A subfamily of the small heat shock proteins of the marine red alga Neopyropia yezoensis localizes in the chloroplast.

Small heat shock proteins (sHSPs) play a crucial role under abiotic stress and are present in all organisms, from eukaryotes to prokaryotes. However, studies on the sHSP gene family in red alga are limited. In this study, we aimed to identify and characterize NysHSP genes from the genome of N. yezoensis, a marine red alga adapted to the stressful intertidal zone. We identified seven NysHSP genes distributed on all three chromosomes. Expression analysis revealed that all NysHSP genes responded to H2O2 and heat stress in the gametophytic thalli, but these genes responded only to heat stress in the sporophytic conchocelis. NysHSP20.3, which has an acidic isoelectric point (pI) and short N-terminal region, was localized as granules in the cytosol. Fluorescence imaging of the NysHSP25.8-GFP and NysHSP28.4-GFP fusion proteins revealed that these proteins were located in the chloroplast. Based on their characteristics and cellular localization, the NysHSPs are divided into two subfamilies. Subfamily I includes four sHSP genes that strongly respond to heat stress and encode a protein localized in the cytosol. The NysHSP gene of subfamily II encodes a polypeptide with a long N-terminal region located in the chloroplast. This study provides insights into the evolution and function of the sHSP gene family of the marine red alga N. yezoensis and how it adapts to the stressful intertidal zone.

Arabidopsis AtMPV17, a homolog of mice MPV17, enhances osmotic stress tolerance.

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.

PtDRG1, a Desiccation Response Gene from Pyropia tenera (Rhodophyta), Exhibits Chaperone Function and Enhances Abiotic Stress Tolerance.

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.

Transcriptome-Based Identification of the Desiccation Response Genes in Marine Red Algae Pyropia tenera (Rhodophyta) and Enhancement of Abiotic Stress Tolerance by PtDRG2 in Chlamydomonas.

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.

Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii.

Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii. The ω-3 fatty acid deficiency in strain CC-620, which contains a putative missense mutation at Thr286 of CrFAD7, was recovered by the overexpression of CC-125 CrFAD7. A Ser substitution in position 286 was able to partially complement the phenotype of the ω-3 fatty acid deficiency, but other substitution variants, such as Tyr, His, Cys, and Gly, failed to do so. Prediction of the phosphorylation target site revealed that Thr286 may be phosphorylated. Analysis of the structural conformation of CC-620 CrFAD7 via topology prediction (and bends in the helix) shows that this missense mutation may collapse the catalytic structure of CrFAD7. Taken together, this study suggests that Thr286 is essential for the maintaining the catalytic structure of CrFAD7.

Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts.

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.

Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl.

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.

Heat shock protein gene family of the Porphyra seriata and enhancement of heat stress tolerance by PsHSP70 in Chlamydomonas.

Heat shock proteins and molecular chaperones are key components contributing to survival in the abiotic stress response. Porphyra seriata grows on intertidal rocks exposed to dynamic environmental changes associated with the turning tides, including desiccation and heat stress. Analysis of the ESTs of P. seriata allows us to identify the nine HSP cDNAs, which are predicted to be PsHSP90, three PsHSP70, PsHSP40 and PsHSP20, and three 5'-truncated HSP cDNAs. RT-PCR results show that most of the PsHSP transcripts were detected under normal cell growth conditions as well as heat stress, with the exception of two cDNAs. In particular, PsHSP70b and PsHSP20 transcripts were upregulated by heat stress. When the putative mitochondrial PsHSP70b was introduced and overexpressed in Chlamydomonas, transformed Chlamydomonas evidenced higher rates of survival and growth than those of the wild type under heat stress conditions. Constitutive overexpression of the PsHSP70b gene increases the transcription of the HSF1 as well as the CrHSP20 and CrHSP70 gene. These results indicate that PsHSP70b is involved in tolerance to heat stress and the effects on transcription of the CrHSP20 and CrHSP70 genes.

IDENTIFICATION OF THE HIGH-TEMPERATURE RESPONSE GENES FROM PORPHYRA SERIATA (RHODOPHYTA) EXPRESSION SEQUENCE TAGS AND ENHANCEMENT OF HEAT TOLERANCE OF CHLAMYDOMONAS (CHLOROPHYTA) BY EXPRESSION OF THE PORPHYRA HTR2 GENE(1).

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.

