Plant Physiol. (1975) 56, 676-679 Suaeda monoica, a C4 Plant without Typical Bundle Sheaths Received for publication May 19, 1975 and in revised form July 15, 1975 ADIVA SHOMER-ILAN, SVEN BEER, AND YOAV WAISEL Department of Botany, Tel Aviv University, The Dr. George S. Wise Centerfor Life Sciences, Tel Aviv, Israel (9, 13, 14, 20). The latter, frequently called the Kranz anatomy, consists of a layer of large cells which usually envelope the Suaeda monoica Forssk. ex J. F. Gmel was found to possess vascular bundles and which are densely packed with chloroplasts. the C4 pathway of photosynthesis. The succulent leaves of This bundle sheath formation is surrounded by chlorenchymatous Suaeda lack a green bundle sheath formation but have a mesophyll cells. The existence of such differentiated chlorenlayer of chlorenchyma, containing large and centripetally chyma, with the inner layer attached to the vascular bundles, is arranged chloroplasts, which surrounds the water tissue. commonly believed to be necessary for the operation of the C4 We suggest that the proximity of a chlorenchymatous cell carbon fixation pathway. layer to the vascular bundles is not necessary for the operaThe characterization of C4 plants by the above mentioned tion of the C4 pathway. features has been generally recognized. Since it was shown that the balance between C3 and C4 carboxylation could be altered by factors such as salinization, ontogeny, and leaf age (10, 11, 16), it seemed that the correlation between the appearance of a chlorenchymatous bundle sheath layer and the C4 carbon fixation pathway was not of a basic nature (4). The possibility of finding a C4 plant which lacks these typical structural features Since the discovery of the C4 carbon fixation pathway (7, 12), was therefore very likely. an increasing number of plants possessing this pathway have been recognized. It was found that in addition to the primary fixation MATERIALS AND METHODS of CO2 into 4-carbon acids, C4 plants possess certain other charSuaeda monoica Forssk. ex J. F. Gmel plants were grown on acteristics, i.e. a low CO2 compensation point (5), low discrimination against 'IC (3, 17), and a specific anatomical leaf structure aqueous solutions in a growth chamber (16 or 9 hr light period; ABSTRACT ..: .0, J""' .. V-01-4 47 k. e 47chi .JL. i. ... v. 1. -d' ,: t '.': .:;. ..0, jdt AOWL, EN ' * A W ~~';^s FIG. 1. Cross section of Suaeda monoica leaf (190 X). e: k is + ^ ~A v Mt epidermis; cho: outer layer of chlorenchyma; chi: inner layer of chlorenchyma; water tissue; v: vascular bundle. 676 w: Downloaded from https://academic.oup.com/plphys/article/56/5/676/6074695 by guest on 06 July 2023 .... Plant Physiol. Vol. 56, 1975 C4 PLANT WITHOUT BUNDLE SHEATHS 677 V... ...A; e FiG. 2. Magnified section of the leaf shown in Fig. 1 (740 X). e: epidermis; cho: outer layer of chlorenchyma; chi: inner layer of chlorenchyma with centripetally arranged chloroplasts; w: water tissue. 1.8 X 104 ergs X cm-2 X sec-' light intensity, Sylvania white and cool white VHO lamps supported by incandescent light; 25 C day and 18 C night temperature; 70% relative humidity). Two types of nutrient solutions were used: (a) full strength Hoagland solution (8) and (b) the same solution to which NaCl was added to a final concentration of 100 mm. The solutions were constantly aerated. Three- to four-month-old plants were used in the following analysis. Phosphoenolpyruvate Carboxylase Activity. PEP-case' from 5 g leaf material was extracted and assayed as previously described (2). The assay mixtures were free of sodium chloride. 