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An increase in seawater temperature owing to global warming is expected to substantially limit the growth of marine algae, including Pyropia yezoensis, a commercially valuable red alga. To improve our knowledge of the genes involved in the acquisition of heat tolerance in P. yezoensis, transcriptomes sequences were obtained from both the wild-type SG104 P. yezoensis and heat-tolerant mutant Gy500. We selected 1,251 differentially expressed genes that were up- or downregulated in response to the heat stress condition and in the heat-tolerant mutant Gy500, based on fragment per million reads expression values. Among them, PyHRG1 was downregulated under heat stress in SG104 and expressed at a low level in Gy500. PyHRG1 encodes a secretory protein of 26.5 kDa. PyHRG1 shows no significant sequence homology with any known genes deposited in public databases to date. However, PyHRG1 homologs were found in other red algae, including other Pyropia species. When PyHRG1 was introduced into the single-cell green alga Chlamydomonas reinhardtii, transformed cells overexpressing PyHRG1 showed severely retarded growth. These results demonstrate that PyHRG1 encodes a novel red algae-specific protein and plays a role in heat tolerance in algae. The transcriptome sequences obtained in this study, which include PyHRG1, will facilitate future studies to understand the molecular mechanisms involved in heat tolerance in red algae.

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.

Water temperature is one of the major factors that impacts the growth and life cycle of Pyropia tenera, one of the most valuable and cultivated marine red algae belonging to Bangiales (Rhodophytes). We analyzed transcriptome from gametophyte of P. tenera under normal and high temperature conditions, and identified four small heat shock proteins (sHSPs). They have no significant amino acid sequence homology with known proteins in public databases except PhsHSP22 from Pyropia haitanensis. PtsHSP19.3 gene responded to high temperature but slightly or not to desiccation, freezing or high salt condition. When the PtsHSP19.3 gene was overexpressed in Chlamydomonas reinhardtii, transformed Chlamydomonas lines revealed much higher growth rate than that of control cells under heat stress condition. Transformed cells also grew well in those of the control cell onto the medium containing high salt or H 2 O 2 . When the PtsHSP19.3 was fused to GFP and introduced into tobacco protoplast, fluorescence was detected at several spots. Results indicate that PtsHSP19.3 may form super-molecular assembles and be involved in tolerance to heat stress.

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.

Plants have developed adaptive strategies to survive under different abiotic stressors. To identify new components involved in abiotic stress tolerance, we screened unannotated expressed sequence tags (ESTs) and evaluated their cold or drought response in Arabidopsis. We identified a drought response gene (DRG) encoding a 39.5-kDa polypeptide. This protein was expressed specifically in siliques and was induced by drought stress in most tissues. When a DRG-GFP construct was introduced into Arabidopsis protoplasts, GFP signals were detected only in the nucleus. The drg mutant plant was more sensitive to mannitol-induced osmotic stress in agar plates and to drought or freezing stress in soil than the wild-type. Activating the DRG restored the normal sensitivity of drg mutants to abiotic stressors. No differences in drought or freezing tolerance were observed between the wild-type and transgenic plants overexpressing the DRG. When DRG was expressed in a cold-sensitive Escherichia coli strain BX04, the transformed bacteria grew faster than the untransformed BXO4 cells under cold stress. These results demonstrate that DRG is a nuclear protein induced by abiotic stresses and it is required for drought and freezing tolerance in Arabidopsis.

Pyropia are commercially valuable marine red algae that grow in the intertidal zone. They are extremely tolerant to desiccation stress. We have previously identified and reported desiccation response genes (DRGs) based on transcriptome analysis of P. tenera. Among them, PtDRG1 encodes a polypeptide of 22.6 kDa that is located in the chloroplast. PtDRG1 does not share sequence homology with any known gene deposited in public database. Transcription of PtDRG1 gene was upregulated by osmotic stress induced by mannitol or H2O2 as well as desiccation stress, but not by heat. When PtDRG1 was overexpressed in Escherichia coli or Chlamydomonas, transformed cells grew much better than control cells under high temperature as well as osmotic stress induced by mannitol and NaCl. In addition, PtDRG1 significantly reduced thermal aggregation of substrate protein under heat stress condition. These results demonstrate that PtDRG1 has a chaperone function and plays a role in tolerance mechanism for abiotic stress. This study shows that red algae have unknown stress proteins such as PtDRG1 that contributes to stress tolerance.

