Planta

Planta (1983)159:336-341

9 Springer-Verlag 1983

Ion compartmentation in Porphyra umbilicMis determined by electron-probe X-ray microanalysis Christian Wiencke 1, Ralf Stelzer 1 and Andr6 Lfiuchli z 1 Botanisches Institut der Tier/irztlichen Hochschule, Biinteweg 17 D, D-3000 Hannover 71, Federal Republic of Germany, and z Department of Land, Air and Water Resources, University of California, Davis, CA 95616, USA

Abstract. The ion composition of cell compartments in the intertidal red alga Porphyra umbiliealis adapted for two weeks in 3.5 x artificial seawater was determined by X-ray microanalysis of unfixed, frozen, bulk specimens. A procedure is described for the calculation of ion concentrations in the main cell compartments, cytoplasm, vacuoles and plastid. The results indicate high K + 9 and low Na + concentrations in cytoplasm and plastid. Sodium ions are preferentially localized in vacuoles. Both, vacuoles and plastid contain high C1- concentrations. Key words: Ion compartmentation - Osmotic stress - Porphyra - X-ray microanalysis.

ume with increasing external salinity from 1 to 3.5x seawater (Wiencke and L/iuchli 1980). In particular, from changes in intracellular C1- and K + concentrations with variations in the salinity level, it was inferred that C1- is mainly located in the vacuoles, whereas K + occurs predominantly in cytoplasm and, to a smaller extent, in vacuoles (Wiencke and L/iuchli 1981). In order to investigate experimentally ion compartmentation in Porphyra, electron-probe X-ray microanalysis of unfixed, frozen, hydrated pieces of thallus was used (compare Pitman et al. 1981; Stelzer 1981; Storey et al. 1983; Yeo et al. 1977). Furthermore, attempts were made to calculate ion concentrations in cell compartments using data of X-ray microanalyses and of intracellular ion concentrations derived from chemical analyses.

Introduction

Osmotic regulation in algae can be achieved by changes in internal concentrations of inorganic ions and low-molecular-weight organic solutes (Bisson and Kirst 1979; Gutknecht et al. 1978; Hellebust 1976; Kauss 1978; Zimmermann 1978). As regards the intracellular distribution of these solutes, it is generally assumed that - in algae as well as in higher plants - inorganic ions are mainly localized in vacuoles, whereas low-molecular-weight organic solutes may be predominant in the cytoplasm (Flowers et al. 1977; Greenway and Munns 1980; Kirst and Bisson 1979; Wyn Jones et al. 1977). In Porphyra umbilicalis, an intertidal red alga, CI-, K + and the ~-galactosylglycerols floridoside and isofloridoside serve as the main osmotic solutes during hyperosmotic stress (Wiencke and L/iuchli 1981). It was suggested that inorganic ions are accumulated in the vacuoles, as the vacuolar system of Porphyra increases considerably in vol-

Material and methods Porphyra umbilicalis (L.) J. Ag. was collected at Helgoland (North Sea). Culture conditions, freeze-fracture technique and volume measurements of cells and cell compartments have been published previously (Wiencke and L/iuchli 1980). Measurements of osmotic solute contents by chemical analysis, nonosmotic volume and calculation of intracellular concentrations have also been described elsewhere (Wiencke and L/iuchli 1981). For X-ray microanalysis, pieces of unfixed thallus were mounted on a copper holder, frozen in melting N 2 and fractured transversely under liquid N 2 (Pitman et al. 1981; Yeo et al. 1977). The frozen specimens were transferred to the cold stage ( - 1 8 6 ~ C) of a scanning electron microscope (ETECAutoscan Hayward, Calif., USA). For analysis the electron beam was focussed in the reduced-area mode onto an area of less than 1 gm 2. The accelerating voltage was 10 kV. The specimen current was approx. 10 - 8 - 10 - 9 A. Emitted X-rays were analysed with a KEVEX (Burlingame, Calif., USA) energy dispersive X-ray detector. The peak intensities were obtained by use of a basic TN 2000 MCA data processing function. Basic data for calculations were the number of counts minus background for each element in each spectrum. In order to correct the Na values for lower counting efficiency, an aqueous solution of known composition was frozen and

C. Wiencke et al. : Ion compartmentation in

Porphyra

337

Fig. 1. Scanning electron micrograph of a frozen and crossfractured Porphyra thallus cultivated for two weeks in 3.5 x artificial seawater medium ASP1z. (P: protoplast; CW: cell wall). x310

analysed. Compared with the heavier elements C1 and K, Na count rates had to be multiplied by 2.66. The corrected count rates for each spectrum were expressed as percentage of the elements : Na + Mg + P + S + C1 + K = 100%. For calculation of concentrations of monovalent ions, however, Na + C1 + K were set to 100%.

