Streptomyces liangshanensis sp. nov., a novel actinomycete isolated from rhizosphere soil of Fagopyrum tataricum.

A novel actinomycete strain, designated strain QMT-12T, was isolated from the rhizospheric soils of Fagopyrum tataricum and characterized using a polyphasic approach. Strain QMT-12T was found to have morphological features typical of the genus Streptomyces. The predominant fatty acids included C18:1 cis9 (35.9%), Summed feature 6 (C18:2 cis9, 12/C18:0 a or C18:0 anteiso/C18:2 c) (30.6%) and C16:0 (16.3%). The whole-cell sugars were arabinose and glucose. The whole-cell-wall amino acids included alanine, aspartate, glutamic acid, glycine and LL-diaminopimelic acid. The menaquinones were MK-9, MK-9(H2), MK-9(H4), MK-9(H6) and MK-9(H8). The diagnostic phospholipids consisted of diphosphatidyl glycerol, phosphatidylethanolamine, phosphatidyl methyl ethanolamine, phospholipids, phosphotidyl inositol, phosphotidylinositol mannosides, and phospholipids of unknown structure containing glucosamine. The full-length 16S rRNA gene sequence analysis showed that strain QMT-12T belonged to the genus Streptomyces and had 98.2, 98.1, 98.1 and ≤ 98.0% similarities to Streptomyces camponoticapitis 2H-TWYE14T, Streptomyces scopuliridis NRRL B-24574T, Streptomyces inhibens NEAU-D10T and other Streptomyces species with validly published and correct names, respectively. Phylogenetic analysis indicated that strain QMT-12T was closely related to Streptomyces inhibens NEAU-D10T. However, the average nucleotide identity value and the digital DNA-DNA hybridization value between strain QMT-12T and S. inhibens NEAU-D10T were 85.0 and 22.3%, respectively, well below 95-96% and 70% cut-off point recommended for delineating species. Based on its phenotypic and genotypic characteristics, strain QMT-12T (= CICC 11056T = JCM 33963T) represents the type strain of a novel species, for which the name Streptomyces liangshanensis sp. nov. is proposed.

Comparative Analysis of Four Buckwheat Species Based on Morphology and Complete Chloroplast Genome Sequences.

Buckwheat is a nutritional and economically crop belonging to Polygonaceae, Fagopyrum. To better understand the mutation patterns and evolution trend in the chloroplast (cp) genome of buckwheat, and found sufficient number of variable regions to explore the phylogenetic relationships of this genus, two complete cp genomes of buckwheat including Fagopyrum dibotrys (F. dibotrys) and Fagopyrum luojishanense (F. luojishanense) were sequenced, and other two Fagopyrum cp genomes were used for comparative analysis. After morphological analysis, the main difference among these buckwheat were height, leaf shape, seeds and flower type. F. luojishanense was distinguishable from the cultivated species easily. Although the F. dibotrys and two cultivated species has some similarity, they different in habit and component contents. The cp genome of F. dibotrys was 159,320 bp while the F. luojishanense was 159,265 bp. 48 and 61 SSRs were found in F. dibotrys and F. luojishanense respectively. Meanwhile, 10 highly variable regions among these buckwheat species were located precisely. The phylogenetic relationships among four Fagopyrum species based on complete cp genomes was showed. The results suggested that F. dibotrys is more closely related to Fagopyrum tataricum. These data provided valuable genetic information for Fagopyrum species identification, taxonomy, phylogenetic study and molecular breeding.

Research on Root Responses to Pb and Zn Combined Stress of Carex putuoshan.

