Prolonged Survival of CAM-Mode Mesembryanthemum crystallinum in Darkness and its Possible Dependence on Malate

Transcription

1 Plant CellPhysiol. 29(1): (1988) JSPP 1988 Prolonged Survival of CAM-Mode Mesembryanthemum crystallinum in Darkness and its Possible Dependence on Malate Y. Sanada 1, K. Nishida 1-3 and G. Edwards 2 1 Botanical Institute, Faculty of Sciences, Kanazawa University, Kanazawa 920, Japan 2 Botany Department, Washington State University, Pullman, Washington , U.S.A. A remarkable difference was found in the survival of leaves of Mesembryanthemum crystallinum with plants grown in the C 3 versus the CAM mode. With excised leaves (petiole in solution) of C 3 -mode plants subjected to 6 days of darkness, there was a large reduction in the chlorophyll content of the leaf and leaf turgor had decreased. By day 9, the chlorophyll had disappeared, except at the major veins, and the leaf tip had dried and turned brown. In contrast, the leaf tissue in the CAM mode showed only a partial loss of chlorophyll during the same period, and even after 17 days of darkness, the tissue at the base was still alive. Similarly, intact plants grown in the C 3 mode deteriorated much faster during 20 days of darkness than did plants grown in the CAM mode. Chlorophyll content, chlorophyll a/b ratio, phosphoenolpyruvate carboxylase, NADP-malic enzyme, malate and starch content were measured. In both C 3 - and CAM-mode plants, the starch content decreased rapidly during the dark period and was nearly depleted after two days. In the CAM-mode tissue, there was a relatively high level of malate during prolonged darkness (up to 17 days), with a transitory rise early in the dark period. In contrast, the malate content was low and rapidly depleted in the C 3 -mode leaves kept in darkness. These findings suggest that malate may be an important source of carbon for sustaining leaves of CAM-mode M. crystallinum during prolonged darkness. Key words: Crassulacean acid metabolism Malate Mesembryanthemum crystallinum The CAM mode of photosynthesis can be induced in darkness has not been examined. Mesembryanthemum crystallinum by the addition of a In the present study, the malate, starch and Chi conhigh concentration of salt (0.4 M NaCl) to the nutrient tents and the activities of PEP carboxylase and NADP medium of plants growing in the C 3 mode (Winter and malic enzyme were measured in leaves of C 3 - and CAM-M. Willert 1972, Winter and Liittge 1976). Thus, this species crystallinum in darkness for up to 20 days. The results is very useful for comparative studies of the physiology and suggest that malate plays an important role in maintaining biochemistry of C 3 and CAM plants (e.g. Holtum and leaf tissue of CAM-M. crystallinum in prolonged darkness. Winter 1982, Winter et al. 1982, Winter 1985). We have found that whenm. crystallinum plants grow-.,,.,.»,... _,,, t, t /,, Materials and Methods ing in each of these two separate modes are placed m darkness, those in the CAM mode survive markedly longer Growth of plants Seeds of Mesembryanthemum than those in the C 3 mode. Vickery (1952) measured the crystallinum L. were germinated in a mixture of soil and rise and fall of starch, malate and other compounds in sand in a growth cabinet. They were grown under a 12-h leaves of the CAM plant Bryophyllum calycinum during light period (light intensity 400 fimol quantam~ 2 s~' be- 8 days of darkness. However, the question of how CAM tween nm at soil level) at 23 C and a 12-h dark leaf tissue survives longer than C 3 leaf tissue in prolonged period at 16 C at 50-60% relative humidity. Plants were watered once every two days with half-strength Hoag- Abbreviations: CAM, Crassulacean acid metabolism; PEPC, land'nutrient solution. For induction of CAM, NaCl was phosphoenolpyruvate carboxylase. added to the nutrient medium. After about 6 weeks, 3 To whom requests for reprints should be addressed. nutrient medium containing 50 nm NaCl was given to the 117

