PORPHYRIN BIOSYNTHESIS IN THE SOYBEAN CALLUS TISSUE LEVELS CYSTHATIONASE, RHODANESE, AMINOLEVULINATE SYNTHETASE AND AMINOLEVULINATE DEHYDRATASE IN CLONES OF DIFFERENT AGE

Abstract 1. 1. The activity of Succinyl CoA Synthetase (Suc CoA-S), Cysthationase, Rhodanese, Aminolevulinate Synthetase (ALA-S) and Aminolevulinate Dehydratase (ALA-D) was studied in old (405–407 subcultures) and young (34–36 subcultures) soybean callus clones as a function of the days of growing. 2. 2. Suc CoA-S, ALA-S and ALA-D activities were much lower in old than in young callus, while the activity of Cysthationase and Rhodanese was higher in old callus. 3. 3. ALA-S reached its maximum activity when Rhodanese and Cysthationase their minimum, on the 11th day of growth. It is suggested that the cellular content of a possible thio-compound which would regulate ALA-S activity, is controlled through its degradation by Rhodanese.


INTRODUCTION
For many years we have been interested in the study of tetrapyrrole biosynthesis in soybean callus tissue cultures, a highly dividing system which either dark or light-grown fails to synthesize chlorophyll in amount equivalent to that found in mature leaves (Batlle et al., 1975 and references there in;Wider de Xifra & Batlle. 1976, 1978Batlle et al., 1976a,b;Wider de Xifra et al., 1978).
One of the important findings that has emerged from these studies has been the direct measurement of Aminolevulinate Synthetase (EC 2.3.1.37) (ALA-S) in a vegetable like tissue. It was found that enzyme activity reached a sharp maximum on the 11 th day of growth (Wider de Xifra er nl.. 1971). Some properties of the soybean callus ALA-S resemble those of the Rh. spheroides enzyme (Marriot et al., 1969;Tuboi et al., 1969) and the presence of a compound which seems to control ALA-S activity was also detected in these cultured cells, which could account for the changes in activity during aging of crude extracts or supernatants.
On the other hand, it has been demonstrated that in Rh. spheroides, cystine trisulphide is an activator of ALA-S, that both Cysthationase (EC 4.2.1.15) and Rhodanese (EC 2.8.1.1) are involved in the formation and degradation respectively on this trisulphide and consequently on the control of ALA-S activity (Wider de Xifra et al., 1976).
Furthermore, after studying these tissue cultures for nearly 15 hr, we have observed that some of their properties were modified as the clone of callus was physiologically older; it was found that the longer the time colourless callus was cultured in the dark, the less was the activity of the enzymes involved in porphyrin biosynthesis.
Therefore, it was considered appropiate to reinvestigate in older callus, some of the phenomena already studied and compare with data obtained from younger callus.
In this paper we will then report some results, studying the activity of Sue CoA-S, Cysthationase, Rhodanese, ALA-S and ALA-D in both old and young soybean callus clones, as a function of the days of growing, with the view of correlating the levels of ALA-S, Cysthationase and Rhodanese activities in clones of different age and their role, if any. in the regulation of soybean callus porphyrin biosynthesis.

MATERIALS AND METHODS
Unless otherwise indicated all chemicals were purchased from Sigma Chemical Co.

Doys of growing
As can be seen in Fig. 1, Sue CoA-S and ALA-D activities were much lower in old than in young callus; they were also dependent on the days of growing; Sue CoA-S reached its maximum on the days 7th and 8th for young and old callus respectively, while ALA-D activitiy was highest on the 10th and 8th day of growth. Maxima for young callus do not apparently show any clear correlation; however those for old callus were coincident, which might suggest that they could be related to some extent with the content of ALA in the cell. One possible explanation for this phenomenon is that Sue CoA-S was assayed as described by Wider de Xifra & Tigier (1971).
ALA-S was assayed according to Wider de Xifra er al. (1971).
ALA-D was measured as described by Tigier et al. (1970).
Cysthationase and Rhodanese were assayed following the procedures described by Wider de Xifra et al. (1976) slightly modified.
Enzyme units 1 unit of enzyme activity is defined as the amount of enzyme which catalyses the formation of 1 nmol of product in 60 set under the standard conditions. Enzymic activities are expressed as units per mg of protein.  7th days in old and young callus respectively. A very Vol. 6,p. 196. Springer Verlag,Berlin. interesting finding was that in all tissues tested, the These results might indicate. that a similar scheme 3ioph~s. 130,92-100. to that proposed by Wider de Xifra et al. (1976)  Finally, these findings have also confirmed early work indicating that metabolic changes occur in these tissues cultures after prolonged culture; here, it was found that the length of time previously spent by the callus undergoing subculture diminishes its capacity to synthesize the enzymes involved in porphyrin biosynthesis but increases that reiated to the enzymes responsible for sulphur metabolism. Therefore when working with plant tissue cultures it is important to establish the age of the cultures and if possible to perform comparative studies in clones of different age.