Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in Φ_PSII and q _L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.     

Production of glycyrrhizin in callus cultures of licorice

Licorice plants, Glycyrrhiza glabra, G. uralensis, and G. inflata, were investigated for callus induction using Murashige and Skoog (MS) medium combined with auxins and cytokinins. After 4 weeks of culture, 33-100% of leaf or stem explants formed calli. Maximum of shoot induction from callus cultures was achieved by G. inflata stem explants cultured on MS medium supplemented with 1 mg/l alpha-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzyladenine (BA) (67%) which also gave maximum shoot formation per explant (two shoots per explant). These results indicated that all three Glycyrrhiza species regenerated shoots from callus cultures on MS medium combined with NAA and BA or only thidiazuron (TDZ; 0.1 and 0.5 mg/l). Glycyrrhizin contents of G. uralensis calli induced using MS medium in combination with NAA and BA [(27.60 +/- 8.47) microg/g DW] or TDZ alone [(36.52 +/- 2.45) microg/ g DW] were higher than those found in other combinations.     

Profound difference of metabolic pharmacokinetics between pure glycyrrhizin and glycyrrhizin in licorice decoction

To investigate the difference of metabolic pharmacokinetics between pure glycyrrhizin (GZ) and GZ in licorice decoction, six New Zealand White rabbits were orally given pure GZ and licorice decoction containing equivalent content of GZ in a randomized crossover design. HPLC methods were used for the quantitation of GZ and glycyrrhetic acid (GA) in serum. The results indicated that the areas under curves (AUCs) of GZ and GA after administration of licorice decoction were significantly higher than those after pure GZ. This result was contradictory with that obtained in rats. To explore the mechanism of the pharmacokinetic difference, feces of rabbits, rats, pigs and humans were used to investigate the presystemic metabolism of pure GZ and GZ in licorice decoction. The results indicated that pure GZ was hydrolyzed to GA more rapidly and to a greater extent than that in licorice decoction by various feces. In addition, when pure GZ was fermented, the metabolic profiles of GA and 3-dehydroGA in rabbit feces were quite different from other feces. In conclusion, the bioavailabilities of GZ and GA are significantly better from licorice than from pure GZ in rabbits but the presystemic metabolism of pure GZ in rabbit is rather different from that in rat, pig and human.     

Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.     

A plant-transformation-competent BIBAC library of ginseng (Panax ginseng C. A. Meyer) for functional genomics research and characterization of genes involved in ginsenoside biosynthesis

Ginseng (Panax ginseng C. A. Mey.) is widely used as a major medicinal herb and as a feedstock for the medicine, beverage, food, cosmetic, etc. industries, in China and several other Asian countries. However, limited research has been accomplished into its genetics, genomics and breeding. To clone, characterize and utilize the genes of economic importance in the species, we have developed a large-insert plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library for Jilin ginseng cv. Damaya. The library contains 141,312 clones, with an average insert size of 110 kb, each likely containing approximately 20–30 genes. The clones of the library have all been arrayed in 384-well microplates and permanently archived. We screened the library and identified BIBAC clones containing nine genes likely involved in the biosynthesis pathway of ginsenosides—the major medicinally effective compounds of ginseng—with approximately four BIBACs per gene. This result further verified the quality of the library and demonstrated its utility in cloning, characterization and utilization of economically important genes in ginseng. Furthermore, since the library is cloned in a plant-transformation-competent BIBAC vector (pCLD04541) that can be directly transformed in a variety of plants via both the Agrobacterium-mediated method and the particle bombardment method, we have also demonstrated the stability of large-insert ginseng DNA BIBACs in different Agrobacterium strains, which is crucial to large-insert BIBAC transformation in plants. Therefore, the Jilin ginseng BIBAC library provides resources and tools useful for functional genomics research, and cloning, characterization and utilization of economically important genes in the species.     

香蕉品质相关功能基因组学的研究进展

香蕉是重要的热带水果之一,是热区人民脱贫致富的主要经济来源。香蕉品质一直是人们关注的焦点。文章综述了近年来香蕉果实品质如成熟、软化、糖代谢及香气相关功能基因分离和鉴定等方面的最新研究进展,将有助于从源头上对香蕉进行创新性的探索与研究,支撑香蕉品质改良和新品种培育。     

Research progress on banana functional genomics involved in fruit quality

Banana is one of the most important tropical fruits and main economical resource for tropical people. Banana quality is always becoming a focus for people to follow with interest. Here, we reviewed recent research progresses on isolation and identification of banana genes involved in fruit quality such as ripening, softening, glycometabolism, and scent, which will help us explore their functions and facilitate banana quality improvement.     