Results

Plant material adapted for two weeks in 3.5 x seawater medium ASP12 (Provasoli 1964) was used in all experiments. Maximum vacuolation occurs in Porphyra at this hyperosmotic stress (Wiencke and Lfiuchli 1980) and, hence, X-ray microanalytical measurements in vacuoles, cytoplasm and plastid can be accomplished successfully. Figure 1 shows a scanning electron micrograph of a frozen and cross-fractured Porphyra thallus commonly used in this study. At a higher magnification (Fig. 2), cell walls and the major cell compartments of plastid, cytoplasm and vacuoles can be clearly distinguished. The identification of the cell compartments was easy as the fine-structural organization of the Porphyra cell was known from transmission electron microscopic studies. For comparison, Fig. 3 shows the ultrastructure of a

Porphyra cell as revealed by freeze-fracture electron microscopy. X-ray microanalyses were made after exact optical identification of the cell compartments. The relative peak intensities of the different elements in the compartments cytoplasm, vacuoles and plastid are given in Table 1. In the cytoplasm (compare Fig. 4A) the predominant element is K, followed by P. The relative amounts of S and C1 in the cytoplasm are much lower and cytoplasmic Na and Mg contents are negligible. In the vacuoles (compare Fig. 4B) the main elements are C1 and Na. The other elements are low in this compartment. In the plastid, K and C1 and, to a lesser extent, S and P are the most dominant elements. The contents of Na and Mg are very low in this compartment. It should be noted that the elemental composition of the plastid (mainly with regard to S and P) is by no means as homogeneous as in the other compartments, which presumably is a result of subcompartmentation (e.g. pyrenoid, stroma, thylakoids; compare Fig. 4 C, D). When X-ray microanalytical data are used for the estimation of concentrations of inorganic ions, one must realize that not all of the measured ele-

C. Wiencke et al. : Ion compartmentation in Porphyra

338

Fig. 2. Scanning electron micrograph of frozen, fractured Porphyra cells (cultivation as described in legend of Fig. 1). Cell walls (CW), protoplasts and the compartments, plastid (p/), vacuoles (vat) and cytoplasm (cyt) can be distinguished easily, x 4300 Fig. 3. Freeze-fracture electron micrograph of a Porphyra cell. The same plant material was used as for the scanning-electronmicroscope preparations, x 8300

Table 1. Elemental distribution in cell compartments of Porphyra measured by X-ray microanalysis (Na + Mg + P + S + C1+ K = 100% ; means • SEM 9-12 measurements per compartment) Compartment

Na

Mg

P

S

C1

K

Cytoplasm Vacuoles Plastid

4.0 • 1.4 39.8 _+4.9 3.3 _+5.8

2.8 _ 0.8 4.6 _+1.4 1.6 • 0.4

21.2 + 3.8 3.5 • 0.7 7.8 •177 3.2

12.2 _+1.8 2.9 • 0.6 15.1 • 6.6

14.4 _+1.5 46.1 _+4.3 31.8 • 6.7

45.3 _+2.9 3.2_+ 0.9 40.4 • 5.9

merits are present in ionic form. H o w e v e r , the relative a m o u n t s o f the elements N a , C1 a n d K in the X - r a y spectra are considered to c o r r e s p o n d to ion c o n c e n t r a t i o n s and, hence, the calculations o f concentrations were restricted to these elements. T h e following e q u a t i o n was used: VOteyt X [ion]cyt + volva r • [ion]v,r + vOlpl x [ion], 1= [ion]r162

(1)

volcyt, VOlva e a n d VOlpl are the v o l u m e s o f c y t o p l a s m (34%), vacuoles (36%) and plastid ( 3 0 % ) expressed as the p e r c e n t a g e o f p r o t o p l a s m i c v o l u m e ; [ion]r [ion]var a n d [ion]N are the relative ion contents in the respective c o m p a r t m e n t s (Table 2);

[ion]ceu is the intracellular c o n c e n t r a t i o n o f each ion m e a s u r e d by chemical analysis a n d expressed in m m o l 1-1 o s m o t i c v o l u m e (Table 3, "cell "). T h e calculated ion c o n c e n t r a t i o n s are presented in T a ble 3. T h e d a t a indicate t h a t K + is the p r e d o m i n a n t ion in the c y t o p l a s m a n d t h a t the c o n c e n t r a tion o f N a + is very low in this c o m p a r t m e n t . T h e vacuoles are characterized b y high C1- a n d N a + concentrations. Chloride is also a c c u m u l a t e d in the plastid together with K + as its counter• It should be p o i n t e d out t h a t the i o n - c o m p o s i t i o n d a t a f r o m chemical analysis c o r r e s p o n d well with those derived f r o m X - r a y m i c r o a n a l y s i s (Table 3, "cell").