1 Abbreviations: PEP-case: phosphoenolpyruvate carboxylase; IRGA: infrared gas analyzer; CAM: Crassulacean acid metabolism. Carbon Isotope Ratios. The 'IC/12C ratios (613C) were measured using a mass spectrometer. Values were calculated relative to a PDB limestone standard. Malate Content. Malate was extracted by crushing and boiling 5 g of leaf material in 10 ml of H20 for 20 min; 1.5 ml of the extract was assayed in 0.47 M glycine, 0.20 M hydrazine sulfate, 0.23 mM EDTA, 0.03 M NAD, and 50 IU malate dehydrogenase (Sigma). The reaction mixture was kept at pH 9.5. The final reaction volume was 3.2 ml. The reduction of NAD was measured spectrophotometrically at 340 nm. CO2 Compensation Point. Compensation points were measured using an IRGA (Beckman Model 865) at saturating light intensities. Short Time Light Fixation. Branches of Suaeda monoica were Downloaded from https://academic.oup.com/plphys/article/56/5/676/6074695 by guest on 06 July 2023 w 678 Plant Physiol. Vol. 56, 1975 SHOMER-ILAN, BEER, AND WAISEL Table I. Some Characteristics of Carbon Metabolism of Suaeda monoica and Chloris gayana Data are means, in some cases i standard deviation. Suaeda monoica Chloris gayana Characteristics PEP-case activity (jumoles CO2 X mg protein-' X min-') CO2 compensation Concentration (,.l/l) 651C (0/00) High salt Low salt 0.49 0.34 <5 5 <5 -15.89 0.51 -17.02 - 14.84 to 10 440 12 360 75 60 exposed to "CO2 for 5 sec. The plants were then flushed with '2CO2 for different time periods. Leaves were killed with liquid nitrogen. Labeled compounds were extracted in 80% ethanol at pH 4 and then in H20. Extract constituents were separated by TLC according to Feige et al. (6). The chromatographic plates were then autoradiographed. The specific spots were removed, and 'IC activity was determined by liquid scintillation spectrophotometry. RESULTS Leaf Anatomy. Suaeda monoica plants have succulent and semicylindrical leaves. Two different types of chlorenchyma were recognized underneath the epidermis: an outer layer with relatively small chloroplasts and an inner chlorenchymatous layer which contains many large chloroplasts in a centripetal arrangement (Figs. 1 and 2). The center of the leaf was comprised of several layers of a parenchymatous water tissue embedding the vascular bundles. No bundle sheath formation was observed. The cells of the water tissue contained few scattered chloroplasts which were smaller than those of the inner layer of the chloren- Low salt plants High salt plants 100 12 co 2 x- ._ U 50 7 > a: Ala. D / 5 - Suc 15 30 45 Time (sec.) I- 60 -- -A 5 _ 15 , Z S.oh. 30 45 60 Time (sec.) FIG. 3. Time course of 14C labeling pattern. Approximately 20,000 cpm were applied to each plate. Data presented in fractions of the total recovered 14C. Asp: aspartate; Mal: malate; S.ph.: phosphorylated metabolites; Ala: alanine; Suc: sucrose; Ser + Gly: serine + glycine. Downloaded from https://academic.oup.com/plphys/article/56/5/676/6074695 by guest on 06 July 2023 -17.62 Malate content (,urnoles X g dry weighrt) 125 Light (6 hr) 120 Dark (6 hr) chyma, but resembled those of the outer chlorenchymatous layer. No structural dimorphism between chloroplasts of the different green layers could be seen. Electron micrographs showed that both types of chloroplasts contained grana and accumulated starch. Phosphoenolpyruvate Carboxylase. The initial carbon fixation reaction in C4 plants is catalyzed by PEP-case. As it was shown that the pH optimum for this enzyme varied in halophytes under different salt treatments (18, 19), it was necessary to determine the pH optimum in Suaeda before measuring enzyme activity. Optimal activities of PEP-case extracted from salt-treated plants were obtained at pH 7.9, whereas values obtained for saltdepleted plants were lower (pH 7.5). By comparing the specific activity of PEP-case extracted from salt-treated Suaeda monoica leaves with that extracted from Chloris gayana, a known C4 plant, Andrews et al. (1) showed that