Strigolactones are a recently discovered class of plant hormone involved in branching, leaf senescence, root development, and plant-microbe interactions. They are carotenoid-derived lactones, synthesized in the roots and transported acropetally to modulate axillary bud outgrowth (i.e., branching). However, a receptor for strigolactones has not been identified. We have identified the DAD2 gene from petunia, an ortholog of the rice and Arabidopsis D14 genes, and present evidence for its roles in strigolactone perception and signaling. DAD2 acts in the strigolactone pathway, and the dad2 mutant is insensitive to the strigolactone analog GR24. The crystal structure of DAD2 reveals an α/β hydrolase fold containing a canonical catalytic triad with a large internal cavity capable of accommodating strigolactones. In the presence of GR24 DAD2 interacts with PhMAX2A, a central component of strigolactone signaling, in a GR24 concentration-dependent manner. DAD2 can hydrolyze GR24, with mutants of the catalytic triad abolishing both this activity and the ability of DAD2 to interact with PhMAX2A. The hydrolysis products can neither stimulate the protein-protein interaction nor modulate branching. These observations suggest that DAD2 acts to bind the mobile strigolactone signal and then interacts with PhMAX2A during catalysis to initiate an SCF-mediated signal transduction pathway.

The gene for one postulated enzyme that converts GDP-L-galactose to L-galactose-1-phosphate is unknown in the L-galactose pathway of ascorbic acid biosynthesis and a possible candidate identified through map-based cloning is the uncharacterized gene At4g26850. We identified a putative function for At4g26850 using PSI-Blast and motif searching to show it was a member of the histidine triad superfamily, which includes D-galactose uridyltransferase. We cloned and expressed this Arabidopsis gene and the homologous gene from Actinidia chinensis in Escherichia coli and assayed the expressed protein for activities related to converting GDP-L-galactose to L-galactose-1-P. The expressed protein is best described as a GDP-L-galactose-hexose-1-phosphate guanyltransferase (EC 2.7.7.), catalyzing the transfer of GMP from GDP-Lgalactose to a hexose-1-P, most likely D-mannose-1-phosphate in vivo. Transient expression of this A. chinensis gene in tobacco leaves resulted in a >3-fold increase in leaf ascorbate as well as a 50-fold increase in GDP-L-galactose-D-mannose-1-phosphate guanyltransferase activity.GDP-L-galactose pyrophosphorylase ͉ gold kiwifruit ͉ VTC2 H umans and all animals depend on ascorbate (vitamin C) as an essential antioxidant. Humans and several other mammalian species are unable to synthesize ascorbate because of an inactivation of the last gene in the pathway, and so we are dependent on dietary vitamin C, mainly from vegetables and fruits. A significant route for ascorbate biosynthesis in plants is through the L-galactose pathway (1-5), although other pathways may also be operating (6, 7). To date, all of the genes encoding enzymes, and their associated enzymatic activities, for the L-galactose pathway have been identified and at least partially characterized, except for one, an enzyme proposed to convert GDP-L-galactose to L-galactose-1-phosphate. The characterized genes and enzyme activities include the GDP-D-mannose pyrophosphorylase (1,8,9), the GDP-D-mannose 3Ј,5Ј-Epimerase (10-12), the L-galactose-1-P phosphatase (13, 14), L-galactose dehydrogenase (5,15,16), and L-galactono-1,4-lactone dehydrogenase (17, 18). The missing enzyme, which has not been reported as being assayed either as an extracted or purified enzyme activity or as an expressed gene, catalyzes the second committed step to ascorbic acid biosynthesis.The VTC2 mutant of Arabidopsis thaliana was first identified in a screen for mutants with increased sensitivity to ozone and was characterized as showing especially low ascorbic acid levels (19). The mutated gene was cloned by using a map-based approach (20) and identified as a gene (At4g26850) encoding a novel protein. However, the genes reported to show most similarity to At4g26850 were the similarly uncharacterized Arabidopsis gene At5g55120 and other uncharacterized genes from nonplant species. The authors were therefore unable to identify a specific role for Atg26850 in a regulatory or biosynthetic pathway leading to reduced vitamin C levels in the VTC2 mutants (20).We investigated the f...

Anthocyanins, the red/blue pigments found in plants, are polyphenolic compounds consumed by humans and are part of a normal diet. Recent studies have shown that anthocyanins have substantial bioactivity including antioxidant activity and therefore may have beneficial effects on human health. Anthocyanins are a group of over 500 compounds of diverse structures containing different core phenolic aglycons and conjugated with sugars in a variety of glycosylation patterns. In this study, we have investigated the bioabsorption of 15 anthocyanins with structures containing different aglycons and conjugated sugars extracted from blueberry, boysenberry, black raspberry, and blackcurrant in both humans and rats. Intact and unmetabolized anthocyanins were detected in urine of rats and humans following dosing for all molecular structures investigated, thus demonstrating that anthocyanins with diverse molecular structure and from different dietary sources are bioavailable at diet relevant dosage rates. In addition, the relative concentrations of anthocyanins detected in urine following dosing varied, indicating that differences in bioavailability are due to variations in chemical structure. Our results suggest that the nature of the sugar conjugate and the phenolic aglycon are both important determinants of anthocyanin absorption and excretion in rats and humans.

Summary• High-temperature, low-light (HTLL) treatment of 35S:PAP1 Arabidopsis thaliana over-expressing the PAP1 (Production of Anthocyanin Pigment 1) gene results in reversible reduction of red colouration, suggesting the action of additional anthocyanin regulators. High-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and Affimetrix ® -based microarrays were used to measure changes in anthocyanin, flavonoids, and gene expression in response to HTLL.• HTLL treatment of control and 35S:PAP1 A. thaliana resulted in a reversible reduction in the concentrations of major anthocyanins despite ongoing over-expression of the PAP1 MYB transcription factor. Twenty-one anthocyanins including eight ciscoumaryl esters were identified by LCMS. The concentrations of nine anthocyanins were reduced and those of three were increased, consistent with a sequential process of anthocyanin degradation. Analysis of gene expression showed down-regulation of flavonol and anthocyanin biosynthesis and of transport-related genes within 24 h of HTLL treatment. No catabolic genes up-regulated by HTLL were found.• Reductions in the concentrations of anthocyanins and down-regulation of the genes of anthocyanin biosynthesis were achieved by environmental manipulation, despite ongoing over-expression of PAP1. Quantitative PCR showed reduced expression of three genes (TT8, TTG1 and EGL3) of the PAP1 transcriptional complex, and increased expression of the potential transcriptional repressors AtMYB3, AtMYB6 and AtMYBL2 coincided with HTLL-induced down-regulation of anthocyanin biosynthesis.• HTLL treatment offers a model system with which to explore anthocyanin catabolism and to discover novel genes involved in the environmental control of anthocyanins.

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