C. Wiencke et al. : Ion compartmentation in Porphyra

K

4A

9"P

, ..

339

[

"

4C

~a'

[ $

P

:':

.- i .,-. i'-. -r

i

i

O1~0

$ 120

IIFS=~

f

O.Ol~

t!r

4B

5 I20

4D $

S:

:i

"

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=0~

~$=I~

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-,

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Fig. 4A-D. Representative energy dispersive X-ray spectra from three major cell compartments of Porphyra. A cytoplasm, B vacuoles, C and D plastid. For comparison with the other elements, the count rates of Na have to be multiplied by 2.66

Table 2. Distribution of monovalent ions in cell compartments of Porphyra measured by X-ray microanalysis (Na § + K § C1- = 100% ; means _+SEM) Compartment

Na +

K+

C1

Cytoplasm Vacuoles Plastid

6.3___2.0 44.3_+5.4 4.0_+3.2

70.8--2.1 3.8_+1.2 53.2_+3.7

24.2--2.8 51.9_+4.8 42.7_+6.9

Table 3. Ion concentrations in cells and cell compartments of Porphyra and comparison of the calculated ion composition (as percentage of concentration data) with that measured by X-ray microanalysis Compartment

Ion

Concerttration, mM, in osmotic volume

Ion composition, percentage of concentration data

Ion composition, X-ray data, % (see Table 2)

Cytoplasm

Na + K+ C1-

57 a 751 a 311"

5.1 67.1 27.8

6.3 70.8 24.2

Vacuoles

Na + K+ CI-

405 a 41 a 668"

36.3 3.6 60.0

44.3 3.8 51.9

Piastid

Na + K+ C1-

36 a 564 a 549"

3.2 49.1 47.8

4.0 53.2 42.7

Cell

Na + K+ C1

176 b 439 b 5/1 b

15.6 40.0 45.4

19.0 41.0 40.0

a Calculated by use of equation 1 b Chemical analysis, data from Wiencke and L/iuchli (1981)

Discussion

In the present report, data on ion relations and ion concentrations in the three major compartments of a marine algal cell are given. One difficulty which arose during the evaluation of the Xray microanalytical data needs to be discussed briefly. Like all plant cells, the Porphyracell shows large differences in density and chemical composition of the individual cell compartments. The different matrix substances, being more proteinaceous in the cytoplasm and plastid compared with the vacuoles, lead to different interactions of the electron beam with the sample and, hence, to different X-ray yields in the individual compartments (compare Anderson 1967; Eshel 1974). Therefore, the ion content of the vacuoles (mainly of Na § tends to be underestimated compared with that of the cytoplasm and plastid which tends to be overestimated. As no accurate correction factors are available at present, this factor has to be kept in mind when X-ray microanalytical data are quantitatively interpreted. Another problem has been indicated above: among the measured elements, only Na, C1 and K are present as inorganic ions. With regard to the other elements, only a fraction of the detected P represents inorganic phosphate, the remaining P is most probably associated with phosphorylated sugars and nucleotides. Thus, the relatively high P content in cytoplasm and plastid becomes understandable. Part of the detected S in the plastid may