the values were approximately equal. The activity in saltdepleted Suaeda plants was about 15 % lower (Table I). Carbon Isotope Ratios. Examination of the '3C/'2C ratios in Suaeda monoica leaves following different salt treatments and at different leaf ages yielded values between -15 and -17 6'3C %,. Similar values were obtained for Chloris plants grown under the same conditions. Malate Content. The possibility of Suaeda monoica being a CAM plant was investigated by analyzing the variation in malate content of the leaves during consequent periods of light and dark. The results presented in Table I show that no excess malate had accumulated during the dark period. This pattern was unchanged in plants grown under long (16 hr) or short (9 hr) photoperiods. CO2 Compensation Point. Measurements showed that saltdepleted Suaeda monoica plants had a CO2 compensation point of 5 ,ul/l. The compensation point of salt-treated plants was even lower (Table I). Short Time Light "4CO2 Fixation. The best proof for the existence of the C4 pathway is the determination of the primary products of photosynthesis. Results of short time light fixation experiments in salt-treated Suaeda plants show that after 5-sec exposure to 14CO2 a typical C4 pattern of labeling was found: 85 %xC of the label was found in aspartate, 8%o in malate, and 3% in alanine and serine (Fig. 3). Different chase periods in '2CO2 following the 5-sec pulse of "CO2 resulted in a rapid decrease in the Plant Physiol. Vol. 56, 1975 C4 PLANT WITHOUT BUNDLE SHEATHS content of labeled aspartate and in the appearance of the label in phosphorylated metabolites and sucrose. Acknowledgments-Measurements of 13C/12C ratios were made by Dr. A. Nissenbaum of the Weizman Institute, Rehovot, Israel. The authors are grateful for his kind cooperation in this project. Sincere thanks are also due to our colleague Dr. Y. Samish for measuring the C02 compensation points. LITERATURE CITED 1. ANDREWS, T. J., H. S. JOHNSON, C. R. SLACK, AND M. D. HATCH. 1971. Malic enzyme and amino transferases in relation to 3-PGA formation in plants with the C4-dicarboxylic acid pathway of photosynthesis. Phytochemistry 10: 20052013. 2. BEER, S., A. SHOMER-ILAN, AND Y. WAIsEL. 1975. Salt-stimulated phosphoenolpyruvate carboxylaae in Cakile maritima. Physiol. Plant. 34: 293. 3. BENDERx M. M. 1971. Variations in the 13C/12C ratios of plants in relation to the pathway of photosynthetic carbon dioxide fixation. Phytochemistry 10: 12391244. 4. BROWN, W. V. 1975. Variations in anatomy, association, and origins of Kranz tissue. Am. J. Bot. 62: 395-402. 5. DOWNTON, W. J. S. AND E. B. TREGUNNA. 1968. Carbon dioxide compensationits relation to photosynthetic carboxylation reactions, systematics of the graminae, and leaf anatomy. Can. J. Bot. 46: 207-215. 6. FEIGE, B., H. GIMMLER, W. D. JESCHKE, AND W. SIMONIS. 1969. Eine Methode zur dunnschichtchromatographischen Auftrennung von 14C- und P - markierten Stoffwechselprodukten. J. Chromatog. 41. 8090. 7. HATCH, M. D. AND C. R. SLACK. 1966. Photosynthesis by sugarcane leaves. A new carboxylation reaction and the pathway of sugar formation. Biochem. J. 101:103-111. 8. HOAGLAND, D. R. AND D. J. ARNON. 1950. The water culture method for growing plants without soil. Calif. Agric. Exp. Sta. Circ. 347: 1-32. 9. JOHNsoN, H. S. AND M. D. HATICH. 1968. Distribution of the C4-dicarboxylic acid pathway of photosynthesis and its occurrence in dicotyledonous plants. Phytochemistry 7: 375-380. 10. KENNEDY, R. A. AND W. M. LAETFcCH. 1973. Relationships between leaf development and primary photosynthetic products in the C4 plant Portulaca oleracea L. Planta 115: 113-124. 11. KEANNA, R. AND S. K. SINHA. 1973. Change in the predominance from C4 to Ca pathway following anthesis in Sorghum. Biochem. Biophys. Res. Commun. 