Biosci. Biotechnol. Biochem., 69 (6), 1156–1161, 2005
Changes in the Composition of Xylem Sap during Development of the Spadix of Skunk Cabbage (Symplocarpus foetidus) Yoshihiko O NDA and Kikukatsu I TOy Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan Received January 28, 2005; Accepted March 23, 2005
The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of postthermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31:2 24:7 l h 1 g 1 ) and post-thermogenic (50:5 30:4 l h 1 g 1 ) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus. Key words:
skunk cabbage; spadix; heat production; energy source; xylem sap
Certain primitive plants increase their own temperature by considerably accelerating aerobic respiration. These thermogenic plants include several species of the arum lily family,1–3) palms,4–6) lotus,7–9) cycads,10) and water lilies.11,12) To date, however, only three plant species, Symplocarpus foetidus,3,13–18) Philodendron selloum,19–23) and Nelumbo nucifera8,9,24) have been shown to be homeothermic. Interestingly, homeothermic heat production exhibits an inverse relationship between the level of respiration and changes in ambient temperature. The spadix of S. foetidus has been shown to increase its respiratory rate under low ambient temperature.3,18) Furthermore, oxygen consumption in P. selloum has also been shown to increase as ambient temperature decreases.22) These results suggest that a large amount of respiratory substrate is required to increase heat production, which is induced by decreased ambient temperatures. Indeed, the major energy source y
for S. foetidus and P. selloum have been shown to be carbohydrates3) and lipids21,25) respectively. Therefore, a continuous supply of substrates from the xylem sap, which is produced and blended in the root and transported via xylem vessels, appears to play a crucial role in homeothermic heat production in plants. Interestingly, when the spadix of S. foetidus is removed from the rest of the plant, heat production terminates immediately.13,16) This observation suggests the presence of a pathway for the translocation of an energy source from the roots to the spadix in S. foetidus. Specifically, it is probable that carbohydrates, such as sugars that are catabolized from starch in the roots,13) are transported to support homeothermic heat production and the development of the spadix. However, whether the root controls the physiological state of the thermogenic spadix via compositional changes in the xylem sap in S. foetidus is poorly understood. Moreover, no evidence has been presented for changes in carbohydrates or amino acids in xylem sap, both of which are potentially utilized for energy metabolism in the spadix of S. foetidus. To clarify the mechanism for maintaining stigma stage-specific heat production in S. foetidus, we analyzed the levels of sugars, organic acids, and amino acids in xylem sap from the roots using thermogenic (stigma stage) and post-thermogenic (male stage) plants. Because the protogynous spadix of S. foetidus exhibits stigma stage-specific homeothermic heat production,3,15,16,18) our analysis of the xylem sap from various spadix stages enabled us to show the potential cross-talk between the roots and the spadix in S. foetidus. This is the first report to identify the developmental changes in the composition of xylem sap in thermogenic plants.
Materials and Methods Plant materials, collection of xylem sap, and measurement of temperature. The experiments with Symplocarpus foetidus plants were carried out on a natural population in the village of Hakuba, Nagano Prefecture in central Japan (36 390 N, 137 500 E) between 18:00 and 19:00 on April 22, 2002. Plants with thermogenic
To whom correspondence should be addressed. Tel/Fax: +81-19-621-6143; E-mail:
[email protected]
Composition of Xylem Sap in Skunk Cabbage
