Wheat Embryo Cell-free System Research Articles

The application of biostimulants Regoplant and Stimpo to increase wheat resistance to salinity conditions

aim. Investigation of bioprotective action of polycomponent biostimulants of natural origin Regoplant and Stimpo according to physiological and molecular genetic parameters of resistance of winter wheat plants (Triticum aestivum L.) varieties Antonivka ta Hoduval'nytsya odes'ka to the salinity conditions. Methods. The physiological parameters of resistance of experimental wheat plants obtained from seeds treated and untreated with solutions of Regoplant and Stimpo biostimulants used at concentrations of 50 μl/L and 75 μl/L grown under salt stress on 0,1 M sodium chloride solution were studied. Using Dot blotting method the degree of hybridization between cytoplasmic si/miRNA isolated from cells of experimental wheat plants grown under salinity conditions and mRNA of control plants grown in the absence of salinity conditions was determined. In the wheat embryo cell-free system of protein synthesis in vitro the silencing activity of si/miRNA on the template of mRNA isolated from cells of experimental wheat plants grown under salt stress conditions and control plants was studied. Results. It was found that the use of biostimulants Regoplant and Stimpo by soaking the seeds of wheat plants for 24 hours at concentrations of 50 μl/L and 75 μl/L improved the physiological parameters of wheat plants grown under salt stress conditions, which increased on average: seed germination – up to 69-75%, the length of seedlings – up to 28-37%, the length of roots – up to 41-42%, leaf area – up to 25-38%, the raw weight of seedlings – up to 48-53%, the dry weight of seedlings – up to 37-46 %, respectively, compared to control. The Dot-blot hybridization method showed that the largest decrease in the percentage of hybridized mRNA and si/miRNA molecules was observed in experimental wheat plants grown from seeds treated with Regoplant and Stimpo biostimulants under salt stress conditions - up to 66-75%, respectively, compared to control. The increase of silencing activity of si/miRNA on the template of mRNA isolated from experimental wheat plants obtained from seeds treated with Regoplant and Stimpo biostimulants and grown under salt stress conditions was shown to be 39-42%, respectively, compared to control. Conclusions. The use of biostimulants Regoplant and Stimpo in concentrations of 50 μl/L and 75 μl/L for presowing seed treatment contributes to the increase of the resistance of winter wheat plants to salinity conditions.
 Keywords: Triticum aestivum L., polycomponent biostimulants, resistance of wheat to salinity conditions, RNA interference.

Impact of New Natural Biostimulants on Increasing Synthesis in Plant Cells of Small Regulatory si/miRNA With High Anti-Nematodic Activity

Plant endoparasitic cyst nematode Heterodera schachtii Schmidt, gallic nematode Meloidogyne incognita and stem nematode Ditylenchus destructor damage various agricultural crops. The application of ecologically safe natural biostimulants with bioprotective properties is a newer approach for increasing plant resistance to parasitic nematodes. The molecular-genetic analysis of biostimulants action on plant genome is necessary for creation of new effective bioregulators for plant protection against phytopathogenic organisms. In our field and greenhouse experiments, we investigated the influence of new natural biostimulants Avercom and its derivatives on plant protection against nematodes Meloidogyne incognita and Ditylenchus destructor. Considerable increase of resistance to nematodes and productivity of cucumber and potato were observed for plants treated by biostimulant Avercom and its derivatives. Impact of biostimulants Radostim-super and Avercom on increase of resistance of sugar beet and cucumber sprouts to nematodes Heterodera schachtii and Meloidogyne incognita was studied in the laboratory conditions. Comparative analysis of morpho-physiological signs of control and experimental plants showed that plants treated by Radostim-super and Avercom were more viable and resistant to these nematodes as compared to control sprouts. In the molecular-genetic experiments, we studied the impact of these biostimulants on inducing synthesis of small regulatory si/miRNA, which plays key role in plant immune protection. Using method Dot-blot hybridization we studied degree of homology between si/miRNA with mRNA populations, isolated from plants untreated and treated with new natural biostimulants. We found considerable difference in the degree of homology (6-28%) between populations of mRNA and si/miRNA from nematode-infected plants that were either untreated or treated with biostimulants. We have also investigated silencing of translation of mRNA activity of si/miRNA in the wheat embryo cell-free system of protein synthesis. In these experiments, we found high inhibitory activity (38-65%) of si/miRNA from plants treated by biostimulants as compared to low inhibitory activity (15-20%) of si/miRNA from untreated plants. Obtained differences in the degree of homology between populations of mRNA and si/miRNA from untreated and treated with biostimulants plants, which were infected by nematode, and also the high inhibitory activity of si/miRNA from plants treated by biostimulants confirm that these biostimulants induce synthesis of anti-nematodic si/miRNA in plants, resulting in considerable increase of their resistance to these phytopathogens.

