Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.).

BACKGROUND: 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway, and is the target of the herbicide glyphosate. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the allohexaploid wheat EPSPS gene were cloned and characterized. METHODS: Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated using PCR and inverse PCR approaches from soft white spring "Louise'. Development of genome-specific primers allowed the mapping and expression analysis of TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1 on chromosomes 7A, 7D, and 4A, respectively. Sequence alignments of cDNA sequences from wheat and wheat relatives served as a basis for phylogenetic analysis. RESULTS: The three genomic copies of wheat EPSPS differed by insertion/deletion and single nucleotide polymorphisms (SNPs), largely in intron sequences. RT-PCR analysis and cDNA cloning revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at much lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Phylogenetic analysis of 1190-bp cDNA clones from wheat and wheat relatives revealed that: 1) TaEPSPS-7A1 is most similar to EPSPS from the tetraploid AB genome donor, T. turgidum (99.7 % identity); 2) TaEPSPS-7D1 most resembles EPSPS from the diploid D genome donor, Aegilops tauschii (100 % identity); and 3) TaEPSPS-4A1 resembles EPSPS from the diploid B genome relative, Ae. speltoides (97.7 % identity). Thus, EPSPS sequences in allohexaploid wheat are preserved from the most two recent ancestors. The wheat EPSPS genes are more closely related to Lolium multiflorum and Brachypodium distachyon than to Oryza sativa (rice). CONCLUSIONS: The three related EPSPS homoeologues of wheat exhibited conservation of the exon/intron structure and of coding region sequence, but contained significant sequence variation within intron regions. The genome-specific primers developed will enable future characterization of natural and induced variation in EPSPS sequence and expression. This can be useful in investigating new causes of glyphosate herbicide resistance.

In vitro inhibitory effects on free radicals, pigmentation, and skin cancer cell proliferation from Dendrobium hybrid extract: A new plant source of active compounds.

Orchidaceae are diverse plants whose bioactive compounds have various biological activities. New hybrids of Dendrobium have been generated to gain characteristics shared with their ancestors. Dendrobium Pearl Vera (designated as DH) is derived from parents used for dermatological treatments and cosmetics. However, the phytoconstituents and biological properties of DH have not been reported. The current study investigated extracts from DH plants using four solvents (water, methanol, ethanol, or 2-propanol). The propanolic extract (DH-P) contained the highest phenolic and flavonoid contents, along with a high scavenging performance for free radicals. In total, 25 tentative constituents in the DH-P matrix were identified, consisting of amino acids, nucleotides, and three types of secondary metabolites: furan, phenolics, and alkaloids. The DH-P inhibited human tyrosinase in vitro in a concentration-dependent manner of the phenolic content. Furthermore, there was no significant difference between DH-P with 10 µg/ml phenolic content and 0.75 mM kojic acid (a commercial whitening agent) on the inhibition of human tyrosinase. Incubation with DH-P containing at least 15 µg/ml phenolic content greatly inhibited the proliferation of human melanoma; however, the cell viability was not affected by the phenolic content at 5 µg/ml or less. The half-maximal inhibitory concentration (IC50) of the phenolic content in DH-P on melanoma viability was 12.90 ± 1.04 µg/ml. Melanin production in vivo by human melanoma incubated with 5 µg/ml phenolic content in DH-P was reduced significantly, compared to 2.5 µg/ml phenolic content in DH-P, 100 µg/ml arbutin, and in control. The identified components, including 5-hydroxymethyl-2-furaldehyde, salicylic acid, nicotinamide, acetophenone, cytidine, adenosine, proline, or valine, have been reported to be associated with depigmentation, antioxidant, and anticancer. This research revealed, for the first time, the tentative phytoconstituents of Dendrobium Pearl Vera and their biological activities, thus demonstrating the potential use of DH-P in dermal applications.

Nutritional Profiles, Phytochemical Analysis, Antioxidant Activity and DNA Damage Protection of Makapuno Derived from Thai Aromatic Coconut.

Makapuno is a natural mutant coconut cultivar with jelly-like endosperm. Here, we investigated the nutritional compositions, active ingredients, and antioxidant activities of Makapuno meat and water. The contents of macronutrients, sugars, vitamins, amino acids, and fatty acids were reported. We found that Makapuno meat has higher dietary fiber with lower protein and fat content compared to normal coconut meat. Medium-chain fatty acids were the major fat component of Makapuno meat and water. Phytochemical analysis revealed that while flavonoid content was lower, the total phenolic, alkaloid, and tannin contents of Makapuno meat were comparable with those of mature coconut. However, Makapuno water contained higher alkaloid content when compared to mature and young coconuts. The antioxidant activities, as examined by DPPH, FRAP, and ABTS assays, showed that Makapuno meat and water had antioxidant activities, and Makapuno water exhibited protective activity against DNA damage. Hence, this research provides the nutraceutical importance of Makapuno, which could be used in the food industry.