(stigma stage) and post-thermogenic (male stage) spadices were selected. Neither thermogenic nor postthermogenic plants had expanded leaves. Xylem sap was collected over a 1-h period immediately after cutting spadices at the upper part of the central stalk using stainless-steel razor blades.16) Measurements of the spadix and ambient air temperatures were made just before the spadix was cut. The total volume of the collected xylem sap was measured and each sample was stored at 20 C until analysis. The temperature was measured using an automatic recording thermometer (TR-52; T and D, Nagano, Japan), as described in our previous report.16) The mean air temperature in the present study was 9:1 0:2 C. Sugar analysis. Sugars (sucrose, glucose, and fructose) in the xylem saps were analyzed by high-performance liquid chromatography (HPLC) using KS801 and KS802 columns (both columns were 8:0 300 mm; Shodex, Tokyo) warmed to 80 C.16) Deionized water was used as the mobile phase at a flow rate of 0.4 ml min1 , and peaks were detected with a refractive index detector. The detected peaks were identified and quantified using a standard solution consisting of sucrose, glucose, and fructose. Organic acid and amino acid analysis. Xylem saps were introduced into a cation exchange column (Dowex 50 W 4 resin, Dow Chemical, Auburn Hills, MI), and an aliquot of the effluent from the column was subsequently introduced into an anion exchange column (Amberlite IRA-400 resin, Rohm and Hass, Philadelphia, PA). Absorbed substances in the cation and anion exchange resins were eluted with 1 M ammonia and 1 M formate solutions respectively. The eluted fractions from the anion exchange resin were further analyzed for organic acids by HPLC (LC-4; Shimadzu, Kyoto, Japan) using a SCR-102 (H) column (600 mm long column; Shimadzu, Kyoto, Japan) warmed to 65 C. Phosphoric acid, adjusted to pH 1.8, was used as an eluting solvent at a flow rate of 0.25 ml min1 . The substances eluted from the cation exchange resin were analyzed and quantitated on a fully automated amino acid analyzer (flow rate 0.1 ml min1 ) (JLC-500/ V; JEOL, Tokyo) according to the manufacture’s instructions.
1157
of the spadix at various stages. Although we tried to include samples from the early stigma stage with prethermogenic plants, almost all pre-thermogenic plants were under the ground, and hence we could not study those plants for further analysis. The temperature of the spadix at the stigma and male spadix stages were 22:5 1:1 C and 9:1 0:4 C respectively (Fig. 1). Because the average ambient temperature during the experiment was 9:1 0:2 C (Fig. 1), these results clearly indicate that our sample plants exhibited stigma stage-specific heat production. Hereafter, we refer to plant at the stigma and male spadix stages as thermogenic and post-thermogenic respectively. Volume of xylem sap Figure 2 shows the mean volume of sap collected during the 1-h period after decapitation of the spadix. Because most of the plants had no expanded leaves and the spadix was the only sink organ for xylem sap in a plant, data are expressed as a volume per fresh weight of the spadix (Fig. 2). No significant difference was observed in the total volume of xylem sap per fresh
Fig. 1. Developmental Stage-Specific Heat Production of S. foetidus. The spadix temperatures of thermogenic (stigma stage) and postthermogenic (male stage) plants were measured just before removing the spadix (n ¼ 12). Ambient air temperatures are also presented (n ¼ 60). Each bar represents mean SD. Bars with a different letter are significantly different (P < 0:05).
Statistical analysis. Analysis of variance was performed using the SAS program (SAS Institute, Cary, NC). Obtained values were expressed as mean SD.
Results Stigma stage-specific heat production in the spadix To determine whether the protogynous spadix exhibited stigma stage-specific heat production when the xylem saps were collected, we measured the temperature
Fig. 2. Total Volume of Xylem Saps from Thermogenic and PostThermogenic Plants of S. foetidus. Xylem saps were collected over a 1-h period immediately after removing the spadices. Each bar represents the mean SD (n ¼ 12). No significant difference was observed (P < 0:05).
1158
Y. ONDA and K. ITO
weight of the spadix between thermogenic (31:2 24:7 ml h1 g1 ) and post-thermogenic (50:5 30:4 ml h1 g1 ) plants. The fresh weights of the spadices of thermogenic and post-thermogenic plants were 5:6 1:4 g and 8:6 3:2 g respectively. Analysis of sugars As shown in Fig. 3, the concentrations of three major sugars in the xylem sap (sucrose, glucose, and fructose) were significantly higher in thermogenic plants. Other neutral sugars such as arabinose, xylose, mannose, and galactose were not detectable in our assay. Sucrose from thermogenic plants exhibited the highest concentration (4:5 0:4 mM g1 ) among the detected sugars in xylem saps. Analysis of organic acids Figure 4 shows the levels of three organic acids (citrate, malate, and succinate) in the xylem saps. These molecules are potential substrates of the tricarboxylic acid (TCA) cycle within mitochondria. The levels of
Table 1. Concentrations of Amino Acids in Xylem Sap from Thermogenic and Post-Thermogenic Plants of S. foetidus Amino acid Asp Thr Ser Asn Glu Gln Gly Ala Val Ile Leu Tyr Phe
Thermogenic
Post-thermogenic
(mM/g spadix fresh weight) 0:12 0:14 a 0:02 0:02 b 0:11 0:07 a 0:06 0:09 a 0:10 0:06 a 0:06 0:07 a 1:85 0:89 a 0:61 0:53 b 0:12 0:06 a 0:04 0:03 b 0:58 0:36 a 0:22 0:36 b 0:03 0:02 a 0:01 0:01 b 0:12 0:13 a 0:02 0:01 b 0:28 0:55 a 0:16 0:17 a 0:02 0:01 a 0:05 0:06 a 0:03 0:01 a 0:03 0:04 a 0:01 0:01 a 0:01 0:01 a 0:02 0:01 a 0:02 0:03 a
Each datum represents the mean SD (thermogenic, n ¼ 11; post-thermogenic, n ¼ 12). Values followed by a different letter are significantly different (P < 0:05).
citrate concentration were lower than those of malate or succinate, and no significant difference was observed between thermogenic and post-thermogenic plants (Fig. 4). On the other hand, the concentrations of malate and succinate in the thermogenic plants were significantly higher (Fig. 4). Analysis of amino acids Among the detected amino acids, Asn and Gln, which contain amide nitrogen, were the primary amino acids in xylem sap in both thermogenic and post-thermogenic plants (Table 1). Concentrations of Asp, Asn, Glu, Gln, Gly, and Ala were significantly lower in post-thermogenic plants, whereas the levels of Thr, Ser, Val, Ile, Leu, Tyr, and Phe showed no significant changes.
Fig. 3. Concentrations of Sugars in Xylem Saps from Thermogenic and Post-Thermogenic Plants of S. foetidus. Each bar represents a mean SD (thermogenic, n ¼ 12, postthermogenic, n ¼ 12). Bars with different letters are significantly different (P < 0:05).
Fig. 4. Concentrations of Organic Acids in Xylem Saps from Thermogenic and Post-Thermogenic Plants of S. foetidus. Each bar represents a mean SD (thermogenic, n ¼ 11, postthermogenic, n ¼ 12). Bars with different letters are significantly different (P < 0:05).
Discussion Thermogenesis during the stigma stage of S. foetidus is terminated immediately after the spadix is removed from the rest of the plant.13,16) Furthermore, because the respiration quotient of the thermogenic spadix is 1.0 and there are no leaves during flowering of S. foetidus, a continuous supply of carbohydrates, reserved as starch in the roots, is necessary for stigma stage-specific heat production.3,14) If the mechanism that maintains such stigma stage-specific heat production is controlled, in part, by the energy source from the roots, the composition of xylem sap, especially carbohydrates, should differ between thermogenic and post-thermogenic plants. In the present study, we focused accordingly on the composition of the xylem saps obtained from thermogenic and post-thermogenic plants. Xylem sap is often used to estimate the interaction between roots and above-ground parts,26,27) mineral absorption,28) and nitrogen assimilation and translocation.29–32) In the protogynous spadix, homeothermic heat production has been shown to disappear at the male stage
Composition of Xylem Sap in Skunk Cabbage
when pollen is shed from the entire surface of the spadix.3,13) In our experience, however, weak heat production was often observed during the early stage in male plants, although pollen appeared on the entire surface of the spadix (data not shown). For this reason, plants were chosen for further analysis after confirmation of complete termination of heat production by careful measurement of spadix temperature in a cold environment (Fig. 1). No significant difference in sap volume per unit weight of the spadix was observed, however, between thermogenic and post-thermogenic plants (Fig. 2). These results suggest that root pressures, which were created by the osmotic pressure of xylem sap, between thermogenic and post-thermogenic plants were relatively constant irrespective of the developmental stage of the spadix. There was, however, a significant change in component concentrations in the xylem sap. Namely, the