Single-cell saps were ex- tracted from cells of the upper epidermis and from cells. Abbreviations: EDX (micro)analysis, energy dispersive x-ray analy- sis; TNSC ...
Plant Physiol. (1994) 104: 1201-1208
Cells of the Upper and Lower Epidermis of Barley (Hordeum vulgare 1.) Leaves Exhibit Distinct Patterns of Vacuolar Solutes’ Wieland Fricke*, Jeremy Pritchard, Roger A. Leigh, and A. Deri Tomos School of Biological Sciences, University College of North Wales, Bangor, Gwynedd, LL57 2UW, Wales, United Kingdom (W.F., J.P., A.D.T.); and Biochemistry and Physiology Department, Agricultura1 and Food Research Councii lnstitute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, Uriited Kingdom (R.A.L.) ~
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analysis of frozen tissue (Leigh et al., 1986; Williams et al., 1991; Leigh and Storey, 1993), and single-cell sampling and analysis (Malone et al., 1991; Fricke et al., 1994a, 1994b; P. Hinde, R.A. Leigh, and A.D. Tomos, unpublished data), have confirmed that solutes are differentially distributed between epidermal and other leaf cells. Of these various techniques, only single-cellsampling and analysis, which is based on the pressure probe technique, allows the combined measurement of vacuolar solute concentrations and water relations parameters (osmolalityand turgor) of individual cells in situ (Malone et al., 1991; Tomos et al., 1994).Thus, the data obtained from the solute analysis of individual cells provide information not only about the intercellular distribution and transport of solutes within and between leaf tissues but also about the way these cells regulate their osmolality and turgor. Recently, we used single-cell sampling to measure the solute concentrations in saps taken from individual upper epidermal, mesophyll, and bundle sheath cells of barley leaves (Fricke et al., 1994a). This showed that Caz+ was present at osmotically significant concentrations (25 m) only in sap from epidermal cells, whereas high concentrations (>2 mM) of P, sugars, amino acids, and malate were present only in sap from bundle sheath and mesophyll cells. Chloride concentrations were highest in epidermal cells, whereas the concentrations of both NOs- and K+ were similar in a11 three cell types. However, the work also indicated that there must be considerable further heterogeneity in cell solute concentrations that was not accounted for by measurements made on the three different cell types. One possible source of this heterogeneity is differences between the upper and lower epidermal cell layers. Previous studies had suggested that ion concentrations were similar in each of these layers in barley leaves (Leigh and Storey, 1993), although differences have been reported in sorghum and lupin leaves (Treeby and van Steveninck, 1988; Boursier and Lauchli, 1989). . Here we report comparisons of the solute concentrations and osmolality of cells in the upper and lower epidermal layers of the third leaf of barley. Single-cell saps were extracted from cells of the upper epidermis and from cells
Vacuolar saps were extracted from individual, anatomically uniform cells of the upper (adaxial) and lower (abaxial) epidermis of the third leaf of barley (Hordeum vulgare 1.) using a modified pressure probe. Saps (volume 80-200 pl) were sampled at various times between 3 d before and 7 d after full-leaf expansion and were analyzed for their osmolality and their concentrations of NO3-, malate, CI-, K+, and CaZ+. The osmolalities of upper and lower epidermis both increased with time but were similar to each other. In young leaves, K+ and CaZ+were evenly distributed between the two epidermal layers, but as the leaf aged, the upper epidermis accumulated high (40-100 mM) Caz+, whereas cells of the lower epidermis accumulated K+ instead. Nitrate concentration was 100to 150 mM higher i n the upper than in the lower epidermis, whereas CI- was 50 to 120 mM higher i n the lower epidermis. lhese differences did not depend on the leaf developmental stage. l h e uneven distribution of epidermal NO3- and CI- was maintained over a wide range of epidermal sap concentrations of these ions and was not affected by NO3- or CI- starvation or by an increase i n the light intensity from 120 to 400 amo1 m-* s-’. However, the latter did cause a decrease in epidermal NO3- and the appearance and accumulation of epidermal malate, particularly in the upper epidermis. l h e physiological implications of the results for solute storage in leaves and for the pathways of ion distribution to the epidermis are discussed.
