Functional Characterization of a Novel Glycosyltransferase (UGT73CD1) from Iris tectorum Maxim. for the Substrate promiscuity.

Glycosylflavonoids are a class of natural products with multiple pharmacological activities and a lot of glycosyltransferases from various plant species have been reported that they were involved in the biosynthesis of these phytochemicals. However, no corresponding glycosyltransferase has been identified from the famous horticultural and medicinal plant Iris tectorum Maxim. Here, UGT73CD1, a novel glycosyltransferase, was identified from I. tectorum. based on transcriptome analysis and functional identification. Phylogenetic analysis revealed that UGT73CD1 grouped into the clade of flavonoid 7-OH OGTs. Biochemical analysis showed that UGT73CD1 was able to glycosylate tectorigenin at 7-OH to produce tectoridin, and thus assigned as a 7-O-glycosyltransferase. In addition, it also possessed robust catalytic promiscuity toward 12 structurally diverse flavonoid scaffolds and 3, 4-dichloroaniline, resulting in forming O- and N-glycosides. This work will provide insights into efficient biosynthesis of structurally diverse flavonoid glycosides for drug discovery.

Adaptive flexibility of enzymatic antioxidants SOD, APX and CAT to high light stress: The clonal perennial monocot Iris pumila as a study case.

High solar radiation has been recognized as one of the main causes of the overproduction of reactive oxygen species (ROS) and oxidative stress in plants. To remove the excess of ROS, plants use different antioxidants and tune their activity and/or isoform number as required for given light conditions. In this study, the adaptiveness of light-induced variation in the activities and isoform patterns of key enzymatic antioxidants SOD, APX and CAT was tested in leaves of Iris pumila clonal plants from two natural populations inhabiting a sun exposed dune site and a forest understory, using a reciprocal-transplant experiment. At the exposed habitat, the mean enzymatic activity of total SODs was significantly greater than that in the shaded one, while the amount of the mitochondrial MnSOD was notably higher compared to the plastidic Cu/ZnSOD. However, the number of Cu/ZnSOD isoforms was greater in the forest understory relative to the exposed site (three vs. two, respectively). An inverse relationship recorded between the quantities of MnSOD and Cu/ZnSOD in alternative light habitats might indicate that the two enzymes compensate each other in maintaining intracellular ROS and redox balance. The adaptive population differentiation in APX activity was exclusively recorded in the open habitat, which indicated that the synergistic effect of high light and temperature stress could be the principal selective factor, rather than high light alone. The enzymatic activity of CAT was similar between the two populations, implicating APX as the primary H2O2 scavenger in the I. pumila leaves exposed to high light intensity.

Molecular cloning and characterization of O-methyltransferases from the flower buds of Iris hollandica.

In plants, O-methyltransferases (OMTs) play an important role in methylation of secondary metabolites, especially flavonoids and other phenylpropanoids, and two cDNA clones, IhOMT1 and IhOMT2 (Iris hollandica OMT), encoding OMTs were successfully isolated from a cDNA library of flower buds of I. hollandica. IhOMT1 encodes an open reading frame (ORF) of 365 amino acids with calculated molecular mass of 40,193Da and isoelectric point (pI) of 5.54, while IhOMT2, which shares 31.5% amino acid sequence identity with IhOMT1, encodes 369 amino acids with calculated molecular mass of 40,385Da and pI of 5.50. In addition, the molecular masses of both recombinant IhOMT1 and IhOMT2 proteins were estimated to be about 40kDa by protein gel blot analysis. Characterization of the enzymatic properties using the recombinant IhOMT1 protein confirmed that IhOMT1 cDNA encodes a S-adenosyl-L-methionine (SAM)-dependent caffeic acid 3-OMT, which catalyzes the transfer of the methyl moiety from SAM to caffeic acid to form ferulic acid. Its optimum activity was observed at pH 7.5-8.0 and at 35 degrees C. This is the first report of the isolation and characterization of a COMT cDNA clone involved in the phenylpropanoid biosynthesis of Iridaceae plants. In contrast, IhOMT2 showed no activity in SAM-dependent assays for various phenylpropanoids.

Delay of Iris flower senescence by protease inhibitors.

Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than half to serine proteases, with a minor role of metalloproteases. Treatment of isolated tepals with the purported serine protease inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] or DFP (diisopropyl-fluorophosphate) prevented the increase in endoprotease activity and considerably delayed or prevented the normal senescence symptoms. The specific cysteine protease-specific E-64d reduced maximum endoprotease activity by 30%, but had no effect on the time to visible senescence. Zinc chloride and aprotinin reduced maximum endoprotease activity by c. 50 and 40%, respectively, and slightly delayed visible senescence. A proteasome inhibitor (Z-leu-leu-Nva-H) slightly delayed tepal senescence, which indicates that protein degradation in the proteasome may play a role in induction of the visible senescence symptoms. It is concluded that visible senescence is preceded by large-scale protein degradation, which is apparently mainly due to cysteine- and serine protease activity, and that two (unspecific) inhibitors of serine proteases considerably delay the senescence symptoms.