Control of translational initiation in the wheat-embryo cell-free protein expression system for producing homogenous products

Wheat-embryo cell-free protein expression system allows efficient production of a wide variety of proteins. Homogeneity of the end products is an important characteristic of an advanced cell-free system that will be used in a field of protein science such as structural biology. A translation enhancer such as the omega sequence derived from tobacco mosaic virus, that allows cap-independent translation of the mRNA in the cell-free system, is required for low-cost preparation of template mRNAs in the cell-free translation system. However, the use of translational enhancers often leads to unexpected byproducts. Several AUU codons in the omega sequence can potentially function as translation initiators. We confirmed that the in-frame AUU in the omega sequence functions as a non-canonical start codon and results in the extension of the N-terminus of the target protein in some cases. Investigation of the selectivity of non-canonical initiation codon under the control of omega sequence in the wheat-embryo cell-free system revealed that seven non-AUG codons, CUG, AUA, AUU, GUG, ACG, AUC, and UUG, are recognized as translation initiators. We found that the introduction of an in-frame stop codon just upstream of the target open reading frame is an efficient way to avoid unexpected byproducts. This minor but effective modification facilitates production of homogeneous proteins within the wheat-embryo cell-free protein expression system at the preparative scale.

A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 microM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B(1-320)) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26 degrees C.

In vitro selection of scFv and its production: an application of mRNA display and wheat embryo cell-free and E. coli cell production system

Synthetic DNA libraries encoding human antibody V(L) and V(H) fragments were designed, constructed, and enriched using mRNA display. The enriched libraries were then combined to construct a scFv library for mRNA display. Sequencing revealed that 46% of the library coded for full-length scFvs. Considering the number of molecules used in mRNA display, the size of the library displayed was calculated to be >10(10). To verify this, we tried to isolate a scFv against human RANK. A scFv was successfully isolated in the sixth round of panning and was synthesized in wheat embryo cell-free (WE) and Escherichia coli cell systems. In the WE system, even though the production level was high, the product was almost soluble. However, in the E. coli system, it was over-produced as inclusion bodies. The inclusion bodies were successfully refolded and showed approximately the same binding affinity as the WE product. These results demonstrate that using mRNA display with synthetic libraries and WE and E. coli cell production systems, a system for in vitro selection and small- to large-scale production of scFvs has been established.

A completely in vitro system for obtaining scFv using mRNA display, PCR, direct sequencing, and wheat embryo cell-free translation

Using mRNA display followed by in vitro sequencing and translation, a complete in vitro system for obtaining scFv has been developed. An mRNA display library for synthetic scFv was panned against human TNF receptor (TNFR). The nucleotide portion of the enriched molecules was subjected to limiting dilution, and PCR-amplified. Three of the proteins encoded by the amplified fragments were synthesized in a wheat embryo (WE) cell-free system using a batch method. They were shown to bind TNFR by ELISA. One of their sequences was identified in vitro. The identified clone was further synthesized at approx. 0.5 mg/ml reaction mixture in a WE system with dialysis as a totally soluble protein.

Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system

Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (∼10 μg protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system.

Structure-Based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway

Rice (Oryza sativa) anthranilate synthase alpha-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.

Arrangement of lambda light chain genes in mutant clones of the MOPC 315 mouse myeloma cells.

The synthesis of lambda light chains and the arrangement of the lambda-chain genes was examined in cells of the mouse myeloma MOPC 315, which is an alpha lambda 2 producer, and in several mutants derived from it. The mutants produce lambda 2 chains only (MOPC 315.26, MOPC 315.34, and MOPC 315.37) or fail to produce alpha and lambda 2 chains (MOPC 315.25 and MOPC 315.36). Messenger RNA from the lambda 2 chain-producing cells directed the synthesis of a lambda 2 chain precursor and a fragment of the lambda 1 chain (lambda 1 F) in a wheat embryo cellfree system, whereas mRNA from the cells that do not produce lambda 2 chains directed the synthesis of lambda 1 F only. DNA from the parental MOPC 315 cells and from the lambda 2 chain-producing cells contained discrete EcoRI restriction fragments coding for rearranged lambda 1 and lambda 23 chain genes and their respective germ-line V and J-C regions. DNA from the no-Ig-producing cells contained fragments coding for the rearranged lambda 1 chain gene and the germ-line V lambda 2 region, but it lacked the sequences coding for the rearranged lambda 2 chain gene and the germ-line V lambda 1 and J-C lambda 1 regions. These results suggest that rearrangements of the lambda 1 and lambda 2 chain genes occur on different chromosomes in MOPC 315 cells and imply that rearrangements of the lambda 1 and lambda 2 chain genes on the same chromosome may be mutually exclusive.

