoplast to an average of 20 mM. However, after 2 h of feeding [14C]mannitol, the average guard cell apoplastic [14C]mannitol concentration was ∼300 mM, implying not only continuity of the apoplast, but also the potential for transpiration-linked accumulation of bulk leaf apoplastic solutes (Fig. 6). Altogether, these results proved that the bulk leaf apoplast of basil is continuous, just as it is in broad bean (Ewert et al., 2000). Guard cell apoplastic photosynthate accumulation was limited or absent in basil As the essence of this study, the photosynthate concentrations in the guard cell apoplast were determined during the middle of the day, when transpiration was at a maximum. Complementarily, photosynthates in the guard cell symplast were also assayed. The source of transpiration-linked photosynthate accumulation in the guard cell apoplast is the bulk leaf apoplast (see kinetics analysis of Lu et al., 1997; Kang et al., 2007). Thus, the initial focus was to measure RFOs, the most abundant sugars in the bulk leaf apoplast, in the guard cell apoplast. Neither stachyose nor raffinose was detectable in guard cells dissected from freeze-dried whole leaf or rinsed epidermis (HPLC method, up to 1000 individually dissected pairs of guard cells pooled for each measurement). These results place the upper limit of these compounds at 0.006 pmol per whole guard cell pair and 2 mM in the guard cell symplast (based on a volume of 4.5×10−15 m3). It is implausible that sugars would accumulate in the guard cell apoplast, but be absent in the symplast (where they would be metabolized). However, in this extreme scenario—if RFOs were at the detection limit and were restricted only to the aqueous volume of the guard cell wall—the maximum concentration in the guard cell wall would be only an osmotically insignificant 10 mM. Two methods were used for assay of the sucrose content of the guard cell apoplast and of the guard cell symplast of basil during transpiration. First, using quantitative histochemistry, the sucrose content of the guard cell apoplast was 0.018±0.02 pmol guard cell pair−1 (n=47) and that of the symplast was 0.36±0.03 pmol guard cell pair−1 (n=47). These values were almost identical to values obtained with HPLC from extracts of pooled guard cells (0.004±0.1 and 0.45±0.13 pmol guard cell pair−1 for the apoplastic and symplastic compartments, respectively). The reducing sugars were at a lower concentration (Fig. 7; glucose, 0.05±0.03 and 0.16±0.05 pmol guard cell pair−1 and fructose, 0.01±0.03 and 0.07±0.03 pmol guard cell pair−1 for the apoplastic and symplastic compartments, respectively). In summary, the sucrose concentration in the guard cell symplast in basil (∼80 mM, calculated) was similar to that in broad bean (∼110–150 mM, Lu et al., 1997; Outlaw and De Vlieghere-He, 2001; Kang et al., 2007). However, the basil guard cell apoplastic sucrose concentration was not detectable [i.e. 15±17 mM (histochemical method); cf. broad bean ≥150 mM; see next paragraph]. Fig. 7. Open in new tabDownload slide Sugar content (mean ±SE) in the basil guard cell symplast and guard cell apoplast at maximum leaf conductance (11.00 h) Sucrose was measured with both HPLC (extracts of 120 individually dissected guard cell pairs per analysis; n=5 plants, two growth lots) and with histochemical methods (n=47 single extracts from individually dissected pairs of guard cells from six plants, two growth lots); glucose and fructose were measured with the HPLC method (n=5 plants, two growth lots) only. RFOs (not shown) were not detectable (<10 mM) in pooled extracts of whole guard cells (up to 1000 individually dissected per analysis.) Fig. 7. Open in new tabDownload slide Sugar content (mean ±SE) in the basil guard cell symplast and guard cell apoplast at maximum leaf conductance (11.00 h) Sucrose was measured with both HPLC (extracts of 120 individually dissected guard cell pairs per analysis; n=5 plants, two growth lots) and with histochemical methods (n=47 single extracts from individually dissected pairs of guard cells from six plants, two growth lots); glucose and fructose were measured with the HPLC method (n=5 plants, two growth lots) only. RFOs (not shown) were not detectable (<10 mM) in pooled extracts of whole guard cells (up to 1000 individually dissected per analysis.) Overall, the sugar concentration in the basil guard cell apoplast was low, i.e. the upper limit of the RFO concentration was 10 mM, and neither sucrose, glucose, nor fructose was detectable there (P-values >0.2, compared with concentration zero). In contrast, the broad bean guard cell apoplastic sucrose concentration was very high, ≥150 mM (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001; Kang et al., 2007), calculated according to Ewert et al. (2000), during transpiration. Thus, the guard cell apoplastic photosynthate concentration was significantly lower in basil, our model for a symplastic phloem loader, than in broad bean, our model for an apoplastic phloem loader. Discussion Basil, a model symplastic phloem-loading species for guard cell studies Strict symplastic phloem loaders have not been used for guard cell studies (see Introduction). The Lamiaceae generally are herbaceous symplastic phloem loaders (Gamalei, 1991), and have been studied for this reason (Büchi et al., 1998). Following comparisons of taxonomically diverse symplastic phloem loaders (not shown), a member of this family, basil, emerged as the choice for the present studies. Basil primarily transports RFOs (Fig. 1), indicating that this species is a symplastic phloem loader (cf. Büchi et al., 1998). In general, basil is a good physiological model for guard cell studies: (i) a compact erect plant, it produces several mature leaves within 1 month from seed; (ii) flat, homobaric, and opposite pairs of leaves facilitate porometry, peeling of epidermis, and ensure uniform illumination for control and treatments; (iii) long petioles (∼2.5 cm) simplify use of a pressure chamber; (iv) sufficiently large guard cells facilitate precise aperture measurements and dissection; and (v) daily changes in leaf conductance, stomatal aperture size, guard cell starch content, and potassium content are typical (Fig. 2B). Role of the apoplast, with emphasis on effector accumulation around guard cells The bulk leaf apoplast plays important and diverse roles in a plant's physiology. Water and nutrient transport are, of course, essential functions (Sattelmacher and Horst, 2007). By diverse means, processes in and beyond the apoplast regulate plant–plant (Holdaway-Clarke and Hepler, 2003), symbiotic (Paszkowski, 2006), and pathogenic interactions (Kamoun, 2006). Endogenous integrative signalling substances also move in the apoplast and inform cells of biotic or abiotic stresses (Mittler, 2002; Davies et al., 2005). As a special case, the guard cell apoplast is particularly important because guard cells are symplastically isolated at maturity (Wille and Lucas, 1984, and references therein) and the bulk leaf apoplast is continuous (Figs. 5, 6; Ewert et al., 2000), with the guard cell apoplast being the terminal point in the evaporative pathway. These facts have several implications. For example, in broad bean, xylem source ABA and apoplastic photosynthate accumulate in the guard cell apoplast and result in stomatal closure (Lu et al., 1995, 1997; Zhang and Outlaw, 2001a, b; Outlaw and De Vlieghere-He, 2001; Kang et al., 2007). It was hypothesized that sucrose accumulation in the guard cell apoplast is a signal that integrates the rates of transpiration, of photosynthesis, and of translocation in apoplastic phloem loaders (see Introduction). This mechanism is not exclusive and, indeed, would not seem to account for the transient increase in conductance when ambient humidity is lowered (Buckley, 2005) or humidity responses in plants under non-photosynthetic (Kaiser and Legner, 2007), and perhaps other conditions. On the other hand, the essential elements of the mechanism are firmly established, including—importantly—that petiolar-fed mannitol mimics stomatal closure caused by sucrose accumulation around guard cells (i.e. possible changes in the turgor of epidermal cells do not override the osmotic effect on guard cells). Redundancy in mechanisms that regulate stomatal responses to modest hydraulic perturbations is anticipated and reminiscent of the environmentally dependent, species-specific, overlapping, and independent molecular stomatal responses to ABA (Outlaw, 2003; Israelsson et al., 2006; Marten et al., 2007). Previous work on photosynthate accumulation in the guard cell apoplast has been conducted on broad bean, a strict apoplastic phloem loader. In contrast to those species, symplastic phloem loaders (Lalonde et al., 2004) have a low bulk leaf apoplastic photosynthate concentration (≤1 mM, Voitsekhovskaja et al., 2000), which was hypothesized to constrain accumulation of photosynthate in the guard cell apoplast as described in the following. In symplastic phloem-loading species, sucrose is transported from mesophyll cells into the companion cell–sieve tube element complex symplastically, and then is used to synthesize RFOs in the companion cells (Turgeon, 2006). Therefore, translocate is mostly RFOs, not sucrose. As mentioned, it was hypothesized that photosynthesis-dependent transpiration-linked accumulation of bulk leaf apoplastic photosynthate in the guard cell apoplast of symplastic phloem loaders does not occur. The results reported here support this hypothesis as summarized here and expanded in subsequent sections. In basil, a model species characterized for this study, RFOs were not detected in the guard cell apoplast (total <10 mM, cf. ≥150 mM sucrose in broad bean, Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001; Kang et al., 2007) calculated according to Ewert et al. (2000). Neither sucrose, fructose, nor glucose was detected (P >0.2) in the guard cell apoplast. Thus, guard cell apoplastic photosynthates do not exert a biologically significant osmotic effect on guard cells of basil, implying that the integrative theory developed by study of broad bean does not apply