Pb hyper-accumulated Carex putuoshan was taken as experimental material and subjected to combined stress of Pb and Zn. The differential expression of proteins in their roots were analyzed by Proteomic Approach. The protein that was directly involved in the cellular defense under the Pb and Zn combined stress was separated, and expression of those genes was analyzed with Carex Evergold as control. The results were obtained by MALDI-TOF/MS analysis. After applying Pb and Zn combined stress, the expression of 9 protein spots (including 7 different proteins, 2 identical proteins, 1 unknown protein) in Carex putuoshan root was found to be significantly up-regulated. Five proteins were obtained from the 9 proteins related to carbohydrate metabolism, including malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, frutose-1,6-bisphosphate aldolase, enolase, and 6- phosphogluconate dehydrogenase. Two proteins were related to protein biosynthesis, including isoflavone reductase and phytochelatin synthase (PCS). From these proteins, the most important protein is PCS, which is a key enzyme in the synthesis of phytochelatins (PCs) and plays an important role in chelation. It is directly involved in cellular defense under Pb and Zn stress. After Pb and Zn combined stress, the CpPCS in Carex putuoshan was cloned. The full length of cDNA is 1461 bps, and it encodes 486 amino acids with molecular weight of 53.86 kD and pI value of 6.12. Two typical phytochelatin synthase subfamily domains constitute CpPCS protein, which includes three adjacent Cys-Cys elements in the C-terminal region. Phylogenetic analysis of PCS proteins from different species showed that it had the closest relationship with the Oryza sativa and Triticum aestivum. Real-time quantitative PCR analysis indicated that CpPCS and CePCS (Carex Evergold) genes were expressed in the root. The CpPCS and CePCS genes were up-regulated by Pb and Zn treatments. The expression of CpPCS was higher than that of CePCS under the same condition. The study found that CpPCS expression was increased by Pb and Zn stress in the Carex putuoshan enrichment process of Pb, which lead to high expression of PCS protein. CpPCS improved the accumulation ability and resistance of Carex putuoshan to heavy metals with the expression level of glucose metabolism related proteins increasing after Pb and Zn stress.

Overexpression of the Lotus corniculatus Soloist Gene LcAP2/ERF107 Enhances Tolerance to Salt Stress.

The AP2/ERF play a key role in multiple stress responses in plants. we here report a novel salt stress-related gene, LcAP2/ERF107 that encodes an AP2/ERF protein in Lotus corniculatus cultivar Leo. LcAP2/ERF107 was classified into the soloist subfamiliy based on phylogenetic relationship. The transcription of LcAP2/ERF107 were strongly induced by salt and other phytohormones (ABA, ACC, MeJA). A subcellular localization experiment indicated that LcAP2/ERF107 is a nuclear protein that activates transcription. LcAP2/ERF107 overexpression in Arabidopsis resulted in pleiotropic phenotypes, including higher seed germination rate and transgenic plants with enhanced tolerance to salt stress. Further, under salt tolerance the transgenic lines elevated the relative moisture content; however, the relative electrolyte leakage was lower than in control plants. The expression levels of indicative genes RD22, RD29A, LEA4-5, P5CS1 and P5CS2 were found to be increased in the transgenic plants compared with the WT plants. These results indicated that LcAP2/ERF107 play an important role in the responses of plant to salt stress.

Genetic Engineering of Alfalfa (Medicago sativa L.).

Alfalfa is excellent perennial legume forage for its extensive ecological adaptability, high nutrition value, palatability and biological nitrogen fixation. It plays a very important role in the agriculture, animal husbandry and ecological construction. It is cultivated in all continents. With the development of modern plant breeding and genetic engineering techniques, a large amount of work has been carried out on alfalfa. Here we summarize the recent research advances in genetic engineering of alfalfa breeding, including transformation, quality improvement, stress resistance and as a bioreactor. The review article can enables us to understand the research method, direction and achievements of genetic engineering technology of Alfalfa.

Plantlet Regeneration of Tartary Buckwheat (Fagopyrum tataricum Gaertn.) in Vitro Tissue Cultures.

