The Potential of Minor Ginsenosides Isolated from the Leaves of Panax ginseng as Inhibitors of Melanogenesis

Three minor ginsenosides, namely, ginsenoside Rh6 (1), vina-ginsenoside R4 (2) and vina-ginsenoside R13 (3), were isolated from the leaves of hydroponic Panax ginseng. The chemical structures were determined based on spectroscopic methods, including fast atom bombardment mass spectroscopy (FAB-MS), 1D-nuclear magnetic resonance (NMR), 2D-NMR, and, infrared (IR) spectroscopy. The melanogenic inhibitory activity of compounds 1, 2 and 3 was 23.9%, 27.8% and 35.2%, respectively, at a concentration of 80 µM. Likewise, the three compounds showed inhibitory activity on body pigmentation on a zebrafish model, which is commonly used as a model for biomedical or cosmetic research. These results from in vitro and in vivo systems suggest that the three aforementioned compounds isolated from Panax ginseng may have potential as new skin whitening compounds.     

A Simple Strategy for Development of Single Nucleotide Polymorphisms from Non-Model Species and Its Application in Panax

Single nucleotide polymorphisms (SNPs) are widely employed in the studies of population genetics, molecular breeding and conservation genetics. In this study, we explored a simple route to develop SNPs from non-model species based on screening the library of single copy nuclear genes (SCNGs). Through application of this strategy in Panax, we identified 160 and 171 SNPs from P. quinquefolium and P. ginseng, respectively. Our results demonstrated that both P. ginseng and P. quinquefolium possessed a high level of nucleotide diversity. The number of haplotype per locus ranged from 1 to 12 for P. ginseng and from 1 to 9 for P. quinquefolium, respectively. The nucleotide diversity of total sites (π_T) varied between 0.000 and 0.023 for P. ginseng and 0.000 and 0.035 for P. quinquefolium, respectively. These findings suggested that this approach is well suited for SNP discovery in non-model organisms and is easily employed in standard genetics laboratory studies.     

Full-Length cDNA Cloning,Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries

Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR) of 24 bp, a 3′-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.     

Molecular Cloning and Characterization of DXS and DXR Genes in the Terpenoid Biosynthetic Pathway of Tripterygium wilfordii

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879187","term_id":"871331037","term_text":"KM879187"}}KM879187), 2148 bp (TwDXS2, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879186","term_id":"871331021","term_text":"KM879186"}}KM879186), 1410 bp (TwDXR, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879185","term_id":"871330999","term_text":"KM879185"}}KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g⁻¹, a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f.     

Regulation of MIR Genes in Response to Abiotic Stress in Hevea brasiliensis

Increasing demand for natural rubber (NR) calls for an increase in latex yield and also an extension of rubber plantations in marginal zones. Both harvesting and abiotic stresses lead to tapping panel dryness through the production of reactive oxygen species. Many microRNAs regulated during abiotic stress modulate growth and development. The objective of this paper was to study the regulation of microRNAs in response to different types of abiotic stress and hormone treatments in Hevea. Regulation of MIR genes differs depending on the tissue and abiotic stress applied. A negative co-regulation between HbMIR398b with its chloroplastic HbCuZnSOD target messenger is observed in response to salinity. The involvement of MIR gene regulation during latex harvesting and tapping panel dryness (TPD) occurrence is further discussed.     

Cloning,Characterization and Expression Pattern Analysis of a Cytosolic Copper/Zinc Superoxide Dismutase (SaCSD1) in a Highly Salt Tolerant Mangrove (Sonneratia alba)

Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1) cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1). SaCSD1 comprised a complete open reading frame (ORF) of 459 bp which encoded a protein of 152 amino acids. Its mature protein is predicted to be 15.32 kDa and the deduced isoelectric point is 5.78. SaCSD1 has high sequence similarity (85%–90%) with the superoxide dismutase (CSD) of some other plant species. SaCSD1 was expressed with 30.6% yield regarding total protein content after being introduced into the pET-15b (Sma I) vector for expression in Rosetta-gami and being induced with IPTG. After affinity chromatography on Ni-NTA, recombinant SaCSD1 was obtained with 3.2-fold purification and a specific activity of 2200 U/mg. SaCSD1 showed good activity as well as stability in the ranges of pH between 3 and 7 and temperature between 25 and 55 °C. The activity of recombinant SaCSD1 was stable in 0.25 M NaCl, Dimethyl Sulphoxide (DMSO), glycerol, and chloroform, and was reduced to a great extent in β-mercaptoethanol, sodium dodecyl sulfate (SDS), H₂O_2, and phenol. Moreover, the SaCSD1 protein was very susceptive to pepsin digestion. Real-time Quantitative Polymerase Chain Reaction (PCR) assay demonstrated that SaCSD1 was expressed in leaf, stem, flower, and fruit organs, with the highest expression in fruits. Under 0.25 M and 0.5 M salt stress, the expression of SaCSD1 was down-regulated in roots, but up-regulated in leaves.     

A Novel Thylakoid Ascorbate Peroxidase from Jatrophacurcas Enhances Salt Tolerance in Transgenic Tobacco

Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. In the present study, a novel APX gene (JctAPX) was cloned from Jatropha curcas L. The deduced amino acid sequence was similar to that of APX of some other plant species. JctAPX has a chloroplast transit peptide and was localized to the chloroplasts by analysis with a JctAPX-green fluorescent protein (GFP) fusion protein. Quantitative polymerase chain reaction (qPCR) analysis showed that JctAPX was constitutively expressed in different tissues from J. curcas and was upregulated by NaCl stress. To characterize its function in salt tolerance, the construct p35S: JctAPX was created and successfully introduced into tobacco by Agrobacterium-mediated transformation. Compared with wild type (WT), the transgenic plants exhibited no morphological abnormalities in the no-stress condition. However, under 200 mM NaCl treatment, JctAPX over-expressing plants showed increased tolerance to salt during seedling establishment and growth. In addition, the transgenic lines showed higher chlorophyll content and APX activity, which resulted in lower H₂O₂ content than WT when subjected to 400 mM NaCl stress. These results suggest that the increased APX activity in the chloroplasts from transformed plants increased salt tolerance by enhancing reactive oxygen species (ROS)-scavenging capacity under short-term NaCl stress conditions.     

Towards the Identification of New Genes Involved in ABA-Dependent Abiotic Stresses Using Arabidopsis Suppressor Mutants of abh1 Hypersensitivity to ABA during Seed Germination

Abscisic acid plays a pivotal role in the abiotic stress response in plants. Although great progress has been achieved explaining the complexity of the stress and ABA signaling cascade, there are still many questions to answer. Mutants are a valuable tool in the identification of new genes or new alleles of already known genes and in elucidating their role in signaling pathways. We applied a suppressor mutation approach in order to find new components of ABA and abiotic stress signaling in Arabidopsis. Using the abh1 (ABA hypersensitive 1) insertional mutant as a parental line for EMS mutagenesis, we selected several mutants with suppressed hypersensitivity to ABA during seed germination. Here, we present the response to ABA and a wide range of abiotic stresses during the seed germination and young seedling development of two suppressor mutants—soa2 (suppressor of abh1 hypersensitivity to ABA 2) and soa3 (suppressor of abh1 hypersensitivity to ABA 3). Generally, both mutants displayed a suppression of the hypersensitivity of abh1 to ABA, NaCl and mannitol during germination. Both mutants showed a higher level of tolerance than Columbia-0 (Col-0—the parental line of abh1) in high concentrations of glucose. Additionally, soa2 exhibited better root growth than Col-0 in the presence of high ABA concentrations. soa2 and soa3 were drought tolerant and both had about 50% fewer stomata per mm² than the wild-type but the same number as their parental line—abh1. Taking into account that suppressor mutants had the same genetic background as their parental line—abh1, it was necessary to backcross abh1 with Landsberg erecta four times for the map-based cloning approach. Mapping populations, derived from the cross of abh1 in the Landsberg erecta background with each suppressor mutant, were created. Map based cloning in order to identify the suppressor genes is in progress.     

Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula

Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA⁻). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1–MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum.