Dieter Joachim von Willert

NaCl-induzierter crassulaceensäurestoffwechsel bei Mesembryanthemum crystallinum

Summary Mesembryanthemum crystallinum grown under saline conditions in NaCl medium shows the typical gas exchange reactions known from CAM plants, exhibiting a clear CO 2 uptake in the dark. This is accompanied by an increase of malate content in the leaves. Neither CO 2 uptake in the dark nor increase in malate content is found in plants grown in the absence of NaCl. These results, suggesting a drastic metabolic change caused by NaCl, are discussed.

Der einfluß von NaCl auf die atmung und aktivität der malatdehydrogenase bei einigen halophyten und glykophyten

This paper reports the effects of sodium chloride on the in vitro activity of malate dehydrogenase and on the specific activity of malate dehydrogenase isolated from various halophytes and glycophytes grown under saline and non-saline conditions. The influence of substrate salinity on respiration and dark CO2 fixation was also studied. The species used were the halophytesAster tripolium, Atriplex spongiosa, Mesembryanthemum crystallinum, Plantago maritima, Salicornia brachystachya, andSpergularia salina, and the glycophytesAster amellus, Plantago major, andSpergularia rubra.When added to the test solutions, sodium chloride stimulates the activity of malate dehydrogenase. Further addition of sodium chloride results in rapid decline of activity at salt concentrations which vary with the species. Malate dehydrogenase isolated from both glycophytes and halophytes do not show any significant difference in their response to sodium chloride, indicating salt tolerance. Enzyme isolated from halophytes grown under saline conditions is as sensitive to sodium chloride as enzymes isolated from the same plants grown in the absence of additional NaCl. A double reciprocal plot shows a competitive interaction between sodium chloride and oxaloacetate. The addition of sucrose during the in-vitro assay of malate dehydrogenase results in a stronger inhibition than that caused by isosmotic NaCl solutions.Enzymes extracted from seedlings of halophytes and glycophytes grown at various levels of sodium chloride showed important changes in specific activity of malate dehydrogenase (tested without additional sodium chloride in an in-vitro assay). In glycophytes, specific activity decreases with increasing substrate salinity, whereas in halophytes specific activity first increases, to a symptote and finally decreases. A double reciprocal plot shows a non-competitive interaction between sodium chloride and oxaloacetate indicating that the ratio of malate dehydrogenase to total extractable protein is altered by sodium chloride. The respiration of these seedlings runs parallels the specific activity of the malate dehydrogenase whereas CO2 dark fixation declines with increasing substrate salinity.The results are discussed with respect to compensatory reactions rather than compartmentalisation due to the addition of sodium chloride.This paper reports the effects of sodium chloride on the in vitro activity of malate dehydrogenase and on the specific activity of malate dehydrogenase isolated from various halophytes and glycophytes grown under saline and non-saline conditions. The influence of substrate salinity on respiration and dark CO2 fixation was also studied. The species used were the halophytesAster tripolium, Atriplex spongiosa, Mesembryanthemum crystallinum, Plantago maritima, Salicornia brachystachya, andSpergularia salina, and the glycophytesAster amellus, Plantago major, andSpergularia rubra.When added to the test solutions, sodium chloride stimulates the activity of malate dehydrogenase. Further addition of sodium chloride results in rapid decline of activity at salt concentrations which vary with the species. Malate dehydrogenase isolated from both glycophytes and halophytes do not show any significant difference in their response to sodium chloride, indicating salt tolerance. Enzyme isolated from halophytes grown under saline conditions is as sensitive to sodium chloride as enzymes isolated from the same plants grown in the absence of additional NaCl. A double reciprocal plot shows a competitive interaction between sodium chloride and oxaloacetate. The addition of sucrose during the in-vitro assay of malate dehydrogenase results in a stronger inhibition than that caused by isosmotic NaCl solutions.Enzymes extracted from seedlings of halophytes and glycophytes grown at various levels of sodium chloride showed important changes in specific activity of malate dehydrogenase (tested without additional sodium chloride in an in-vitro assay). In glycophytes, specific activity decreases with increasing substrate salinity, whereas in halophytes specific activity first increases, to a symptote and finally decreases. A double reciprocal plot shows a non-competitive interaction between sodium chloride and oxaloacetate indicating that the ratio of malate dehydrogenase to total extractable protein is altered by sodium chloride. The respiration of these seedlings runs parallels the specific activity of the malate dehydrogenase whereas CO2 dark fixation declines with increasing substrate salinity.The results are discussed with respect to compensatory reactions rather than compartmentalisation due to the addition of sodium chloride.SummaryThis paper reports the effects of sodium chloride on the in vitro activity of malate dehydrogenase and on the specific activity of malate dehydrogenase isolated from various halophytes and glycophytes grown under saline and non-saline conditions. The influence of substrate salinity on respiration and dark CO2 fixation was also studied. The species used were the halophytesAster tripolium, Atriplex spongiosa, Mesembryanthemum crystallinum, Plantago maritima, Salicornia brachystachya, andSpergularia salina, and the glycophytesAster amellus, Plantago major, andSpergularia rubra.When added to the test solutions, sodium chloride stimulates the activity of malate dehydrogenase. Further addition of sodium chloride results in rapid decline of activity at salt concentrations which vary with the species. Malate dehydrogenase isolated from both glycophytes and halophytes do not show any significant difference in their response to sodium chloride, indicating salt tolerance. Enzyme isolated from halophytes grown under saline conditions is as sensitive to sodium chloride as enzymes isolated from the same plants grown in the absence of additional NaCl. A double reciprocal plot shows a competitive interaction between sodium chloride and oxaloacetate. The addition of sucrose during the in-vitro assay of malate dehydrogenase results in a stronger inhibition than that caused by isosmotic NaCl solutions.Enzymes extracted from seedlings of halophytes and glycophytes grown at various levels of sodium chloride showed important changes in specific activity of malate dehydrogenase (tested without additional sodium chloride in an in-vitro assay). In glycophytes, specific activity decreases with increasing substrate salinity, whereas in halophytes specific activity first increases, to a symptote and finally decreases. A double reciprocal plot shows a non-competitive interaction between sodium chloride and oxaloacetate indicating that the ratio of malate dehydrogenase to total extractable protein is altered by sodium chloride. The respiration of these seedlings runs parallels the specific activity of the malate dehydrogenase whereas CO2 dark fixation declines with increasing substrate salinity.The results are discussed with respect to compensatory reactions rather than compartmentalisation due to the addition of sodium chloride.