concentration of sugars, organic acids, and amino acids decreased remarkably in post-thermogenic plants. In thermogenic plants, concentrations of sucrose, glucose, and fructose in xylem sap were in the 2–5 mM range for each spadix fresh weight (Fig. 3). These values, especially the sucrose concentration, appear extraordinarily high relative to the levels in the xylem sap of nonthermogenic plants. For example, it has been reported that the level of sucrose in the xylem sap of tobacco and squash is undetectable.26,33) Moreover, the level of glucose and fructose in the xylem sap of squash have been shown to be 64.3 mM,26) which represents as much as 1% of the total hexose concentration found in skunk cabbage. Therefore it is probable that the fairly high levels of soluble sugars in xylem sap provide a potential energy source for stigma stage-specific heat production. This concept is also supported by the fact that the levels of sucrose, glucose, and fructose dramatically decreased when heat production was terminated (Fig. 3). A previous report indicated that sugar concentrations in xylem sap are relatively constant during the stigma stage and do not respond directly to external ambient temperature.16) It appears that fine control of homeothermic heat production against ambient temperature changes was conducted by uncoupling proteins34,35) and/or alternative oxidase,36,37) whose expression is potentially regulated by temperature changes.34,38) Therefore, a remarkable decrease in sugars in post-thermogenic plants as shown in this study suggest that sugars in xylem saps play an important role in supporting stigma stage-specific basal heat production of the spadix. Oxygen consumption during the stigma stage of spadix has been shown to increase during heat production,3,13,18) which suggests that the respiratory pathways, including glycolysis, the TCA cycle, and the mitochondrial electron transport chain are simultaneously stimulated during heat production. As described above, the initial substrates of glycolysis (i.e., sucrose, glucose, and fructose) appeared to be imported from the roots to the spadix via xylem sap. However, whether or not
1159
substrates for the TCA cycle are also supplied from the roots is unknown. Hence we examined the level of three organic acids (citrate, malate, and succinate), which are potentially utilized in the TCA cycle within mitochondria. Concentrations of malate and succinate in thermogenic plants were significantly higher than in post-thermogenic plants (Fig. 4). Malate is catalyzed to oxaloacetate by a malate dehydrogenase in the TCA cycle.39) Alternatively, malate is oxidized to pyruvate, which is subsequently utilized in the TCA cycle, by NADþ malic enzyme that exists in plant mitochondria.40) Furthermore, succinate is provided as a substrate for succinate dehydrogenase in the TCA cycle and a mitochondrial respiratory chain.39,41) It was also found that the concentration of citrate was extremely low, and no significant difference was observed between the concentrations in thermogenic and post-thermogenic plants (Fig. 4). These results clearly suggest that citrate in xylem sap is not preferentially utilized as a major energy source in the spadix of skunk cabbage in the thermogenic and post-thermogenic stages. Because it has been shown that the TCA cycle in the mitochondria is potentially stimulated by any organic acids constituting the pathway,42) it is possible that both malate and succinate are translocated from the roots and catabolized in the increased oxidative reactions in the thermogenic spadix. Finally, our analysis of the amino acid composition of xylem sap revealed that Asn and Gln are the major amino acids. Furthermore, the levels of Asn and Gln, together with Asp, Glu, Gly, and Ala, were significantly higher in thermogenic than in post-thermogenic plants (Table 1). Because both Asn and Gln contain amide nitrogen, xylem sap appears to be a source of nitrogen for the spadix. Previous studies have shown that spadix weight increases during the period of heat production,3,18) which was also confirmed in the present study (see ‘‘Results’’). Therefore, it is also probable that a supply of nitrogen in the form of Asn and Gln, together with sugars and organic acids as an energy source, is, in part, necessary for the basal growth of the spadix. In conclusion, our results clearly indicate that the composition of xylem sap differs between thermogenic and post-thermogenic plants in S. foetidus. These results strongly suggest that developmental termination of the stigma stage-specific heat production in S. foetidus is, at least in part, controlled by root activities via decreased levels of energy source in the xylem sap. Further studies are necessary to show conclusively such a developmentally regulated cross-talk between the roots and the spadix.
