A new gene coding for <Emphasis Type="Italic">p</Emphasis>-coumarate 3-hydroxylase from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

p-Coumarate 3-hydroxylase (C3H) is a rate-limiting enzyme involved in monolignol biosynthesis. The full-length cDNA from Ginkgo biloba and genomic DNA sequence encoding C3H (designated as GbC3H) were cloned and characterized for the first time by rapid amplification of cDNA ends technique. The full-length cDNA of GbC3H was of 1860 bp containing a 1527 bp open reading frame encoding a cytochrome P450 protein of 508 amino acids with a calculated mol wt of 57.46 kD and an isoelectric point of 7.09. Two introns were present in the GbC3H gene. Comparative and bioinformatic analyses revealed that GbC3H had close similarity with C3Hs from other species and contained a conserved cytochrome P450 cysteine heme-iron ligand signature. Phylogenetic analysis indicated that GbC3H shared a common evolutionary origin based on sequence and had the closest relationship to C3H from gymnosperm species. Southern blot analysis indicated that GbC3H belonged to a small-gene family. Tissue expression pattern analysis revealed the highest expression of GbC3H in roots followed by leaves, and no expression was detected in stems. Only a few proteins of this class have been found, so the cloning and characterization of GbC3H will be useful in understanding the role of C3Hs in the lignin biosynthesis at the molecular level. This text was submitted by the authors in English.     

Cloning,characterization and functional analysis of a flavonol synthase from <Emphasis Type="Italic">Vaccinium corymbosum</Emphasis>

Key message

VcFLS from Vaccinium corymbosum promoted myricetin biosynthesis in Arabidopsis thaliana and VcFLS expression was induced by salicylic acid.

Abstract

Flavonoids are polyphenols with important functions in pigmentation, UV filtration, and symbiotic nitrogen fixation. Flavonols are a class of flavonoids that are produced by the desaturation of dihydroflavanols in a reaction that is catalyzed by flavonol synthase (FLS). In the study reported here, we cloned the full-length cDNA of FLS (designated as VcFLS) from Vaccinium corymbosum (blueberry) using rapid amplification of cDNA ends (RACE). The cDNA contained a 1005-bp open reading frame that encoded a 334-amino acid protein. Phylogenetic analysis showed that VcFLS was closely related to FaFLS, a flavonol synthase that catalyzed the formation of kaempferol and had little effect on the formation of quercetin. Quantitative RT-PCR analysis demonstrated that VcFLS was expressed in all of the tissues tested, with particularly high expression in the petals and young leaves (both green and red). The flavanols myricetin and quercetin also occurred in all of these tested tissues, with the highest levels detected in mature leaves. The expression of VcFLS was not consistent with the accumulation of quercetin and myricetin in different tissues, nor were the expressions of VcFLS, VcPAL, VcCHS, VcF3H, and VcF3′5′H consistent with the accumulation of the quercetin during fruit development. However, the change in the trend of VcCHS and VcF3H expression was similar with myricetin accumulation during fruit development. Expression profiling analysis revealed that VcFLS expression was induced by salicylic acid, a phytohormone involved in plant defense against pathogens, and was suppressed by gibberellic acid, a phytohormone involved in various aspects of plant development. Heterologous expression of VcFLS in Arabidopsis thaliana increased the content of myricetin, but did not affect quercetin content. Thus, we conclude that VcFLS is a key enzyme in the flavonol biosynthetic pathway and would appear to be involved in the plant defense response.

     

<Emphasis Type="Italic">Streptomyces ginkgonis</Emphasis> sp. nov., an endophyte from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

A novel endophytic actinomycete strain, designated KM-1-2^T, was isolated from seeds of Ginkgo biloba at Yangling, China. A polyphasic approach was used to study the taxonomy of strain KM-1-2^T and it was found to show a range of phylogenetic and chemotaxonomic properties consistent with those of members of the genus Streptomyces. The diamino acid of the cell wall peptidoglycan was identified as LL-diaminopimelic acid. No diagnostic sugars were detected in whole cell hydrolysates. The predominant menaquinones were identified as MK-9(H₆) and MK-9(H₈). The diagnostic phospholipids were found to be phosphatidylethanolamine and phosphatidylcholine. The DNA G + C content of the novel strain was determined to be 72.9 mol%. The predominant cellular fatty acids (> 10.0?%) were identified as iso-C_14?:?0, iso-C_16?:?0, C_16?:?0 and C_17?:?0 cyclo. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is closely related to Streptomyces carpaticus JCM 6915^T (99.3%), Streptomyces harbinensis DSM 42076^T (98.9%) and Streptomyces cheonanensis JCM 14549^T (98.5%). DNA-DNA hybridizations with these three close relatives gave similarity values of 39.1 ± 1.9, 35.8 ± 2.3, and 47.4 ± 2.7%, respectively, which indicated that strain KM-1-2^T represents a novel species of the genus Streptomyces. This is consistent with the morphological, physiological and chemotaxonomic data. Cumulatively, these data suggest that strain KM-1-2^T represents a novel Streptomyces species, for which the name Streptomyces ginkgonis sp. nov. is proposed, with the type strain KM-1-2^T (= CCTCC AA2016004^T = KCTC 39801^T).     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">polyfermenticus</Emphasis> isolated from <Emphasis Type="Italic">Meju,</Emphasis> Korean soybean fermentation starter

Bacteria of the Bacillus species have been reported as an important microorganism in fermented soybean products. In the present study, thirty Bacillus isolates were screened from Meju, a Korean soybean fermentation starter. The comparative analysis of 16S rDNA sequences, 16S-23S internal transcribed spacer sequences, phenotypic, and biochemical characterizations revealed three phylogenetically distinct groups namely Bacillus atrophaeus, Bacillus polyfermenticus and Bacillus subtilis. The isolates were assayed for poly-γ-glutamate production and fibrinolytic activity. Among the isolates, B. polyfermenticus exhibited maximum poly-γ-glutamate production and fibrinolytic activity. Moreover, the soybean products fermented by B. polyfermenticus have increased the time taken for coagulation and hemorrhage in mice. The results of the present study clearly indicate the functional role of B. polyfermenticus in fermented soybean products.     

Isolation and identification of endophytic fungus SX01, a red pigment producer from <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L

Fungal pigments are a potential resource as natural food colorant. Endophytic fungus SX01, which is able to produce abundant soluble red pigments, was isolated from the twigs of Ginkgo biloba L. For further research and utilization of this strain and its secondary metabolites, morphological and molecular characteristics of SX01 was identified. Morphological identification which employed light microscope and scanning electron microscope showed that SX01 was a species of genera Penicillium. ITS1-5.8S-ITS2 region and 18S rRNA gene were then cloned, and the molecular phylogeny of these two sequences proved that strain SX01 was Penicillium purpurogenum.     

A simple method for cryopreservation of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> callus

Two-years-old Ginkgo biloba cell culture initiated from cotyledonary explants was cryopreserved by a simple desiccation method. Preliminary incubation of callus clumps on MS preculture medium supplemented with 100 g l⁻¹ sucrose and 2 mg l⁻¹ ABA for 7 and 14 days resulted in accumulation of endogenous soluble sugars and was essential for cell culture post-cryopreservation survival. The optimal time for the preculture on sucrose-and-ABA containing medium was found to be 14 days. The sufficient desiccation duration was determined as 150 min. FCM profiles of calli maintained for 2 years remained stable and were not affected by cryopreservation.