Physiological and molecular characterization of habituated and non- habituated soybean callus lines (Glycine max (L.) Merr cv. Acme)

Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

A cytokinin habituated soybean callus has been isolated, utilizing the cytokinin soybean bioassay. The habituated callus line was subsequently characterized with a non-habituated callus line in relationship to levels of endogenous growth substances, ultrastructure, nitrogen metabolism and pattern of gene expression. The cytokinin habituated soybean callus contained a higher level of endogenous cytokinin-like activity in comparison to the non-habituated callus. This higher level of cytokinin present is in part due to a lower rate of degradation. The habituated callus tissue produced very low levels of ethylene, while the non-habituated callus produced ethylene at a much higher rate (57 fold higher), than the habituated callus. In contrast to what was found in habituated sugarbeet callus, only low levels of putrescine could be detected in both callus types. The putrescine content of habituated callus tissue was lower than that of non-habituated callus tissue. The ultrastructure of habituated callus cells exhibited several differences to what was observed in the non-habituated callus. Habituated callus cells appeared to have a thinner cell wall than that of the non-habituated callus cells. The cristae of the mitochondria in habituated cells were thicker than that of the non-habituated callus cells, indicating a lower metabolic activity. On day 14 of the growth period the nuclei of habituated callus demonstrated active RNA synthesis as indicated by the presence of several vacuolated nucleoli. Although no significant differences between proline levels of habituated callus and proline levels of non-habituated callus were observed, it was demonstrated that there was a difference in proline metabolism between the habituated and non-habituated calli. Utilizing an inhibitor of OAT, gabaculine, it was shown that in habituated callus tissue proline originated from ornithine during the first 14 days of growth. During the second half of the growth period, which characteristically consists of tissue with low biosynthetic activity, proline originated from glutamate. The production of proline in habituated callus from ornithine also corresponded to a period of high NH₄⁺ content in both callus types, while the production of proline from glutamate corresponded to a period of low NH₄⁺ content in the cells of both callus types. No such correlation was observed in proline metabolism of non-habituated callus. A similar turning point was observed in the activity of OAT of both callus types. Although the specific activity of OAT in both callus types mirrored their changes in RNA concentration, the percentage inhibition of OAT by gabaculine was not significant from day 14 in both callus types. This may indicate a change in the catalyzing properties of OAT in both callus types. It was further demonstrated that the non-habituated callus tissue contained some inhibitor inactivating OAT activity. With the use of gabaculine it was further shown that, in contrast to what was found in other habituated calli, there is no metabolic link between proline metabolism and putrescine synthesis. Both the habituated callus and the non-habituated callus exhibited a high nitrogen influx during the first 14 days of the growth period. The low NH₄⁺ content present in both callus types during the second half of the growth period coincided with higher levels of amino acids present in both callus types. The levels of precursor amino acids (glutamate, aspartate and alanine) did not fluctuate during the growth period, indicating a tight control on amino acid pools. Levels of amino acids further down the path of metabolism did not fluctuate drastically and there appeared to be very little difference between the levels of different amino acids measured in the habituated and nonhabituated calli. Serine was the dominant amino acid in both callus types. Total RNA concentrations of habituated callus were low in comparison to that of the non-habituated callus, except for a striking 12 fold increase on day 14 of the growth period. RNA concentrations of non-habituated callus increased gradually during the growth period and the highest concentration was recorded 21 days after subculturing. Several polypeptides were observed in the habituated callus that were not present in the non-habituated callus, utilizing IEF. Three polypeptides exhibited a change in concentration from day 6 to day 14 of the growth period in both the habituated and nonhabituated callus. These polypeptides appeared to decrease in nonhabituated callus, while they increased in the habituated callus. A complete cDNA library was constructed for both of the habituated and nonhabituated callus lines. Six different clones, that were over expressed in the habituated callus tissue, were isolated via subtractive techniques. One clone was characterized and showed homology to the glutamate/aspartate transport protein, the membrane component, of E. coli.