DISTRIBUTION OF ATPASE IN CELLS OF SALICORNIA PACIFIC A VAR. UTAHENSIS AS DETERMINED BY LEAD PHOSPHATE PRECIPITATION AND X-RAY MICROANALYSIS

Size: px

Start display at page:

Download "DISTRIBUTION OF ATPASE IN CELLS OF SALICORNIA PACIFIC A VAR. UTAHENSIS AS DETERMINED BY LEAD PHOSPHATE PRECIPITATION AND X-RAY MICROANALYSIS"

Percival Watts
6 years ago
Views:

1 JVJ» Phytol (198) 84, DISTRIBUTION OF ATPASE IN CELLS OF SALICORNIA PACIFIC A VAR. UTAHENSIS AS DETERMINED BY LEAD PHOSPHATE PRECIPITATION AND X-RAY MICROANALYSIS BY D.J.WEBER, W. M. HESS AND CHONG KYUN KIM Department of Botany and Range Science, Brigham Young University, Provo, Utah {Accepted 3 May 1979) SUMMARY Salicornia pacifica stem tissues were treated with ATP, Ca(NO3)2 and Pb(NO3)2 to localize ATPase. ATPase activity was present along the plasma membranes and in the cytoplasm of the cortical palisade, inner cortex and vascular cells of young shoots. ATPase was also present in the plasma membranes of tracheids which are present in the palisade region. Ouabain did not inhibit ATPase, but NaF", crown ethers, and iv,iv'-dicyclohexyl carbodiimide (DCCD) did so in vitro. Only DCCD significantly inhibited (82 "^o) ATPase in tissues processed for electron microscopy. Energy dispersive microanalysis was used to verify that the precipitates were Pb phosphate. INTRODUCTION The halophyte, Salicornia pacifica var. utahensis is a pioneer plant in salt playas and can grow in highly saline soils. Salicornia shoots contain high concentrations of salts, although the key photosynthetic enzyme, ribulose-l,5-bisphosphate carboxylase is salt sensitive (Weber et al., 1977o). Weber, Rasmussen and Hess (1977ft), using an electron microprobe, demonstrated that the concentration of NaCl in the palisade region was lower than that in the inner cortex of young shoots. The inner cortex cells lack chloroplasts. These observations indicate that a mechanism exists for compartmentalization of the NaCl. Silver chloride precipitation was used by Hess, Hansen and Weber (1975) to locate the chloride ions found in -S. pacifica cells. Chloride ions were present in all eel! types; their concentration was low or absent in the chloroplasts and high in the vacuoles. One mechanism for salt concentration may be compartmentalization by a Na+, K+ ATPase pumping system. ATPase in animals functions as a Na+, K+ pump to move ions across membranes (Skou, 1974). The activity of the Na+ K+ ATPase in animal systems is readily inhibited by ouabain (Skou, 1974). However, the investigation of Na+, K+ ATPases in plant cells is more complex because it is difficult to obtain sufficient plasma membrane to extract ATPase; also, plant Na+, K+ ATPase does not appear to be ouabain sensitive. Still, there have been several reports which indicate that Na+, K+ ATPases are present in higher plants (Jennings, 1976; Malone, Burke and Hanson, 1977; Winter-Sluiter, Lauchli and Kramer, 1977). The Na+, K+ ATPase pump may be of significance in ion movement in higher plants, particularly halophytes (Kylin, 1973). Jennings (1969) proposed that reversal of a Na+ pump, which pumps sodium out of the cell across the plasmalemma X/8O/O2O285-H3 $2./ 198 The New Phytologist

286 D.J.WEBER, W. M. HESS AND CHONG KYUN KIM against an electrical chemical potential gradient, would cause ATP to be synthesized when the concen:tration of sodium in the external medium is increased.

