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Pyropia yezoensis is the most widely cultivated and economically important marine red alga. However, due to its simple morphology, it is not easy to identify P. yezoensis cultivars by phenotype. In the present study, simple sequence repeats (SSRs) were identified in the scaffold-genome sequence of P. yezoensis cultivar SG104. These SSRs were used to develop SSR markers that could be used to study genetic diversity of P. yezoensis and identify P. yezoensis cultivars. A total of 23,120 SSRs that contained di-, tri-, tetra-, penta-, or hexa-nucleotide repeat motifs were identified. Of the 353 SSR markers that were selected for further analysis, 12 SSRs were selected for their ability to distinguish cultivars and assess cultivar genetic diversity. The 12 SSR loci yielded a total of 30 alleles, ranging from two to three alleles per locus. With the exception of cultivars HP2 and HG, all cultivars generated a single allele for each SSR locus. As such, these results demonstrate that these cultivars are double haploid lines. Cluster and STRUCTURE analyses of the SSR marker data indicated that the studied cultivars could be separated into two main clusters, which have different blade types. These SSR markers will be useful for assessing the genetic diversity of P. yezoensis germplasm collections and can be used for the identification and future registration of new cultivars. The findings of the present study will facilitate the fine genetic mapping and quantitative trait loci analysis of P. yezoensis, and, as such, may provide a framework for the molecular breeding of new P. yezoensis cultivars.

Mycosporine-like amino acids (MAAs) are small secondary metabolites produced by some marine species. These compounds absorb ultraviolet (UV) light, typically between 310 and 362 nm, and protect the producers from UV-associated damage. They also have anti-photoaging, anticancer, and anti-inflammatory properties, which has generated considerable interest in the cosmetic, pharmaceutical, biotechnology, and materials fields. Commercial use has been limited due to its reliance marine organisms for production, resulting in low and inconsistent yields. This study was undertaken to increase production of MAAs. We generated transgenic lines of the Nannochloropsis salina that carry and express the MAA biosynthesis genes from red alga Pyropia yezoensis. We characterized their MAA yield and the properties of MAAs produced by these lines. When exposed to UV light, the transgenic lines generated fewer reactive oxygen species compared to wild-type. The yield of porphyra-334 in one of the transgenic lines was 25 mg g−1 dry cell weight, the greatest reported. We hope these results will support the use of MAAs in numerous applications and increase our understanding of their biosynthetic pathways.

Mutation in the human MPV17 gene or the functional yeast orthologue SYM1 result in mitochondrial DNA depletion. MPV17 homologs are also found in plants including Arabidopsis, but the function of these genes remain unclear. Arabidopsis genome contains 10 MPV17 homologs. Among these, the AtMPV17 protein was localized in mitochondria as MPV17 and SYM1. The yeast sym1 knock out mutant cannot grow on ethanol-containing medium at 37 °C. AtMPV17 complements the ethanol growth defection of sym1 yeast MPV17 ortholog cells at 37 °C, suggesting that AtMPV17 is a functional ortholog of SYM1. AtMPV17 knock out mutant, atmpv17 show similar growth and seed development to those of the wild-type plant on normal growth condition. However, atmpv17 mutant is more sensitive to ABA and mannitol during germination and seedling growth than wild type plants. Growth retardation of the atmpv17 knock out mutant on medium containing ABA and mannitol is complemented by AtMPV17 overexpression. These results suggest that the AtMPV17 contributes to osmotic stress tolerance in plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-020-00834-x) contains supplementary material, which is available to authorized users.