Studies of the ionic and water relations of leaf epidermal cells have largely been restricted to stomatal guard cells (Mansfield et al., 1990) because of their importance in gas exchange. There is little information about the water and solute relations of other epidermal cells even though these are likely to be important in regulating the water and solute relations of the stomatal complex and may also be important sites of nutrient storage (Penny and Bowling, 1974; Leigh and Tomos, 1993). Epidermal cells contain a large central vacuole and occupy about 30% of the total leaf symplastic volume in barley (Dietz et al., 1992a). Studies of barley (Hordeum vulgare L.) leaves using a variety of techniques, including protoplast isolation (Martinoia et al., 1981; Granstedt and Huffaker, 1982; Dietz et al., 1992a, 1992b), EDX
’
Abbreviations:EDX (micro)analysis,energy dispersive x-ray analysis; TNSC, total negative solute charge; TPSC, total positive solute charge.
This work was supported by a grant (LR5/521) from the Agricultural and Food Research Council. * Corresponding author; fax 44-248-370731. 1201
Fricke et al.
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located directly opposite in the lower epidermis. Both upper and lower epidermal cells were equivalent with respect to their distances from the corresponding adjacent lateral and intermediate veins. The results indicate that there can be considerable differences in the solute content of equivalent cells in the upper and lower epidermis and that they have physiologically distinct behaviors. MATERIALS AND METHODS Plant Material and Growth Conditions
Barley (Hordeum vulgare L. cv Klaxon) seedlings were grown hydroponically in nutrient solution [l m MgS04, 4 m Ca(NO&, 4 mM KN03, 1 mM NH4HzP04, 1 m (NH4)2HP04,2 m NaCl, and the following micronutrients (PM):12.5 B, 1.0 Mn, 1.0 Zn, 0.25 Cu, 0.038 Mo, and 36 Fel under a 16-h light (120 Pmol m-2 s-')/8-h dark regime as described previously (Fricke et al., 1994a). Plants received fresh nutrient solution at the time the third leaf emerged (1517 d after germination). In some experiments this second nutrient solution was modified as follows. Low-concentration NOS- medium contained 2 m Nos- (instead of the usual 12 mM) and 12 mM C1-, and low-concentration C1- medium contained 40 p~ C1- (instead of the usual 2 mM) and 13 mM NO3-, of which 1 m was supplied as NaN03. The concentrations of the other macro and micro elements remained unchanged. When plants were transferred from low- (120 rmol m-2 s-') to high-light conditions (400 pmol m-' s-'; referred to as high-light plants), this occurred concomitantly with the change of nutrient solution. The nutrient solution remained unchanged throughout the rest of the experimental period, and cells were analyzed at the times specified in the text and figure legends. Extraction and Microanalysis of Epidermal Cell Saps and Bulk Leaf Extracts
Vacuolar saps (80-200 pL) were extracted from single epidermal cells with a silicone-oil-filled microcapillary (Malone et al., 1989, 1991). Unless othenvise indicated a11 cells sampled were from the third leaf. Cells analyzed in the upper epidermis were located within a 5-mm-wide trough region (fourth trough from midrib) midway along the leaf blade. Cells analyzed in the lower epidermis were located directly opposite these upper epidermal cells. In the experiment in Figure 4, C and D, the equivalent cells from the fourth leaf were analyzed. In a11 experiments three to four cells were individually sampled and analyzed per epidermal layer and leaf. The results were not affected by the order in which the epidermal layers were sampled. Previous studies have shown that leaf epidermal cell extracts are vacuolar in origin and devoid of cytoplasmic contamination (Fricke et al., 1994a). Thus, their analysis provides information about the composition of vacuoles in individual epidermal cells. The techniques used to analyze single-cell sap samples have been described in detail elsewhere (Fricke et al., 1994a; Tomos et al., 1994). Osmolalities were measured with a modified (Malone et al., 1991; Malone and Tomos, 1992) nanoliter osmometer (Clifton Technical Physics, Hartford, NY), and elemental concentrations of K, C1, Ca, and Na were
Plant Physiol. Vol. 'I 04, 1994
determined by EDX microanalysis using a Hitachi Si520 scanning electron microscope equipped with a Link Analytical QX 2000 x-ray analyzer. The concentrations of malate and NO3- were measured by enzyme-linked microassays using a fluorescence rnicroscope (Leitz MI'V Compact 2 Microscope Fluorometer, equipped with filter block A and MPV software; Leitz, Wetzlar, Germany). Pssay components were pipetted with constriction microcapillaries of approximate volumes (0.5 and 5 nL). Droplets of 5 nL containing a11 assay components except the reaction starter (see below) were placed in rows on a microscope slide, within a 3-mm-deep aluminum ring filled with water-saliurated liquid paraffin. The composition of the 5-nL droplets was as follows: malate assay, 30 