2 286 D.J.WEBER, W. M. HESS AND CHONG KYUN KIM against an electrical chemical potential gradient, would cause ATP to be synthesized when the concen:tration of sodium in the external medium is increased. By the action of the ATPase pump the sodium moves into the cell down the electrical chemical potential gradient of the ion involved. The minimum requirements for this hypothesis are a sodium activated ATPase at the plasmalemma, a low passive permeability of the ion at this membrane, and a sodium pump at the tonoplast directed toward transporting ions into the vacuole. While evidence for this hypothesis is still being accumulated, there is evidence that Na+ activated ATPase exists in higher plant cells. Jennings (1969) also suggested that if the ATP is synthesized by the reversible action of this sodium pump, it may be utilized in making the cell wall more flexible which could increase turgor and the volume of the cell. ATPase releases inorganic phosphate when converted to ADP. This phosphate reacts with Pb to form a Pb phosphate complex which precipitates and may be visualized in the electron microscope. The presence of Pb phosphate can be verified by X-ray microanalysis. The purpose of this investigation was to determine the location of ATPase by Pb phosphate precipitation in S. pacifica, with particular emphasis upon palisade, inner cortex, and vascular cells. MATERIALS AND METHODS Young plants of Salicornia pacifica Standl var. utahensis (Tidestrom) Munz were collected from salt playas near Goshen, Utah. The plants were sub-irrigated by placing the pots in shallow pans with a constant water level. Developing young shoots were collected and processed for electron microscopy according to Hess (1969). The procedures for Pb phosphate precipitation followed Gilder andcronshaw (;1973) as modified by Rasmussen (pers. comm.), which involved incubation of tissues in ATP, Pb(NO3)2, and Ca(NO3)2. Crown ethers [1,4,7,1,13-pentaoxacyclopentadecane-2,6-dione (O^s), 1,4,7,1,13,16-hexaoxacyclononadecane-17,19-dione (M^g), didenzo-18-crown-6 (B^g) and dicyclohexano-18-crown-6 (Cjg)], NaF- and iv,a^'-dicyclohexyl carbodiimide (DCCD) were added with the ATP to inhibit ATPase. Preparation of A TPase Water rinsed tissues (15 to 2 g fresh wt) were chopped into small segments and ground in an ice-jacketed mortar and pestle. The grinding medium consisted of -25 M sucrose, 3 mm EDTA and 25 mm Tris-MES, ph 7-2. Eour ml of the medium were used per g fresh wt of tissue. The debris was filtered through four layers of cheesecloth and centrifuged at 13^ for 15 min. The supernatant was then centrifuged at 8^ for 3 min, the pellet suspended in 2 ml of fresh grinding medium and centrifuged again at 8^ for 3 min. The pellet was suspended in -25 M sucrose in 1 mm Tris-MES, ph 7-2, and this preparation was assayed for ATPase activity. Assay of ATPase activity A modified Lowry method was used (Lowry, 1957). The final concentrations of constituents in the standard assay were: ATP, 3 mm; Tris-MES, 3 mm; MgS4, 3mM; and KCl, 5 mm. The assay medium (2 ml) was incubated at 37 C for 3 min and the reaction then stopped by adding -4 ml of 3% ice-cold trichloroacetic acid. The tissue was centrifuged to remove denatured protein, then the

3 ATPase distribution in Salicornia 287 inorganic phosphate was assayed in -4 ml of the supernatant fluid. The difference in /<mol of inorganic phosphate hberated in the presence and absence of ATPase inhibitors is referred to as ATPase activity. X-ray microanalysis Energy-dispersive X-ray microanalysis was conducted with an ED AX 7 series interfaced with a Data General NOVA 2 computer and a Texas Instrument Silent 7 ASR. The ED AX X-ray spectrometer was interfaced with a Philips EM4 transmission electron microscope with a high tilt goniometer stage. Sections for analysis were approximately 13 nm thick. Analysis time was 8 s at 8 to 16 kev, with 18 tilt. Analysis was at 4 kv with a condenser 1 spot size of 1 /^m and emission of 25 [ih.. The Ly^^ peak for lead was used for comparison of counts as there was no overlap of other peaks. Data was taken from 9 channels with 126 as the central channel, and background was stripped in all instances. Sections were not post section stained with lead salts. RESULTS Histochemistry and electron microscopy Tissues which were processed without ATP solution or inhibitors did not contain electron-dense precipitates along the membranes or in the cytoplasm (Plate 1), ahhough membranes were distinctly visible. When Pb(NO3)2 and Ca(NO3)2, but no ATP, were added to the fixation solutions, there was little or no Pb precipitation along the membranes or in the cytoplasm (Plate 2). This indicates that little free phosphate is present in the cytoplasm. When ATP, Pb(NO3)2, and Ca(NO3)2 were present during fixation, Pb phosphate precipitation was readily evident along the plasma membranes and in the cytoplasm (Plate 3, No. 1). The Pb precipitate was also present in abundance along the membranes around the wall thickenings of tracheid cells (Plate 3, No. 2). In the inner cortex, the Pb phosphate precipitate was also present along the plasma membranes and in the cytoplasm (Plate 4, No. 1). Vascular cells also had Pb phosphate precipitate along the membranes and in the cytoplasm (Plate 4, No. 2). In vitro assay of inhibitors of A TPase and electron microscopy The precipitation of a Pb phosphate complex indicates the presence of an ATPase, whilst the absence of a precipitate in the presence of an ATPase inhibitor would confirm that ATPase activity was inhibited. The action of various concentrations of ouabain up to 5 mm was tested on ATPase from Salicornia and no inhibition occurred; this accords with the findings of Leonard and Hotchkiss (1976) that ouabain does not inhibit plant ATPase. Brentwood and Cronshaw (1978) used NaF to inhibit ATPase in phloem of Pisum sativum and a similar effect was found with the ATPase isolated from S. pacifica (Table 1); however, when NaF was added to thefixation solutions for electron microscopy, little inhibition was evident. Apparently the NaF complexed with materials or was lost during the fixation process. Table 2 shows that while some inhibition of the ATPase occurred with all the crown ethers, Cjg crown ether was the most inhibitory. However, when the crown ethers were added to the fixation solutions for electron microscopy, although the Pb phosphate precipitate was reduced, it was not to the extent suggested by the w vitro assay.