Pyropia yezoensis is the most cultivated marine red algae in East Asia and is desiccation tolerant. PyMPV17, a desiccation stress response gene isolated from P. yezoensis, shares significant sequence similarity with mouse MPV17. A yeast knock-out mutant for SYM1, an MPV17 ortholog, could not grow on ethanol-containing medium at 38°C. Expression of PyMPV17 in yeast sym1 cells complemented the ethanol growth defect at 38°C, which suggests that PyMPV17 is a functional ortholog of SYM1. PyMPV17-green fluorescent protein (GFP) fluorescence was detected in mitochondria and PyMPV17 transcription was upregulated under desiccation stress in P. yezoensis gametophytes. Transcription of the PyMPV17 gene also increased in response to H2O2 and abscisic acid treatments. When PyMPV17 was introduced into the green alga Chlamydomonas, transgenic cells grew better than control cells on agar plates containing mannitol. Total malondialdehyde content in the transgenic cells was lower than that in the control cells under stress conditions. These results suggest that PyMPV17 reduces oxidative damage and contributes to the mechanism underlying tolerance to osmotic stress in red algae.

Pyropia is a genus of red algae that is cultivated as a commercial food and is a model organism in marine biotechnology. Its habitat is threatened by global climate change, resulting in considerable interest in developing productive strains that are tolerant to the changing climate. Such strains will be valuable only if they are genetically stable, but introduced genetic traits often are either weakened or completely lost in progeny. To determine and understand the likely fate of a newly acquired trait in Pyropia, we generated PyGUS transgenic P. tenera and analyzed the expression of the transgene and its copy number through its sexual life cycle. The vegetative and diploid cells of parent lines showed stable expression of the PyGUS gene, but approximately 30% of progeny had no histochemical staining for GUS and reduced levels of PyGUS mRNA, even though the transgene was present. Genomic qPCR analysis revealed that the copy number of the PyGUS gene was reduced in gametophyte thalli, which were generated after meiosis. We conclude that the PyGUS gene was markedly unstable during meiosis, and that loss of gene copies and the altered expression of the transgene occurred mostly during meiotic recombination. We anticipate that these results will benefit understanding of molecular mechanisms that confer stability to genetic traits in P. tenera and related species.

The marine red alga Pyropia tenera (Rhodophyta) is one of the most valuable and widely cultivated Pyropia species. Temperature is a major factor that affects the growth and life cycle of Pyropia. Small heat-shock proteins (sHSPs) play a crucial role in heat stress response. Among sHSPs isolated from P. tenera, PtsHSP19.6 showed the strongest response to heat stress but not to other abiotic stresses such as desiccation and freezing. Although an α-crystallin domain was found in PtsHSP19.6, its amino acid sequence showed low homology with known sHSPs, except those from red algae. This led us to investigate whether PtsHSP19.6 from red algae has chaperone activity and is involved in high-temperature tolerance. PtsHSP19.6 significantly reduced the thermal aggregation of alcohol dehydrogenase used as a substrate. When PtsHSP19.6 was introduced into Escherichia coli, the transformed cells grew better than the control cells under high-temperature conditions. Fluorescence of the PtsHSP19.6-GFP fusion protein was detected from granules in the cytoplasm. The in vitro analysis data showed that PtsHSP19.6 forms oligomers with a molecular weight greater than 240 kDa in solution. This study showed that PtsHSP19.6 from P. tenera forms oligomers in solution and plays a role in heat tolerance, with a chaperone function similar to that observed in green plants.

High-temperature stress related to global warming reduces the growth and productivity of seaweeds. Thus, the development of new strains is urgently required for maintaining or even enhancing the productivity of useful seaweeds such as red alga Pyropia yezoenesis in an increasingly warmer sea environment. To develop competitive high-temperature-tolerant strains of P. yezoensis (Sugwawon no. 104), we screened libraries of gamma-irradiated strains and identified high-temperature-resistant (HTR) mutants. Our results showed that HTR-1 and HTR-2 grew well at higher temperatures that inhibited the growth of the wild-type strain. The efficiency of conchosporangium maturation and conchospore release of HTR-1 was similar to or higher than the wild-type strain. Moreover, thallus growth, pigment content, photosynthetic efficiency, and monospore release from the growing thallus in HTR-1 could be maintained even at high temperature. Taken together, our data demonstrate that HTR-1 may be suitable for industrial cultivation at sea, even at elevated temperatures.

