Plant Physiol. (1989) 89, 539-5430032-0889/89/89/0539/05/$01 .00/0

Received for publication July 5, 1988and in revised form August 30, 1988

Effect of Sodium Nutrition on the Ultrastructure ofChloroplasts of C4 Plants

Christopher P. L. Grof, Mark Johnston, and Peter F. Brownell*Department of Botany, James Cook University of North Queensland, Townsville 4811, Queensland, Australia

ABSTRACT

Mesophyll chloroplasts from sodium-deficient compared tonormal plants of the C4 species Kochia childsii and Amaranthustricolor were found to have significantly less stacking in theirgrana. On the other hand, no marked difference of thylakoidarrangement between bundle sheath chloroplasts from sodium-deficient and normal plants of A. tricolor were observed.

Sodium is unique as a micronutrient for higher plants inthat it is required only by plants possessing the C4' C02-concentrating appendage (6, 7). The mesophyll cells are im-plicated as the site of the lesion since increased ambient CO2concentration alleviated the signs of sodium-deficiency with-out restoring the lower Chl a/b ratio, characteristic ofsodium-deficiency, to that of normal plants (13-15).

Increased concentrations of alanine (9, 18) and pyruvateand decreased concentrations ofPEP, OAA, malate, aspartate,and 3-PGA (M Johnston, CPL Grof, PF Brownell, unpub-lished observations) have been found in leaves of sodium-deficient compared to normal C4 plants, suggesting a limita-tion in the conversion of pyruvate to PEP.

It is unlikely that this limitation is due to reduced activityof pyruvate orthophosphate dikinase, the enzyme catalyzingthis reaction (4, 8). The transport of pyruvate into the meso-phyll chloroplast and its subsequent conversion to PEP maybe limited by the supply of ATP from photosynthetic phos-phorylation. Decreased Chl a/b ratios ( 13), and fluorescenceratios (10) observed in sodium deficiency are consistent withdysfunction of the light reactions. These differences betweensodium-deficient and normal plants suggest that the ultra-structure ofmesophyll and/or bundle sheath chloroplasts mayalso be affected by sodium nutrition.

MATERIALS AND METHODS

Amaranthus tricolor L. (nonpigmented form) and Kochiachildsii Hort. were used. The procedures for the germinationand growth of plants under low sodium conditions have beendescribed previously (5). The concentration of sodium as animpurity in the complete culture solution was approximately0.08 ,uM. Normal plants were obtained by supplying appro-

' Abbreviations: C4, C4 dicarboxylic photosynthetic pathway; PEP,phosphoenolpyruvate; OAA, oxaloacetate; 3-PGA, 3-phosphoglycer-ate; LHC, light harvesting complex.

priate cultures with NaCl to give a final concentration of0.1 mM.

Leaves of K. childsii and A. tricolor were cut into thinsections in a fixative containing 3% glutaraldehyde in 0.05 Mcacodylate (pH 7.0). The sections were rotated in a smallvolume of fixative at room temperature for 1 h, rinsed in 0.05M cacodylate twice, and then left to stand in 0.05 M cacodylateovernight. The following day, the sections were postfixed in1% OS04, in 0.05 M cacodylate for 1 h at room temperature.The OS04 was removed and replaced by distilled water. Theleaf sections were then subjected to the following dehydrationsteps: 30% ethanol, 2 min; 50% ethanol, 2 min; 70% ethanol,2 min; 80% ethanol, 5 min; 85% ethanol, 5 min; 90% ethanol,5 min; 95% ethanol, 5 min; 100% ethanol, 10 min; 100%ethanol, 10 min; 100% ethanol (dehydrated), 10 min; 1:1,100% ethanol: Spurr's resin, 2 h; 1:3, 100% ethanol: Spurr'sresin, 3 h; 100% Spurr's resin, overnight; 100% Spurr's resin,

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Figure 1. Ultrastructural features of mesophyll chloroplasts from amature sodium-deficient (a) and a normal (b) leaf of K. childsii stainedwith uranyl acetate and lead citrate. G, grana; P, peripheral reticulum.

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Plant Physiol. Vol. 89,1989

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Figure 2. Ultrastructural features of mesophyll chloroplasts from a

mature sodium-deficient (a) and a normal (b) leaf of A. tricolor stainedwith uranyl acetate and lead citrate. G, grana; P, peripheral reticulum;S, starch.

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2 h. The sections were then transferred to embedding mouldscontaining 100% Spurr's resin and left overnight to cure at60°C. Ultrathin sections (70-90 nm) were cut on an LKBNova ultramicrotome using a diamond knife, stained withuranyl acetate (saturated in 50% acidified ethanol) for 6 min,and lead citrate (19) for 1 min, then examined using a JEOLFX2000 electron microscope.Numerous sections were cut from a minimum offour leaves

from three separate plants of each treatment. Once a partic-ular section was photographed on account of its distinguish-able detail within the chloroplast, a minimum of 5 Atm wascut offthe face ofthe block to ensure that the same chloroplastwas not photographed again. Generally, the block was entirelyrefaced prior to more ultrathin sections being cut. Prints(10.15 x 12.70 cm) were made of each chloroplast. Thethylakoids making up each granal stack were counted using astereo dissecting microscope. The chloroplasts selected forcounting were chosen on the basis of clarity to ensure aminimum of counting errors.A chi-square analysis was carried out on the frequencies of

granal stacks comprised of different numbers of thylakoids.Ten representative mesophyll and bundle sheath chloroplastsfrom both sodium-deficient and normal leaves of K. childsiiand A. tricolor were examined.