4 288 D.J.WEBER, W. M. HESS AND CHONG KYUN KIM Leonard and Hotchkiss (1976) reported that -1 M DCCD inhibited ATPase activity in a membrane preparation from corn roots. Some inhibition of S. pacifica ATPase was evident with -2 mm DCCD (Table 3), whilst 1 mm added to the fixation solutions produced a marked reduction in the amount of Pb phosphate precipitate associated with the membrane and the cytoplasm (Plates 5, 6), although not complete (compare Plate 3, No. 1 with Plate 6, No. 1). The DCCD results strongly indicate that the precipitate of Pb phosphate in tissues treated with ATP, C(NO)2 and Pb(NO3)2 (Plates 3, 4) is due to ATPase activity. Table 1. Effect of NaF on ATPase activity in presence and absence of NaCl. Incubation condition: Tris-MES, ph6-5, 3 mm; ATP, 3 mm: MgSO^, 3 mm; 5 mm; and Na+, 5 mm. Specific activity of control was 8-18 /imol Pi mg protein-^ h~^ Concentration of NaF (mm) Specific activity (% standard assay) + NaCl 4 5 Specific activity (% standard assay) -NaCl 1 Table 2. Effect of crown ethers on ATPase activity. Incubation condition: Tris-MES, ph6-5, 3 mm; ATP, 3 mm: MgSO^, 3 mm; K+, 5 mm; and Na+, 5 mm. Specific activity of control was 8-18 fimol Pi mg protein'^ h~^ Concentration of crown ethers (mm) Specific activity (% standard assay) Table 3. Effect of DCCD on ATPase activity in the presence and absence of NaCl. Incubation condition: Tris-MES, ph6-5, 3 mm; ATP, 3 mm: MgSO^, 3 mm; K+, 5 mm; and Na+, 5 mm. Specific activity of control was 8-18 fimol Pi mg protein'^ h"^ Concentration of DCCD (mm) Specific activity (% standard assay) + NaCl Specific activity (% standard assay) -NaCl,

5 ATPase distribution in Salicornia 289 X-ray microanalysis The Pb phosphate precipitate is evidence that ATPase splits ATP into ADP and phosphate. X-ray microanalysis was used to establish that Pb was indeed present in the precipitate. In the ATP, Ca(NO3)2, Pb(NO3)2 treated tissue the counts for the hpi peak for lead averaged 477 compared to 83 for the same treatment with DCCD as an inhibitor (Fig. 1). The concentration of Pb was associated with the localization of the precipitate along the membranes. Electron density was not necessarily positively correlated with the number of counts because of the presence of osmic acid. Fig. 1. X-ray microanalysis of Pb phosphate precipitation in tissues of Salicornia pacifica. Analysis was along the membranes adjacent to the cell wall. The spot is the average of 1 analyses. Note that the vertical scale is logarithmic. A, ATP, Ca(NO3)2, Pb(NO3)2 treated. B, Vacuole of ATP, Ca(NO3)2, Pb(NO3)2 treated. C, ATP, Ca(NO3)2, Pb(NO3)2, N,N'- dicyclohexyl carbodiimide treated. D, Vacuole of ATP, Ca(NO3)2, iv.at'-dicyclohexyl carbodiimide treated. E, Standard fixation. DISCUSSION Hall and Davie (1975) found ATPase localized predominately in the plasmalemma and in the cell walls of leaf cells of the halophyte, Suaeda maritima. The in vitro and electron microscopy assays indicate that an ATPase is also present in S. pacifica. The thin section studies suggest that ATPase is localized primarily along the plasma membrane, though it is also present in the cytoplasm. One reason why the amount of inhibitor needed in the thin section assay is higher than the in vitro assay could he because of possible interaction of the inhibitor with other materials in the fixation solutions. Malone, Burke and Hanson (1977) also used DCCD to inhibit ATPase

29 D. J. WEBER, W. M. HESS AND CHONG KYUN KIM activity in an electro microscopy study of corn roots and found that DCCD reduced ATPase activity, but did not inhibit it completely.