Feinstruktur und Crassulaceen-Säurestoffwechsel in Blättern von Mesembryanthemum crystallinum während natürlicher und NaCl-induzierter Alterung

SummaryNaCl-treated young plants of Mesembryanthemum crystallinum undergo specific cytological changes which are accompanied by distinct alterations in CO2 gas exchange reactions. Prior to salt treatment mesophyll cells are quite normal. Only lomasome-like structures extrude from the cytoplasm into the vacuole. CO2 gas exchange is that of a Calvin-plant. Later on NaCl-treated plants differ from control plants in several respects. They show the CO2 gas exchange reactions typical for CAM-plants. Electron microscopy clearly shows the formation of new vacuole-like spaces just under the chloroplasts between plasmalemma and cell wall, which are mostly filled with structures similar to “Hechtsche Fäden” of plasmolyzed cells. In many cases a large amount of vesicles and membranes can be observed in the central vacuole, at least some of which are in connection with cytoplasm and even chloroplasts. Chloroplasts themselves sometimes seem to be damaged by high concentrations of NaCl. While having a more distinct crassulacean acid metabolism, old NaCl-treated plants exhibit features of aged cells: cytoplasm becomes empty and vacuolized, stoma thylacoids are reduced.Not only the described changes in fine structure but also the altered CO2 gas exchange reactions take place when glycophytic control plants grow older, thus indicating that NaCl may accelerate the normal ageing process in Mesembryanthemum crystallinum.

Die Bedeutung des anorganischen Phosphats für die Regulation der Phosphoenolpyruvat Carboxylase von Mesembryanthemum crystallinum L.