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.

Summary The SNAT gene of the red alga Pyropia yezoensis is chloroplast encoded, suggesting that plant SNAT genes evolved directly from cyanobacteria to red algae via endosymbiosis and thereafter were transferred to the nucleus., Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min–1 mg–1 of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g–1 of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green 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.

Pyropia tenera (Kjellman) are marine red algae that grow in the intertidal zone and lose more than 90% of water during hibernal low tides every day. In order to identify the desiccation response gene (DRG) in P. tenera, we generated 1,444,210 transcriptome sequences using the 454-FLX platform from the gametophyte under control and desiccation conditions. De novo assembly of the transcriptome reads generated 13,170 contigs, covering about 12 Mbp. We selected 1160 differentially expressed genes (DEGs) in response to desiccation stress based on reads per kilobase per million reads (RPKM) expression values. As shown in green higher plants, DEGs under desiccation are composed of two groups of genes for gene regulation networks and functional proteins for carbohydrate metabolism, membrane perturbation, compatible solutes, and specific proteins similar to higher plants. DEGs that show no significant homology with known sequences in public databases were selected as DRGs in P. tenera. PtDRG2 encodes a novel polypeptide of 159 amino acid residues locating chloroplast. When PtDRG2 was overexpressed in Chlamydomonas, the PtDRG2 confer mannitol and salt tolerance in transgenic cells. These results suggest that Pyropia may possess novel genes that differ from green plants, although the desiccation tolerance mechanism in red algae is similar to those of higher green plants. These transcriptome sequences will facilitate future studies to understand the common processes and novel mechanisms involved in desiccation stress tolerance in red algae.

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high-temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high-temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up-regulated as the result of high-temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild-type strain after high-temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high-temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high-temperature tolerance in marine algae, as well as in Porphyra.

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.

The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

Agrobacterium tumefaciens-mediated cotyle-donary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high-temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high-temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up-regulated as the result of high-temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild-type strain after high-temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high-temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high-temperature tolerance in marine algae, as well as in Porphyra.

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.

Hypericin, a naphthodianthrone, has been identified as the principal active compound found in St. John’s wort (Hypericum perforatum L.). To generate a gene resource for hypericin and other valuable metabolites, we generated expressed sequence tags (ESTs) from H. erectum. Analyses of the ESTs enabled us to select three cDNAs, HeHyp1, HeHyp2, and HeHyp3, evidencing significant sequence homology to Hyp-1 that were involved in hypericin biosynthesis from H. erectum. The deduced amino acid sequence of HeHyp1 cDNA exhibits 95 % identity with Hyp-1. The HeHyp2 and HeHyp3 polypeptides also exhibit 81.1 % identity with Hyp-1. The transcripts of HeHyp1, HeHyp2, and HeHyp3 were detected in the root, stem, leaf, flower, and callus cells. Study using recombinant protein suggests that Hyp-1, HeHyp2, and HeHyp3 may be involved in the biosynthetic of hypericin or other emodin derivatives.

To develop transgenic cucumber tolerant to abi-otic stress, a cotyledonary-node explants were co-cultivated with Agrobacterium tumefaciens (EHA101) carrying TPSP gene (pHC30-TPSP). After transfer to fresh medium every two week for eight weeks, putative transgenic plants were selected when shoots grown a length greater than 3 cm from the cotyledonary-node explants on selection medium supple-mented with 5 mgl -1 phospinotricin as selectable agent. The confirmation of transgenic cucumber was based on the North-ern blot analysis. Thirty four shoots (5.2%) with resistance to phospinotricin were obtained from 660 explants inoculated. Of them, transformants were only confirmed from 11 plants (1.7%). Transgenic cucumber expressing TPSP gene was more synthesized at 3.8 times amounts of trehalose (0.014 mg g fresh wt -1 ) than non-transformants (0.0037 mg g fresh wt -1 ). However, all of transgenic plants showed abnormal morphology, including stunted growth ( height 150 cm) under the same growth envi-ronment. These results lead us to speculate that the over-production of trehalose was toxic for cucumber, even though that had known for rice as non-toxic.

