Since Green Fluorescent Protein (GFP) from Aequorea jellyfish emits green fluorescence without any additional substrates or co-factors, it has emerged as a powerful new reporter for use in a variety of organisms. However, improvements such as the alteration of the excitation spectrum and the elimination of a cryptic intron site were necessary for its efficient use in plants. An engineered synthetic GFP with a S65T mutation (replacement of the serine in position 65 with a threonine) in the chromophore has provided up to 100-fold brighter fluorescent signals than the wild-type jellyfish GFP sequence without any toxic effects. The sGFP(S65T) has been widely used for studies such as localization analysis and promoter assays. This sGFP(S65T) is also an ideal candidate for the development of transformation methods for crops and trees in which it has previously proved difficult to obtain transformants via conventional methods. A non-invasive, quantitative detection technique for this GFP has been achieved in conjunction with a fluorescent imaging system.
Full-length cDNAs of ArPHYAs were fused with the 35S promoter of cauliflower mosaic virus (CaMV35SArPHYAs) and introduced into horseradish hairy roots. The phytochrome levels in hairy roots that had been transformed with CaMV35SArPHYAs were about 2-3 times higher than those in normal hairy roots. Though ArphyAs were photochemically active in horseradish hairy roots, the efficiency of ArphyAs on shoot formation was different among them. The efficiency of light-induced formation of adventitious shoots in horseradish hairy roots was greatest for ArphyA3 and smallest for ArphyA2. These results indicated that ArPHYAs isolated from horseradish encoded photochemically active phytochromes and that they were biologically active in horseradish.
Carrot callus cells showed a positive reaction to phloroglucinol-HCl after eliciting with hot-water extract from conidia of Botrytis cinerea. The positive reaction reflected increases in the content of thioglycolic acid lignin and in the activity of phenylalanine ammonia-lyase. Alkaline nitrobenzene oxidation of thioglycolic acid lignin eliminated both ρ-hydroxybenzoic acid and vanillin. The amount of ρ-hydroxybenzoic acid increased after eliciting with hot-water extract of conidia, but the amount of vanillin did not increase. The only phenolic substance liberated from thioglycolic acid lignin by alkaline hydrolysis was ρ-hydroxybenzoic acid and the amount was also increased by elicitation. The phenolic substances in dioxane lignin were ρ-hydroxybenzoic acid, vanillin, vanillic acid, and syringaldehyde, but only the amount of ρ-hydroxybenzoic acid was increased after elicitation. Phenolic substances liberated from cell wall materials by alkaline hydrolysis were predominantly ρ-hydroxybenzoic acid; ρ-hydroxybenzoic acid in particular increased in cell wall materials from cells elicited. The increased ρ-hydroxybenzoic acid formed ester-bonds via its hydroxyl and carboxy groups to cell wall materials. Induced lignification in cultured carrot callus cells indicated an increase of ester-bonded ρ-hydroxybenzoic acid in lignin precursor or wall polysaccharides rather than de novo synthesis of lignin.
Expression of the Ids2 (iron deficiency-specific clone 2) gene was compared between native barley and heterogenous tobacco regarding spatial specificity and iron-deficiency responsiveness. The results of Northern blot and in situ hybridization analyses demonstrated that the Ids2 expression in barley was specific to the endodermis and/or pericycle and in the cortex of roots, and responded exactly to iron starvation. Analysis of the GUS activity regulated by eight 5’-deletion clones of the Ids2 promoter in tobacco exhibited that every clone could promote root specific and iron-deficiency responsive expression. Aspects of the spatial specificity and iron-deficiency responsiveness (e.g. sensitivity, stability and returnability) in tobacco were also similar to those in barley, although a few variations were recognized. It is probable that even the shortest Ids2 promoter region in this experiment (-272/-47) contains the iron-deficiency-inducible and the root-specific cis-element(s), which is recognized by two different types of iron-acquisition strategy plants, barley and tobacco.
A 5822bp long cDNA clone encoding the full length ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 184.108.40.206) protein was isolated from roots of rice (Oryza sativa L. cv. Sasanishiki). Its sequence was identical to those of partial cDNAs for Fd-GOGAT from green leaves and shoots of rice. The predicted open reading frame (4848bp) encodes a 1616 amino acid protein with a molecular mass of 175034 Da that includes a 96-amino acid presequence. The combined nucleotide sequence of genomic clones for Fd-GOGAT isolated from rice was 20899 bp long and contained an entire structural gene, a 5672bp 5’-upstream region from the first methionine and a 779bp 3’-downstream region from the stop codon. The predicted transcribed region (15.4kb) consisted of 33 exons separated by 32 introns. The evolution among diverse GOGAT proteins, expression of Fd-GOGAT gene and intracellular localization of this protein in rice roots are also described.