2 118 Y. Sanada, K. Nishida and G. Edwards plants. This was increased to 400 nim over approximately one week, by which time the extractable activity of PEPC had increased and reached its maximum. Following the induction of CAM in some of the plants, leaf tissues of plants in the two photosynthetic modes were placed in prolonged darkness (following the normal light period) at 23 C under two sets of conditions. In one set of experiments, the second and third leaves were excised with a knife where the petiole joins the stem. The petioles of C 3 -mode leaves were immersed in half-strength Hoagland'nutrient solution, while the petioles of CAMmode leaves were immersed in half-strength Hoagland's solution containing 0.4 M NaCl. These tissues were used for the experiments described below (test for Chi composition, enzyme activities, malate and starch contents) in order to avoid potential effects from translocation in intact plants. In the other set of experiments, intact plants grown in the two modes were placed in prolonged darkness and visual observations were made of the leaves. Enzyme extraction Leaves of M. crystallinum were cut into slices about 5 mm thick with razor blade, and about 2 g fresh weight of tissue was ground for 60 s with a mortar and pestle with 4 ml of grinding medium. The medium contained 0.15 M Tris-HCl (ph7.6), lmm EDTA, 0.5% Na-ascorbate, 5% polyvinylpyrrolidone and 30 mm 2-mercaptoethanol. The homogenate was quickly filtered through two layers of gauze, and a sample was used for the determination of Chi. The crude extract was centrifuged at 10,000 xg for lomin, and then desalted by passage through a Sephadex G-25 column. The eluate was used for enzyme assay. The above procedures were carried out at 4 C. Enzyme assays The enzymes were assayed at 25 C by following the change in absorbance of a pyridine nucleotide at 340 nm in 3.0 ml of reaction medium, as described below. In each case, the reaction was initiated by the compound added last. PEPC: 0.05 M Tris-HCl (ph 8.0), 0.1 mm EDTA, 10 mm MgCl 2, 10 mm NaHCO 3, 0.1 mm NADH, 150fi\ of enzyme extract and 3 mm PEP. The assay was linked to endogenous NAD-malate dehydrogenase. NADP-malic enzyme: 0.05 M Tris-HCl (ph8.0), 0.1 mm EDTA, 5 mm 2-mercaptoethanol, 0.5 mm NADP, 20 mm MgCl 2, 150 ^/l of enzyme extract and 10 mm Namalate. Malate and starch determination The leaf for assay was collected on a given day during the dark treatment and cut with a knife into two sections along the midrib. One section was used for measuring starch content and the other for measuring malate content. After measurement of the fresh weight, the leaf section for measurement of malate content was ground for 2min with a mortar and pestle after the addition of 5 ml of boiling water. The homogenate was centrifuged for lomin at 10,000xg. This process was repeated twice to ensure complete extraction of malate. The supernatants were pooled and used for determining the amount of L-malate by the method of Hohorst (1970). The other leaf section, used for measurement of starch content, was ground with a mortar and pestle for 2 min with 5 ml of 80% ethanol. The homogenate was centrifuged for 10 min at 10,000 x g. After removal of the supernatant, the pellet was extracted twice with 80% ethanol to ensure complete removal of soluble sugars. The starch content in the pellet was assayed using an enzyme kit purchased from Boehringer (Mannheim, FRG). Chlorophylls-Chl was measured by the method of Arnon (1949) and the Chi a/b ratio was calculated. Results Visual observations Fig. 1 shows photographs of excised leaves of C 3 - and CAM-mode M. crystallinum kept in the dark for various periods of time. By day 6, the Chi content was remarkably reduced in the C 3 plants and the leaf turgor had decreased, although this latter fact is not apparent in the photograph. By day 9, most of the Chi had disappeared from the leaf, although some was retained around the major veins. At the margins of the leaf, particularly at the tip, the tissue was dry and dead. By day 11, the leaf was completely dead. In contrast, after 6 to 9 days in the dark, the CAM-mode leaf appeared healthy, although some loss of Chi was apparent. By day 11, the leaf exhibited a condition similar to that of the C 3 -mode leaf after only 6 days. Even after 17 days, the basal portion of the CAM-mode leaf retained water and appeared viable. Similar observations were made of intact plants: plants in the CAM mode survived longer than those in the C 3 mode. Interestingly, with the plants in the C 3 mode, the lower leaves senesced and became very thin sooner than the younger leaves. After 20 days of darkness, the second and third leaves of intact plants became dark grey, whereas the excised leaves in the C 3 mode (Fig. 1) became yellow during senescence. The intact plants in the CAM mode retained water, and appeared viable after 20 days of darkness even though the leaves had lost dark-green color. Similar results were obtained when entire flats of plants growing in the C 3 or the CAM mode were placed in the dark (data not shown). Chi content The Chi contents and Chi a/b ratios in excised leaves of dark-treated plants of C 3 - and CAMmode M. crystallinum are shown in Fig. 2. With tissue in the C 3 mode, the Chi content was severely depleted after 9 days. In contrast, about half of the Chi of tissue in the CAM mode was still retained after 9 days. After 14 days the CAM-mode tissue had lost much of its Chi. In both the C 3 and the CAM tissue, the Chi a/b ratio remained constant till day 4, after which the ratio decreased, especially

3 Dark effect on C r and CAM-mode Mesembryanthemum 119 Days in dark CAM Fig. 1 Photographs of excised leaves of C 3 - and CAM-mode M. crystallinum which were placed in the dark for the number of days indicated.