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.

Activation tagging that uses T-DNA vectors con-taining multimerized transcriptional enhancers from the cau-liflower mosaic virus (CaMV) 35S gene is a powerful tool to determine gene function in plants. This approach has been successfully applied in screening various types of mutations and cloning the corresponding genes. We generated an acti-vation tagged hairy root pool of ginseng ( Panax ginseng C.A. Meyer) in an attempt to isolate genes involved in the bio-synthetic pathway of ginsenoside (triterpene saponin), which is known as the major active ingredient of the root. Quanti-tative and qualitative variation of ginsenoside in activation tagged hairy root lines were profiled using LC/MS. Metabolic profiling data enabled selection of a specific hairy root line which accumulated ginsenoside at a higher level than other lines. The relative expression level of several genes of tri-terpene biosynthetic pathway in the selected hairy root line was determined by real time RT-PCR. Overall results suggest that the activation tagged ginseng hairy root system described in this study would be useful in isolating genes involved in a complex metabolic pathway from genetically intractable plant species by metabolic profiling.

SUMMARY Colpomenia sinuosa is an annual brown algal species that occurs in temperate to tropical waters of the world. In order to examine the genetic diversity among populations of the species and to discuss its current distribution, we analyzed the nuclear ribosomal DNA internal transcribed spacer (ITS) region from 18 specimens of C. sinuosa, and, for comparison, plastid rbcL from the same specimens. The C. sinuosa ITS region (2141–2534 base pairs) is approximately 2.2 times the length of most other brown algae. We found a long repeated sequence of approximately 190 base pairs in the first half of the ITS1 region; five repeats in Northern Hemisphere collections and three in those from the Southern Hemisphere, which result in ITS length variation. The unequal occurrence of tandem repeats of C. sinuosa corresponds to the geographical distribution of the species. The rbcL sequences from all the specimens of C. sinuosa, except the Canary Island samples, were identical, indicating that they indeed belong to the same species.

One of the limitation for Agrobacterium-mediated transformation via organogenesis from cotyledon explants routinely in cucumber is the production of chimeric plants. To overcome the limitation, Agrobacterium-mediated transformation system via somatic embryogenesis from hypocotyl explants of cucumber (c.v., Eunsung) on the selection medium with paromomycin as antibiotics was developed. The hypocotyl explants were inoculated with Agrobacterium tumefaciens strain EHA101 carrying binary vector pPTN290; then were subsequently cultured on the following media: co-cultivation medium for 2 days, selection medium for days, and regeneration medium. The T-DNA of the vector (pPTN290) carried two cassettes, Ubi promoter-gus gene as reporter and 35S promoter-nptll gene conferring resistance to paromomycin as selectable agent. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to paromomycin indicated by the growth of putative transgenic calli on selection medium amended with 100mg/L paromomycin, and GUS gene expression. Forty eight clones (5.2%) with GUS gene expressed of 56 callus clones with resistance to paromomycin were independently obtained from 928 explants inoculated. Of 48 clones, transgenic plants were only regenerated from 5 clones (0.5%) at low frequency. The histochemical GUS assay in the transgenic seeds () also revealed that the gus gene was successfully integrated and segregated into each genome of transgenic cucumber.

Laccase (EC 1.10.3.2) is a small group of enzymes that catalyze the oxidation of a broad range of phenolic compounds including hazardous and recalcitrant pollutants in the environment. This study attempted to develop an efficient system for production of a recombinant laccase by chloroplast genetic transformation of tobacco. Chloroplast transformation vector was constructed and introduced into the tobacco chloroplast genome using particle bombardment. Chloroplast-transformed plants were subsequently regenerated. PCR and southern blot analyses confirmed stable integration of the laccase gene into the chloroplast genome. Northern blot analysis revealed that mRNA of the laccase gene was highly expressed in chloroplast-transformed plants.

