Characterization of p‐coumarate accumulation,p‐coumaroyl transferase,and cell wall changes during the development of corn stems

BACKGROUND: Developmental changes occur in corn (Zea mays L.) stems from cell initiation to fully mature cell types. During cell wall maturation the lignin is acylated with p‐coumarates (pCA). This work describes characterization studies of the p‐coumaroylation process in relation to corn stem development. RESULTS: Corn plants from three locations were harvested and tissues were analyzed from all nodes and even‐numbered internodes above soil line. Changes in carbohydrates reflect a shift to lignification at the expense of structural polysaccharide synthesis. Accumulation of pCA paralleled the incorporation of lignin while ferulate (FA) remained relatively constant as a proportion of the cell wall (5–7 g kg^?1 CW). The p‐coumaroyl transferase (pCAT), which is responsible for attaching pCA to lignin monomers, displayed maximum levels of activity in the middle region of the stem (internodes 10–12, 2–3 nmol L^?1 min^?1 mg^?1). The syringyl content as a proportion of the total lignin did not change significantly with cell wall maturation although there was a trend towards increased amounts of syringyl units in the more mature cell walls. CONCLUSIONS: Incorporation of pCA into corn cell walls not only mirrored lignification but the pCAT activity as well. Levels of pCAT activity may be an indicator of rapid lignification specifically for syringyl type lignin. Copyright © 2008 Society of Chemical Industry     

Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3-beta-glucan synthesis.

Selection of an extragenic suppressor of fks1-1154 Deltafks2, mutations in the catalytic subunits of yeast 1,3-beta-glucan synthase (GS) conferring temperature-sensitivity, led to the LRG1 gene, which was originally identified as a LIM-RhoGAP homologous gene. Mutations in the LRG1 gene restore impaired 1,3-beta-glucan synthesis in the fks1-1154 Deltafks2 mutant as well as that in rho1-2, a temperature-sensitive mutant of Rho-type GTPase that functions as a regulatory subunit of GS. Two-hybrid analyses of Lrg1p, which contains a sequence conserved among Rho GTPase-activating proteins (GAPs), revealed its specific interactions with the active form of Rho1p. Among eight potential yeast RhoGAPs, Lrg1p is the only member that negatively regulates GS activity: mutations in the rest of GAPs, including bem2, Deltabem3, Deltasac7, Deltabag7, Deltarga1, Deltarga2 and Deltargd1, do not suppress impairment of 1,3-beta-glucan synthesis. Analyses of Mpk1p phosphorylation revealed the inability of Lrg1p to regulate the Pkc1p-MAP kinase cascade, a distinct Rho1p-regulating signalling pathway known to be affected by the GAPs, Bem2p and Sac7p. Thus, different groups of Rho1p GAPs control the activity of different Rho1p-effector proteins.     

The p53-, Bax- and p21-dependent inhibition of colon cancer cell growth by 5-hydroxy polymethoxyflavones

Scope: Previously, we reported that 5‐hydroxy polymethoxyflavones (5OH‐PMFs) isolated from orange, namely 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone, 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF) and 5‐hydroxy‐6,7,8,4′‐tetramethoxyflavone (5HTMF), potently induced apoptosis and cell‐cycle arrest in multiple human colon cancer cells. Herein, using isogenic variants of HCT116 human colon cancer cells, we investigated the effects of p53, Bax and p21 on the apoptosis and cell‐cycle arrest induced by different 5OH‐PMFs. Methods and results: Annexin V/PI co‐staining assay demonstrated that 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (p53^+/+) cells but not in HCT116 (p53^?/?) cells. Furthermore, 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (Bax^+/?) cells, whereas their pro‐apoptotic effects on HCT116 (Bax^?/?) cells were marginal. All three 5OH‐PMFs increased G0/G1 cell population of HCT116 (p53^+/+) cells, and these effects were abolished in HCT116 (p53^?/?) and HCT116 (p21^?/?) cells. Immunoblotting analysis showed that 5HHMF and 5HTMF increased the levels of cleaved caspase‐3, cleaved PARP in both HCT116 (p53^+/+) and HCT116 (Bax^+/?) cells and these effects were much weaker in HCT116 (p53^?/?) and HCT116 (Bax^?/?) cells. Conclusion: Our results demonstrated that 5OH‐PMFs, especially 5HHMF and 5HTMF, induce apoptosis and cell‐cycle arrest by p53‐, Bax‐ and p21‐dependent mechanism.     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

Proteolytic processing of certain CaaX motifs can occur in the absence of the Rce1p and Ste24p CaaX proteases

The CaaX motif directs C‐terminal protein modifications that include isoprenylation, proteolysis and carboxylmethylation. Proteolysis is generally believed to require either Rce1p or Ste24p. While investigating the substrate specificity of these proteases, using the yeast a‐factor mating pheromone as a reporter, we observed Rce1p‐ and Ste24p‐independent mating (RSM) when the CKQQ CaaX motif was used in lieu of the natural a‐factor CVIA motif. Uncharged or negatively charged amino acid substitutions at the a₁ position of the CKQQ motif prevented RSM. Alanine substitutions at the a₂ and X positions enhanced RSM. Random mutagenesis of the CaaX motif provided evidence that RSM occurs with approximately 1% of all possible CaaX motif permutations. Combined mutational and genetic data indicate that RSM‐promoting motifs have a positively charged amino acid at the a₁ position. Two of nine naturally occurring yeast CaaX motifs conforming to this pattern promoted RSM. The activity of the isoprenylcysteine carboxyl methyltransferase Ste14p was required for RSM, indicating that RSM‐promoting CaaX motifs are indeed proteolysed. RSM was enhanced by the overexpression of Axl1p or Ste23p, suggesting a role for these M16A subfamily metalloproteases in this process. We have also determined that an N‐terminal extension of the a‐factor precursor, which is typically removed by the yeast M16A enzymes, is required for optimal RSM. These observations suggest a model that involves targeting of the a‐factor precursor to the peptidosome cavity of M16A enzymes where subsequent interactions between RSM‐promoting CaaX motifs and the active site of the M16A enzyme lead to proteolytic cleavage. Copyright © 2009 John Wiley & Sons, Ltd.     