Acknowledgments We are grateful to Atsuo Shirakawa for permission to study S. foetidus on his property. We thank Y. Abe for her excellent assistance, and Y. Kato, T. Ito, M. Otsuka, T. Sato, K. Matsukawa, S. Kawai, and M. Uemura for
1160
Y. ONDA and K. ITO
17)
stimulating discussion. We also thank Professor R.S. Seymour (University of Adelaide, Australia) for his encouragement. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and a Grantin-Aid for the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
18)
References
20)
1)
2)
3)
4) 5) 6)
7)
8) 9)
10)
11)
12)
13)
14) 15)
16)
Gottsberger, G., and Amaral, A., Pollination strategies in Brazilian Philodendron species. Ber. Dtsch. Bot. Ges., 97, 391–410 (1984). Meeuse, B. J. D., and Raskin, I., Sexual reproduction in the arum lily family, with emphasis on thermogenicity. Sex. Plant Reprod., 1, 3–15 (1988). Seymour, R. S., and Blaylock, A. J., Switching off the heater: influence of ambient temperature on thermoregulation by eastern skunk cabbage Symplocarpus foetidus. J. Exp. Bot., 50, 1525–1532 (1999). Schroeder, C. A., Temperature elevation in palm inflorescences. Principles, 22, 26–29 (1978). Gottsberger, G., Flowers and beetles in the South American tropics. Acta Bot., 103, 360–365 (1990). Listabarth, C., Pollination of Bactris by Phyllotrox and Epurea. Implications of the palm breeding beetles on pollination at the community level. Biotropica, 28, 69– 81 (1996). Miyake, K., Some physiological observations on Nelumbo nucifera, Gaertn. Bot. Mag. Tokyo, 12, 112– 117 (1898). Seymour, R. S., and Schultze-Motel, P., Thermoregulating lotus flowers. Nature, 383, 305 (1996). Seymour, R. S., Schultze-Motel, P., and Lamprecht, I., Heat production by sacred lotus flowers depends on ambient temperature, not light cycle. J. Exp. Bot., 49, 1213–1217 (1998). Skubatz, H., Tang, W., and Meeuse, B. J. D., Oscillatory heat-production in the male cones of cycades. J. Exp. Bot., 44, 489–492 (1993). Prance, G. T., and Arias, J. R., A study of the floral biology of Victoria amazonica (Poepp.) Sowerby (Nymphaeaceae). Acta Amazonica, 5, 109–139 (1975). Skubatz, H., Williamson, P. S., Schneider, E. L., and Meeuse, B. J. D., Cyanide-insensitive respiration in thermogenic flowers of Victoria and Nelumbo. J. Exp. Bot., 41, 1335–1339 (1990). Knutson, R. M., Heat production and temperature regulation in eastern skunk cabbage. Science, 186, 746–747 (1974). Knutson, R. M., Plants in heat. Nat. Hist., 88, 42–47 (1979). Uemura, S., Ohkawara, K., Kudo, G., Wada, N., and Higashi, S., Heat-production and cross-pollination of the Asian skunk cabbage Symplocarpus renifolius (Araceae). Amer. J. Bot., 80, 635–640 (1993). Ito, K., Onda, Y., Sato, T., Abe, Y., and Uemura, M., Structural requirements for the perception of ambient temperature signals in homeothermic heat production of skunk cabbage (Symplocarpus foetidus). Plant Cell Environ., 26, 783–788 (2003).
