Dynamic metabolic changes in seeds and seedlings of Brassica napus (oilseed rape) suppressing UGT84A9 reveal plasticity and molecular regulation of the phenylpropanoid pathway.

In Brassica napus, suppression of the key biosynthetic enzyme UDP-glucose:sinapic acid glucosyltransferase (UGT84A9) inhibits the biosynthesis of sinapine (sinapoylcholine), the major phenolic component of seeds. Based on the accumulation kinetics of a total of 158 compounds (110 secondary and 48 primary metabolites), we investigated how suppression of the major sink pathway of sinapic acid impacts the metabolome of developing seeds and seedlings. In UGT84A9-suppressing (UGT84A9i) lines massive alterations became evident in late stages of seed development affecting the accumulation levels of 58 secondary and 7 primary metabolites. UGT84A9i seeds were characterized by decreased amounts of various hydroxycinnamic acid (HCA) esters, and increased formation of sinapic and syringic acid glycosides. This indicates glycosylation and ß-oxidation as metabolic detoxification strategies to bypass intracellular accumulation of sinapic acid. In addition, a net loss of sinapic acid upon UGT84A9 suppression may point to a feedback regulation of HCA biosynthesis. Surprisingly, suppression of UGT84A9 under control of the seed-specific NAPINC promoter was maintained in cotyledons during the first two weeks of seedling development and associated with a reduced and delayed transformation of sinapine into sinapoylmalate. The lack of sinapoylmalate did not interfere with plant fitness under UV-B stress. Increased UV-B radiation triggered the accumulation of quercetin conjugates whereas the sinapoylmalate level was not affected.

Functional characterization and analysis of the Arabidopsis UGT71C5 promoter region.

In the present study, we isolated an Arabidopsis promoter, UGT71C5, and analyzed its role in the regulation of the light response mechanism. We constructed a fusion vector pBI121-pU-GUS by integrating the UGT71C5 promoter upstream of the GUS reporter gene in pBI121, and then transferred this vector into Arabidopsis plants. The GUS activity of the transgenic plants was detected using a spectrophotometer under normal growth conditions as well as under light, drought, and ABA stress-treatments. The obtained results indicated that the GUS activity of transgenic plants ranged in between the activities observed in wild-type and 35S transgenic plants, which were used as positive control. Light stress for 8 and 12 h increased the GUS activity in transgenic plants by 3 and 4 times, respectively, compared to the activity in these plants under normal conditions. No such change in the GUS activity was observed under drought and ABA-treated conditions. This suggests that the UGT71C5 promoter is light inducible. Our study provides helpful insights into the elucidation of inducible promoters in Arabidopsis and the molecular mechanisms of light response.

Formation of cyclodextrins with cyclodextrin glucosyltransferase stimulated with polarized light.

After illumination with white, linearly polarized light (WLPL), cyclodextrin glycosyltransferase produced mixture of alpha-, beta-, and gamma-cyclodextrins (CD) with higher overall yield than did that enzyme when nonilluminated. The illumination also influenced the ratio of those CD and that effect depended on concentration of enzyme and illumination time. At a high enzyme concentration (0.64 U/cm(3)), regardless the illumination time, formation of beta-CD predominated. The highest yield of beta-CD was afforded after 1 h illumination and 2 h illumination led to a significant increase in the yield of gamma-CD. Three-month storage of enzyme illuminated with WLPL did not reduce its enhanced activity.

Functional molecular size and structure of dextransucrase by radiation inactivation and gel electrophoresis.

Robyt et al. have proposed a mechanism for dextransucrase in which dextran is synthesized by the cooperative action of two equivalent nucleophiles (Robyt, J.F., Kimble, B.K. and Walseth, T.F. (1974) Arch. Biochem. Biophys. 165, 634-640). To distinguish between the possibilities that the enzyme is a monomer bearing both nucleophiles, or a dimer with each subunit bearing one nucleophile, the molecular weight of the enzyme was determined by SDS-polyacrylamide gel electrophoresis and by radiation inactivation. Two major forms of dextransucrase from Leuconostoc mesenteroides NRRL B-512F were found on SDS-polyacrylamide gel electrophoresis, with Mr 177 000 and 158 000, and sometimes a minor form with Mr 168 000. No form of dextransucrase smaller than Mr 158 000 was found, either in the presence or absence of dextran T10, although levansucrase was detected at Mr 92 000 and 116 000. On irradiation with 60Co, dextransucrase behaved as a single species with a maximum size of Mr 201 000. Because Mr 201 000 is much smaller than the minimum dimer size of Mr 316 000 (= 2 X 158 000), it is concluded that both nucleophiles are probably located on the same peptide, rather than one on each subunit of a dimer, and that peptide association is probably not required for dextran synthesis.