340

be related to sulphur-containing enzyme proteins such as ferredoxin and thioredoxin (Buchanan et al. 1979; Lara et al. 1980) and to sulpholipids as membrane constituents. The method for the calculation of ion concentrations in cell compartments carried out in this study is based on intracellular X-ray microanalyses but also requires determination of relative volumes of the cell compartments by morphometric analysis and of intracellular ion concentrations by chemical analysis. Its application is limited to single cells and tissues consisting only of one type of cell. The data thus obtained (Table 3) complement and considerably expand the conclusions on ion compartmentation in Porphyra based on gross chemical analyses (Wiencke and L/iuchli 1981). In particular, the compartmentation of Na § in the vacuoles and of CI- and K § in the plastid are results not obtainable by chemical analysis. The X-ray microanalyses do confirm our earlier conclusions that C1- is the major inorganic ion in the vacuoles and that the K § concentration in the cytoplasm is very high. On the other hand, it is also demonstrated that K § does not contribute significantly to the ionic composition of the vacuoles. The pattern of ion compartmentation in Porphyra obtained in this study appears to be in general agreement with published data for other algae. As already pointed out by Gutknecht et al. (1978) and Kirst and Bisson (1979), C1- in general is the predominant vacuolar anion, Na § and-or K § are involved as counterions. For instance, the giant alga Halicystis parvula (Graves and Gutknecht 1976) shows a composition of the cell sap very close to that of Porphyra. In the same alga, an accumulation of K § in the cytoplasm to 420 m M was calculated from the difference between K § concentration in whole cells and isolated cell sap (Graves and Gutknecht 1976). Larkum (1968) demonstrated accumulation of C1- and K § in non-aqueously isolated chloroplasts of Tolypella. In a recent investigation of Porphyra purpurea by Reed and Collins (1980) using radioisotope equilibration techniques, the intracellular concentrations of K § Na § and CI- were estimated to be 356mM, 18 m M and 246mM, respectively, when the alga was kept in seawater. However, the occurrence of a nonosmotic volume (see Wiencke and Lfiuchli 1981) was not taken into consideration by these authors, and subcellular ion compartmentation was not attempted in their study. The latter was carried out, however, in a subsequent paper (Reed and Collins 1981) using the kinetics of ~6Rb+ exchange in Porphyra purpurea to obtain an estimate of the K § concentration in the cytoplasm and in a second compartment (vacuoles and

C. Wiencke et al. : Ion compartmentation in Porphyra

plastid) of cells bathed in seawater. Their indirectly obtained data cannot be compared with our results, however, as in the present study, the three compartments cytoplasm, vacuoles and plastid were directly analysed and, moreover, the alga was adapted in 3.5 x seawater. Our results (Table 3) indicate a very high concentration of K § and a 13 times lower Na § concentration in the cytoplasm of Porphyra. Is this compatible with our knowledge of the ion sensitivity of cytoplasmic enzymes in plant cells? Johnson et al. (1968) showed in Dunaliella viridis, a halophilic green alga, that several enzymes were inhibited in vitro by NaC1 concentrations far lower than those present in the culture medium (3.75 M NaC1). Comparable results were obtained on Dunaliella parva by Ben Amotz and Avron (1972). In contrast to the response to Na +, many enzymes are activated by K § (reviewed by Lfiuchli and Pfl/iger 1979). For example, pyruvate kinase, a cytoplasmic enzyme, is stimulated by K § Maxim u m activation of pyruvate kinase from plant sources is achieved at about 50 m M K § (Miller and Evans 1957; Wildes and Pitman 1975). Preliminary results on pyruvate kinase from Porphyra indicate a considerable in-vitro activation of this enzyme by high KC1 concentrations but no stimulation of the enzyme activity by NaC1 concentrations higher than found in the cytoplasm. Thus, our results on K + and Na § compartmentation in Porphyra (Table 3) are in line with preliminary enzymic data from this alga and with a general hypothesis on cytoplasmic osmoregulation (Wyn Jones et al. 1977). The work was supported by a grant from the Deutsche Forschungsgemeinschaft. We wish to thank the Biologische Anstalt Helgoland, Meeresstation Helgoland, for providing guest laboratory space to C.W. and the Study Group of Electron Microscopy at the Tier/irztliche Hochschule Hannover for providing electron microscopical facilities.

References Andersen, C.A. (1967) An introduction to the electron probe microanalyzer and its application to biochemistry. Methods Biochem. Anal. 15, 147-270 Ben-Amotz, A., Avron, M. (1972) Photosynthetic activities of the halophilic alga Dunaliella parva. Plant Physiol. 49, 240-243 Bisson, M.A., Kirst, G.O. (1979) Osmotic adaptation in the marine alga Griffithsia monilis (Rhodophyceae): the role of ions and organic compounds. Aust. J. Plant Physiol. 6, 523-538 Buchanan, B.B., Wolosiuk, R.A., Schiirmann, P. (1979) Thioredoxin and enzyme regulation. Trends Biochem. Sci. 4, 93 96 Eshel, A. (1974) Quantitative electron probe microanalysis of biological specimens. I. A theoretical analysis of some variables involved. Micron 5, 11-19