Characterization of a maturation-specific mRNA in dry mung bean embryonic axes

The poly(A)-rich RNA from dry mung bean (Vigna radiata [L.] Wilczek) embryonic axes has been isolated and translated in a wheat embryo cell-free system, and the products were analyzed on sodium dodecyl sulfate-polyacrylamide gels. The fluorographyic patterns showed a heavy band at approximately MW 12,000. The messenger RNA coding for this polypeptide disappeared in the course of early germination. This messenger is translated in vivo but simultaneously degrades when the axes imbibe. The poly(A)-rich RNA from dry axes has been fractionated on sucrose-dimethyl sulfoxide gradients, and this messenger has been found to be distributed largely in the 9-14 S region. The polypeptide synthesized in vitro has been immunoprecipitated, using the antiserum raised against this protein purified from dry axes.

Continuous cultures: a tool for studying mutagenesis in eukaryotes

Immunoadsorbents and purified antibodies, prepared by affinity chromatography, have been used to specifically precipitate polysomes synthesising histone F2c from chicken reticulocytes. The 5–18S RNA fraction from these polysomes has been shown to programme the synthesis of F2c as the major detectable product in a wheat embryo cell-free translation system. The drug Trichodermin, an inhibitor of termination of eukaryotic protein synthesis increases the yield of immunoadsorbed messenger RNA.

Isolation of globin mRNA from spleen of anemic rabbits and its translation in wheat embryo cell-free system

Isolation of globin mRNA from spleen of anemic rabbits and its translation in wheat embryo cell-free system

Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide.

A tridecamer oligodeoxynucleotide, d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to reiterated 3'- and 5'-terminal nucleotides of Rous sarcoma virus 35S RNA, is an efficient inhibitor of the translation of proteins specified by the viral RNA in the wheat embryo cell-free system. The inhibition specificity for oncornavirus RNA is greater than for rabbit reticulocyte mRNA or brome mosaic virus RNA. Other oligodeoxynucleotides of similar size have little or no specific effect on the RNA-directed translation. The tridecamer acts as a primer for the avian myeloblastosis virus DNA polymerase when Rous sarcoma virus heated 70S RNA is used as a template, offering evidence that it can hybridize to the RNA. The possible use of such an oligodeoxynucleotide hybridization competitor to inhibit Rous sarcoma virus replication is described in the preceding paper [Zamecnik, P. C. & Stephenson, M. L. (1978) Proc. Natl. Acad. Sci. USA. 75, 280--284].

Translation of exogenous mRNA in a wheat embryo cell-free system; Effect of composition of cell-free system

Translation of exogenous mRNA in a wheat embryo cell-free system; Effect of composition of cell-free system

Cucumber mosaic virus RNA 5 Partial characterization and evidence for no large sequence homologies with genomic RNAs

Cucumber mosaic virus (CMV) contains four major RNA species (RNAs 1,2,3 and 4) the largest three of which are necessary for infectivity [ 1,2] . RNA 4 serves as a messenger for synthesis of coat protein in a wheat embryo cell-free system [3] . The nucleotide sequence of RNA 4 must also be present in RNA 3 since it is known that RNA 3 carries the information for coat protein synthesis in vivo [4,5] . In addition to the genome RNAs and RNA 4, a fifth RNA component (RNA 5) of low molecular weight (100 000) has been detected in CMV preparations [ 1,6] . Interest in RNA 5 was enhanced by the finding that, even though the small RNA is apparently dependent upon the CMV genome RNAs for replication [7] , the relative proportions of RNA 5 and RNA l-4 is tied to the nature of the plant host of viral propagation and, furthermore, the presence of RNA 5 can dramatically alter the response of certain hosts to CMV infection [8,9]. The above findings make it unlikely that RNA 5 is a host RNA normally present in the plant which is entrapped during virus assembly, but they do not in themselves permit us to distinguish between two further possibilities: (1) RNA 5 originates from the CMV RNA but is nonessential for infectivity, i.e., a deletion fragment or partial transcription product of one of the genome RNAs, or (2) RNA 5 is a ‘satellite’ RNA, that is, a parasite RNA with little or no sequence homology with the helper genome. As a prelude to a more detailed sequence analysis, we have determined some of the properties of RNA 5. In particular, the 5’ and 3’ extremities of the RNA

Two fractions of rough endoplasmic reticulum from rat liver. II. Cytoplasmic messenger RNA's which code for albumin and mitochondrial proteins are distributed differently between the two fractions.

