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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.

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 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.

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.

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.

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.

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.

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.

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.

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.

Cotyledonary explants from immature zygotic embryos of each 85 recombinant inbred lines (RILs) were cultured on medium containing MS salts, B5 vitamins, 40 mg 2,4-dichlorophenoxyacetic acid and 30 g sucrose. Frequency of somatic embryo formation on cotyledonary explants showed in thirty-six lines(＜10%), in thirty-seven lines (11~49%), in nine lines (50~89%), and in three lines(>90%), respectively, The highest frequency (up to 90%) and number (6.36 per cotyledon) of somatic embryos were obtained from lines of KM1010, KM1032 and KM1064. Primary somatic embryos produced from three lines produced numerous secondary somatic embryos on the surfaces, which were subcultured for over one year. Upon transfer to maturation and conversion medium (Komatsuda, 1992), somatic embryos converted to plantlets at a frequency of approximately 25%.

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.

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.

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.

A plastid transformation vector was constructed for dicistronic expression of the aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp) genes under the control of the plastid rrn promoter. Gold particles coated with the vector DNA were bombarded onto tobacco leaf explants using a particle delivery system. Leaf explants produced adventitious shoots when cultured on shoot-inducing medium containing 500 mg l(-1) spectinomycin. Shoots that exhibited green fluorescence under UV light were selected. Southern blot analysis detected the presence of the aadA and gfp genes between trnA and trnI in the plastid genome. Northern blot analysis revealed that the aadA and gfp genes were both properly transcribed into a dicistronic transcriptional unit. The expression of the gfp gene in the plastid enabled separation of transformed chloroplasts from wild-type chloroplasts in the protoplast under a fluorescent microscope. The overall results indicate that dicistronic expression of the aadA and gfp genes in the plastid simplifies gene manipulation, facilitating selection and tracking of plastid-transformed cells.

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.