4 120 Y. Sanada, K. Nishida and G. Edwards L i i CAM Total chl > ^ r a/b Chl a/b Days in dark Fig. 2 Changes in Chl content and Chl a/b ratio after excised leaves of C 3 - and CAM-mode M. crystallinum were placed in the dark. c wt/m * moles \ > CAM ^ \ O in the C 3 tissue. PEPC and NADP-malic enzyme activities As expected, the activity of PEPC was much higher in the CAM leaves at the end of the light period than in the C 3 -leaf tissue. During the prolonged treatment in the dark, the activity decreased rapidly, and after 14 days the PEPC activity was very low on a fresh weight basis (Fig. 3). The activity of NADP-malic enzyme was lower than that of PEPC, and it remained constant on a fresh weight basis during the dark treatment. In C 3 leaf tissue, the activities of PEPC and NADP-malic enzyme were quite low, and after one day of darkness the activity was negligible compared to that in tissue in the CAM mode. Starch and malate contents Fig. 4 shows that the starch content of the leaves in the light was nearly the same in both C 3 - and CAM-mode M. crystallinum. When leaves were placed in the dark, the starch content decreased rapidly during the first 24 h; after 6 days most of the starch had disappeared in both photosynthetic types Days in dark Days in dark Fig. 3 Changes in PEPC and NADP-malic enzyme activities after excised leaves of C 3 - and CAM-mode M. crystallinum were Fig. 4 Changes in starch and malate contents in excised leaves placed in the dark. of C 3 - and CAM-mode M. crystallinum in the dark.

5 Dark effect on C 3 - and CAM-mode Mesembryanthemum 121 Prior to the dark period, the malate content in the CAM leaf was higher than that in the C 3 (Fig. 5). During the dark period, there was a rapid transient increase in malate in the CAM leaf, which reached a peak after 24 h. The malate content then decreased rapidly until day 6, and more gradually thereafter. Even after 17 days, a significant level of malate remained in the leaf. The malate content in the C 3 leaf was quite low at the end of the light period, and was largely depleted during the first few days of darkness. Discussion In the present study, excised leaves and potted plants of CAM-Af. crystallinum survived longer in the dark than did those of CyM. crystallinum. In prolonged darkness, there was a more rapid loss of Chi and decrease in the Chi a/b ratio in the C 3 than in the CAM tissue, both of which are indicators of senescence. The loss of Chi in the dark or during senescence has been well studied in C 3 plants (e.g. Chichester and Nakayama 1965, Thimann et al. 1977, Grover et al. 1986). A decrease in the Chi a/b ratio is common in older leaves (Sestak 1985) and during senescence, although it remains constant during short periods in the dark (Grover et al. 1986). The far red-absorbing forms of Chi a were particularly sensitive and were lost during senescence of wheat leaves (Grover et al. 1986). The activity of PEPC was high in the CAM leaf at the end of the light period, but it decreased markedly during prolonged darkness. Winter (1980) found that the activity of this enzyme decreased substantially during 4 days of darkness. Further studies are required to determine whether this loss of activity is associated with a loss in protein or its conversion to an inactive form. PEPC has been found to undergo some changes in vivo during day/night cycles, since it is phosphorylated in the dark and dephosphorylated in the light in the CAM plant Bryophyllum fedtschenkoi (Nimmo et al. 1986) and undergoes interconversion of oligomeric forms in Crassula argenta (Wu and Wedding 1985). In contrast to PEPC, the activity of NADP-malic enzyme in the CAM tissue remained constant on a fresh weight basis during the dark treatments. In the present study with M. crystallinum kept in prolonged darkness, stach was rapidly depleted, and in the CAM tissue malate initially increased and then decreased. Vickery (1952) found that leaves of Bryophyllum calycinum accumulated starch and lost organic acids (including malic acid) during prolonged darkness. This discrepancy may be due to the time of leaf sampling. In the present study, leaves were sampled well into the light period, and the initial changes in the dark (starch depletion and malate accumulation) are well-known features of CAM (Kluge and Ting 1978). Vickery sampled leaves at daybreak, when the plants were just entering the light period. Thus, the changes (starch accumulation and malate depletion) in the tissue in the dark may have mimicked those expected for the normal light period. The differences in the changes in malate and starch contents during darkness in the C 3 and CAM tissue of M. crystallinum are of particular interest. After 24 h the starch was largely depleted and the malate content had peaked in the CAM tissue. Although there would be some net carbon gain by the CAM tissue in the dark due to CO 2 fixation into malate, this in itself would not change the energy content of the tissue, since NADH is required to synthesize malate from oxaloacetate and NAD(P)H would be regenerated as the malate is utilized through malic enzyme. Subsequently, malate was metabolized rapidly for up to 6 days in the dark. During this period, the leaf tissue retained a substantial amount of Chi and appeared reasonably healthy. In contrast, the rapid depletion of both starch and malate contents in the C 3 leaves in darkness suggests the reserves had already been depleted, and thus the leaf senesced more rapidly. During carbon assimilation in CAM plants in the dark, starch provides 3C precursors through glycolysis for PEP carboxylase. Thus, once the starch is depleted, there is presumably no further potential for net synthesis of malate. After malate reaches a maximum level, its decline and rate of utilization is much slower than the initial loss of starch. This may be because the plant is normally "programmed" to degrade starch and accumulate malate in the dark. The longer survival of the CAM tissue in the dark may be due both to the conversion of starch to malate and to the slower rate of degradation of malate. Senescence of the leaf tissue in the dark is probably the result of depletion of carbon reserves necessary for maintenance respiration. Malate may serve as a source of carbon for biochemical functions of the tissue in prolonged darkness. In addition to its much higher level in the CAM tissue, malate may also be a better source of carbon for dark respiration than starch. The TCA cycle can't function effectively with only 3C precursors (e.g. those from starch) as the carbon source if metabolites are removed from the cycle (utilization of acetyl Co A depends on regeneration of oxaloacetate), whereas malate can provide both 4C and 3C precursors (the latter through NADP or NAD-malic enzyme). This work was supported by the U.S.-Japan Cooperative Research Program (The Japan Society for the Promotion of Science, NSF Grant Int ). References Arnon, D. I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: Chichester, C. O. and Nakayama, T. O. (1965) Pigment changes