The use of functional genomics for the identification of a gene cluster encoding for the biosynthesis of an antifungal tambjamine in the marine bacterium Pseudoalteromonas tunicata

A large insert library was created in Escherichia coli from the DNA of the surface-associated marine bacterium Pseudoalteromonas tunicata. Screening of the library for antifungal activity resulted in the detection and identification of a large gene cluster encoding for the biosynthesis of an antifungal tambjamine. A biosynthetic pathway has been proposed based on analysis and annotation of the gene cluster.     

Functional identification of rub52 gene involved in the biosynthesis of rubradirin

An open reading frame, rub52, has been identified as a gene encoding thymidine diphospho-glucose 2,3-dehydratase by sequence analysis of the rubradirin biosynthetic gene cluster of Streptomyces achromogenes var. rubradiris NRRL3061.The gene codes for a protein consisting of 458 amino acids with calculated molecular mass of 50862 Da. The gene was amplified and heterologously expressed in Escherichia coli as a soluble His-tagged fusion protein. C-2 deoxygenation functionality of thymidine diphospho-4-keto-6-deoxyglucose was assigned to the rub52 gene product from in vitro enzyme assay.     

A factor analysis model for functional genomics

Background  

Expression array data are used to predict biological functions of uncharacterized genes by comparing their expression profiles to those of characterized genes. While biologically plausible, this is both statistically and computationally challenging. Typical approaches are computationally expensive and ignore correlations among expression profiles and functional categories.     

Characterization and functional identification of a novel plant 4,5-extradiol dioxygenase involved in betalain pigment biosynthesis in Portulaca grandiflora

Betalains are pigments that replace anthocyanins in the majority of families of the plant order Caryophyllales. Betalamic acid is the common chromophore of betalains. The key enzyme of the betalain biosynthetic pathway is an extradiol dioxygenase that opens the cyclic ring of dihydroxy-phenylalanine (DOPA) between carbons 4 and 5, thus producing an unstable seco-DOPA that rearranges nonenzymatically to betalamic acid. A gene for a 4,5-DOPA-dioxygenase has already been isolated from the fungus Amanita muscaria, but no homolog was ever found in plants. To identify the plant gene, we constructed subtractive libraries between different colored phenotypes of isogenic lines of Portulaca grandiflora (Portulacaceae) and between different stages of flower bud formation. Using in silico analysis of differentially expressed cDNAs, we identified a candidate showing strong homology at the level of translated protein with the LigB domain present in several bacterial extradiol 4,5-dioxygenases. The gene was expressed only in colored flower petals. The function of this gene in the betalain biosynthetic pathway was confirmed by biolistic genetic complementation in white petals of P. grandiflora genotypes lacking the gene for color formation. This gene named DODA is the first characterized member of a novel family of plant dioxygenases phylogenetically distinct from Amanita sp. DOPA-dioxygenase. Homologs of DODA are present not only in betalain-producing plants but also, albeit with some changes near the catalytic site, in other angiosperms and in the bryophyte Physcomitrella patens. These homologs are part of a novel conserved plant gene family probably involved in aromatic compound metabolism.     

Structural and functional analysis of validoxylamine A 7'-phosphate synthase ValL involved in validamycin A biosynthesis

Validamycin A (Val-A) is an effective antifungal agent widely used in Asian countries as crop protectant. Validoxylamine A, the core structure and intermediate of Val-A, consists of two C(7)-cyclitol units connected by a rare C-N bond. In the Val-A biosynthetic gene cluster in Streptomyces hygroscopicus 5008, the ORF valL was initially annotated as a validoxylamine A 7'-phosphate(V7P) synthase, whose encoded 497-aa protein shows high similarity with trehalose 6-phosphate(T6P) synthase. Gene inactivation of valL abolished both validoxylamine A and validamycin A productivity, and complementation with a cloned valL recovered 10% production of the wild-type in the mutant, indicating the involvement of ValL in validoxylamine A biosynthesis. Also we determined the structures of ValL and ValL/trehalose complex. The structural data indicates that ValL adopts the typical fold of GT-B protein family, featuring two Rossmann-fold domains and an active site at domain junction. The residues in the active site are arranged in a manner homologous to that of Escherichia coli (E.coli) T6P synthase OtsA. However, a significant discrepancy is found in the active-site loop region. Also noticeable structural variance is found around the active site entrance in the apo ValL structure while the region takes an ordered configuration upon binding of product analog trehalose. Furthermore, the modeling of V7P in the active site of ValL suggests that ValL might have a similar SNi-like mechanism as OtsA.