to symplastic phloem loaders. Relevance of sugars in the bulk leaf apoplast of symplastic phloem loaders to photosynthesis-dependent transpiration-linked sugar accumulation in the guard cell apoplast The few studies (e.g. Voitsekhovskaja et al., 2000) of bulk leaf apoplastic sugars of symplastic phloem loaders indicate only micromolar concentrations of sucrose, hexoses, and RFOs (as well as galactinol, an RFO precursor; Turgeon, 2006). Similar results were obtained for basil, in which the sum of sugars was <300 μM (Fig. 3). The most abundant sugars in the bulk leaf apoplast of basil were stachyose and glucose (Fig. 3). Only these two sugars increased from the onset of illumination to midday (Fig. 3) and therefore only they are candidates for photosynthesis-dependent sugar accumulation in the guard cell apoplast in this species. In particular, stachyose is important because RFOs at least partially originate from leakage from the phloem in the leaf (Fig. 2 of Ayre et al., 2003), although, in some species, enzyme distributions indicate they may originate elsewhere too (referecnces in van Bel, 1993). However, the potential for a role for stachyose accumulation in the guard cell apoplast, analogous to that of sucrose in apoplastic phloem loaders, is small because of its low absolute concentration in the bulk leaf apoplast [100 μM (Fig. 3) versus 2–6 mM sucrose in apoplastic phloem loaders (Lu et al., 1997; Voitsekhovskaja et al., 2000; Lohaus et al., 2001)]. The potential for a role for glucose is also small because the absolute concentration in the basil apoplast is four times less than that in broad bean (Lohaus et al., 2001; Kang et al., 2007), in which the glucose content of the guard cell apoplast is small and statistically independent of the rate of photosynthesis (Kang et al., 2007). Whereas the preceding indirectly precludes an osmotically important photosynthate accumulation in the guard cell apoplast, a transpiration-linked accumulation of all solutes merits consideration independently. The potential for this simpler mechanism is also limited. In broad bean, only sucrose accumulates to an osmotically important level in the guard cell apoplast, where it is maximally ∼100 times that of the bulk leaf apoplast (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001). In the unlikely scenario that all the solutes in the bulk leaf apoplast of basil accumulated to this maximum extent, the sum change in osmolality would be minor, <40 mM, compared with the value in broad bean. Absence of guard cell apoplastic photosynthate accumulation in symplastic phloem-loading plant basil Despite the negative evaluation of the hypothesis in the preceding section, two compelling facts implied the value of a direct test. First, the bulk leaf apoplast of basil (Fig. 6) is continuous and thus provides a structural pathway for solute movement to the guard cell apoplast. Secondly, transpirational flux, if calculated on a guard cell basis, is possibly substantial [0.34 pmol (total solutes) stoma−1 h−1, in the extreme case in which the entire unaltered transpiration stream reaches guard cells], but still is only 0.25 times that of potential sucrose delivery to guard cells in broad bean (Outlaw and De Vlieghere-He, 2001). The direct test comprised analyses of the RFO, sucrose, and hexose contents of individually dissected guard cells harvested from leaves that were under conditions of high transpiration and photosynthesis. RFOs in the guard cell apoplast of basil were not detected, indicating that the maximum concentration could not have exceeded 10 mM (P <0.001). Neither sucrose, fructose, nor glucose was detected (P >0.2) in the guard cell apoplast of basil, but biological variability in these sugars in the guard cell symplast unavoidably compromised the precision of these assays (Fig. 7). However, with similar limitation (80 mM sucrose in the guard cell symplast of basil, and ∼110 mM in broad bean (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001), the high sucrose content of the guard cell apoplast of broad bean was easily measured (e.g. SE/x, ∼0.2; Outlaw and De Vlieghere-He, 2001). Altogether, these facts indicate the absence of transpiration-linked, photosynthesis-dependent photosynthate accumulation in the guard cell apoplast of basil. The differences in the in planta environment of guard cells of apoplastic and symplastic phloem loaders must be incorporated into explanations of stomatal regulation. We thank Paul Burress for help with the liquid scintillation measurement of [14C]mannitol, and Xixi Jia for help with microscopy. 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This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) © 2007 The Author(s).
TI - Guard cell apoplastic photosynthate accumulation corresponds to a phloem-loading mechanism
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erm262
DA - 2007-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/guard-cell-apoplastic-photosynthate-accumulation-corresponds-to-a-TlhTwmvbgE
SP - 4061
EP - 4070
VL - 58
IS - 15-16
DP - DeepDyve
ER -