The activity of the phosphoenolpyruvate (PEP) carboxylase isolated from non-saline grown plants of the salt-tolerant plant Mesembryanthemum crystallinum is strongly inhibited by malate. This inhibition was found to depend on the pH (rising with increasing H(+)-concentrations) and on the concentration of malate used. The addition of inorganic phosphate (appropriate concentration 30 mM) to the in-vitro enzyme assay prior to malate addition results in a remarkable compensation of malate-caused inhibition of the enzyme activity. Again a dependency upon the pH can be observed. The ability of inorganic phosphate to restore malate-caused inhibition of the PEP-carboxylase increases with increasing pH.Another potent inhibitor of the PEP-carboxylase is NaCl, which shows a minimum inhibition at pH 7. At this pH a concentration of more than 60 mM NaCl is needed to reduce the activity of the enzyme below the control level with a 50% inhibition is reached at 150 mM. If the addition of NaCl is performed in the presence of 30 mM inorganic phosphate the inhibition is less pronounced. The enzyme now tolerates about 100 mM higher concentrations of NaCl without being inhibited.NaCl-treatment of Mesembryanthemum crystallinum plants results in an increase of inorganic phosphate in the cells with a concomitant establishment of a Crassulacean acid metabolism. The present results support evidence for a protective function of inorganic phosphate (compensation of NaCl-induced enzyme inhibition), possibly a commen reaction involved in the question of salt tolerance, and a more specific function (restriction of malatecaused inhibition of the PEP-carboxylase) providing the enzymatic background for the malate accumulation in Mesembryanthemum crystallinum.The activity of the phosphoenolpyruvate (PEP) carboxylase isolated from non-saline grown plants of the salt-tolerant plant Mesembryanthemum crystallinum is strongly inhibited by malate. This inhibition was found to depend on the pH (rising with increasing H(+)-concentrations) and on the concentration of malate used. The addition of inorganic phosphate (appropriate concentration 30 mM) to the in-vitro enzyme assay prior to malate addition results in a remarkable compensation of malate-caused inhibition of the enzyme activity. Again a dependency upon the pH can be observed. The ability of inorganic phosphate to restore malate-caused inhibition of the PEP-carboxylase increases with increasing pH.Another potent inhibitor of the PEP-carboxylase is NaCl, which shows a minimum inhibition at pH 7. At this pH a concentration of more than 60 mM NaCl is needed to reduce the activity of the enzyme below the control level with a 50% inhibition is reached at 150 mM. If the addition of NaCl is performed in the presence of 30 mM inorganic phosphate the inhibition is less pronounced. The enzyme now tolerates about 100 mM higher concentrations of NaCl without being inhibited.NaCl-treatment of Mesembryanthemum crystallinum plants results in an increase of inorganic phosphate in the cells with a concomitant establishment of a Crassulacean acid metabolism. The present results support evidence for a protective function of inorganic phosphate (compensation of NaCl-induced enzyme inhibition), possibly a commen reaction involved in the question of salt tolerance, and a more specific function (restriction of malatecaused inhibition of the PEP-carboxylase) providing the enzymatic background for the malate accumulation in Mesembryanthemum crystallinum.SummaryThe activity of the phosphoenolpyruvate (PEP) carboxylase isolated from non-saline grown plants of the salt-tolerant plant Mesembryanthemum crystallinum is strongly inhibited by malate. This inhibition was found to depend on the pH (rising with increasing H+-concentrations) and on the concentration of malate used. The addition of inorganic phosphate (appropriate concentration 30 mM) to the in-vitro enzyme assay prior to malate addition results in a remarkable compensation of malate-caused inhibition of the enzyme activity. Again a dependency upon the pH can be observed. The ability of inorganic phosphate to restore malate-caused inhibition of the PEP-carboxylase increases with increasing pH.Another potent inhibitor of the PEP-carboxylase is NaCl, which shows a minimum inhibition at pH 7. At this pH a concentration of more than 60 mM NaCl is needed to reduce the activity of the enzyme below the control level with a 50% inhibition is reached at 150 mM. If the addition of NaCl is performed in the presence of 30 mM inorganic phosphate the inhibition is less pronounced. The enzyme now tolerates about 100 mM higher concentrations of NaCl without being inhibited.NaCl-treatment of Mesembryanthemum crystallinum plants results in an increase of inorganic phosphate in the cells with a concomitant establishment of a Crassulacean acid metabolism. The present results support evidence for a protective function of inorganic phosphate (compensation of NaCl-induced enzyme inhibition), possibly a commen reaction involved in the question of salt tolerance, and a more specific function (restriction of malatecaused inhibition of the PEP-carboxylase) providing the enzymatic background for the malate accumulation in Mesembryanthemum crystallinum.