Tartary buckwheat is an ancient annual dicotyledonous herb, which is widely distributed around the world, specifically in the high altitude area of southwestern China and in the hill region of Himalayan. The plantlet regeneration of tartary buckwheat via somatic embryogenesis or multiple shoot induction was investigated in two different tartary buckwheats, Yuanzi and Xichang. The regeneration ability of Yuanzi was better than Xichang tartary buckwheat, and the hypocotyls were better than cotyledons as tartary buckwheat plantlet regeneration explants via somatic embryogenesis. The most suitable medium for callus induction was Murashige and Skoog basal medium added 2 mg/L 2, 4- dichlorophenoxyacetic acid and 1 mg/L Kinetin, which could reach up to 98.96% callus induction percentage. The plantlet regeneration percentage from callus of tartary buckwheat could reach up to 55.77%, which induced on 2.0 mg/L Benzyladenine and 1.0 mg/L KT in MS basal medium. In addition, maximum of multiple shoot induction percentage was 69.05%, which was observed in case of Yuanzi tartary buckwheat in MS basal medium with added 3.0 mg/L 6-BA and 1.0 mg/L Thidiazuron. Roots induction of regenerated plants were achieved on 1/2 MS basal medium with added 1mg/L Indole-3-Butytric acid, which has 75% survival after transferred regenerated plants to soil under field conditions.

Genome-wide analysis of AP2/ERF family genes from Lotus corniculatus shows LcERF054 enhances salt tolerance.

Lotus corniculatus is used in agriculture as a main forage plant. Members of the Apetala2/ethylene response factor (AP2/ERF) family play important roles in regulating gene expression in response to many forms of stress, including drought and salt. Here, starting from database of the L. corniculatus var. japonicus genome, we identified 127 AP2/ERF genes by insilico cloning method. The phylogeny, gene structures, and putative conserved motifs in L. corniculatus var. japonicus ERF proteins were analyzed. Based on the number of AP2/ERF domains and the function of the genes, 127 AP2/ERF genes from L. corniculatus var. japonicus were classified into five subfamilies named the AP2, dehydration-responsive element binding factor (DREB), ERF, RAV, and a soloist. Outside the AP2/ERF domain, many L. corniculatus var. japonicus-specific conserved motifs were detected. Expression profile analysis of AP2/ERF genes by quantitative real-time PCR revealed that 19 LcERF genes, including LcERF054 (KJ004728), were significantly induced by salt stress. The results showed that the LcERF054 gene encodes a nuclear transcription activator. Overexpression of LcERF054 in Arabidopsis enhanced the tolerances to salt stress, showed higher germination ratio of seeds, and had elevated levels of relative moisture contents, soluble sugars, proline, and lower levels of malondialdehyde under stress conditions compared to wild-type plants. The expression of hyperosmotic salinity response genes COR15A, LEA4-5, P5CS1, and RD29A was found to be elevated in the LcERF054-overexpressing Arabidopsis plants compared to wild type. These results revealed that the LcERF genes play important roles in L. corniculatus cv Leo under salt stress and that LcERFs are attractive engineering targets in applied efforts to improve abiotic stress tolerances in L. corniculatus cv Leo or other crops.

Nicotianamine synthase gene family as central components in heavy metal and phytohormone response in maize.

Nicotianamine (NA) is an important divalent metal chelator and the main precursor of phytosiderophores. NA is synthesized from S-adenosylmethionine in a process catalyzed by nicotianamine synthase (NAS). In this study, a set of structural and phylogenetic analyses have been applied to identify the maize NAS genes based on the maize genome sequence release. Ten maize NAS genes have been mapped; seven of them have not been reported to date. Phylogenetic analysis and expression pattern from microarray data led to their classification into two different orthologous groups. C-terminal fusion of ZmNAS3 with GFP was found in the cytoplasm of Arabidopsis leaf protoplast. Expression analysis by reverse transcription polymerase chain reaction revealed ZmNAS genes are responsive to heavy metal ions (Ni, Fe, Cu, Mn, Zn, and Cd), and all 10 ZmNAS genes were only observed in the root tissue except of ZmNAS6. The promoter of ZmNAS genes was analyzed for the presence of different cis-element response to all kinds of phytohormones and environment stresses. We found that the ZmNAS gene expression of maize seedlings was regulated by jasmonic acid, abscisic acid, and salicylic acid. Microarray data demonstrated that the ZmNAS genes show differential, organ-specific expression patterns in the maize developmental steps. The integrated comparative analysis can improve our current view of ZmNAS genes and facilitate the functional characterization of individual members.