본 연구는 아그로박테리움 공동배양법을 이용한 기능획득 인삼 모상근의 대량생산을 위한 조건 확립에 대한 것이다. 일반적으로, 인삼과 같이 형질전환을 통한종자의 확보가 어려운 식물에서는 loss-of-function을 이용한 기능유전체 연구에 한계가 있다. 한편, 유전자의 기능을 활성화시키는 방법 (gain-of-function)인 activation tagging 기술은 이러한 문제점을 극복할 수 있는 대안이 될 수 있으며, Agrobacterium rhizogenes를 이용한 모상근 생산 시스템은 대량의 돌연변이체를 안정적으로 용이하게 얻을 수 있다는 점에서 최적의 시스템이라고 할 수 있다. 본 연구에서는 activation-tagging된 효율적인 형질전환 모상근 생산에 있어서의 최적의 아그로박테리움 균주 및 인삼조직, 배지조성 등에 대한 조건을 확립하였으며, 다양한 배지에서의 형질전환 모상근의 생장률 및 분지율, 표현형 등을 조사하였다. 엽병 절편을 activation-tagging vector pKH01을 가지고 있는 A. rhizogenes R1000와 공동배양하였을 때 배양 4주후 85.9%의 빈도로 모상근이 생산되었다. 모상근의 최대 생장과 분지도를 나타내는 배양조건을 조사한 바 엽병절편을 1/2 SH 배지에서 4주 배양하였을 때 왕성하게 생장하였으며 2.6의 분지도를 보여주었다. 최종적으로 1,989개체의 독립적인 형질전환 모상근 line을 생산하였으며, 이들 모상근 line은 인삼 진세노사이드 생합성 관련 유전자의 발굴 및 기능해석에 유용하게 쓰일 것이다. 【This study describes conditions for the mass production of activation-tagged mutant hairy root lines of ginseng by cocultivation with Agrobacterium rhizogenes. Because it is not currently possible to produce progeny from transgenic ginseng, a loss-of-function approach for functional genomics cannot be appliable to this species. A gain-of-function approach is alternatively the choice and hairy root production by cocultivation of A. rhizogenes would be most practical to obtain a large number of mutants. Various sources of explants were subjected to genetic transformation with various strains of A. rhizogenes harboring the activation-tagging vector pKH01 to determine optimum conditions for the highest frequency of hairy root formation on explants. Petiole explants cocultivated with A. rhizogenes R1000 produced hairy roots at a frequency of 85.9% after 4 weeks of culture. Conditions for maximum growth or branching rate of hairy roots were also investigated by using various culture media. Petiole explants cultured on half strength Schenk and Hildebrandt medium produced vigorously growing branched roots at a rate of 2.6 after 4 weeks of culture. A total of 1,989 lines of hairy root mutants were established in this study. These hairy root lines will be useful to determine functions of genes for biosynthesis of ginsenosides.】

Sorbitol is the primary product of photosynthesis and the major translocation form of photosynthate in the Rosaceae family, which includes pear plants. Therefore, the biosynthesis and breakdown of sorbitol is an important step in the carbohydrate metabolism of pear. In sorbitol metabolism, sorbitol-6-phosphate dehydrogenase (S6PDH) and NAD-sorbitol dehydrogenase (NAD-SDH) play key roles in biosynthesis and breakdown of sorbitol, respectively. We isolated full-length cDNAs encoding S6PDH and NAD-SDH from pear (Pyrus pyrifolia N. cv Niitaka). PyS6PDH cDNA encodes a polypeptide consisting of 311 amino acid residues. PyNAD-SDH cDNA encodes a 371 amino acid polypeptide. The PyS6PDH gene was expressed at high levels in leaves at 30 day after full bloom (DAFB), but its expression was decreased later. Conversely, the transcripts of the PyNAD-SDH gene were detected at low levels in young leaves, but increased with age. These genes may be regulated by the transcriptional level.