RESULTS AND DISCUSSION

Mesophyll Chloroplasts

From Figures 1 and 2, it is evident that the amount ofstacking in the grana of sodium-deficient mesophyll chloro-plasts was markedly less than in the normal chloroplasts ofboth Kochia childsii and Amaranthus tricolor. This is sup-ported by the statistical treatment of the data which showsignificant differences (P < 0.01, K. childsii; P < 0.001, A.

Number of thylakoids per granumFigure 3. The mean frequency of grana containing 2 to 30 thylakoids per stack in mesophyll chloroplasts from mature sodium-deficient (hatchedcolumns) and normal (open columns) leaves of K. childsii.

540 GROF ET AL.

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SODIUM AND C4 CHLOROPLAST STRUCTURE

> 2200

Number of thylakoids per granumFigure 4. The mean frequency of grana containing 2 to 34 thylakoids per stack in mesophyll chloroplasts from mature sodium-deficient (hatchedcolumns) and control (open columns) leaves of A. tricolor.

tt,',:....&+E> ............tricolor) between sodium-deficient and norrnal chloroplastsR

}; *i ~~~~~~~~~~~~in their arrangement of thylakoids into grana (Figs. 3 and 4).

A Bundle Sheath Chloroplasts

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Number of thylakdNo markeddiffgerences were observed between the thyla-Fiur.hemg anactai 2 t 3 koidairangement ofbundle sheath chloroplasts from sodium-

tdeficientandnormalplantsoofdA. t ricolo r(Fig. 5). Further-"in'':'''.<\'.'. xmore, the frequency of thylakoid stackingt n the grana d4d

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n o tdiffer significantly (Fig. 6). The bundle sheath chloroplasts7 M ' 'frombothsodium-deficientand normal leavesof K.childs5i~ ^^ ,-- / +were essentially agranal or paucigranal as has been previously

^, :45<~ ~ reported in other NADP-ME types of C4 plants (3).

40 '5 m><x^ Functional Importance of Granal StackingWeier and Benson (22) suggested that the accumulation of

carotenoid pigments as a result of stacking may protect theChl pigments from bleaching by light at high intensities. Theconcentration of photosynthetic pigments between the bilayermembranes could also facilitate energy transfer between mol-ecules. Gunning and Steer (11) proposed that the greater

'V stacking of grana may confer an advantage by providing asuitable environment for energy transfer between PSII andPSI. Kaplan and Arntzen (16) suggested that stacking hasresulted in improved control of light harvesting and photo-chemical operations.Murata (17) proposed a mechanism in intact algae which

controls the relative proportions of light energy being directedXX̂,,E.,: + to PSI and PSII due to structural rearrangements of the

-} @.pigment antennae in response to a change in light conditions.This optimises the energy utilization.The physical mechanisms which control granal stacking are

described by Staehelin (20) and Barber (1). The physical-:--.0 _> changes responsible for the direction of the energy involve the

phosphorylation/dephosphorylation of a mobile Chl a/b LHCFigure 5. Ultrastructural features of bundle sheath chloroplasts from (2, 21). This affects the membrane charge distribution anda mature sodium-deficient (a) and a normal (b) leaf of A. tricolor consequently the distance ofthe Chl a/b LHC from either thestained with uranyl acetate and lead citrate. G, grana; M, mitochon- PSII-rich stacked or the PSI-rich unstacked regions (21). Thedrion; S, starch. decreased amount of granal stacking observed in sodium-

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Plant Physiol. Vol. 89, 1989

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Number of thylakoids per granumFigure 6. The mean frequency of grana containing 2 to 22 thylakoids per stack in bundle sheath chloroplasts from mature sodium-deficient(hatched columns) and control (open columns) leaves of A. tricolor.

deficient mesophyll chloroplasts suggests that a decreased PSIIactivity may be observed as energy would be directed towardPSI in these plants. It was found that PSII activity was

markedly lower in mesophyll thylakoids extracted from so-

dium-deficient plants representing each of the groups of C4plants, K. childsii (NADP-ME type), C. gayana (PCK-type),and A. tricolor (NAD-ME type). PSI activity of mesophyllthylakoids was unaffected by sodium-deficiency in K. childsii;however, it was greater in sodium-deficient than control plantsof A. tricolor. Bundle sheath thylakoids were relatively unaf-fected by sodium-deficiency (M Johnston, CPL Grof, PFBrownell, unpublished observations). Thus, structural differ-ences in sodium-deficient chloroplasts may be responsible forobserved differences in fluorescence (10) and electron trans-port rates (M Johnston, CPL Grof, PF Brownell, unpublishedobservations).The possible reason why there is a difference in response to

sodium between chloroplasts of C3 plants and mesophyllchloroplasts of C4 plants is that within the C3 plant there isno physiological counterpart to the mesophyll chloroplasts ofC4 plants. These chloroplasts have the major specialized roleof converting pyruvate to PEP whereas the chloroplasts of C3plants and bundle sheath cells of C4 plants have the functionof reducing CO2 via the PCR cycle to photosynthates (12).