Einfluß von NaCl und blattalter auf die CO2-dunkelfixierung bei halophyten — Vergleichende untersuchung bei Mesembryanthemum crystallinum und Atriplex spongiosa

Summary Species used in this investigation were Atriplex spongiosa and Mesembryanthemum crystallinum. Mesembryanthemum-plants having 3 successive leaf pairs (numbered with ascending insertion) were treated with 150 and 300 mM NaCl-solutions, respectively, while Atriplex-plants having 7 successive leaves and branches originated in the axils of the leaves were only treated with 300 mM NaCl. During the following 20–30 days the increase of NaCl in the leaves and the incorporation of 14CO2 in the dark was determined. The experiment showed that the middle leaves, before they had reached their approximate final size, had the highest rates of 14CO2-incorporation in the dark. In the course of the experiment the, optimal CO2 dark fixation rate shifted towards the tip of the plants. This was the case in treated and untreated Mesembryanthemum-plants and in untreated Atriplex plants. Leaves of treated Atriplex plants wilted and were killed within 20 days of NaCl-treatment. In contrast, leaves of the branches were not affected by NaCl. During the first days of NaCl-treatment 14CO2 fixation declined. The lower CO2 incorporation of the branches of Atriplex was not restored as was the case in Mesembryanthemum-leaves. Here the fixation rate of the treated plants reached the level of the untreated ones within 13 days. Thereafter a drastic increase in 14CO2 dark fixation occurred. At that time plants exhibit a Crassulacean acid metabolism, as was reported earlier. The lag phase between the beginning of the NaCl treatment and the first appearence of increased dark fixation is not affected by the concentration of NaCl in the growing solution. But leaf age and NaCl-concentration influence the magnitude of dark fixation. The flexibility and possibility of adaptation to changed environment conditions decreases with increasing leaf age. The first leaf pair of Mesembryanthemum was killed by NaCl, the increase in dark fixation of the second leaf pair was half of that exhibited by the third leaf pair. The results are discussed on a morphogenetic and oecophysiological basis.

Sukkulenten und ihr Überleben in Wüsten

Succulents grow preferentially in such arid areas where periodical rainfall prevails and rainless periods are not too extended. It is the benefit of succulence and not of the mode of photosynthesis which allows a positive carbon balance during a period of desiccation. Nocturnal CO2 uptake and overnight malate accumulation depend mainly on water availability in the soil and atmospheric humidity. For the dominating CAM-succulents in the Southern Namib nocturnal malate accumulation seems to be directly linked to an improvement of the water status of the plant. S ukkulenten k6nnen wenige Millimeter klein sein (z.B. Conophytum comptonii), sie k6nnen aber auch 15 m und mehr hoch werden (z.B. Cereus giganteus). Es gibt Blattsukkulenten (z.B. Crassulaceae, Mesembryanthemaceae, Zygophyllaceae), Stammsukkulenten (z.B. Asclepiadaceae, Cactaceae, Euphorbiaceae) und solche, die sukkulente Stfimme und B1/itter haben (z.B. Asteraceae, Crassulaceae, Portulacaceae). Sukkulenten kommen in unterschiedlichen Wuchsformen vor. Es gibt annuelle und ausdauernde Formen. Die ausdauernden Sukkulenten k6nnen immergrfin, aber auch wechselgriin sein. Die Standorte, die Sukkulenten besiedeln, reichen von Spalten im nackten Fels fiber steinigen Untergrund bis zu Sand oder Schluff, der Wurzelraum kann basisch, sauer oder salzhaltig sein. Es gibt also nicht die Sukkulente, sondern es gibt zahlreiche, sehr verschiedene sukkulente Pflanzen. Eine Auswahl verschiedener Lebensformen zeigt Fig. 1. Gemeinsam ist ihnen nur die Sukkulenz.