Ginseng ESTs allowed us to identify an unknown transcript which is highly abundant in rhizomes and seeds. We called the cDNA ginseng-specific abundant protein (GSAP), and identified three homologues, GSAP1, GSAP2, and GSAP3. GSAP cDNAs encode a small polypeptide consisting of 121 or 117 amino acids, and GSAP3 shows 87.6% amino acid sequence homology with GSAP1. GSAP transcripts were detected in most plant tissues, but GSAP3 is highly expressed in seeds, and is up-regulated under stressed conditions, water deficit. GSAP3-GFP fusion protein is located in the cell wall when expressed in onion epidermis cells. The transgenic Arabidopsis seedlings which over-expressed GSAP3 grew faster than those of the wild-type plant on the medium containing 300 mM mannitol and 100 mM NaCl. GSAP3 may play a role in altering the characteristics of the cell wall to allow for more tolerance of water deficit stress under abiotic stress conditions.

Human serum albumin (HSA) is the most abundant protein in plasma and is the most often used intravenous protein in many human therapies. However, HSA is currently extracted only from plasma because commercially feasible recombinant expression systems are not available. This study attempted to develop an efficient system for recombinant HSA production by chloroplast transformation of tobacco. A HSA cDNA was isolated from a cDNA library constructed with human liver tissue. Chloroplast transformation vectors were constructed by introducing various regulatory elements to HSA regulatory sequences. Vectors were delivered by particle bombardment into leaf explants and chloroplast-transformed plants were subsequently regenerated into whole plants. Southern blot analysis confirmed that the HSA cDNA was incorporated between rps12 and orf70B of the chloroplast genome as designed. Western blot analysis revealed that hyper-expression and increasing the stability of HSA were achieved by modification of the regulatory sequences using the psbA5`UTRs in combination with elements of the 14 N-terminal amino acids of the GFP and the FLAG tag. However, only plants transformed with the vector containing all of these elements were able to accumulate HSA.

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.

A hundred and ninety-three hairy root lines were produced from Korea ginseng ( P. ginseng ). These hairy root lines were classified into five different types, depending on their phenotypes. Metabolic profiling by 1 H NMR analysis indicated that the glutamine concentration was about five-fold higher in hairy root types with sluggish growth rates and low branching phenotypes than other hairy root types. Glutamine was detected at much higher levels in root tip zones when compared to those of root elongation zones. When the segments of the high branching hairy root were cultured in a medium containing more than 30 mM glutamine, root growth and lateral root formation were significantly reduced. It is known that nitrate is a major nitrogen source and can act as regulatory signal on the root development. In this study, we identify and suggest that glutamine plays an important role in root growth and phenotypes in ginseng hairy roots.

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.

Agrobacterium tumefaciens-mediated cotyledonary node transformation was used to produce transgenic soybean. Cotyledonary node explants of three cultivars and one genotype were co-cultivated with strains Agrobacterium (LBA4404, GV3101, EHA101, C58) containing the binary vectors (pCAMBIA3301 and pPTN289) carrying with CaMV 35S promoter-GUS gene as reporter gene and NOS promoter-bar gene conferring resistance to glufosinate (herbicide Basta) as selectable marker. There was a significant difference in the transformation frequency depend on bacteria strain. The EHA101 strain of the bacterial strains employed gave the maximum efficiency (3.6%). One hundred-six lines transformed showed the resistance in glufosinate. Histochemical GUS assay showed that at least 11 plants transformed with the GUS gene were positive response. The soybean transformants were obtained from the Thorne (5 plants), 1049 (5 plants) and Bakun (1 plant), respectively. Southern blot analysis and leaf painting assay revealed that the GUS and bar gene segregated and expressed in their progeny.

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.

Biotechnology in the 21st century will be driven by three emerging technologies: genomics, high-throughput biology, and bioinformatics. These technologies are complementary to one another. A large number of economically important crops are currently subjected to whole genome sequencing. Functional genomics for determining the functions of the genes comprising the given plant genome is under progress by using various means including phenotyping data from transgenic mutants, gene expression profiling data from DNA microarrays, and metabolic profiling data from LC/mass analysis. The aim of plant molecular breeding is shifting from introducing agronomic traits such as herbicide and insect resistance to introducing quality traits such as healthful oils and proteins, which will lead to improved and nutritional food and feed products. Plant molecular breeding is also expected to aim to develop crops for producing human therapeutic and industrial proteins.