Pyropia tenera is a marine red alga that grows in the intertidal zone and may lose more than 90% of its water during twice-daily low tides. Based on the differentially expressed genes (DEGs) identified from the desiccation transcriptome of P. tenera gametophyte, a complementary DNA was isolated that upregulated strongly under desiccation stress and encoded a polypeptide of 591-amino acid residues showing a sequence homology with α-1,2-fucosyltransferase, named PtFUT. Fucosyltransferases transfer an l-fucose residue to various acceptor molecules, but information of their activity in the red algae is limited. PtFUT showed upregulation in transcription in gametophytes of P. tenera under desiccation and osmotic stress conditions induced by mannitol. Transcription of the PtFUT gene was also increased by abscisic acid as well as H2O2 treatment. The presence of a signal peptide for nucleus localization and fluorescence of the PtFUT-GFP fusion proteins in tobacco protoplasts indicated that PtFUT is located in the nucleus. When PtFUT was overexpressed in Chlamydomonas, the PtFUT improved the growth of transgenic cells under osmotic stress. These results suggest that PtFUT may play a role in tolerance to desiccation stress and provide insights into the molecular functions of the fucosyltransferases and understanding the desiccation tolerance mechanisms in marine red algae growing in intertidal zones.

Many ginsenoside biosynthesis-related genes have been identified in Panax ginseng. However, there is no report of the analysis of changes in gene expression induced methyl jasmonic acid (MeJA) using DNA microarray analysis. To identify the genes related to ginsenoside biosynthesis, we harvested P. ginseng adventitious roots at 0, 24, 72 and 120 h after MeJA treatment. At 120 h after MeJA elicitation, the contents of all ginsenosides increased compared to the control. We analyzed gene expression patterns in ginseng adventitious roots treated with MeJA using DNA microarray analysis and selected candidate genes related to ginsenoside biosynthesis, including genes encoding squalene synthase (SQS), squalene epoxidase (SE), dammarenediol-II synthase (DS), cytochrome P450 oxidase (CYP) and glycosyltransferase (GT). The expression patterns of these genes in MeJA-treated ginseng adventitious roots obtained by quantitative RT-PCR were consistent with those obtained by microarray analysis. Therefore, DNA microarray analysis is an efficient tool for selecting candidate genes associated with secondary metabolite biosynthesis in plants.

The gene expression at the branch point of chlorophyll and heme synthesis in the model microalga, Chlamydomonas reinhardtii, is different from that of higher plants. Another green alga, Arctic Chlorella, was recently isolated from Arctic sea ice and may be a promising candidate for a biofuel. To understand the chlorophyll metabolic pathway and relevant nuclear gene expression in Chlorella sp., we characterized chlorophyll-deficient mutants of the Arctic Chlorella sp. ArM0029B. First, we characterized the chlorophyll and heme biosynthetic pathways based on genes identified by bioinformatics analysis of the genome of Arctic Chlorella sp. ArM0029B. Then, we isolated and analyzed nine chlorophyll-deficient mutants that showed reduced expression of the ChlM gene, which encodes Mg-protoporphyrin methyltransferase. Expression of 5-amino levulinic acid dehydratase (encoded by ALAD) and glutamyl-tRNA reductase (encoded by HemA) was reduced in all nine independent mutants compared to wild type. These results indicated that Arctic Chlorella ArM0029B may have a regulatory mechanism of gene expression at earlier steps of the Mg-porphyrin branch that is more similar to higher plants than to the microalga C. reinhardtii. This study provides useful insight into the regulation of porphyrin precursor formation in Chlorella and related microalgae.