6 122 Y. Sanada, K. Nishida and G. Edwards in senescent and stored tissue. In Chemistry and Biochemistry of Plant Pigments. Edited by Goodwin, T. W. pp Academic Press, London and New York. Grover, A., Sabat, S. C. and Mohanty, P. (1986) Relative sensitivity of various spectral forms of photosynthetic pigments to leaf senescence in wheat (Triticum aestivum L.). Photosynthesis Res. 10: Hohorst, H. J. (1970) L-(-)-Malat. In Methoden der enzymatischen Analyse. Bd. 2. Edited by Bergmeyer, H. U. pp Verlag, Chemie, Weinheim. Holtum, J. A. M. and Winter, K. (1982) Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L. Planta 155: Kluge, M. and Ting, I. P. (1978) Crassulacean Acid Metabolism. pp Springer-Verlag, Berlin. Nimmo, G. A., Nimmo, H. G., Hamilton, I. D., Fewson, C. A. and Wilkins, M. B. (1986) Purification of the phosphorylated night form and dephosphorylated day form of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi. Biochem. J. 239: Sestak, Z. (1985) Photosynthesis during Leaf Development, pp Dr. W. Junk, Dordrecht. Thimann, K. V., Tetley, R. M. and Krivak, B.M. (1977) Metabolism of oat leaves during senescence. V. Senescence in light. Plant Physiol. 59: Vickery, H. B. (1952) The formation of starch in leaves of Bryophyllum calycinum cultured in darkness. Plant Physiol. 27: Winter, K. (1980) Day/night changes in the sensitivity of phosphoenolpyruvate carboxylase to malate during Crassulacean acid metabolism. Plant Physiol. 65: Winter, K. (1985) Crassulacean acid metabolism. In Photosynthetic Mechanisms and the Environment. Edited by Barber, J. and Baker, N. R. pp Elsevier, Amsterdam. Winter, K., Foster, J. G., Edwards, G. E. and Holtum, J. A. M. (1982) Intracellular localization of enzymes of carbon metabolism in Mesembryanthemum crystallinum exhibiting C 3 photosynthetic characteristics or performing Crassulacean acid metabolism. Plant Physiol. 69: Winter, K. and Luttge, U. (1976) Balance between C 3 and CAM pathway of photosynthesis. In Water and Plant Life, Ecological Studies. Vol. 19. Edited by Lange, O. L., Kappen, L. and Schulze, E. D. pp Springer-Verlag, Berlin. Winter, K. and von Willert, D. J. (1972) NaCl-induzierter Crassulacean-saurestoffwechsel bei Mesembryanthemum crystallinum. Z. Pflanzenphysiol. 67: Wu, M-X. and Wedding, R. T. (1985) Regulation of phosphoenolpyruvate carboxylase from Crassula by interconversion of oligomeric forms. Arch. Biochem. Biophys. 240: (Received May 20, 1987; Accepted October 23, 1987)