6 29 D. J. WEBER, W. M. HESS AND CHONG KYUN KIM activity in an electro microscopy study of corn roots and found that DCCD reduced ATPase activity, but did not inhibit it completely. Lead was easily detected in the precipitates by X-ray microanalysis although it was necessary to use the Ly^j peak for comparative counts because of overlap of elements with other lead peaks. In spite of the use of a relatively small peak the inhibition of ATPase by DCCD was approximately 82% (Fig. 1). It is interesting that electron density alone cannot be used as an indication of lead precipitate when osmic acid is used for an electron stain. X-ray microanalysis has also been used by Van Steveninek et al. (1976) to localize the chloride ion after AgCl precipitation. It is recognized that there may be more than one ATPase involved in the Pb phosphate precipitation reaction. For example, one ATPase may be localized on the plasma membrane, another in the cytoplasm and another on the tonoplast. More sophisticated fractionation will be needed to establish whether more than one ATPase exists. Questions that need to be considered further are whether the ATPase is functioning as a Na+, K+ pump in keeping the salt concentration more concentrated in the inner cortex cells in the young shoots, and whether ATP is produced if the Na is pumped toward the vacuoles, or away from the palisade cells. If the Na pump requires ATP, much energy would probably be required to keep the salt compartmentalized in the inner cortex or even in the vacuole. ACKNOWLEDGEMENTS We wish to express our appreciation to Connie Swensen and Jim Allen for technical assistance. This research was supported in part by funds from National Science Foundation Grant No. BMS and Brigham Young University Research Division. REFERENCES BENTWOOD, B. J. & CRONSHAW, J. (1978). Cytochemical localization of adenosine triphosphate in the phloem of Pisum sativum and its relation to the function of transfer cells. Planta 14, 111. GILDER, J. & CRONSHAW, J. (1973). The distribution of adenosine triphosphate activity in differentiating and mature phloem cells of Nicotiana tabacum and its relationship to phloem transport. Journal of Ultrastructural Research, 44, 388. HALL, J. L. & DAVIE, C. A. M. (1973). Fine structure and localization of adenosine triphosphatase in the halophyte Suaeda maritima. Protoplasma, 83, 29. HESS, W. M. (1966). Fixation and staining of fungus hyphae and host plant root tissue for electron microscopy. Stain Technology, 41, 27. HESS, W. M., HANSEN, D. J. & WEBER, D. J. (1975). Light and electron microscopy localization of chloride ions in cells of Salicornia pacifica var. Utahensis. Canadian Journal of Botany, 53, JENNINGS, D. H. (1976). The effect of sodium chloride on higher plants. Biological Reviews, 51, 453. JENNINGS, D. H. (1968). Halotypes. Succulence and sodium in plants - a united theory. New Phytologtst, 67, 899. KYLIN, A. (1973). Adenosine triphosphatases stimulated by sodium and potassium. Biochemistry and possible significance for salt resistance. In: Ion Transport in Plants (Ed. by W.P.Anderson), pp Academic Press, New York. LEONARD, R. T. & HOTCHKISS, C. W. (1976). Cation-stimulated adenosine triphosphate activity and cation transport in corn roots. Plant Physiology, 58, 331. LOWRY, O. H. (1957). Micromethods for the assay of enzymes. Meth. Enzymology, 4, 366. MALONE, C. P.. BURKE, J. J. & HANSON, J. B. (1977). Histochemical evidence for the occurrence of oligomycin-sensitive plasmalemma ATPase in corn roots. Plant Physiology, 6, 916. SKOU, J. C. (1974). The (Na+, K+) activated enzyme and its relationship to the transport of sodium and potassium. Quarterly Review of Biophysics, 7, 41.