Der Einfluß von Verapamil auf die nächtliche Malatakkumulation bei Bryophyllum tubiflorum

Excised phyllodia of Bryophyllum tubiflorum standing in phosphate buffer to which verapamil is added undergo specific alterations in their gaseous exchange reactions as well as in the amount of malate accumulated in the dark period. Inhibition of accumulation of malate increases with increasing concentrations of verapamil. The effects caused by verapamil are reversible. During the dark period plasmalemma under the chloroplasts detaches itself from the cell wall, forming a vacuole-like space between plasmalemma and cell wall which disappears in the following light period. In the presence of verapamil this formation of a vacuole-like structure does not occur.Verapamil is thought to influence the permeability of plasma membranes, thus decreasing the capacity of the vacuole to accumulate malate in the dark. Our interpretation of the results seems to demonstrate that the occurence of crassulacean acid metabolism is dependent on the integrity of the plasma membranes. Further details are discussed.Excised phyllodia of Bryophyllum tubiflorum standing in phosphate buffer to which verapamil is added undergo specific alterations in their gaseous exchange reactions as well as in the amount of malate accumulated in the dark period. Inhibition of accumulation of malate increases with increasing concentrations of verapamil. The effects caused by verapamil are reversible. During the dark period plasmalemma under the chloroplasts detaches itself from the cell wall, forming a vacuole-like space between plasmalemma and cell wall which disappears in the following light period. In the presence of verapamil this formation of a vacuole-like structure does not occur.Verapamil is thought to influence the permeability of plasma membranes, thus decreasing the capacity of the vacuole to accumulate malate in the dark. Our interpretation of the results seems to demonstrate that the occurence of crassulacean acid metabolism is dependent on the integrity of the plasma membranes. Further details are discussed.SummaryExcised phyllodia of Bryophyllum tubiflorum standing in phosphate buffer to which verapamil is added undergo specific alterations in their gaseous exchange reactions as well as in the amount of malate accumulated in the dark period. Inhibition of accumulation of malate increases with increasing concentrations of verapamil. The effects caused by verapamil are reversible. During the dark period plasmalemma under the chloroplasts detaches itself from the cell wall, forming a vacuole-like space between plasmalemma and cell wall which disappears in the following light period. In the presence of verapamil this formation of a vacuole-like structure does not occur.Verapamil is thought to influence the permeability of plasma membranes, thus decreasing the capacity of the vacuole to accumulate malate in the dark. Our interpretation of the results seems to demonstrate that the occurence of crassulacean acid metabolism is dependent on the integrity of the plasma membranes. Further details are discussed.

Der einfluß von NaCl auf den Vorgang der nächtlichen Malatanhäufung in Blattstreifen des Halophyten Mesembryanthemum crystallinum

Summary NaCl-treated Mesembryanthemum plants exhibit the characteristics of a Crassulacean acid metabolism (CAM). Leaf slices taken from these plants show a pronounced accumulation of malate in aqueous solution in the dark with a maximum at pH 6.5. The activity of the Phosphoenolpyruvate (PEP) carboxylase — the main enzyme envolved in dark CO 2 -fixation — follows the curve of the malate formation in leaf slices. In contrast, plants grown under non-saline conditions show no significant alteration in malate content during the night. However, if leaf slices from these plants are incubated in solution they can synthesize and store malate showing a maximum accumulation at pH 8. The same dependency on the pH is found for the activity of the PEP-carboxylase in untreated plants. These results indicate that different substrates are involved in the process of dark acidification, CO 2 being the substrate in NaCl-treated and bicarbonate in untreated plants. When leaf slices of untreated plants are subjected to increasing concentrations of NaCl in the solution total malate accumulation increases and maximum accumulation is shifted from pH 8 to pH 7. This alteration in the accumulation pattern is not accompanied by a change in the response of the activity of the PEP-carboxylase to pH indicating that specific ionic effects may be involved in this shifting. In an in vitro assay the activity of the PEP carboxylase is strongly inhibited by malate. If prior to the addition of malate, NaCl is added to the assay, malate caused inhibition is restricted. This compensatory effect of NaCl in concentrations not inhibitory to the enzyme (up to 70 mM) is found only at pH 7. Thus, NaCl not only interacts with the substrate but also with a potent inhibitor of the PEP carboxylase. Since Mesembryanthemum crystallinum is a halophilic CAM plant, the latter reaction may be of great adaptive significance. The presented results are discussed in terms of the question wether or not results obtained from enzymatic in vitro assay reflect what happens in the living cell.