Transgene silencing is a common phenomenon in higher plants and microalgae. In the present study, we analyzed transgene silencing in the macroalga Pyropia yezoensis. This is the first report of transgene silencing in macroalgae. Transgenic lines were generated by transformation with the PyGUS gene and a hygromycin resistance gene (PtAph7-1). Histochemical GUS staining detected PyGUS expression in only three out of 12 lines. Southern and northern blotting, polymerase chain reaction (PCR), and reverse transcription (RT)-PCR analyses indicated that the PyGUS gene was silenced in the other 9 lines. Interestingly, PyGUS gene silencing in transgenic line 7 was repeatedly initiated at a later stage, in the developmental process from monospore to thallus through asexual reproduction. We identified genes encoding Argonaute and Dicer-like protein, the major components of the RNA-silencing pathway, in the genome and transcripts of P. yezoensis. Interestingly, we found the DDA motif in the PIWI domain of the AGO, suggesting that translational repression may be the major gene-silencing pathway in P. yezoensis. In this study, the observation of PyGUS gene silencing and identification of RNA-silencing components indicate that the gene (transgene) silencing machinery functions actively in the macroalga P. yezoensis. These results will be useful to study the regulation of gene expression and RNA-silencing mechanisms in Pyropia species and related macroalgae.

Cyclophilins (CYPs) are ubiquitous in all subcellular compartments, possess peptidyl-prolyl cis-trans isomerase (PPIase) activity and are present in prokaryotes and eukaryotes. Their physiological functions are various such as protein folding, symbiotic relationships, disease or plant responses to abiotic stresses. Pyropia seriata (Bangiales, Rhodophyta) is a marine red alga that is cultivated as commercially valuable seaweed. By analyzing transcriptome data, we identified six full coding sequences of CYPs from P. seriata. Among them, only PsCYP1 showed upregulation of transcription in responded to high temperature stress. PsCYP1 belongs to the single domain form of cytosolic cyclophilin and contains the typical 12 conserved amino acid residues for PPIase activity. Despite no detected nuclear localization signal (NLS), a chimeric PsCYP1 protein with green fluorescent protein (GFP) was detected predominantly in nucleus. When PsCYP1 was introduced into the green alga, Chlamydomonas, the introduced PsCYP1 conferred salt and heat stress tolerance in transgenic Chlamydomonas. Especially the transgenic Chlamydomonas cells exhibited a salt-tolerant phenotype. The cyclophilin genes from P. seriata will facilitate future studies of the molecular function of cyclophilin, and the mechanisms of abiotic stress tolerance in red algae.

Pyropia seriata, a marine red alga belonging to the order Bangiales (Rhodophyta), is one of the most valuable and widely cultivated Pyropia species. Water temperature and salinity are major factors affecting the growth and yield of Pyropia cultivation. Currently, there is limited data regarding the regulatory pathways and molecular mechanisms of the abiotic stress responses in red algae. In this study, we generated 1,403,321 expression sequence tags (ESTs) using 454 sequencing technology from the gametophyte thalli under control and high temperature conditions to identify the abiotic stress response genes. De novo assembly of the transcriptome reads generated 11,218 contigs, whereas 135,292 sequences remained as unassembled reads. Approximately 61.9 % of the contigs shared significant homology with known genes in our database, including the NCBI RefSeq database, plus Pyropia and Porphyra sequences. Sequence analyses demonstrated that the Pyropia transcriptome has a relatively high guanine-cytosine (GC) content with predominant trinucleotide repeats (93.9 %). GGC was the most common motif in identified simple sequence repeats (SSRs). We selected 754 differentially expressed genes (DEGs) response to abiotic stress based on reads per kilobase per million reads (RPKM) values, many of which have no significant homology with known sequences in public database. These transcriptome sequences will facilitate future studies to understand the common processes and novel mechanisms involved in abiotic stress tolerance in red algae.