7 New Phytologist, Vol. 84, No. 2 Plate 1 O I. WEBER, W. M. HESS AND C. K. KIM (Facing p. 29)

8 The Nezv Phytotogist, Vol. 84, No. 2 Plat. 2 D. J. WEBER, W. M. HESS AND C. K. KIM

9 . * ; 7.. (? New Phytologisl, Vol 84, No. 2 Plate 3 I f. WEBER, W. M. HESS AND C. K. KIM

10 The New Phytologist, Vol. 84, No. 2 Plat 4 D. J. WEBER, W. M. HESS AND C. K. KIM

$\ -, \ ri^e New Phytologist, Vol. 84, No. 2 Plate 5 - TT- 2 i- ' ' 15.$

11 \ -, \ ri^e New Phytologist, Vol. 84, No. 2 Plate 5 - TT- 2 i- ' ' 15." - 1 > s - f% } " p "^ U /.^ ) fi 1 % WEBER, W. M. HESS AND C. K. KIM

12 The New Phytologist, Vol. 84, No. 2 Plat 6 f % 't^''i?#. 4:».' ^ ^ ^ ^ ^^"%-i^ % *. D. J. WEBER, W. M. HESS.WD C. K. KIM

13 ATPase distribution in Salicornia 291 VAN STEVENINCK, R. F. M., VAN STEVENINCK, M. E., PETERS, P. D. & HALL, T. A. (1976). Ultrastructural localization of ions. IV. Localization of chloride and bromide in Nitella translucens and X-ray energy spectroscopy of silver precipitation products. Journal of Experimental Botany, 27, WEBER, D. J., ANDERSEN, \V. R., HESS, S., HANSEN, D. J. & GUNASEKARAN, M. (1977a). Ribulose-1,5- bisphosphate carboxylase from plants adapted to extreme environments. Plant and Cell Physiology, 18, 693. WEBER, D. J., RASMUSSEN, H. P. & HESS, W. M. (1977 b). Electron microprobe analyses of salt distribution in the halotype Salicornia pacifica var. Utahensis. Canadian Journal of Botany, 55, WINTER-SLUITER, E., LAUCHLI, A. & KRAMER, D. (1977). Cytochemical localization of K+-stimulated adenosine Triphosphatase activity in Xylem Parenchyma cells of barley roots. Plant Physiology, 6, 923. EXPLANATION OF PLATES PLATE 1 Thin sections of Salicornia pacifica cells that were not incubated with ATP, Ca(NO3)2, and Pb(NO3)2. No. 1. Cross-section of portions of palisade cells in a Salicornia shoot showing cell wall and cytoplasm. X No. 2. Cross-section of a portion of a tracheid from the palisade region of a young shoot showing wall thickenings, x 2. PLATE 2 Thin sections of Salicornia pacifica cells incubated with Ca(NO3)2 and Pb(NO3)2 but not ATP. No. 1. Cross-section of palisade cells showing cell walls and cytoplasm, x No. 2. Cross-section of palisade cells showing a portion of a tracheid with wall thickenings (left) and cytoplasm, x 2. Note the absence of lead phosphate precipitate. PLATE 3 Thin sections of Salicornia pacifica cells treated with ATP, Ca(NO3)2, and Pb(NO3)2. No. 1. Cross-section of portions of two palisade cells showing a chloroplast with a starch grain (upper right). Note the accumulation of lead phosphate along the membranes and in the cytoplasm, x No. 2. Cross-section of portions of a palisade cell (lower) and a tracheid with wall thickenings, x 85. Note the accumulation of lead phosphate along the membranes. PLATE 4 Thin sections of Salicornia pacifica cells treated with ATP, Ca(NO3)2, and Pb(NO3)2. No. 1. Cross-section of inner cortex cells showing lead phosphate deposits along the membranes and in the cytoplasm, x 73. No. 2. Cross-section of vascular cells in a young shoot showing lead phosphate deposits along the membranes and in the cytoplasm, x 86. PLATE 5 Thin sections of Salicornia pacifica cells treated with ATP, Ca(NO3)2, Pb(NO3)2 and iv,iv'-dicyclohexyl carbodiimide. No. 1. Cross section of a portion of a tracheid cell in the palisade region. Note that lead phosphate deposition is minimal, x 12. No. 2. Cross section of vascular cells in a young shoot showing minimal lead phosphate deposition along the membranes and in the cytoplasm, x 65. PLATE 6 Thin sections of Salicornia pacifica cells treated with Ca(NO3)2, ATP and iv^,iv'-dicyclohexyl carbodiimide. No. 1. Cross-section of portions of palisade cells showing reduced lead phosphate deposition along the membranes and in the cytoplasm, x 2. No. 2. Cross section of portions of vascular cells in a young shoot showing minimal deposition of lead phosphate along the membranes, x 16.

Translocation 11/30/2010. Translocation is the transport of products of photosynthesis, mainly sugars, from mature leaves to areas of growth and

Translocation 11/30/2010. Translocation is the transport of products of photosynthesis, mainly sugars, from mature leaves to areas of growth and Translocation Translocation is the transport of products of photosynthesis, mainly sugars, from mature leaves to areas of growth and storage. Phloem is the tissue through which translocation occurs. Sieve