Cloning, Expression, and Functional Characterization of Two Highly Efficient Flavonoid-di-O-glycosyltransferases ZmUGT84A1 and ZmUGT84A2 from Maize (Zea mays L.).

The maize (Zea mays L.) glycosyltransferase family 1 comprises many uridine diphosphate glycosyltransferase (UGT) members. However, UGT activities and biochemical functions have seldom been revealed. In this study, the genes of two flavonoid di-O-glycosyltransferases ZmUGT84A1 and ZmUGT84A2 were cloned from maize plant and expressed in Escherichia coli. Phylogenetic analysis showed that the two enzymes were homologous to AtUGT84A1 and AtUGT84A3. The two recombinant enzymes showed a high conversion rate of luteolin to its glucosides, mainly 4',7-di-O-glucoside and minorly 3',7-di-O-glucoside in two-step glycosylation reactions in vitro. Moreover, the recombinant ZmUGT84A1 and ZmUGT84A2 had a broad substrate spectrum, converting eriodictyol, naringenin, apigenin, quercetin, and kaempferol to monoglucosides and diglucosides. The highly efficient ZmUGT84A1 and ZmUGT84A2 may be used as a tool for the effective synthesis of various flavonoid O-glycosides and as markers for crop breeding to increase O-glycosyl flavonoid content in food.

Efficient and genotype independent maize transformation using pollen transfected by DNA-coated magnetic nanoparticles.

Current gene delivery methods for maize are limited to specific genotypes and depend on time-consuming and labor-intensive tissue culture techniques. Here, we report a new method to transfect maize that is culture-free and genotype independent. To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%, transfection was performed at cool temperature using pollen pretreated to open the germination aperture (40%-55%). Magnetic nanoparticles (MNPs) coated with DNA encoding either red fluorescent protein (RFP), ß-glucuronidase gene (GUS), enhanced green fluorescent protein (EGFP) or bialaphos resistance (bar) was delivered into pollen grains, and female florets of maize inbred lines were pollinated. Red fluorescence was detected in 22% transfected pollen grains, and GUS stained 55% embryos at 18 d after pollination. Green fluorescence was detected in both silk filaments and immature kernels. The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts (29%-74%) quantitated by polymerase chain reaction, and 5%-16% of the T1 seedlings showed immunologically active EGFP or GUS protein. Moreover, 1.41% of the bar transfected T1 plants were glufosinate resistant, and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization. These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.

Natural variation of ZmCGT1 is responsible for isoorientin accumulation in maize silk.

Maize (Zea mays L.) silk contains high levels of flavonoids and is widely used to promote human health. Isoorientin, a natural C-glycoside flavone abundant in maize silk, has attracted considerable attention due to its potential value. Although different classes of flavonoid have been well characterized in plants, the genes involved in the biosynthesis of isoorientin in maize are largely unknown. Here, we used targeted metabolic profiling of isoorientin on the silks in an association panel consisting of 294 maize inbred lines. We identified the gene ZmCGT1 by genome-wide association analysis. The ZmCGT1 protein was characterized as a 2-hydroxyflavanone C-glycosyltransferase that can C-glycosylate 2-hydroxyflavanone to form flavone-C-glycoside after dehydration. Moreover, ZmCGT1 overexpression increased isoorientin levels and RNA interference-mediated ZmCGT1 knockdown decreased accumulation of isoorientin in maize silk. Further, two nucleotide polymorphisms, A502C and A1022G, which led to amino acid changes I168L and E341G, respectively, were identified to be functional polymorphisms responsible for the natural variation in isoorientin levels. In summary, we identified the gene ZmCGT1, which plays an important role in isoorientin biosynthesis, providing insights into the genetic basis of the natural variation in isoorientin levels in maize silk. The identified favorable CG allele of ZmCGT1 may be further used for genetic improvement of nutritional quality in maize.

Synthesis and anti-hyperglycemic activity of hesperidin derivatives.

A series of hesperidin derivatives were prepared and identified by IR, (1)H NMR, and MS spectra. These compounds were evaluated in vitro and in vivo based on α-glucosidase inhibition, glucose consumption of HepG2 cells, and blood glucose level in streptozotocin-induced diabetic mice. The results revealed that all the compounds exhibited anti-hyperglycemic activities. The inhibition at 10(-3) M of compounds 3 and 7a on α-glucosidase were 55.02% and 53.34%, respectively, as compared to 54.80% by acarbose. Treated by compound 3 and the reference drug metformin, glucose consumption of HepG2 cell were 1.78 and 2.11 mM, respectively. After the streptozotocin-induced diabetic mice were oral administrated with compound 3 at 100 mg kg(-1) d(-1) for 10 days, the blood glucose level of 3 treated mice (13.23 mM, P<0.05) showed significant difference when compared to model control (23.03 mM). Thus, compound 3 exhibited promising anti-hyperglycemic activity.