Complete genome sequence of a putative novel alphaendornavirus isolated from Fagopyrum esculentum in South Korea.

The complete genome sequence of a putative new virus isolate, provisionally named "Fagopyrum esculentum endornavirus 2" (FeEV2), is 15,706 nucleotides long with a single, large open reading frame and a typical endornavirus genome organization. FeEV2 shares 19.4%-22.1% nucleotide sequence identity with other known endornavirus genome sequences. The putative polyprotein, RNA-dependent RNA polymerase (RdRp), helicase, and glycosyltransferase (GT) share 10.6%-24.3%, 30.4%-66.1%, 16.3%-45.7%, and 10.1%-21.6% amino acid sequence identity, respectively, with the homologous sequenced proteins from known endornaviruses. This suggests that it is a member of a new, distinct species. Phylogenetic analysis of RdRp sequences places FeEV2 with other Alphaendornavirus genus members (family Endornaviridae). This is the first report of the complete genome sequence of FeEV2, which was isolated from Fagopyrum esculentum in South Korea.

Temporally distinct regulatory pathways coordinate thermo-responsive storage organ formation in potato.

Tuberization is an important developmental process in potatoes, but it is highly affected by environmental conditions. Temperature is a major environmental factor affecting tuberization, with high temperatures suppressing tuber development. However, the temporal aspects of thermo-responsive tuberization remain elusive. In this study, we show that FT homolog StSP6A is suppressed by temporally distinct regulatory pathways. Experiments using StSP6A-overexpressing plants show that post-transcriptional regulation plays a major role at the early stage, while transcriptional regulation is an important late-stage factor, suppressing StSP6A at high temperatures in leaves. Overexpression of StSP6A in leaves restores tuber formation but does not recover tuber yield at the late stage, possibly because of suppressed sugar transport at high temperatures. Transcriptome analyses lead to the identification of potential regulators that may be involved in thermo-responsive tuberization at different stages. Our work shows that potato has temporally distinct molecular mechanisms that finely control tuber development at high temperatures.

Complete genome sequence of artemisia virus B, a new polerovirus infecting Artemisia princeps in South Korea.

The complete genome sequence of a new polerovirus found naturally infecting Artemisia princeps, artemisia virus B (ArtVB), was determined using high-throughput sequencing. The ArtVB genome comprises 6,141 nucleotides and contains six putative open reading frames (ORF0 to ORF5) with a genome structure typical of poleroviruses. A multiple sequence alignment showed that the complete ArtVB genome shares 50.98% nucleotide sequence identity with ixeridium yellow mottle virus 1 (IxYMaV-1, GenBank accession no. KT868949). ArtVB shares the highest amino acid sequence identity in P0 and P3-P5 (21.54%-51.69%) with other known poleroviruses. Phylogenetic analysis indicated that ArtVB should be considered a member of a new species within the genus Polerovirus, family Luteoviridae.

High-throughput sequencing and de novo assembly of Brassica oleracea var. Capitata L. for transcriptome analysis.

BACKGROUND: The cabbage, Brassica oleracea var. capitata L., has a distinguishable phenotype within the genus Brassica. Despite the economic and genetic importance of cabbage, there is little genomic data for cabbage, and most studies of Brassica are focused on other species or other B. oleracea subspecies. The lack of genomic data for cabbage, a non-model organism, hinders research on its molecular biology. Hence, the construction of reliable transcriptomic data based on high-throughput sequencing technologies is needed to enhance our understanding of cabbage and provide genomic information for future work. METHODOLOGY/PRINCIPAL FINDINGS: We constructed cDNAs from total RNA isolated from the roots, leaves, flowers, seedlings, and calcium-limited seedling tissues of two cabbage genotypes: 102043 and 107140. We sequenced a total of six different samples using the Illumina HiSeq platform, producing 40.5 Gbp of sequence data comprising 401,454,986 short reads. We assembled 205,046 transcripts (≥ 200 bp) using the Velvet and Oases assembler and predicted 53,562 loci from the transcripts. We annotated 35,274 of the loci with 55,916 plant peptides in the Phytozome database. The average length of the annotated loci was 1,419 bp. We confirmed the reliability of the sequencing assembly using reverse-transcriptase PCR to identify tissue-specific gene candidates among the annotated loci. CONCLUSION: Our study provides valuable transcriptome sequence data for B. oleracea var. capitata L., offering a new resource for studying B. oleracea and closely related species. Our transcriptomic sequences will enhance the quality of gene annotation and functional analysis of the cabbage genome and serve as a material basis for future genomic research on cabbage. The sequencing data from this study can be used to develop molecular markers and to identify the extreme differences among the phenotypes of different species in the genus Brassica.

Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene.

Paclitaxel is produced by various species of yew trees and has been extensively used to treat tumors. In our research, a taxadiene synthase (TS) gene from Taxus brevifolia was used to transform the roots of cultured ginseng (Panax ginseng C.A. Meyer) to produce taxadiene, the unique skeletal precursor to taxol. The TS gene was successfully introduced into the ginseng genome, and the de novo formation of taxadiene was identified by mass spectroscopy profiling. Without any change in phenotypes or growth difference in a TS-transgenic ginseng line, the transgenic TSS3-2 line accumulated 9.1 µg taxadiene per gram of dry weight. In response to the treatment of methyl jasmonate for 3 or 6 days, the accumulation was 14.6 and 15.9 µg per g of dry weight, respectively. This is the first report of the production of taxadiene by engineering ginseng roots with a taxadiene synthase gene.

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants.

Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 µM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 µM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.

Enhanced tolerance to methyl viologen-induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD, APX and NDPK2 genes.

Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)-induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non-transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV-treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses.

Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection against various abiotic stresses.

Plants synthesize compatible solutes such as glycinebetaine (GB) in response to abiotic stresses. To evaluate the synergistic and protective effect of GB, transgenic potato plants expressing superoxide dismutase (SOD) and ascorbate peroxidase (APX) targeting to chloroplasts (referred to as SSA plants) were retransformed with a bacterial choline oxidase (codA) gene to synthesize GB in chloroplast in naturally occurring non-accumulator potato plants (including SSA) under the control of the stress-inducible SWPA2 promoter (referred to as SSAC plants). GB accumulation resulted in enhanced protection of these SSAC plants and lower levels of H(2)O(2) compared with SSA and non-transgenic (NT) plants after methyl viologen (MV)-mediated oxidative stress. Additionally, SSAC plants demonstrated synergistically enhanced tolerance to salt and drought stresses at the whole-plant level. GB accumulation in SSAC plants helped to maintain higher activities of SOD, APX and catalase following oxidative, salt and drought stress treatments than is observed in SSA and NT plants. Conclusively, GB accumulation in SSAC plants along with overexpression of antioxidant genes rendered the plants tolerant to multiple environmental stresses in a synergistic fashion.

A novel pepper (Capsicum annuum) receptor-like kinase functions as a negative regulator of plant cell death via accumulation of superoxide anions.

Plant receptor-like kinases belong to a large gene family. The Capsicum annuum receptor-like kinase 1 (CaRLK1) gene encodes a transmembrane protein with a cytoplasmic kinase domain and an extracellular domain. The CaRLK1 extracellular domain (ECD)-green fluorescent protein (GFP) fusion protein was targeted to the plasma membrane, and the kinase domain of the CaRLK1 protein exhibited autophosphorylation activity. CaRLK1 transcripts were more strongly induced in treatment with Xag8ra than in treatment with Xag8-13. Furthermore, infection with incompatible Xanthomonas campestris pv. vesicatoria race 3 induced expression of CaRLK1 more strongly than in the compatible interaction. Cell death caused by both a disease-forming and an HR-inducing pathogen was delayed in the CaRLK1-transgenic plants. Ectopic expression of CaRLK1 also induced transcripts of the lesion stimulating disease (LSD) gene, a negative regulator of cell death. Respiratory burst oxidase homolog (RBOH) genes were up-regulated in the transgenic plants compared with the wild type, as the concentration of the superoxide anion was increased. In contrast, the concentration of H(2)O(2) did not differ between the transgenic and wild-type plants. These results support the theory that the suppression of plant cell death by CaRLK1 is associated with consistent production of the superoxide anion and induction of the RBOH genes and the LSD gene, but not with the concentration of H(2)O(2). Thus, CaRLK1 may be a receptor of an as yet unidentified pathogen molecular pattern and may function as a negative regulator of plant cell death.

Enhanced tolerance of transgenic potato plants overexpressing nucleoside diphosphate kinase 2 against multiple environmental stresses.

In plants, nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes. In this study, we developed transgenic potato plants (Solanum tuberosum L. cv. Atlantic) expressing Arabidopsis NDPK2 (AtNDPK2) gene in cytosols under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SN plants) or enhanced CaMV 35S promoter (EN plants) and evaluated their tolerance to various environmental stress, including methyl viologen (MV)-mediated oxidative stress, high temperature, and salt stress. When 250 muM MV was sprayed to whole plants, plants expressing NDPK2 showed significantly an enhanced tolerance compared to non-transgenic (NT) plants. SN plants and EN plants showed 51% and 32% less visible damage than NT plants, respectively. Transcript level of AtNDPK2 gene and NDPK2 activity in SN plants following MV treatment well reflected the plant phenotype. Ascorbate peroxidase (APX) activity was also increased in MV-treated SN plants. In addition, SN plants showed enhanced tolerance to high temperature at 42 degrees C. The photosynthetic activity of SN plants after treatment of high temperature was decreased by about 10% compared to the plants grown at 25 degrees C, whereas that of NT plants declined by 30%. When treated with 80 mM NaCl onto the plantlets, both SN plants and EN plants also showed a significant reduced damage in root growth. These results indicate that overexpression of NDPK2 under the stress-inducible SWPA2 promoter might efficiently regulate the oxidative stress derived from various environmental stresses.

Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses.

Transgenic potato plants (Solanum tuberosum L. cv. Superior) with the ability to synthesize glycinebetaine (GB) in chloroplasts (referred to as SC plants) were developed via the introduction of the bacterial choline oxidase (codA) gene under the control of an oxidative stress-inducible SWPA2 promoter. SC1 and SC2 plants were selected via the evaluation of methyl viologen (MV)-mediated oxidative stress tolerance, using leaf discs for further characterization. The GB contents in the leaves of SC1 and SC2 plants following MV treatment were found to be 0.9 and 1.43 micromol/g fresh weight by HPLC analysis, respectively. In addition to reduced membrane damage after oxidative stress, the SC plants evidenced enhanced tolerance to NaCl and drought stress on the whole plant level. When the SC plants were subjected to two weeks of 150 mM NaCl stress, the photosynthetic activity of the SC1 and SC2 plants was attenuated by 38 and 27%, respectively, whereas that of non-transgenic (NT) plants was decreased by 58%. Under drought stress conditions, the SC plants maintained higher water contents and accumulated higher levels of vegetative biomass than was observed in the NT plants. These results indicate that stress-induced GB production in the chloroplasts of GB non-accumulating plants may prove useful in the development of industrial transgenic plants with increased tolerance to a variety of environmental stresses for sustainable agriculture applications.

Molecular characterization of a cDNA encoding DRE-binding transcription factor from dehydration-treated fibrous roots of sweetpotato.

A new dehydration responsive element-binding (DREB) protein gene encoding for an AP2/EREBP-type transcription factor was isolated by screening of the cDNA library for dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Its cDNA (referred to as swDREB1) fragment of 1206bp was sequenced from, which a 257 amino acid residue protein was deduced with a predicted molecular weight of 28.17kDa. A search of the protein BLAST database revealed that this protein can be classified as a typical member of a DREB subfamily. RT-PCR and northern analyses revealed diverse expression patterns of the swDREB1 gene in various tissues of intact sweetpotato plant, and in leaves and fibrous roots exposed to different stresses. The swDREB1 gene was highly expressed in stems and tuberous roots. In fibrous roots, its mRNA accumulation profiles clearly showed strong expression under various abiotic stress conditions such as dehydration, chilling, salt, methyl viologen (MV), and cadmium (Cd) treatment, whereas it did not respond to abscisic acid (ABA) or copper (Cu) treatment. The above results indicate that swDREB1 may be involved in the process of the plant response to diverse abiotic stresses through an ABA-independent pathway.

Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature.

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. In order to develop transgenic potato plants with enhanced tolerance to environmental stress, the genes of both Cu/Zn superoxide dismutase and ascorbate peroxidase were expressed in chloroplasts under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants). SSA plants showed enhanced tolerance to 250 microM methyl viologen, and visible damage in SSA plants was one-fourth that of non-transgenic (NT) plants that were almost destroyed. In addition, when SSA plants were treated with a high temperature of 42 degrees C for 20 h, the photosynthetic activity of SSA plants decreased by only 6%, whereas that of NT plants decreased by 29%. These results suggest that the manipulation of the antioxidative mechanism of the chloroplasts may be applied in the development of industrial transgenic crop plants with increased tolerance to multiple environmental stresses.

Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta.

Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.

Transgenic ginseng cell lines that produce high levels of a human lactoferrin.

In order to produce a human lactoferrin (hLf) protein in cultured plant cells, we developed Korean ginseng (Panax ginseng) cell line using an oxidative stress-inducible peroxidase (SWPA2) promoter and characterized the production of human lactoferrin in cultured cells. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to human lactoferrin cDNA under the control of SWPA2 promoter was engineered. Transgenic Korean ginseng cell lines that produced a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analyses. Western blot and ELISA analyses showed that hLf protein was synthesized in the transgenic cells. The production of hLf showed a maximal level (up to 3.0% of total soluble protein) in the stationary phase of callus cultures. These results suggest that the transgenic cell lines in this study will be biotechnologically useful for the commercial production of hLf protein in cell cultures, with no need for purification.