Maize inbred H99 and A188 generally respond well in tissue culture, but only A188 had been efficiently transformed by Agrobacterium tumefaciens by the method previously described. The problem in transformation of H99 appeared to be a negative impact of infection with A. tumefaciens. Various conditions and additives to the culture media to promote callus induction from infected immature embryos were screened. Two major modifications, 1) addition of silver nitrate to the media for selection of transformed cells and 2) use of carbenicillin in place of cefotaxime to eliminate bacteria after infection, turned out to be most effective. A large number of transgenic plants of H99 were produced by the improved protocol based on these modifications, whereas no transformants were created by the original protocol in these genotypes. Integration and inheritance of the transgenes were confirmed by Southern hybridization and genetic analysis. Even the frequency of transformation of A188 was further improved by the new protocol.
The lectin activity of 30-kDa xylem sap protein (XSP30) found in cucumber (Cucumis sativas L.) xylem sap was analyzed by lectin blot coupled with immunological detection of XSP30. XSP30 is homologous to galactose-binding lectin, but it had only weak binding activity against asialofetuin, an animal glycoprotein with a terminal galactose. This reaction was not reduced by galactosidase treatement of asialofetuin. XSP30 bound strongly to soybean agglutinin, whose sugar chains consist solely of mannose and N-acetylglucosamine (GlcNAc), but bound only weakly to soybean peroxidase and γ-globulin, whose GlcNAc is fucosylated. The binding activity was inhibited by tri-N-acetylchitotriose (GlcNAc)3. Leaf parenchyma cells were stained with XSP30 and aniti-XSP30 antibody; this staining was reduced by proteinase treatment of the sections, and XSP30 bound to proteins in leaf particulate fraction. These results suggest that XSP30 transported from root via xylem sap binds to chitobiose, GlcNAc-GlcNAc, in N-linked glycans of leaf glycoproteins.
After the strawberry shoots on culture medium were cold-hardened at 5°C for 20-30 days, shoot tips were dissected and precultured at 5°C for 1 day on MS medium containing 2 M glycerol and 0.3M sucrose. Precultured 10 shoot tips were placed in 2.0ml cryotubes each, and treated with a mixture of 2M glycerol and 0.3M sucrose for 20min at 25°C. They were then treated with PVS2 solution for 50min at 25°C, and immersed into LN. After rapid rewarming in water at 35°C, they were transferred onto the MS medium containing 1gl-1 PVP and 0.2mgl-1 BA and grown under standard conditions. By this protocol, survival rate of cryopreserved “Donner” shoot tips reached 93%. This protocol was successfully applied to 7 other races or lines of strawberry with high survival rates. These results prompt us to setting up large-scale strawberry LN storage system.
To analyze the mechanisms of salt tolerance in the mangrove plant, Bruguiera sexangula, functional screening for mangrove cDNAs encoding proteins essential for salt tolerance was performed using Escherichia coli as the host organism. A transformant expressing a eukaryotic elongation factor 1A homologue (Bs-eEF1A) displayed enhanced tolerance to salt and osmotic stress. This distinct function was not conferred by other plant eEF1A homologues isolated from two halophytes (Suaeda japonica and Salsola komarovii) or Arabidopsis thaliana. Levels of Bs-eEF1A transcripts in cultured B. sexangula cells were enhanced in response to salt stress. These data suggest that Bs-eEF1A plays an important role in the salt-tolerance mechanisms of B. sexangula, in addition to its original function in elongation during translation.
Genetically transformed calli of paulownia (Paulownia fortunei) were generated at high frequency after co-cultivation of petiole segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for GUS and NPT II. The effects of acetosyringone, carbon sources and pH on the transformation of paulownia were examined. The presence of acetosyringone in the co-cultivation medium enhanced the induction efficiency of kanamycin-resistant calli. Sucrose was most suitable carbon source in the medium. The pH of the medium had no significant effect on the efficiency at pH 5 to 8. Successful transformation was confirmed by histochemical analysis of GUS activity in kanamycin-resistant calli, and by the detection of NPT II gene in the genome. Kanamycin-resistant calli induced from petiole segments had the ability of regeneration, however, the frequency of regeneration of shoot was very low.
A 20-kDa protein (p20) having 40% sequence similarity with Kunitz-type soybean trypsin inhibitor (STI) from Glycine max (soybean) cultured cells was expressed in Escherichia coli. The recombinant p20 (rp20) inhibited the activity of trypsin at the same level as STI (rp20, Ki=50nM; STI, Ki=75nM), and both rp20 and STI displayed non-competitive inhibition of trypsin activity. Although STI inhibited the activity of α-chymotrypsin (Ki=140nM) and elastase (Ki=207nM), rp20 did not inhibit the activity of α-chymotrypsin and elastase. These results show that p20 is a novel type of trypsin inhibitor.
Treatment of transformed Atropa belladonna root cultures with high concentrations of salicylic acid (SA) resulted in the production of methylated salicylic acid derivatives but not of glucosylated derivatives. This response was induced by 0.1mM SA whereas treatment with SA at concentrations higher than 1.2mM caused growth retardation. Nontransformed roots of A. belladonna responded to the addition of SA in almost the same manner as transformed roots, which suggests that this is a natural response in A. belladonna. We could not detect methylated SA in cultures of Nicotiana plumbaginifolia and Hyoscyamus niger. Besides SA, several aromatic compounds were methylated by the transformed roots of A. belladonna, and other responses, such as the conversion of benzoic acid to benzyl alcohol, were also observed.