52: 121-124. 12. KORTBCHAK, H. P., C. E. HARTT, AND G. 0. BURR. 1965. Carbon dioxide fixation in sugarcane leaves. Plant Physiol. 40: 209-213. 13. LAETBCH, W. M. 1968. Chloroplast specialization in dicotyledons possessing the C4-dicarboxylic acid pathway of photosynthetic C02 fixation. Am. J. Bot. 55: 875-883. 14. LAETSCH, W. M. 1974. The C4 syndrome: a structural analysis. Annu. Rev. Plant Physiol. 25: 27-52. 15. OLESEN, P. 1974. Leaf anatomy and ultrastructure of chloroplasts in Sal8ola kiali L. as related to the C4-pathway of photosynthesis. Bot. Notiser 127: 352-363. 16. SHoMER-ILAN, A. AND Y. WAISEL. 1973. The effect of sodium chloride on the balance between the C3- and C4-carbon fixation pathways. Physiol. Plant. 29: 190193. 17. SmiTB, B. N. AND S. EPSTEIN. 1971. Two categories of 13C/12C ratios for higher plants. Plant Physiol. 47: 380-384. 18. TREICHEL, S. P.. G. 0. KIRST, AND D. J. v. WILLERT. 1974. Veranderung der Activitalt der Phosphoenolpyruvat Carboxylase durch NaCl bei Halophyten verschidener Biotope. Z. Pflanzenphysiol. 71: 437-449. 19. WAISEL, Y., S. BEER, AND A. SHOMER-ILAN. 1974. The effects of sodium chloride on carbon fixation pathways and productivity of some halophytes. Deutsche Botanische Gesellschaft und Vereinigung fur Angewandte Botanik. Tagung in Wflrzburg. p. 114. 20. WELKIE, G. W. AND M. CALDWELL. 1970. Leaf anatomy of species in some dicotyledon families as related to the Ca and C4 pathways of carbon fixation. Can. J. Bot. 48: 2135-2146. Downloaded from https://academic.oup.com/plphys/article/56/5/676/6074695 by guest on 06 July 2023 DISCUSSION The various characteristics which were investigated are generally believed to be important criteria for proving that a plant is of the C4 type. The low discrimination values against '1C, obviously resulting from the high activity of PEP-case, show that Suaeda monoica could have been either a C4 or a CAM plant. If this species could perform Crassulacean acid metabolism, a considerable accumulation of organic acids, mainly malate, in the dark would be expected. The lack of such accumulation in the dark evidently shows that Suaeda monoica is not a CAM plant. Furthermore, the low CO2 compensation point in light found in Suaeda monoica is another indication that this is a C4 rather than a CAM plant. The labeling pattern which follows short time light fixation is again typical for C4 plants. In this case the flow of carbon seems to go more via aspartate than via malate. A higher content of malate was found in the salt-depleted as compared with the salt-treated plants (Table I). Relatively more malate appeared in the salt-depleted plants also as the first product of light fixation. Thus, NaCl seems to affect the balance between the malate and aspartate formation in Suaeda monoica. It is not known whether the special structural differentiation of the chlorenchyma in Suaeda is what enables the C4 metabolism of this plant. If it does, it is evident that the location of a chlorenchymatous cell layer near the vascular bundles is not essential. C4 metabolism can exist therefore even in plants with such chlorenchyma located at some distance from the vascular bundles. A similar case was reported for the C4 plant Salsola kali (15). The appearance of small vascular bundles in the periphery of the water tissue indicates that, unlike in Suaeda, the inner chlorenchymatous layer of Salsola leaves is a true bundle sheath formation. We conclude that two types of chlorenchymatic cells may be necessary for a successful operation of the C4 carbon fixation pathway. However, there is no absolute requirement for the Kranz cells (4) to be associated with the vascular bundles. The commonly accepted structural description of C4 plants should therefore be somewhat modified. 679