ACKNOWLEDGMENTS

The authors wish to thank Dr. Luong Van-Thinh, Heather Winsor,and Fiona Scott for their helpful advice.

LITERATURE CITED

1. Barber J (1986) Surface electrical charges and protein phospho-rylation. In LA Staehelin, CJ Arntzen, eds, Photosynthesis III.Photosynthetic Membranes and Light Harvesting Systems.Encyclopedia of Plant Physiology (New Series), Vol 19. Sprin-ger-Verlag, Berlin, pp 653-664

2. Bennett J (1963) Regulation of photosynthesis by reversiblephosphorylation ofthe light harvesting chlorophyll a/b protein.Biochem J 212: 1-13

3. Bishop DG, Reed ML (1976) The C4 pathway of photosynthesis:ein kranz-typ wirtschaftswunder? Photochem Photobiol Rev1: 1-69

4. Boag TS (1981) Characterization ofC4 photosynthesis in sodium-deficient plants. PhD thesis, Australian National University,Canberra, Australia

5. Brownell PF (1979) Sodium as an essential micronutrient forplants and its possible role in metabolism. Adv Bot Res 7: 117-224

6. Brownell PF, Crossland CJ (1972) The requirement for sodiumas a micronutrient by species having the C4 dicarboxylic pho-tosynthetic pathway. Plant Physiol 49: 794-797

7. Brownell PF, Crossland CJ (1974) Growth responses to sodiumby Bryophyllum tubiflorum under conditions inducing Cras-sulacean acid metabolism. Plant Physiol 54: 416-417

8. Dorney WJ (1985) Different responses to sodium by phosphoen-olpyruvate carboxykinase type C4 species in relation to twoalternative metabolic schemes. BSc honours thesis, JamesCook University of North Queensland, Townsville, Australia

9. Grof CPL, Johnston M, Brownell PF (1986) Free amino acidconcentrations in leaves of sodium-deficient C4 plants. Aust JPlant Physiol 13: 343-346

10. Grof CPL, Johnston M, Brownell PF (1986) In vivo chlorophylla fluorescence in sodium-deficient C4 plants. Aust J PlantPhysiol 13: 589-595

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SODIUM AND C4 CHLOROPLAST STRUCTURE

11. Gunning BES, Steer MW (1975) Ultrastructure and the Biologyof Plant Cells. Edward Arnold, London

12. Hatch MD (1976) Photosynthesis: the path of carbon. In JBonner, JE Varner, eds, Plant Biochemistry. Academic Press,New York, pp 797-844

13. Johnston M, Grof CPL, Brownell PF (1984) Effect of sodiumnutrition on chlorophyll a/b ratios in C4 plants. Aust J PlantPhysiol 11: 325-332

14. Johnston M, Grof CPL, Brownell PF (1984) Responses to am-bient CO2 concentrations by sodium-deficient C4 plants. AustJ Plant Physiol 11: 137-141

15. Johnston M, Grof CPL, Brownell PF (1986) Sodium-deficiencyin the C4 species Amaranthus tricolor L. is not completelyalleviated by high CO2 concentrations. Photosynthetica 20:476-479

16. Kaplan S, ArntzenCJ (1982) Photosynthetic membrane structureand function. In Govinjee, ed, Photosynthesis, Vol 1. EnergyConversion by Plants and Bacteria. Academic Press, NY, pp65-151

17. Murata N (1969) Control ofexcitation transfer in photosynthesis.

1. Light induced change of chlorophyl a fluorescence in Por-phyridium cruentum. Biochim Biophys Acta 172: 242-251

18. Nable RO, Brownell PF (1984) Effect of sodium nutrition andlight upon the concentrations of alanine in leaves of C4 plants.Aust J Plant Physiol 11: 319-324

19. Reynolds ES (1963) The use of lead citrate at high pH as an

electron-opaque stain in electron microscopy. J Cell Biol 17:108-212

20. Staehelin LA (1986) Chloroplast structure and supra molecularorganization of photosynthetic membranes. In LA Staehelin,CJ Arntzen, eds, Photosynthesis III. Photosynthetic Mem-branes and Light Harvesting Systems. Encyclopedia of PlantPhysiology (New Series), Vol 19. Springer-Verlag, Berlin, pp

1-8421. Staehelin LA, Arntzen CJ (1983) Regulation ofchloroplast mem-

brane function: protein phosphorylation changes the spatialorganization of membrane components. J Cell Biol 97: 1327-1337

22. Weier TE, Benson AA (1967) The molecular organization ofchloroplast membranes. Am J Bot 54: 389-402

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