The haustorium is a specific organ for penetration of host tissue in parasitic plants. To better understand the molecular mechanisms of the establishment of host-parasite relationships using gene expression profiles, early stage haustorium germination from mature mistletoe seeds was examined. We have generated a cDNA library that is an excellent source of expressed sequence tags (ESTs) specifically related to haustorium development and host penetration. We analyzed 4,771 ESTs derived from a mistletoe (Viscum album) haustorium cDNA library. Cluster analysis identified 727 contigs (23.88%) and 2,317 singletons (76.12%), yielding a total of 3,044 unique genes. Among these genes, 1,760 clones (57.8%) showed significant similarity to known genes; the remaining 1,284 clones were novel. Annotation of genes with Gene Ontology indicated that the most abundant ESTs reveals that defense against biotic / abiotic stresses, primary metabolic processes, cell wall loosening and modification is critical for haustorium development and establishment of the host-parasite connection. Especially, it is likely that xyloglucan endotransglycosylases (XETs), glucanase, expansins and other cell wall hydrolases cooperate in cell wall modification during the stages of host-parasite connection. Functional expression of full-length forms corresponding to target ESTs was applied to characterize haustorium genes. This EST information will help gene discovery and characterization and provide promising targets for genetic engineering. The EST analysis presented here represents the first reported transcriptome data for V. album var. coloratum. Although the number of ESTs analyzed may not be sufficient to completely elucidate the mechanism of the host-parasite connection, the integrated approaches reported here represent an essential step toward understanding haustorium development in parasitic plants and provide a valuable resource for defining molecular mechanisms in the host-parasite connection.

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonas reinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropia tenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropia tenera/Chlamydomonas reinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.

The marine red alga Pyropia tenera grows on intertidal rocks, where it undergoes dynamic environmental changes including temperature, desiccation, osmotic shock, and changes in light intensity. Therefore, Pyropia have developed a variety of strategies and mechanisms to overcome those environmental stressors. In an effort to identify the genes involved in the high-temperature tolerance of P. tenera, we generated 368,334 expression sequence tags (ESTs) using 454 sequencing technology and 3,331 ESTs using the Sanger method. Among the total ESTs, 222,024 reads were generated from gametophyte thalli under control condition and 149,641 reads were generated under high temperature condition. These ESTs were assembled into 17,870 contigs consisting of 336,016 reads, whereas 35,924 sequences remained as unassembled ESTs. Only 16.5 % of contigs shared significant similarity with an E value of ≤1E− 10 with UniProt sequence. The 95 different SSR motifs were discovered in 1,586 contigs. Trinucleotide repeat was absolutely predominant (90.2 %) SSR, and GGC was the most common motif. A comparison of the ESTs from gametophyte thalli under normal and heat stress conditions enabled us to identify the transcripts that were up or downregulated by high temperature. Most of transcripts produced under the high temperature condition belong to heat shock protein family and novel transcripts not matched to known genes in current public databases. These ESTs will provide valuable information to identify the DNA markers for the Pyropia species and the genes involved in the molecular mechanism of thermotolerance in red algae.

Porphyra is a commercially valuable source of food and drugs and an important model organism for algal research. However, genetic research on Porphyra tenera has been limited by a lack of a heterologous gene expression system. In this study, we isolated native promoter PtHSP70 for the efficient expression of foreign genes in this organism. This promoter lies approximately 1 kb upstream of the heat shock protein 70 coding sequence and was isolated using adapter ligation-mediated genomic polymerase chain reaction. Promoter activity was evaluated using the synthetic GUS gene (PyGUS) with optimized codons for Porphyra yezoensis. Interestingly, the PtHSP70 promoter allowed the efficient expression of PyGUS in P. tenera and P. yezoensis, whereas the PyGAPDH promoter from P. yezoensis was not fully functional in P. tenera. The PtHSP70 promoter may have a more conserved regulatory mechanism than the PyGAPDH promoter between these species, suggesting that PtHSP70 could serve as a universal promoter for Porphyra species. We also established an efficient transient transformation system for P. tenera by evaluating transformation parameters including gold particle quantity, helium and vacuum pressure, developmental stages of leafy gametophytes, and target distance. Under optimal conditions of transient transformation, the frequency of GUS expression was determined by histochemical staining as 30–50 cells per bombardment. In addition, PyGUS expression was detected during the regeneration of monospores in P. tenera, indicating successful genetic transformation. Therefore, the new transient transformation system using the PtHSP70 promoter can be used for foreign gene expression in P. tenera, which may advance the development of P. tenera as a model organism.