p‐Coumaric acid and its conjugates: dietary sources,pharmacokinetic properties and biological activities

p‐Coumaric acid (4‐hydroxycinnamic acid) is a phenolic acid that has low toxicity in mice (LD₅₀ = 2850 mg kg^?1 body weight), serves as a precursor of other phenolic compounds, and exists either in free or conjugated form in plants. Conjugates of p‐coumaric acid have been extensively studied in recent years due to their bioactivities. In this review, the occurrence, bioavailability and bioaccessibility of p‐coumaric acid and its conjugates with mono‐, oligo‐ and polysaccharides, alkyl alcohols, organic acids, amine and lignin are discussed. Their biological activities, including antioxidant, anti‐cancer, antimicrobial, antivirus, anti‐inflammatory, antiplatelet aggregation, anxiolytic, antipyretic, analgesic, and anti‐arthritis activities, and their mitigatory effects against diabetes, obesity, hyperlipaemia and gout are compared. Cumulative evidence from multiple studies indicates that conjugation of p‐coumaric acid greatly strengthens its biological activities; however, the high biological activity but low absorption of its conjugates remains a puzzle. © 2015 Society of Chemical Industry     

Proteins interacting with Lin 1p,a putative link between chromosome segregation,mRNA splicing and DNA replication in Saccharomyces cerevisiae

Proteins involved in chromosome segregation during mitosis are likely to participate in other cell cycle-coordinated processes. Using a two-hybrid screen we identified a novel nuclear protein, Lin1, interacting with Irr1p/Scc3p, a component of the cohesin complex. The second round of two-hybrid assay with Lin1p as the bait resulted in the identification of six proteins: Prp8, Slx5, Siz2, Wss1, Rfc1 and YIL149w. These proteins have previously been shown to participate in mRNA splicing, DNA replication, chromosome condensation, chromatid separation and alternative cohesion. We propose that Lin1p may constitute a link among these processes.     

Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation

The Cdc48/p97 AAA-ATPase functions in membrane fusion and ubiquitin-dependent protein degradation. Here, we show that, in yeast, Cdc48p interacts with three novel proteins, Cuil-3p, which contain a conserved ubiquitin-related (UBX) domain. Cui2p and Cui3p are closely related, interact with each other, and are localized at the perinuclear membrane. Cdc48p binds directly the UBX domain of Cui3p in vitro. Multiple deletions of the CUI1, CUI2 and CUI3 genes confer deficiency in sporulation and degradation of model ubiquitin-protein fusions. The Cuil-3 proteins were also found to interact with Ufd3p, a WD repeat protein known to associate with Cdc48p. Together, these results indicate that the Cuil-3 proteins form complexes that are components of the ubiquitin-proteasome system.     

Chemical composition and antimicrobial activity of Anzer tea essential oil

Anzer tea (Thymus praecox, subsp. caucasicus var. caucasicus) naturally grows in the eastern Black Sea region of Turkey. Anzer tea, a creeping plant with crimson‐pink flowers, is important for honey production in the region. In the present study, content, composition and antimicrobial properties of Anzer tea's essential oil were investigated. Essential oil content of dried aerial plant parts varied between 1.53% and 2.05%. Essential oil composition was studied by means of gas chromatography–mass spectrometry, and 26 components were identified. The major components were thymol (47.45%), γ‐terpinene (8.73%), p‐cymene (8.30%), terpinyl acetate (4.88%) and carvacrol (4.66%). Essential oil was also screened for its antibacterial activity. In a screen for antibacterial activity, Anzer tea essential oil had significant activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Candida albicans. Copyright © 2007 Society of Chemical Industry     

p‐Coumaric acid in cereals: presence,antioxidant and antimicrobial effects

Coumaric acid is a hydroxy derivative of cinnamic acid and naturally occurs in three isomers (ortho‐, meta‐ and para‐); p‐coumaric acid is one of the most commonly occurring isomer in nature. p‐coumaric acid, classified as a phytochemical and nutraceutical, is found in various edible plants, such as carrots, tomatoes and cereals. p‐coumaric acid (4‐hydroxy‐cinnamic acid) occurs widely in the cell walls of graminaceous plants. It decreases low‐density lipoprotein (LDL) peroxidation, shows antioxidant and antimicrobial activities and plays an important role in human health. It is found in the endosperm of kernels at a limited level; however, the amount of p‐coumaric acid increases significantly in peripheral tissues. In terms of cereal types, it appears that pericarp fractions in barley, wheat, oat and corn are the fractions richest in p‐coumaric acid. It is both a good antioxidant and a good antimicrobial; therefore, it is natural alternative instead of synthetic additives, nowadays.