Feinstruktur und Crassulaceen-Säurestoffwechsel in Blättern von Mesembryanthemum crystallinum während natürlicher und NaCl-induzierter Alterung@@@Ultrastructure and crassulacean acid metabolism in Mesembryanthemum crystallinum leaves during normal and NaCl-induced ageing

SummaryNaCl-treated young plants of Mesembryanthemum crystallinum undergo specific cytological changes which are accompanied by distinct alterations in CO2 gas exchange reactions. Prior to salt treatment mesophyll cells are quite normal. Only lomasome-like structures extrude from the cytoplasm into the vacuole. CO2 gas exchange is that of a Calvin-plant. Later on NaCl-treated plants differ from control plants in several respects. They show the CO2 gas exchange reactions typical for CAM-plants. Electron microscopy clearly shows the formation of new vacuole-like spaces just under the chloroplasts between plasmalemma and cell wall, which are mostly filled with structures similar to “Hechtsche Fäden” of plasmolyzed cells. In many cases a large amount of vesicles and membranes can be observed in the central vacuole, at least some of which are in connection with cytoplasm and even chloroplasts. Chloroplasts themselves sometimes seem to be damaged by high concentrations of NaCl. While having a more distinct crassulacean acid metabolism, old NaCl-treated plants exhibit features of aged cells: cytoplasm becomes empty and vacuolized, stoma thylacoids are reduced.Not only the described changes in fine structure but also the altered CO2 gas exchange reactions take place when glycophytic control plants grow older, thus indicating that NaCl may accelerate the normal ageing process in Mesembryanthemum crystallinum.

Der Einfluß von Verapamil auf die nächtliche Malatakkumulation bei Bryophyllum tubiflorum@@@Effect of verapamil on dark acidification in Bryophyllum tubiflorum

Excised phyllodia of Bryophyllum tubiflorum standing in phosphate buffer to which verapamil is added undergo specific alterations in their gaseous exchange reactions as well as in the amount of malate accumulated in the dark period. Inhibition of accumulation of malate increases with increasing concentrations of verapamil. The effects caused by verapamil are reversible. During the dark period plasmalemma under the chloroplasts detaches itself from the cell wall, forming a vacuole-like space between plasmalemma and cell wall which disappears in the following light period. In the presence of verapamil this formation of a vacuole-like structure does not occur.Verapamil is thought to influence the permeability of plasma membranes, thus decreasing the capacity of the vacuole to accumulate malate in the dark. Our interpretation of the results seems to demonstrate that the occurence of crassulacean acid metabolism is dependent on the integrity of the plasma membranes. Further details are discussed.Excised phyllodia of Bryophyllum tubiflorum standing in phosphate buffer to which verapamil is added undergo specific alterations in their gaseous exchange reactions as well as in the amount of malate accumulated in the dark period. Inhibition of accumulation of malate increases with increasing concentrations of verapamil. The effects caused by verapamil are reversible. During the dark period plasmalemma under the chloroplasts detaches itself from the cell wall, forming a vacuole-like space between plasmalemma and cell wall which disappears in the following light period. In the presence of verapamil this formation of a vacuole-like structure does not occur.Verapamil is thought to influence the permeability of plasma membranes, thus decreasing the capacity of the vacuole to accumulate malate in the dark. Our interpretation of the results seems to demonstrate that the occurence of crassulacean acid metabolism is dependent on the integrity of the plasma membranes. Further details are discussed.SummaryExcised phyllodia of Bryophyllum tubiflorum standing in phosphate buffer to which verapamil is added undergo specific alterations in their gaseous exchange reactions as well as in the amount of malate accumulated in the dark period. Inhibition of accumulation of malate increases with increasing concentrations of verapamil. The effects caused by verapamil are reversible. During the dark period plasmalemma under the chloroplasts detaches itself from the cell wall, forming a vacuole-like space between plasmalemma and cell wall which disappears in the following light period. In the presence of verapamil this formation of a vacuole-like structure does not occur.Verapamil is thought to influence the permeability of plasma membranes, thus decreasing the capacity of the vacuole to accumulate malate in the dark. Our interpretation of the results seems to demonstrate that the occurence of crassulacean acid metabolism is dependent on the integrity of the plasma membranes. Further details are discussed.