Previously, our work with ginseng hairy root shows that the tissue of low-branching and slow-growing phenotype contains high level of glutamine. In order to check if the high glutamine concentration inhibits the root growth, we applied exogenous glutamine or glutamate into growth medium and check the root growth of Arabidopsis. While glutamine did not affect root growth, over 0.1 mM glutamate inhibited severe root growth. However, when the amino acid solution was adjusted to pH 5.7 and added into medium, Arabidopsis seedlings show normal growth pattern on medium containing glutamate or aspartate. These results demonstrated that inhibition of the root growth by high concentration of exogenous glutamate was a result of the low pH toxicity caused by acidic amino acid, although low concentration (0.05 mM) of glutamate has an inhibitory effect on the primary root growth. The application of exogenous glutamine or glutamate increases glutamine concentration within root tissue about 3- to 4-fold. However, concentration of glutamate is not significantly increased. The KO mutant on each of the Gln1_1, Gln1_2, or Glu2 gene was little effective on the root growth. These results indicate that high concentration of endogenous glutamine observed in root tissue does not affect root growth.

The dehydrins (DHN) are a family of late embryo abundant (LEA D-11) proteins, which accumulate during the late stage of seed development or under low temperature or water deficient conditions. They are believed to play a protective role in freezing and drought tolerance. The dehydrin genes exist as multi-gene families. Here, we have identified 9 unique dehydrin genes from Korean ginseng (Panax ginseng C.A. Meyer), a typical medicinal plant Among these,PgDhn1 andPgDhn2 encode for YSK3- and KS-type dehydrins, respectively, and are very abundant. Gene expression analyses revealed that the majority of thePgDhn gene transcripts are detected under cold, as well as dehydration conditions. The exceptions arePgDhn5 and PgDhn9—the former being unresponsive to cold treatment, and the latter exhibiting only seed-specific expression. We also identified an alternative transcript of thePgDhn2 gene that harbors an intron in its 3’-untranslated region. Our results may prove useful in further studies ofDhn genes, including investigations into the mechanisms underlying gene expression, the nature of their variations, and their physiological functions.

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.

Methyl jasmonate (MeJA) treatment increases the levels of plant secondary metabolites, including ginsenosides, which are considered to be the main active compounds in ginseng (Panax ginseng C.A. Meyer). To create a ginseng gene resource that contains the genes involved in the biosynthesis of secondary metabolites, including ginsenosides, we generated 3,134 expression sequence tags (ESTs) from MeJA-treated ginseng hairy roots. These ESTs assembled into 370 clusters and 1,680 singletons. Genes yielding highly abundant transcripts were those encoding proteins involved in fatty acid desaturation, the defense response, and the biosynthesis of secondary metabolites. Analysis of the latter group revealed a number of genes that may be involved in the biosynthesis of ginsenosides, namely, oxidosqualene cyclase (OSC), cytochrome P450, and glycosyltransferase. A novel OSC gene was also identified by this analysis. RNA gel blot analysis confirmed that transcription of this OSC gene, along with squalene synthase (SS) and squalene epoxidase (SE) gene transcription, is increased by MeJA treatment. This ginseng EST data set will also provide important information on the genes that are involved in the biosynthesis of other secondary metabolites and the genes that are responsive to MeJA treatment.

14-3-3 proteins are a family of conserved proteins expressed in all eukaryotes. They function as regulators in signaling pathways through protein-protein interaction. These proteins have been implicated in plant metabolism, development, and responses to abiotic and biotic stresses. Here, we isolated six cDNAs encoding 14-3-3 proteins ofPanax ginseng, designated asPg14-3-3, by searching the ginseng EST database. Their degree of amino acid identity ranged from 53 to 84%. Phylogenetic analysis showed that thePg14-3-3 isoforms can be divided into two groups by early duplication, with one group being further split into three subgroups because of subsequent duplication events.Pg14-3-3s are differentially expressed in various root tissues and leaves, and their transcript levels are up-or down-regulated by either cold or drought stresses. These results suggest a possible role for Pg14-3-3 proteins in abiotic stress responses.