mM glycylglycine/ KOH buffer (pH 10.0), 80 m Glu, 85 units mL-' of malate dehydrogenase (EC 1.1.1.37), 10 units mL-' of glutamate oxaloacetate transaminase (EC 2.6.1.1), 0.1% (w/v) BSA; NO3- as'say, 96 mM triethanolamine/KOH (pH 7.6), 0.1% (w/v) BSA, 1.8 m NADPH, and 4 p~ flavin adenine dinucleotide. Droplets contained BSA to minimize the precipitation of coupling enzymes at the interface with the liquid paraffin. Cell sap samples or standard solutions were added to the assay mixture droplets using the same constriction pipette (approximately 10 pL), and the initial fluorescence was recorded. The reaction was initiated by the addition of 0.5 nL of reaction starter (malate assay, 45 mM IVAD; NO3assay, 0.44 units mL-' of nitrate reductase [EC 1..6.6.2])and the change in NAD(P)H fluorescence was recorded until the malate/N03--dependent reaction was completed. In some experiments, turgor pressure measurements were made before the sap sample was taken (Hiisken et al., 14178). Osmolality measurements were performed on the whole of the single-cell extract. For the remaining analyses, subsamples (5-15 pL) of the extract were used to provide replicate measurements. One single epidermal cell extract (approximately 80-200 pL) yielded enough subsamples for the three to four replicate analyses of its solute contents. Time-course experiments were performed on two independent sets of plants. For each set of plants, leaves from two plants were analyzed per day, and three to four cells were aiialyzed from each epidermal layer of each leaf. Bulk leaf extracts were prepared (Fricke et al., 1994a) from the midregion of the leaf used for single-cell 5,ampling and analyzed for C1- and Nos- by HPLC (Gorham, 1987). Resolution and Replication of Measurements
Und.er the given conditions, the lower limits of resolution of the analytical methods used were as follows: malate and NO3- by fluorometric microanalysis, 2 and 5 m, respec-
tively; K, C1, Ca, and Na by EDX microanalysis, 5 to 10 mM (15-30 mM for Na); osmolality measurements, 5 mosmol kg-'; and C1- and NO3- by HPLC (Dionex), 0.2 to 0.3 m. RESULTS Sap Composition and Osmolality
In both upper and lower epidermal layers, the osmolality increased from about 420 mosmol kg-' 3 d tlefore full leaf expartsion to about 550 mosmol kg-' 5 d after full leaf
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Vacuolar Solutes in Barley Leaf Epidermal Cells expansion. Thereafter, epidermal osmolalities remained constant (Fig. 1).The values and patterns of change were similar in each epidermal layer. In fully expanded leaves, upper and lower epidermal cells had turgor pressures of 9.2 +- 1.5 and 9.7 & 1.5 bar, respectively (means +- SD of four plant analyses, with n = 24 cells analyzed per epidermal layer). Both epidermal layers, therefore, had similar water potentials. Concomitant with the increase in osmolality, there was an increase in (vacuolar) sap NO3- concentrations, but the NO3concentrations in upper epidermal cells were about 100 m~ higher than those in lower epidermal cells (Fig. 2A). C1 concentrations remained relatively constant but, in contrast to Nos- concentrations, were 50 to 150 m~ higher in the lower epidermis (Fig. 2B). Comparison of the C1- and Nosconcentrations indicated that C1- quantitatively replaced Nos- in the lower epidermal cells, whereas the reverse was true in the upper epidermis. Under the low-light conditions used to grow the plants in the above experiment, cells of both epidermal layers contained osmotically insignificant concentrations of those inorganic (S04'-, Pi) and organic solutes (amino acids, malate, citrate) likely to represent major, alternative anions in barley leaves (results not shown). Therefore, the sum of C1- and NO3- concentrations provided a good measure of the TNSC in vacuolar extracts of those epidermal cells. The TNSC of upper and lower epidermal cells was similar until the time of full leaf expansion and increased with leaf age. The increase tended to be larger in the upper epidermis (Fig. 2C). Eight days after full leaf expansion, upper epidermal cells had a TNSC about 100 m~ higher than that in lower epidermal cells. In upper epidermal cells, C1- accounted for about 30% and NO3- for about 70% of the TNSC; these values were reversed for the lower epidermis (Fig. 2D). The distribution of K+ and Ca2+between upper and lower epidermal cells was dependent on the leaf developmental state, although the differences were not as obvious as those for C1- and Nos-. At the time of full leaf expansion, K+
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Figure 2. Concentrations of NO3- (A) and CI- (B), TNSC ([CI-] + [NO3-]) expressed in meq L-' (C), and charge ratio ([CI-I: ([CI-+N03-])) (D) in upper (O) and lower (O)epidermal cells of the third leaf of barley as a function of leaf age. Leaves were analyzed
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at various times preceding and following their full expansion (same experiments as shown in Fig. 1). Other details are as described for Figure 1.