Induction of annexin by heavy metals and jasmonic acid in Zea mays.

Plant annexins are Ca(2+)- and phospholipid-binding proteins forming an evolutionary conserved multi-gene family. They are implicated in the regulation of plant growth, development, and stress responses. With the availability of the maize genome sequence information, we identified 12 members of the maize annexin genes. Analysis of protein sequence and gene structure of maize annexins led to their classification into five different orthologous groups. Expression analysis by RT-PCR revealed that these genes are responsive to heavy metals (Ni, Zn, and Cd). The maize annexin genes were also found to be regulated by Ustilago maydis and jasmonic acid. Additionally, the promoter of the maize annexin gene was analyzed for the presence of different stress-responsive cis-elements, such as ABRE, W-box, GCC-box, and G-box. RT-PCR and microarray data show that all 12 maize annexin genes present differential, organ-specific expression patterns in the maize developmental steps. These results indicate that maize annexin genes may play important roles in the adaptation of plants to various environmental stresses.

Isolation and screening of strains producing high amounts of rutin degrading enzymes from Fagopyrum tataricum seeds.

The rutin degrading enzyme (RDE) was isolated and purified from tartary buckwheat seeds. The RDE was purified about 11.34-fold and its final yield was 3.5%, which was very low, due to our purification strategy of giving priority to purity over yield. The RDE molecular weight was estimated to be about 60 kDa. When rutin was used as substrate, an optimal enzyme activity was seen at around pH 5.0 and 40 °C. Strains isolation strategy characterized by the use of rutin as sole carbon source in enrichment cultures was used to isolate RDE-producing strains. Then the active strains were identified by morphology characterization and 18s rDNA-ITS (Internal Transcribed Spacer) gene sequencing. Three isolates coded as B3, W2, Y2 were successfully isolated from fusty Fagopyrum tataricum flour cultures. Strain B3 possessed the highest unit activity among these three strains, and its total activity reached up to 171.0 Unit. The active isolate (B3) could be assigned to Penicillium farinosum. When the Penicillium farinosum strains were added to tartary buckwheat flour cultures at pH 5.0, 30 °C after 5 days fermentation, the quercetin production raised up to 1.78 mg/l, almost 5.1 times higher than the fermentation without the above active strains. Hence, a new approach was available to utilize microorganism-aided fermentation for effective quercetin extraction from Fagopyrum tataricum seeds.

Aldehyde dehydrogenase protein superfamily in maize.

Maize (Zea mays ssp. mays L.) is an important model organism for fundamental research in the agro-biotechnology field. Aldehydes were generated in response to a suite of environmental stresses that perturb metabolism including salinity, dehydration, desiccation, and cold and heat shock. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)(+)-dependent aldehyde dehydrogenases. Here, starting from the database of Z. mays, we identified 28 aldehyde dehydrogenase (ALDH) genes and 48 transcripts by the in silico cloning method using the ALDH-conserved domain amino acid sequence of Arabidopsis and rice as a probe. Phylogenetic analysis shows that all 28 members of the ALDH gene families were classified to ten distinct subfamilies. Microarray data and quantitative real-time PCR analysis reveal that ZmALDH9, ZmALDH13, and ZmALDH17 genes involve the function of drought stress, acid tolerance, and pathogens infection. These results suggested that these three ZmALDH genes might be potentially useful in maize genetic improvement.

Genome-wide identification of genes involved in raffinose metabolism in Maize.

The raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, are synthesized by a set of distinct galactosyltransferases, which sequentially add galactose units to sucrose. The accumulation of RFOs in plant cells are closely associated with the responses to environmental factors, such as cold, heat and drought stresses. Systematic analysis of genes involved in the raffinose metabolism has not been reported to date. Searching the recently available working draft of the maize genome, six kinds of enzyme genes were speculated, which should encode all the enzymes involved in the raffinose metabolism in maize. Expression patterns of some related putative genes were analyzed. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as galactinol synthase (ZmGolS5, ZmGolS45 and ZmGolS37), raffinose synthase (ZmRS1, ZmRS2, ZmRS3 and ZmRS10), stachyose synthase (ZmRS8) and ß-fructofuranosidase, are encoded by multiple gene members with different expression patterns. These results reveal the complexity of the raffinose metabolism and the existence of metabolic channels for diverse RFOs in maize and provide useful information for improving maize stress tolerance through genetic engineering.