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum and has been widely applied to the discrimination of closely related microbial strains. Leaf samples of six species and one variety of higher plants (Rosa multiflora, R. multiflora var. platyphylla, Sedum kamtschaticum, S. takesimense, S. sarmentosum, Hepatica insularis, and H. asiatica) were subjected to PyMS for spectral fingerprinting. Principal component analysis of PyMS data was not able to discriminate these plants in discrete clusters. However, canonical variate analysis of PyMS data separated these plants from one another. A hierarchical dendrogram based on canonical variate analysis was in agreement with the known taxonomy of the plants at the variety level. These results indicate that PyMS is able to discriminate higher plants based on taxonomic classification at the family, genus, species, and variety level.

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.

Researchers have widely adopted the hairy root culture system as a means for producing secondary metabolites, including ginsenosides from ginseng. Although bacterial genes are involved, the aspects of plant gene expression are unclear. Using a cDNA microarray approach, we identified genes that are differentially expressed in ginseng hairy roots afterAgrobacterium rhizogenes infection. Our goal was to gain an initial understanding of the correlation between hairy root morphology and ginsenoside production. Among the 250 genes analyzed here, 63 (including 14 that are unclassified) were differentially expressed in a hairy root line containing a high level of ginsenosides. Of the genes that had been functionally categorized, 29% and 17% were active in metabolism and stress responses, respectively. Most were primarily associated with ribosomal proteins, thereby functioning in protein synthesis and destination. Their expression was down-regulated in hairy roots having less lateral branching. This phenotype may have resulted from the manipulation of metabolic activities by the translational machinery.

The paradigm of biological research has been changed by recent developments in genomics, high-throughput biology, and bioinformatics. Conventional biology often was based on empirical, labor-intensive, and time-consuming methods. In the new paradigm, biological research e is driven by a holistic approach on the basis of rational, automatic, and high-throughput methods. New functional compounds can be discovered by using high-throughput screening systems. Secondary metabolite pathways and the genes involved in those pathways are then determined by studying functional genomics in conjunction with the data-mining tools of bioinformatics. In addition, these advances in metabolic engineering enable researchers to confer new secondary metabolic pathways to crops by transferring three to five, or more, heterologous genes taken from various other species. In the future, engineering for the production of useful compounds will be designed by a set of software tools that allows the user to specify a cell’s genes, proteins, and other molecules, as well as their individual interactions.

The characterization of a cDNA clone encoding non-specific lipid transfer protein (PvLTP, formerly named PVR3) in the roots of bean seedlings has been previously reported. In this study, we examined the temporal and spatial accumulation of PvLTP mRNA and the effect of the auxin naphthaleneacetic acid (NAA) on the accumulation of PvLTP mRNA during root development. In situ hybridization showed that accumulation of PvLTP mRNA is highly tissue-specific. Accumulation was detected in the cortical tissue, but not in other tissues of root, including the quiescent center and root cap. Within the cortical tissue, accumulation of PvLTP mRNA was developmentally regulated; accumulation of PvLTP mRNA was high in the cortical tissue of the proximal and ground meristem and declined as cortical tissue developed further. Since the appropriate distribution of auxin is an important factor responsible for the maintenance of root meristem organization. We examined effect of auxin on the accumulation of PvLTP mRNA in relation to the development of cortical tissue. In bean seedlings grown on medium supplemented with 5 μM NAA, morphological alternations, including radial root expansion and abnormal tissue organization in the root apical meristem, were observed. Only faint accumulation signals of PvLTP mRNA were observed in the cortical tissue of proximal meristem region, indicating that cortical tissue development was repressed by exogenous NAA. However, our results suggest that the change in accumulation of PvLTP mRNA is not direct regulatory effect but reflective effect of altered development of cortical tissue that was induced by exogenous NAA. The temporal and spatial accumulation of PvLTP mRNA indicates that PvLTP is a useful marker for the development of cortical tissue in the root tip in bean seedlings.

We previously studied the production of shikonin derivatives by cell lines of Lithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 μM NAA and 0.3 μM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds. In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity. The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.