C. Wiencke et al. : [on compartmentation in Porphyra Flowers, T.J., Troke, P.F., Yeo, A.R. (1977) The mechanism of salt tolerance in halophytes. Annu. Rev. Plant Physiol. 28, 8%121 Graves, J.S., Gutknecht, J. (1976) Ion transport studies and determination of the cell wall elastic modulus in the marine alga Halicystis parvula. J. Gen. Physiol. 67, 579-597 Greenway, H., Munns, R. (1980) Mechanisms of salt tolerance in nonhalophytes. Annu. Rev. Plant Physiol. 31, 149-190 Gutknecht, J., Hastings, D.F., Bisson, M.A. (1978) Ion transport and turgor pressure regulation in giant algal cells. In: Membrane transport in biology. III. Transport across multimembrane systems, pp. 125-174, Giebisch, G., Tosteson, D.C., Ussing, H.H., eds. Springer, Berlin Heidelberg New York Hellebust, J.A. (1976) Osmoregulation. Annu. Rev. Plant Physiol. 27, 485-505 Johnson, M.K., Johnson, E.J., MacElroy, R.D., Speer, H.L., Bruff, B.S. (1968) Effect of salts on the halophilic alga Dunaliella viridis. J. Bacteriol. 95, 1461-1468 Kauss, H. (1978) Osmotic regulation in algae. Prog. Phytochem. 5, 1-27 Kirst, G.O., Bisson, M.A. (1979) Regulation of turgot pressure in marine algae: Ions and low-molecular-weight organic compounds. Aust. J. Plant Physiol. 6, 539-556 Lfiuchli, A., Pfliiger, R. (1979) Potassium transport through plant cell membranes and metabolic role of potassium in plants. In: Potassium research - review and trends. Proc. 11th Congr_ Int. Potash Inst. Bern, pp. 111 t63, Gething, A., yon Peter, A., eds. Lara, C., de la Torre, A., Buchanan, B.B. (1980) Ferrialterin: an ion-sulfur protein functional in enzyme regulation in photosynthesis. Biochem. Biophys. Res. Commun. 94, 133%1344 Larkum, A.W.D. (1968) Ionic relations of chloroplasts in vivo. Nature (London) 218, 447-449 Miller, G., Evans, H.J. (1957) The influence of salts on pyruvate kinase from tissues of higher plants. Plant Physiol. 32, 346-354

341 Pitman, M.G., Lfiuchli, A., Stelzer, R. (1981) Ion distribution in roots of barley seedlings measured by electron probe Xray microanalysis. Plant Physiol. 68, 673-679 Provasoli, L. (1964) Growing marine seaweeds. Proc. 4th Int. Seaweed Symp. 1961, pp. 9-17, de Virville, A.D., Feldman, J., eds. Oxford London New York Paris Reed, R.H., Collins, J.C. (1980) The ionic relations of Porphyra purpurea (Roth) C. Ag. (Rhodophyta, Bangiales). Plant Cell Environ. 3, 399-407 Reed, R.H., Collins, J.C. (1981) The kinetics of Rb + and K § exchange in Porphyra purpurea. Plant Sci. Lett. 20, 281-289 Stelzer, R. (1981) Ion localization in the leaves of Puccinellia peisonis. Z. Pflanzenphysiot. 103, 27-36 Storey R., Pitman, M.G., Stelzer, R., Carter, C. (1983) X-ray microanalysis of cells and cell compartments of Atriplex spongiosa. I. Leaves. J. Exp. Bot. 34, 778-794 Wiencke, C., L/iuchli, A. (1980) Growth, cell volume and fine struture of Porphyra urnbilicalis in relation to osmotic tolerance. Planta 150, 303-311 Wiencke, C., L/iuchli, A. (1981) Irforganic ions and floridoside as osmotic solutes in Porphyra umbilicalis. Z. Pflanzenphysiol. 103, 247-258 Wildes, R.A., Pitman, M.G. (1975) Pyruvate kinase activity in roots of barley seedlings in relation to salt status. Z. Pflanzenphysiol. 76, 69-75 Wyn Jones, R.G., Storey, R., Leigh, R.A., Ahmad, N., Pollard, A. (1977) A hypothesis on cytoplasmic osmoregulation. In: Regulation of cell membrane activities in plants, pp. 121136, MarrY, E., Ciferri, O., eds. Elsevier/North-ttolland Biomedical Press, Amsterdam Yeo, A.R., L/iuchli, A., Kramer, D., Gullasch, J. (1977) Ion measurements by X-ray microanalysis in unfixed, frozen, hydrated plant cells of species differing in salt tolerance. Planta 134, 35-38 Zimmermann, U. (1978) Physics of turgor- and osmoregulation. Annu. Rev. Plant Physiol. 29, 121-148 Received 25 April; accepted 19 July 1983