Improvement of drought and salt tolerance in Arabidopsis and Lotus corniculatus by overexpression of a novel DREB transcription factor from Populus euphratica.

A novel DREB (dehydration-responsive element binding) gene, designated PeDREB2a, was isolated from the desert-grown tree, Populus euphratica Oliv. PeDREB2a is classified into the A-5 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. Using semi-quantitative RT-PCR, we found that the PeDREB2a was greatly induced by drought, NaCl, low temperature, 1-naphthaleneacetic acid (NAA), 6-benzyl aminopurine (6-BA) and gibberellic acid (GA3) treatments in P. euphratica seedling. Yeast transactivity assay demonstrated that PeDREB2a gene encodes a transcription activator. Overexpression of PeDREB2a under the stress-inducible rd29A promotor in transgenic Arabidopsis and Lotus corniculatus forage plants resulted in enhanced tolerance to salt and drought stresses. The PeDREB2a overexpressing Arabidopsis lines showed higher root length and plant height and had elevated levels of soluble sugars and lower levels of malondialdehyde under stress conditions compared to control plants. The results revealed that PeDREB2a play an essential role as a DREB transcription factor in regulation of stress-responsive signaling in P. euphratica.

An protocol for genetic transformation of Catharanthus roseus by Agrobacterium rhizogenes A4.

Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. To study and extract catharanthine and other metabolites from C. roseus, a technique was developed for producing hairy root cultures. In this study, the Agrobacterium rhizogenes A4 was induced in the hairy roots from leaf explants, and the concentration of antibiotics (100 mg/L kanamycin) was elucidated for selection after transformation. The polymerase chain reaction amplification of rol genes results revealed that transgenic hairy roots contained rol genes from the root induced (Ri)-plasmid. Catharanthine from C. roseus hairy roots was separated and analyzed using high-performance liquid chromatography. Over-expression of CrOrca3 (octadecanoid-responsive Catharanthus AP2/ERF domain), and cytohistochemical staining methods were used to validate transgenic hairy roots from C. roseus. Hairy root culture of C. roseus is a valuable approach for future efforts in the metabolic engineering of terpenoid indole alkaloids in plants.

Identification and Characterization of a Trypsin Inhibitor from Fagopyrum tataricum Seeds.

This study was aimed at investigating the purification and identification of serine protease inhibitors, F. tataricum trypsin inhibitor (FtTI) from tartary buckwheat (Fagopyrum tataricum) seeds. The FtTI was isolated by anion exchange chromatography, affinity chromatography, and centrifugal ultrafiltration. Under reducing and nonreducing conditions, an SDS-PAGE analysis showed that the isolated protein consists of a single polypeptide chain with a molecular mass of approximately 14 kDa. The two isoforms of FtTI were confirmed by the mass spectrometric profile where the two peaks corresponded to 11.487 and 13.838 kDa. The complete amino acid sequence of FtTI has been established by automatic Edman degradation and mass spectrometry. The molecule of FtTI consists of 86 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. The active site of FtTI contains an Asp66-Arg67 bond. The Ki value was calculated using the equation for slow tight binding inhibition which was 1.6 nM for trypsin. FtTI retained its inhibitory activity over a wide range of pH (3-10) and temperature (20-80 °C). FtTI can be rapidly inactivated by the combination of high temperature and high pressure. An analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor Ι family. Furthermore, FtTI exhibited a strong inhibitory activity against phytopathogenic fungi.

Soybean transcription factor GmMYBZ2 represses catharanthine biosynthesis in hairy roots of Catharanthus roseus.

Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Production of catharanthine in cell cultures or in hairy roots established by transformation with Agrobacterium rhizogenes is of interest because catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. Here, we observed a high amount of catharanthine in hairy roots of C. roseus, established by infecting leaf explants with the A. rhizogenes >agropine-type A4 strain carrying plasmid pRi. T-DNA transfer from plasmid pRi into hairy roots was confirmed by PCR for the essential T-DNA genes rolA and rolB and the agropine synthesis gene ags. The results suggest that integration of T-DNA into the plant DNA plays a positive role on the catharanthine pathway in C. roseus hairy roots. Furthermore, co-transformation with the soybean transcription factor GmMYBZ2 indicated that GmMYBZ2 reduces the catharanthine production by alteration of expression of a number of genes linked to the pathway. Transcription levels of the zinc-finger transcription factor 1 gene ZCT1 were high, and the transcription levels of the anthranilate synthase gene ASα, the strictosidine synthase gene STR, and the key transcription factor gene octadecanoid-responsive Catharanthus APETALA2/ethylene response factor were low. In addition, GmMYBZ2 had a negative effect on the gene expression levels of A-type cyclin CYSA and B-type cyclin CYSB, which was correlated with a reduced growth rate of the hairy roots.

Production and metabolic engineering of bioactive substances in plant hairy root culture.

In the past three decades, hairy roots research for the production of valuable biological active substances has received a lot of attention. The addition of knowledge to enhance the yields of desired substances and the development of novel tools for biomass engineering offer new possibilities for large-scale cultivation of the plant hairy root. Hairy roots can also produce recombinant proteins through the transfer of Agrobacterium T-DNA into the plant genome, and thereby hold immense potential for the pharmaceutical industry. This review highlights some of the significant progress made in the past few years and outlines future prospects for exploiting the potential utility of hairy root cultures as "chemical factories" for producing bioactive substances.

Transcriptional response of the catharanthine biosynthesis pathway to methyl jasmonate/nitric oxide elicitation in Catharanthus roseus hairy root culture.

Jasmonates and nitric oxide (NO) play important roles in the regulation of the signaling network leading to the biosynthesis of plant secondary metabolites. In this work, we explore the effect of constitutive overexpression of CrORCA3 (octadecanoid-responsive Catharanthus AP2/ERF domain), methyl jasmonate (MeJA), and sodium nitroprusside (SNP) on the differentiated tissue of Catharanthus roseus hairy roots. The changes in catharanthine concentration and in the levels of mRNA transcripts of pathway genes and regulators were tracked for 192 h. ORCA3 overexpression led to a slight decrease of the accumulation of catharanthine, while MeJA treatment caused a large increase in the levels of transcripts of pathway genes and the catharanthine concentration. SNP treatment alone or SNP in combination with MeJA treatment caused a dramatic decrease of the cathanranthine concentration, while at the same time the levels of transcripts of zinc finger-binding proteins genes (ZCTs) increased. The latter treatment also caused a decrease of the levels of transcripts of type-I protein prenyltransferase gene (PGGT-I). This response of transcriptional repressors and pathway genes may explain the antagonistic effects of NO and MeJA on catharanthine biosynthesis in C. roseus hairy roots.

Production and metabolic engineering of terpenoid indole alkaloids in cell cultures of the medicinal plant Catharanthus roseus (L.) G. Don (Madagascar periwinkle).

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a plant species known for its production of TIAs (terpenoid indole alkaloids), many of which are pharmaceutically important. Ajmalicine and serpentine are prescribed for the treatment of hypertension, whereas the bisindoles vinblastine, vincristine and 3',4'-anhydrovinblastine are used for their antineoplastic activity in the treatment of many cancers. However, TIAs are produced in small yields in C. roseus, which make them expensive. Cell and metabolic engineering has focused on increasing flux through the TIA pathway by various means, including optimization of medium composition, elicitation, construction of noval culture systems and introduction of genes encoding specific metabolic enzymes into the C. roseus genome. The present review will attempt to present the state-of-the-art of research in this area and provide an update on the cell and metabolic engineering of TIAs in C. roseus. We hope that this will contribute to a better understanding of the ways in which TIA production can be achieved in different C. roseus culture systems.