Janine M. Cooney

DAD2 Is an α/β Hydrolase Likely to Be Involved in the Perception of the Plant Branching Hormone, Strigolactone

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 missing step of the l-galactose pathway of ascorbate biosynthesis in plants, an l-galactose guanyltransferase, increases leaf ascorbate content

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-l-galactose 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.

Environmental regulation of leaf colour in red 35S:PAP1 Arabidopsis thaliana

* 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 cis-coumaryl 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.

A proteomic approach identifies early pregnancy biomarkers for preeclampsia: Novel linkages between a predisposition to preeclampsia and cardiovascular disease

Preeclampsia (PE) is a common, potentially life‐threatening pregnancy syndrome triggered by placental factors released into the maternal circulation, resulting in maternal vascular dysfunction along with activated inflammation and coagulation. Currently there is no screening test for PE. We sought to identify differentially expressed plasma proteins in women who subsequently develop PE that may perform as predictive biomarkers. In seven DIGE experiments, we compared the plasma proteome at 20 wk gestation in women who later developed PE with an appropriate birth weight for gestational age baby (n=27) or a small for gestational age baby (n=12) to healthy controls with uncomplicated pregnancies (n=57). Of the 49 differentially expressed spots associated with PE‐appropriate for gestational age, PE‐small for gestational age or both (p<0.05, false discovery rate corrected), 39 were identified by LC‐MS/MS. Two protein clusters that accurately (>90%) classified women at risk of developing PE were identified. Immunoblots confirmed the overexpression of fibrinogen γ chain and α‐1‐antichymotrypsin in plasma prior to PE. The proteins identified are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity and acute‐phase responses, indicating novel synergism between pathways involved in the pathogenesis of PE. Our findings are remarkably similar to recently identified proteins complexed to high‐density lipoprotein and linked to cardiovascular disease.

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red‐fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.

Short-term blackcurrant extract consumption modulates exercise-induced oxidative stress and lipopolysaccharide-stimulated inflammatory responses.

Exercise-induced oxidative stress is instrumental in achieving the health benefits from regular exercise. Therefore, inappropriate use of fruit-derived products (commonly applied as prophalytic antioxidants) may counteract the positive effects of exercise. Using human exercise and cellular models we found that 1) blackcurrant supplementation suppressed exercise-induced oxidative stress, e.g., plasma carbonyls (0.9 +/- 0.1 vs. 0.6 +/- 0.1 nmol/mg protein, placebo vs. blackcurrant), and 2) preincubation of THP-1 cells with an anthocyanin-rich blackcurrant extract inhibited LPS-stimulated cytokine secretion [TNF-alpha (16,453 +/- 322 vs. 10,941 +/- 82 pg/ml, control vs. extract, P < 0.05) and IL-6 (476 +/- 14 vs. 326 +/- 32 pg/ml, control vs. extract, P < 0.05)] and NF-kappaB activation. In addition to its antioxidant and anti-inflammatory properties, we found that postexercise plasma collected after blackcurrant supplementation enhanced the differential temporal LPS-stimulated inflammatory response in THP-1 cells, resulting in an early suppression of TNF-alpha (1,741 +/- 32 vs. 1,312 +/- 42 pg/ml, placebo vs. blackcurrant, P < 0.05) and IL-6 (44 +/- 5 vs. 36 +/- 3 pg/ml, placebo vs. blackcurrant, P < 0.05) secretion after 24 h. Furthermore, by using an oxidative stress cell model, we found that preincubation of THP-1 cells with hydrogen peroxide (H(2)O(2)) prior to extract exposure caused a greater suppression of LPS-stimulated cytokine secretion after 24 h, which was not evident when cells were simultaneously incubated with H(2)O(2) and the extract. In summary, our findings support the concept that consumption of blackcurrant anthocyanins alleviate oxidative stress, and may, if given at the appropriate amount and time, complement the ability of exercise to enhance immune responsiveness to potential pathogens.

Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple

The dihydrochalcone phlorizin (phloretin 2′‐glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP‐glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [3H]‐UDP‐glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP‐glucose co‐migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP‐glucose, UDP‐xylose and UDP‐galactose. This is the first report of functional characterization of a UDP‐glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.

Blueberry fruit polyphenolics suppress oxidative stress-induced skeletal muscle cell damage in vitro.

Skeletal muscle damage can result from disease and unaccustomed or excessive exercise. Muscle dysfunction occurs via an increased level of reactive oxygen species and hence there is potential in antioxidants as amelioration strategies. We explored the putative benefit of fruit polyphenolic extracts in reducing the susceptibility of skeletal muscle cells to oxidative stress. Muscle myotubes were simultaneously challenged with fruit extracts (1-50 microg/mL) and calcium ionophore (A23187), hydrogen peroxide, or 2,4-dinitrophenol and damage monitored by release of cytosolic enzymes. A blueberry fruit extract displayed a potent and significant dose-dependent protective capacity. Evaluation of the protective capacity of anthocyanin sub-extracts of blueberry fruit and pure individual glycosides, with identification of extract polyphenolic components using MS, suggested that malvidin galactoside and/or glucoside were the active compounds. These in vitro data support the concept that blueberry fruits or derived foods rich in malvidin glycosides may be beneficial in alleviating muscle damage caused by oxidative stress. More research on the benefits of blueberry fruit consumption in human intervention studies is warranted.

Antifungal saponins from Paris polyphylla Smith.

Three steroidal saponins, including one new and two known compounds, were isolated from the rhizomes of Paris polyphylla Smith. One- and two-dimensional NMR, LC-MS, and interpretation of hydrolytic cleavage experiments led to the identification of the structure of the new saponin as ( 25R)-spirost-5-ene-3 beta,17 alpha-diol (pennogenin) 3- O-{ O- alpha- L-rhamnopyranosyl-(1-->2)- O-[ O- beta-xylopyranosyl-(1-->5)- alpha- L-arabinofuranosyl-(1-->4)]- beta- D-glucopyranoside}. The isolated saponins were evaluated for their antifungal activity against Cladosporium cladosporioides and Candida species and showed comparable activity to chemicals used in some commercial products.

Blackcurrant proanthocyanidins augment IFN-γ-induced suppression of IL-4 stimulated CCL26 secretion in alveolar epithelial cells.

Epidemiological studies reveal that fruit consumption reduces the prevalence of airway inflammation and childhood asthma. In particular, blackcurrant polyphenolic extracts have been shown to alleviate lung inflammation. Since IL-4-stimulated eotaxin-3 (CCL26) secretion is a major factor in the continuous eosinophil recruitment observed in atopic asthma, our focus was to evaluate the effectiveness of blackcurrant polyphenolic compounds on CCL26 secretion in human alveolar epithelial cells. Our results indicate that a proanthocyanin-enriched blackcurrant extract (BC-P), but not anthocyanin-enriched blackcurrant extract suppressed both IL-4- and IL-13-stimulated CCL26 secretion in a dose-dependent manner. Furthermore pre-incubation of cells with BC-P caused a time-dependent suppression of IL-4-stimulated CCL26 secretion. Moreover, epigallocatechin (EGC), and to a lesser extent epicatechin, metabolites identified in the proanthocyanidin extract, suppressed IL-4-stimulated CCL26 secretion. EGC was also effective at reducing the cellular phosphorylated STAT-6/STAT-6 ratio. Furthermore, both BC-P and purified EGC potentiated the ability of IFN-gamma to suppress IL-4-stimulated CCL26 secretion. The progression of an allergic immune response is complex, identifying plant compounds that target specific cellular events and complement the bodys own immune actions is important for the development of functional foods. Our findings support the potential for blackcurrant polyphenolic compounds to reduce eosinophil recruitment and alleviate eosinophilic-driven airway inflammation.