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Days after full leaf expansion
Osmolality ,
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Figure 1. Osmolalities in upper (O) and lower (O)epidermal cells of the third leaf of barley as a function of leaf age. Leaves were analyzed at various times preceding and following their full expansion. At each time leaves from two plants were analyzed, and symbols for these two plants are slightly offset horizontally to prevent overlap and to aid comparison. Results are shown as means f SD (omitted if smaller than symbol size) of n = 3 or 4 individual cell analyses per epidermal layer and leaf.
concentrations in the upper epidermis exceeded or were similar to those in the lower epidermis, but 7 d after full leaf expansion they were lower (Fig. 3A). Ca'+ concentrations were initially similar in both cell layers but then increased preferentially in the upper epidermis, eventually reaching about 100 m~ (Fig. 3B). Na concentrations in each epidermal layer were less than 15 to 20 mM (i.e. near the detection limit) under the growth conditions used (not shown). The TPSC,
Plant Physiol. Vol. 'I 04, 1994
Fricke et al.
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Under control conditions, plants were grown under low light and the nutrient solution contained 12 m~ hl03- and 2 r n C1-. ~ Plants starved of NO3- or g o w n under elevated light intensity (400 instead of 120 pmol m-' s-I) for 3 to 4 d prior to analysis had decreased levels of NO3- ir1 both epidermal layers and in bulk leaf extracts (Fig. 4'4). Nitrate decreased by a greater extent in the upper compared with the lower epidermis, but its uneven distribution between the upper and lower epidermis was still maintained. Plants starved of C1- for 4 d showed the same epidennal (and bulk leaf) concentrations of Nos- as control plants supplied with sufficient C1- and NO3- (Fig. 4A). C1- starvation for 4 d did not affect the epidermal concentrations of C1-, although the bulk leaf C1- concentration decreased by 35%1(Fig. 4B).
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Effects of Nitrate or Chloride Starvation and of liicreased Light Intensity
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epidermis but not in the lower epidennis. Consequently, leaf aging was accompanied by the development of a m uneven distribution of K+ and Ca2+between the upper md lower epidermal layers.
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Figure 3. Concentrations of K+ (A) and Ca2+ (B), TPSC (2[Ca2'].'. [K+I) expressed in meq L-' (C), and charge ratio (2[Caz+1:(2[Ca2+]r [K+J)) (D) in upper (O) and lower (O) epidermal cells of the third leaf of barley as a function of leaf age. Leaves were analyzed at various times preceding and following their full expansion (same experiments as shown in Figs. 1 and 2). Other details are as
described for Figure 1.
representing the sum of 2[Ca2+]plus [K+] and expressed in meq L-', increased with leaf age, but the change was similar for upper and lower epidermal cells (Fig. 3C). In young leaves, Caz+accounted for about 20% of the TPSC in cells of both epidennal layers, but this increased to about 60% in the upper epidennis of older leaves and remained at about 30% in the lower epidennis (Fig. 3D). Thus, as the leaf aged, Ca2+ increasingly replaced K+ as the main cation in the upper
CI-
LL 12 mM : 2 mM
LL 2 mM 12 mM
LL 13 mM 40 UM
HL 12 mM 2 mM
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LL 13 mM 40 U M
Growth conditions
Figure 4. Concentrations of NO3- (A and C) and CI- (B and D) in upper (open bars) and lower (hatched bars) epidermal (epid.)cells and in the bulk leaf extracts (solid bars) of the third (A and B) and fourth IC and D) leaves of barley as a function of NO3- and CIsupply and light intensity. Plants were grown under low light (LL, 120 pmol m-' s-') on control medium containing 12 m M NO3- and 2 m M