19)
21)
22)
23)
24) 25)
26)
27)
28)
29)
30)
31)
32)
33)
34)
Ito, K., Ito, T., Onda, Y., and Uemura, M., Temperaturetriggered periodical thermogenic oscillations in skunk cabbage (Symplocarpus foetidus). Plant Cell Physiol., 45, 257–264 (2004). Seymour, R. S., Dynamics and precision of thermoregulatory responses of eastern skunk cabbage Symplocarpus foetidus. Plant Cell Environ., 27, 1014–1022 (2004). Nagy, K. A., Odell, D. K., and Seymour, R. S., Temperature regulation by the inflorescence of Philodendron. Science, 178, 1195–1197 (1972). Seymour, R. S., Bartholomew, G. A., and Barnhart, M. C., Respiration and heat production by the inflorescence of Philodendron selloum Kock. Planta, 157, 336–343 (1983). Seymour, R. S., Barnhart, M. C., and Bartholomew, G. A., Respiratory gas exchange during thermogenesis in Philodendron selloum Koch. Planta, 161, 229–232 (1984). Seymour, R. S., Pattern of respiration by intact inflorescences of the thermogenic arum lily Philodendron selloum. J. Exp. Bot., 50, 845–852 (1999). Seymour, R. S., Diffusion pathway for oxygen into highly thermogenic florets of the arum lily Philodendron selloum. J. Exp. Bot., 52, 1465–1472 (2001). Seymour, R. S., and Schultze-Motel, P., Heat-producing flowers. Endeavour, 21, 125–129 (1997). Walker, D. B., Gysi, J., Sternberg, L., and DeNiro, M. J., Direct respiration of lipids during heat production in the inflorescence of Philodendron selloum. Science, 220, 419–421 (1983). Satoh, S., Iizuka, C., Kikuchi, A., Nakamura, N., and Fujii, T., Proteins and carbohydrates in xylem sap from squash root. Plant Cell Physiol., 33, 841–847 (1992). Schurr, U., and Schulze, E. D., The concentration of xylem sap constituents in root exudate, and in sap from intact, transpiring castor bean plants (Ricinus communis L.). Plant Cell Environ., 18, 409–420 (1995). White, M. C., Decker, A. M., and Chaney, R. L., Metal complexation in xylem fluid. Plant Physiol., 67, 292– 300 (1981). Ivanko, S., and Ingversen, J., Investigation on the assimilation of nitrogen by maize roots and the transport of some major nitrogen compounds by xylem sap. I. Nitrate and ammonia uptake and assimilation in the major nitrogen fractions of nitrogen-starved maize roots. Physiol. Plant., 24, 59–65 (1971). Ivanko, S., and Ingversen, J., Investigation on the assimilation of nitrogen by maize roots and the transport of some major nitrogen compounds by xylem sap. III. Transport of nitrogen compounds by xylem sap. Physiol. Plant., 24, 355–362 (1971). Pate, J. S., Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol. Biochem., 5, 109–119 (1973). Thornton, B., and Macduff, J. H., Short-term changes in xylem N compounds in Lolium perenne following defoliation. Ann. Bot., 89, 715–722 (2002). Hocking, P. J., The composition of phloem exudate and xylem sap from tree tobacco (Nicotiana glauca Grah.). Ann. Bot., 45, 633–643 (1980). Ito, K., Isolation of two distinct cold-inducible cDNAs encoding plant uncoupling proteins from the spadix of skunk cabbage (Symplocarpus foetidus). Plant Sci., 149,
Composition of Xylem Sap in Skunk Cabbage
35)
36) 37)
38)
167–173 (1999). Ito, K., Abe, Y., Johnston, S., and Seymour, R. S., Ubiquitous expression of a gene encoding for uncoupling protein isolated from the thermogenic inflorescence of the dead horse arum Helicodiceros muscivorus. J. Exp. Bot., 54, 1113–1114 (2003). Meeuse, B. J. D., Thermogenic respiration in aroids. Annu. Rev. Plant Physiol., 26, 117–126 (1975). Rhoads, D. M., and McIntosh, L., Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott). Proc. Natl. Acad. Sci. U.S.A., 88, 2122–2126 (1991). Ito, Y., Saisho, D., Nakazono, M., Tsutsumi, N., and Hirai, A., Transcript levels of tandem-arranged alternative oxidase genes in rice are increased by low
39)
40) 41)
42)
1161
temperature. Gene, 203, 121–129 (1997). Schnarrenberger, C., and Martin, W., Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. Eur. J. Biochem., 269, 868–883 (2002). Artus, N. N., and Edwards, G. E., NAD-malic enzyme from plants. FEBS Lett., 182, 225–233 (1985). Affourtit, C., Krab, K., and Moore, A. L., Control of plant mitochondrial respiration. Biochim. Biophys. Acta, 1504, 58–69 (2001). Siedow, J. M., and Day, D. A., Respiration and photorespiration. In ‘‘Biochemistry and Molecular Biology of Plants’’, eds. Buchanan, B. B., Gruissem, W., and Jones, R. L., American Society of Plant Physiologists, Maryland, pp. 679–687 (2000).