Subcellular fractions were obtained from rat liver homogenates under conditions which prevented degradation of polysomes (pH 8.5 and high ionic strength). Rough endoplasmic reticulum (RER) was recovered in high yields from a low-speed nuclear pellet (rapidly sedimenting endoplasmic reticulum, RSER) and from a postmitochondrial supernate (rough microsomes). The polysomal RNA content of these two fractions was very similar. When polyA+-RNA's were translated inthe mRNA-dependent wheat embryo cell-free system, both fractions yielded polypeptide products which had similar electrophoretic patterns on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Activities of messenger RNA's which code for albumin and for polypeptides destined for transport to the inner membrane and matrix of mitochondria (i.e. 'mitoplasts') were assayed by translating in the more active rabbit reticulocyte cell-free system followed by immunoprecipitation of radioactive products and coelectrophoresis with immunoprecipitated marker proteins on SDS-polyacrylamide gels. These tests indicated that albumin mRNA is about equally distributed between the two fractions of RER, or slightly enriched in the RSER fraction when activity is expressed as a percentage of total polypeptide synthesis. Activities of cytoplasmic mRNA's which code for at least some mitoplast proteins could be detected in both fractions, but all were enriched in the rough microsome fraction, not the RSER (two- to threefold when corrected for differences in total polypeptide synthesis in the lysate). Comparisons of mRNA's from free vs. membrane-bound polysomes indicated that most of the albumin mRNA activity (86-91%) and mitoplast protein mRNA activities (75%) were present in the bound fraction. Assuming that RSER and rough microsomes do not derive exclusively from different cells types, the evidence suggests that, compared to albumin and most other membrane-bound mRNA's, cytoplasmic mRNA's coding for mitoplast proteins may be preferentially segregated or compartmentalized within the cell on the microsomal class of RER.

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina.

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.

Intracellular site of synthesis and localization of leghemoglobin in root nodules

A majority of the total protein synthesis in host cell cytoplasm is inhibited by cycloheximide. Because leghemoglobin represents a large proportion of total cellular protein in the nodule, this observation suggests that leghemoglobin may be translated on the 80S-type ribosomes. Analysis of the nascent peptides isolated from free and membrane-bound polysomes showed that free polysomes contain more immunoreactive material against antibodies to leghemoglobin as compared to that of membrane-bound polysomes. When free and membrane-bound polysomes were incubated in a wheat embryo cell-free protein-synthesizing system, a larger percentage of the released polypeptides from free polysomes was found to be immunoreactive. Similar results were obtained by translation in vitro of the poly(A)-containing mRNA isolated from free and membrane-bound polysomes. For immunocytochemical localization of leghemoglobin, the antibodies were conjugated with ferritin. Antibody conjugates were strictly localized in the host cell cytoplasm and adjacent to the outer surface of the membrane surrounding the bacteroids. Ferritin was not found on the inner surface of the membrane or within the membrane sac. These data suggest that leghemoglobin is synthesized preferentially on the free polysomes in the host cell cytoplasm, directed by a poly(A)-containing 9S mRNA that is most likely of plant origin, and that its location is restricted to the host cell cytoplasm.

Translation of nonpolyadenylylated messenger RNA of sea urchin embryos.

A large proportion of newly synthesized polyribosomal RNA of sea urchin blastulae is not polyadenylylated. The size distributions of the polyadenylylated and nonpolyadenylylated RNA are indistinguishable (mean size of 26 S). Upon translation of sea urchin polyribosomal RNA containing poly(A) and that without poly(A) in a wheat embryo cell-free protein-synthesizing system, where polypeptide synthesis is dependent on added messenger, both classes of RNA support peptide synthesis to the same extent. A preliminary analysis of the proteins synthesized in response to added mRNA (polyadenylylated and nonpolyadenylylated) indicates that these classes of mRNA molecules may code for different populations of proteins.

The isolation of chicken histone F2c [formula omitted] messenger RNA by immunoadsorption of F2c-synthesising polysomes

Immunoadsorbents and purified antibodies, prepared by affinity chromatography, have been used to specifically precipitate polysomes synthesising histone F2c from chicken reticulocytes. The 5–18S RNA fraction from these polysomes has been shown to programme the synthesis of F2c as the major detectable product in a wheat embryo cell-free translation system. The drug Trichodermin, an inhibitor of termination of eukaryotic protein synthesis increases the yield of immunoadsorbed messenger RNA.