Biological and biochemical studies of the effects of Gibberellic acid and growth retardants on anthocyanin production by carrot cell suspension cultures

Author

Avihai Ilan

Date of Award

5-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Botany

Major Professor

Donald Dougall

Committee Members

Beth Mullin, Leslie Hickok, Ramond Holton, Dan Roberts, Otto Schawrz

Abstract

The inhibition of anthocyanin accumulation by gibberellic acid (GA) was investigated in carrot cell suspension cultures. Several types of growth retardants including quaternary ammonium compounds (CCC, AMO-1618), substituted pyrimidines (ancymidol), triazoles (paclobutrazol, uniconazole) and a norbomenodiazetine (tetcyclacis) increased anthocyanin production in carrot cell suspensions. The inhibitory effect of exogenously supplied GA₃, which reduced anthocyanin accumulation to 35% of non-treated control, was partially reversed in the presence of paclobutrazol to 77% of the control. The hypothesis that growth retardants exert their effect on anthocyanin production through the inhibition of synthesis and thus decreased levels of endogenous GAs was investigated. An hypothesis for the action of GA₃ can be constructed from the study of Hinderer and Seitz (Archives Biochem Biophys 246:217-224, 1986) which suggested that GA stimulates 3'-nucleotidase activity in carrot cell suspensions. According to this hypothesis, the 3'-nucleotidase dephosphorylates malonyl CoA giving 3'-dephosphomalonyl CoA which inhibits chalcone synthase, an enzyme essential for anthocyanin biosynthesis and as a result, anthocyanin accumulation is suppressed. An investigation of this hypothesis showed that 3'-nucleotidase activity in cell extracts was not greatly altered when cells were grown in the presence of GA₃ or uniconazole. This finding supports the rejection of the above hypothesis and raises the following questions. What step in anthocyanin biosynthesis is inhibited by GA₃ and what is the mechanism of this inhibition? In an attempt to identify the enzymatic step at which GA₃ inhibits anthocyanin synthesis, intermediates of anthocyanin biosynthesis were fed to carrot cell suspension cultures in the presence of GA₃. Naringenin and dihydroquercetin reversed GA₃ inhibition. Malonic acid, a potential precursor for malonyl CoA, and 4-coumaric acid did not restore anthocyanin production to the level of the non-treated control. These findings suggested that anthocyanin biosynthesis is inhibited by GA₃ at the enzymatic step catalyzed by chalcone isomerase or chalcone synthase. A major source for malonyl CoA, which is required for anthocyanin biosynthesis, is acetyl CoA. The conversion of acetyl CoA into malonyl CoA is catalyzed by the enzyme acetyl CoA carboxylase. For this reason, the effects of GA₃ and uniconazole on acetyl CoA carboxylase were also investigated. The activities of chalcone isomerase, chalcone synthase and acetyl CoA carboxylase were measured in extracts from untreated, from GA₃-treated and from uniconazole-treated cells. Acetyl CoA carboxylase activity was the same in extracts from untreated cells and GA₃-treated cells but no activity was found in extracts from uniconazole-treated cells. Chalcone isomerase activity was lower in extracts from GA₃-treated cells and much lower in extracts from uniconazole-treated cells than in extracts of untreated cells. Anthocyanin accumulation was increased in the presence of uniconazole which implies that the low level of chalcone isomerase in this treatment and the higher level in the GA₃ treatment are sufficient for the anthocyanin biosynthesis. The total chalcone synthase activity in extracts from GA₃ and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of non-treated cells while only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃- treated cells did not contain the peak of chalcone synthase activity in untreated cells which preceded the main peak and was about 1/4 of the total activity. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and is the site of GA inhibition of anthocyanin biosynthesis.

Recommended Citation

Ilan, Avihai, "Biological and biochemical studies of the effects of Gibberellic acid and growth retardants on anthocyanin production by carrot cell suspension cultures. " PhD diss., University of Tennessee, 1994.
https://trace.tennessee.edu/utk_graddiss/10377