Expression of barley HvCBF4 enhances tolerance to abiotic stress in transgenic rice.

C-repeat/dehydration-responsive element binding factors (CBF/DREBs) are a family of transcription factors that regulate freezing tolerance in Arabidopsis. As a step towards understanding the stress response of monocotyledonous plants, we isolated a barley gene HvCBF4 whose expression is induced by low-temperature stress. Transgenic over-expression of HvCBF4 in rice resulted in an increase in tolerance to drought, high-salinity and low-temperature stresses without stunting growth. Interestingly, under low-temperature conditions, the maximum photochemical efficiency of photosystem II in the dark-adapted state (F(v)/F(m), where F(v) is the variable fluorescence and F(m) is the maximum fluorescence) in HvCBF4 plants was higher by 20% and 10% than that in non-transgenic and CBF3/DREB1A plants, respectively. Using the 60K Rice Whole Genome microarray, 15 rice genes were identified that were activated by HvCBF4. When compared with 12 target rice genes of CBF3/DREB1A, five genes were common to both HvCBF4 and CBF3/DREB1A, and 10 and seven genes were specific to HvCBF4 and CBF3/DREB1A, respectively. Interestingly, HvCBF4 did not activate Dip1 and Lip5, two important target genes of CBF3/DREB1A, in transgenic rice under normal growth conditions, but their expression was enhanced by HvCBF4 under low-temperature conditions. Our results suggest that CBF/DREBs of barley act differently from those of Arabidopsis in transgenic rice.

A ginseng-specific abundant protein (GSAP) located on the cell wall is involved in abiotic stress tolerance.

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.

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome.

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed. 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.

Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites.

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.

Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis.

To develop an experimental system for studying ginsenoside biosynthesis, we generated thousands of ginseng (Panax ginseng C.A. Meyer) hairy roots, genetically transformed roots induced by Agrobacterium rhizogenes, and analyzed the ginsenosides in the samples. 27 putative ginsenosides were detected in ginseng hairy roots. Quantitative and qualitative variations in the seven major ginsenosides were profiled in 993 ginseng hairy root lines using LC/MS and HPLC-UV. Cluster analysis of metabolic profiling data enabled us to select hairy root lines, which varied significantly in ginsenoside production. We selected hairy root lines producing total ginsenoside contents 4-5 times higher than that of a common hairy root population, as well as lines that varied in the ratio of the protopanaxadiol to protopanaxatriol type ginsenoside. Some of the hairy root lines produce only a single ginsenoside in relatively high amounts. These metabolites represent the end product of gene expression, thus metabolic profiling can give a broad view of the biochemical status or biochemical phenotype of a hairy root line that can be directly linked to gene function.

Comparative DNA sequence analysis of wheat and rice genomes.

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.

The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms.

Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.

Barley Cbf3 gene identification, expression pattern, and map location.

Although cold and drought adaptation in cereals and other plants involve the induction of a large number of genes, inheritance studies in Triticeae (wheat [Triticum aestivum], barley [Hordeum vulgare], and rye [Secale cereale]) have revealed only a few major loci for frost or drought tolerance that are consistent across multiple genetic backgrounds and environments. One might imagine that these loci could encode highly conserved regulatory factors that have global effects on gene expression; therefore, genes encoding central regulators identified in other plants might be orthologs of these Triticeae stress tolerance genes. The CBF/DREB1 regulators, identified originally in Arabidopsis as key components of cold and drought regulation, merit this consideration. We constructed barley cDNA libraries, screened these libraries and a barley bacterial artificial chromosome library using rice (Oryza sativa) and barley Cbf probes, found orthologs of Arabidopsis CBF/DREB1 genes, and examined the expression and genetic map location of the barley Cbf3 gene, HvCbf3. HvCbf3 was induced by a chilling treatment. HvCbf3 is located on barley chromosome 5H between markers WG364b and saflp58 on the barley cv Dicktoo x barley cv Morex genetic linkage map. This position is some 40 to 50 cM proximal to the winter hardiness quantitative trait locus that includes the Vrn-1H gene, but may coincide with the wheat 5A Rcg1 locus, which governs the threshold temperature at which cor genes are induced. From this, it remains possible that HvCbf3 is the basis of a minor quantitative trait locus in some genetic backgrounds, though that possibility remains to be thoroughly explored.

Plant regeneration from callus cultures ofLithospermum erythrorhizon.

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.